Pharmac. Ther. Vol. 48, pp. 189-222, 1990 Printed in Great Britain. All rights reserved
0163-7258/90 $0.00+ 0.50 © 1990 Pergamon Press pie
Specialist Subject Editor: G. Powls
METABOLISM OF PYRIMIDINE ANALOGUES A N D THEIR NUCLEOSIDES GEORGE C. DAHER, BARRY E. HARRIS and ROBERTB. DIASIO Department of Pharmacology, Division of Clinical Pharmacology, University of Alabama at Birmingham, Birmingham, AL 35294, U.S.A. Abstract--The pyrimidine antimetabolite drugs consist of base and nucleoside analogues of the naturally occurring pyrimidines uracil, thymine and cytosine. As is typical of antimetabolites, these drugs have a strong structural similarity to endogenous nucleic acid precursors. The structural differences are usually substitutions at one of the carbons in the pyrimidine ring itself or substitutions at one of the hydrogens attached to the ring of the pyrimidine or sugar (ribose or deoxyribose). Despite the differences noted above, these analogues, can still be taken up into cells and then metabolized via anabolic or catabolic pathways used by endogenous pyrimidines. Cytotoxicity results when the antimetabolite either is incorporated in place of the naturally occurring pyrimidine metabolite into a key molecule (such as RNA or DNA) or competes with the naturally occurring pyrimidine metabolite for a critical enzyme. There are four pyrimidine antimetabolites that are currently used extensively in clinical oncology. These include the fluoropyrimidines, fiuorouracil and fluorodeoxyuridine, and the cytosine analogues, cytosine arabinoside and azacytidine.
CONTENTS 1. Fluoropyrimidines 1.1. Historical perspective 1.2. Chemistry 1.3. Uptake into cells 1.4. Metabolism 1.4.1. Catabolism 1.4.2. Anabolism 1.5. Metabolic site of action 1.5.1. Thymidylate synthase 1.5.2. RNA incorporation 1.5.3. DNA incorporation 1.5.4. Incorporation into cell membranes 1.6. Miscellaneous fluoropyrimidine drugs 2. Arabinosylcytosine 2.1. Historical perspective 2.2. Chemistry 2.3. Uptake into cells 2.4. Metabolism 2.4.1. Catabolism 2.4.2. Anabolism 2.5. Metabolic sites of action 2.5.1. Inhibition of DNA biosynthesis 2.5.2. Inhibition of DNA repair 2.5.3. Incorporation into nucleic acids 2.5.4. Inhibition of membrane precursor synthesis 3. Azacytidine 3.1. Historical perspective 3.2. Chemistry 3.3. Uptake into cells 3.4. Metabolism 3.4.1. Catabolism 3.4.2. Anabolism 3.5. Metabolic sites of action References 189
190 190 190 190 191 192 194 200 200 202 203 203 204 204 204 204 205 206 206 208 210 210 210 210 210 211 211 211 211 211 212 212 213 213
190
G.C. DAHERet al. 1. F L U O R O P Y R I M I D I N E S
Over the past few decades, much information has been accumulated on the metabolism and clinical pharmacology of fluoropyrimidines. 5-Fluorouracil (FUra) and 5-fluoro-2'-deoxyuridine (FdUrd) have been shown to be effective antimetabolites with antitumor activity in a number of different tumors including carcinomas of the gastrointestinal tract, breast, ovary, and skin. Extensive studies have produced a large volume of information on the metabolism of fluoropyrimidines but the relative importance of various metabolites in the antitumor activity of these drugs is still unclear. Also, despite many pharmacokinetic studies the best route or schedule of administration for fluoropyrimidines has not been determined. Several routes of administration are currently used including continuous infusion, bolus intravenous, intraperitoneal, and hepatic arterial infusion.
chemistry especially after it became apparent that FUra and its derivatives showed antitumor activity. In this review, our discussion of the chemistry of FUra will be narrowed to what is pertinent to its role as an anticancer agent. FUra differs from the naturally-occurring nucleic acid precursor, uracil, by the presence of a fluorine on the fifth carbon. In addition, the van der Waal's radius of the fluorine atom (1.35 A) is similar to that of hydrogen (1.2 A), thus minimizing alteration of the structural conformation and insuring that F U r a can be metabolized by the enzymes that metabolize uracil. The consequences of the substitution of the fluorine for the hydrogen at the fifth position of the pyrimidine ring will be discussed in detail in Section 1.5.1. The electronegativity of the fluorine atom on the ring increases the acidity of FUra, which is reflected by a lower pK a as compared to its natural analogue uracil (Berens and Shugar, 1963).
1.1. HISTORICAL PERSPECTIVE The fluoropyrimidine drugs (Fig. 1), represented primarily by the pyrimidine base FUra and its nucleoside FdUrd, were introduced over thirty years ago. In contrast to most chemotherapy agents, these drugs were rationally synthesized. The motivation for the design of these drugs came from an earlier observation that certain tumors (e.g. rat hepatomas) required exogenous uracil to sustain nucleic acid synthesis and, hence, tumor growth. This, in turn, led to the suggestion that pyrimidine analogues could be synthesized with physicochemical properties similar to uracil. Because of their structural similarity, these analogues can be taken up by cells and metabolized. However, the minor structural alteration may interfere with a critical enzymatic step or may replace the naturally-occurring pyrimidine nucleotides. Since most tumors are rapidly dividing these drugs may be selective for tumor cells in comparison to their normal counterparts. 1.2. CHEMISTRY
Since FUra was first synthesized by Duschinsky from acyclic precursors (Duschinsky et al., 1957), considerable attention has been focused on its bio-
0
Uracil 5-Fluorouracil
pKl 9.50 8.00
F U r a is commercially available in solution buffered with NaOH to yield an alkaline solution with a pH of approximately 9.0. Upon ultraviolet exposure, there is no evidence of dimer formation. However, in aqueous solution, FUra undergoes a photochemically induced reaction to form an alkalilabile, acid-soluble compound that has been identified as 5-fluoro-6-hydroxy-5,6-dihydrouracil (Lozeron et al., 1964). Exposure to wavelengths less than 270 nm causes secondary reactions of this initial product releasing the fluorine from the fifth carbon to form barbituric acid.
1.3. UPTAKE INTO CELLS
The pyrimidine base F U r a had long been thought to enter cells through a simple diffusion process achieving equilibrium rapidly between intracellular and extracellular compartments (Jacquez, 1962; Kessel and Hall, 1967). Subsequent studies (Wohlhueter et al., 1980) using rapid sampling techniques suggested that both F U r a and uracil enter the cell via a carrier-mediated transport process which
0
O H
H Uracil
pK2 > 13.0 ~ 13.0
5-Fluorouracll (FUrs)
OH 5 - F l u o r o - 2 ' - d e o x y u rldlne (FdUrd)
FIG. |. Structures of uracil, 5-fluorouracil and 5-fluoro-2'-deoxyuridine.
Pyrimidine analogues and their nucleosides appeared to be saturable and nonconcentrative. Concentrations which resulted in 'half saturation' were relatively high such that entry of either pyrimidine at pharmacological concentrations was first order. At relatively low concentrations of 5-fluorouracil, transport was not rate-limiting to intracellular metabolism of either pyrimidine. The nucleoside F d U r d is believed to enter cells via the same mechanism used by the naturally occurring nucleosides uridine and deoxyuridine. As with studies examining uptake of pyrimidine bases, these studies have been complicated by technical limitations, particularly the need for rapid sampling techniques to discriminate between transport and metabolism. This is due to the fact that the nucleoside is rapidly phosphorylated within the cells and, thus, cannot pass back out of the cell. The studies of Bowen et al. (1978) demonstrated that under pharmacologic conditions, transport of F d U r d was rapid and not ratelimiting. In contrast, metabolism of FdUrd appeared to be the rate-limiting step.
Liver end ..~ extrshepetlc tissues
191
-8O'/,
FUrs
1-3%
Anebollsm
1s-2o%) Urinary sx¢retlon
FIG. 2. Metabolic fate of 5-fluorouracil in humans. 1.4. METABOLISM
Fluoropyrimidines must be initially metabolized before they are clinically effective. Biotransformation may result in the formation of either less toxic catabolites or more toxic anabolites. In this review we will discuss the metabolism of pyrimidine analogues, their nucleosides, as well as, the biochemical modulation of their metabolism. In addition, we will review their sites of action.
H o r (F)
A
NAD(P)H'~
NAD(P)H
NAD(p),dk" ~ [ "~"NAD(P)
q~°'~o O
N.,C~.
FO
© 9
FIG. 3. Catabolism of 5-fluorouracil in humans. (1) Dihydropyrimidine dehydrogenase (DPD); (2) dihydropyrimidinase; (3) fl-alanine synthase; (4) bile acid n-acyl CoA transferase. FUPA = 5-fluoroureidopropionic acid; FBAL = 5-fluoro-fl-alanine; N-cholyI-FBAL = N-cholyl-5-fluoro-fl-alanine. JPT 48~2--F
192
G.C. DAHERet al.
The metabolic fate of administered FUra is given in Fig. 2. The level of FUra available for anabolism is primarily determined by catabolism (Heggie et al., 1987). The importance of catabolism has been demonstrated in clinical studies in which FUra was co-administered with other pyrimidines such as thymine or uracil resulting in increased availability of FUra and, in turn, increased anabolism (Au et al., 1982). Therefore, the balance between catabolism and anabolism is determined by several factors including: (1) the intracellular environment (e.g. presence of NADPH, ATP, PRPP) of the exposed cell; (2) the intracellular concentration of the substrate and/or its analogue; and (3) the activities of anabolic vs catabolic enzymes which are present at variable levels in different cell types. 1.4.1. Catabolbm
Catabolism has a major role in regulating the variability of fluoropyrmidines. This degradative pathway is identical to the reductive pathway of the naturally occurring pyrmidines, uracil and thymine. The presence of a halogen on the fifth carbon of the pyrimidine ring does not impede its catabolism and,
~ 0
H or (F)
surprisingly, FUra is more efficiently degraded than is uracil (Naguib et al., 1985). In this catabolic pathway (Fig. 3), the double bond between C-5 and C-6 of the pyrimidine ring is first reduced in the presence of NADPH. The ring is then hydrolyzed between N-3 and C-4 and the resulting ~-fluoro-flureidopropionate (FUPA) is subsequently cleaved between N-I and C-2 to yield ~-fluoro-fl-alanine (FBAL), carbon dioxide, and ammonia. The degradative pathway consists of three enzymes: (1) dihydropyrimidine dehydrogenase (DPD), (2) dihydropyrimidinase (DHPase); and (3) fl-alanine synthase. Each of these enzymes will be discussed separately. Halogenated pyrimidine analogues are usually dehalogenated in the course of their degradation and excreted in urine. In contrast, catabolism of FUra does not result in defluorination. Our laboratory has examined the clinical pharmacokinetics of 3H.FUra and its metabolites in plasma, urine and bile (Heggie et al., 1987). Over 24 hr, FBAL represented the major metabolite in urine accounting for 50% of the administered dose. Biliary excretion of radioactivity over 24 hr represented 2-3% of the total dose. A significant percentage (i.e. 80-90%) of these biliary metabolites were previously unrecognized (Heggie et al., 1987). These novel metabolites were later identified as conjugates of FBAL and either cholic acid (Sweeny
et al., 1987) or N-chenodeoxycholic acid (Sweeny et al., 1988). This previously unrecognized reaction is
catalyzed by cholyl-CoA-glycine/taurine N-acyltransferase (EC 2.3.1.13). This enzyme is present in the soluble fraction of liver and requires the prior activation of bile acid to a CoA derivative (Vessey et al., 1977). The isolation and characterization of this enzyme are currently in progress in our laboratory. 1.4.1.1. Dihydropyrimidine dehydrogenase. DPD (EC
1.3.1.2) is the initial enzyme in the degradation of the pyrimidine bases uracil and thymine. Specifically, it catalyzes the reversible reduction of the pyrimidine base to its corresponding dihydropyrimidine. This enzyme (eqn 1) also catalyzes the conversion of FUra to dihydrofluorouracil (FUHz). In contrast, cytosine and its analogues are not substrates for DPD. These agents must be deaminated to uracil by cytosine deaminase before their subsequent degradation. It is important to note, however, that cytosine deaminase is restricted only to micro-organisms and is not present in mammalian cells (O'Donovan and Neuhard, 1970).
0
~~L~y or (F) (1)
DPD activity is confined primarily to cytosolic supernatant fraction (Canellakis, 1956; Fritzson, 1960; Shiotani and Weber, 1981). It has been purified to homogeneity from rat liver cytosol (Shiotani and Weber, 1981). This enzyme is composed of two identical subunits with an approximate MW of 110 _+ 3 kDa each. DPD has been detected in normal human liver, pancreas, lung, intestinal mucosa, lymphocytes and in various neoplastic and hematopoietic tumors (Naguib et al., 1985). DPD activity in most tumors has been shown to be equivalent or higher than that determined in normal tissues from which the tumors derive (Maehera et al., 1981; Naguib et al., 1985; Weber, 1980). The pH optimum for rat liver DPD has been shown to be 7.4. In the presence of NADPH, the apparent Km was 2.6 pM for thymine and 1.8 #M for uracil. The apparent Km for NADPH was 15 #M with thymine as a substrate and 11 p M with uracil. The specific activity of DPD at 20 #M FUra was reported to be 47.4#mol/mg protein/hr. In contrast, the specific activity of DPD at the same concentration of uracil was 34.9#mol/mg protein/hr (Shiotani and Weber, 1981). In studies with rat liver, it has been suggested that DPD is the rate-limiting enzyme for pyrimidine catabolism (Canellakis, 1956; Fritzson, 1960, 1962; Queener et al., 1971). Other studies have suggested
Pyrimidine analogues and their nucleosides that dihydropyrimidinase is the rate-limiting enzyme in rat hepatocytes (Mentre et al., 1984; Sommadossi et al., 1982) and in human extrahepatic tissues and hematopoietic neoplasms (Naguib et al., 1985). Still other studies have suggested that fl-alanine synthase is the rate-limiting enzyme in mouse liver (Sanno et al., 1970; Traut and Loechel, 1984). Although it is still unclear which enzyme is rate-limiting, the
193
catalyzes the hydrolysis of a variety of 5,6-dihydropyrimidines (Kim et al., 1976; Wallach and Santiago, 1957), including the reversible conversion of FUH2 to FUPA (eqn 2), as well as catalyzing the hydrolytic ring opening of 5-phenylhydantoin and ~-phenylsuccinamide (Dudley et al., 1974; Maguire and Dudley, 1978). It has also been implicated in the dehalogenation of other pyrimidines (Kim et al., 1976).
O
H~
I
H or (F)
H2N~ ( ~ "~1 % or IF) (2)
presence of DPD activity is critical since its deficiency results in severe neurologic toxicity (Diasio et al., 1988; Tuchman et al., 1985). This toxicity was suggested to be due to an increased anabolism of FUra (Diasio et al., 1988). Inhibition of DPD activity has been shown to potentiate the effect of FUra in vitro and in vivo (Cooper et al., 1972; Desgranges et al., 1986; Masaaki et al., 1988; Matthes et al., 1974). 5-Benzyloxybenzyluracil and 1-deazauracil appear to be the most potent inhibitors of DPD activity with apparent Ki values of 0.2 and 0.5 #M, respectively (Naguib et al., 1989). In contrast the Km value for uracil has been reported to be 9/a M. The potential therapeutic benefit of inhibitors of DPD has been suggested in studies using bromovinyideoxyuridine in the modulation of FUra metabolism (Iigo et al., 1988). However, in light of the severe toxicity in patients with DPD deficiency, extreme caution should be used before administration of these compounds in conjunction with FUra chemotherapy. Further studies on these inhibitors are needed to determine their in vivo metabolism and possible side effects. Varying plasma concentration of FUra has been demonstrated during continuous infusion of FUra (Erlichman et al., 1986; Kawai et al., 1976; Petit et al., 1988). Our laboratory has also demonstrated that DPD activity in rat liver varies over a 24 hr period in association with light~zlark cycle (Harris et al., 1988). This may provide a biochemical explanation for the periodic variation of the plasma FUra level. In patients receiving FUra by protracted continuous infusion (300 mg/m2/day), we demonstrated an apparent inverse relationship between DPD activity in human peripheral blood mononuclear cells and plasma FUra levels (Harris et al., 1990). This association suggests that DPD may be a major determinant of the circadian variation of FUra plasma concentration when this drug is administered by protracted continuous infusion. 1.4.1.2. Dihydropyrimidinase. Dihydropyrimidinase (EC 3.5.2.2) is the second enzyme in the degradative pathway of uracil and thymine. It catalyzes the reversible hydrolysis of dihydrouracil to fl-ureidopropionic acid (fl-UPA). Dihydropyrimidinase also
Dihydropyrimidinase has been partially purified from calf (Wallach and Santiago, 1957) and rat liver (Maguire and Dudley, 1978) and, more recently, the bovine liver enzyme has been purified to homogeneity (Brooks et aL, 1983, 1979; Lee et aL, 1986). It was shown to be a tetramer with a MW of 226 kDa, and subunit molecular weight of 56.5 kDa. This enzyme contains 4 gram atoms of tightly bound Zn 2+ per mole of active enzyme with a Ks >1.3 x 1 0 9 M -1 (Brooks et al., 1983; Lee et al., 1986), presumably one gram atom Zn 2÷ per subunit. Previous work suggested that dihydropyrimidinase is a true Zn 2÷ metalloenzyme, since there exists a linear relationship over an enrichment range of 0 to 4 gram atoms of Zn 2÷ per mole of enzyme (Brooks et al., 1979). Chelation of Zn 2÷ by 2,6-dipicolinic acid, 8-hydroxyquinoline-5-sulfonic acid, or o-phenanthroline will cause the loss of dihydropyrimidinase activity (Brooks et al., 1983). The reaction pathway for this inactivation process involves the formation of a ternary enzyme-metal-chelator complex which then dissociates to yield the apoenzyme and the Zn 2÷chelate binary complex (Lee et al., 1986). Enzymatic activity will only be restored by reconstituting the hoioenzyme with the reincubation of the apoenzyme with Zn 2÷, Co 2÷ or Mn 2÷ (Brooks et al., 1983). Electron paramagnetic resonance studies have shown that substitution of Mn 2÷ for Zn 2÷ increases the specific activity of the enzyme approximately six-fold but only increases the Km value for 5-bromo-5,6-dihydrouracil (BrUH2) two-fold (Lee et aL, 1987). Like other metalloenzymes (e.g. carbonic anhydrase, dihydroorotase), dihydropyrimidinase is inhibited by several substituted sulfonamides. With 4-nitrobenzene sulfonamide, the inhibition was found to be competitive with respect to BrUH2 with a K~ value of approximately 0.6 mM (Brooks et al., 1983). 1.4.1.3. fl-Alanine synthase (fl-ureidopropionase). flAlanine synthase (EC 3.5.1.6), the third enzyme in the catabolism of pyrimidines, irreversibly cleaves flUPA (N-carbamyl-fl-alanine) to fl-alanine and generates carbon dioxide from the second carbon of the pyrimidine ring and ammonia from N-3. In addition, fl-alanine synthase also splits FUPA into FBAL (eqn 3).
194
G. C. DAHERet al. F
FO
(3)
H
fl-Alanine synthase was recently purified to homogeneity from rat liver, and was shown to have a MW in the range of approximately 323 to 327 kDa with a pH optimum for enzyme activity of 7.0 and a pl of 6.4 (Tamaki et al., 1987). Kinetic studies with this enzyme demonstrated that propionic acid, the structural analogue of fl-UPA, acts as an allosteric activator as well as a competitive inhibitor of fl-alanine synthase (Kikugawa et al., 1988). This derivative has a K~ of approximately 0.3 mM at pH 7.0; whereas, the Km for fl-UPA was 0.06 mM. A recent report (Matthews and Traut, 1987) suggested that fl-alanine synthase may exist as three different molecular weight species: a native form in the absence of ligands with a MW of 235 kDa; an active, higher MW form in the presence of its substrate fl-UPA (or its analogues); and an inactive, lower MW form in the presence of its product fl-alanine (or its analogues). It is thought that flalanine synthase contains at least one catalytic and one regulatory site. More work is needed in order to elucidate the complete mechanism of action of this enzyme.
1.4.2. A n a b o l i s m The anabolism of FUra to nucleotides occurs through one of several pathways (Fig. 4). As with catabolism, these pathways are identical to the de nova pathways used by the naturally-occurring pyrimidine, uracil. These include:
(l) a direct conversion catalyzed by pyrimidine phosphoribosyl transferase of FUra to FUMP by the transfer of ribose phosphate from phosphoribosyl pyrophosphate (PRPP) (Reyes, 1969),
(2) a sequential, two-step reaction consisting of the initial addition of a ribose by uridine phosphorylase to yield FUrd, followed by phosphorylation to FUMP by uridine kinase (Skrld, 1958), and (3) the addition of deoxyribose-l-phosphate to FUra by thymidine phosphorylase to yield FdUrd followed by phosphorylation to FdUMP by thymidine kinase (Hartmann and Heidelberger, 1961). 1.4.2. I. Uridine phosphorylase. Uridine phosphorylase (UrdPase; EC 2.4.2.3) catalyzes the conversion of the naturally occurring pyrimidine, uracil, to its nucleoside, uridine. FUra has also been shown to be
a substrate for this enzyme. As shown in eqn 4, UrdPase cleaves inorganic phosphate (P~) from ribose-l-phosphate (R-I-P) and, subsequently, forms a nucleoside bond between the pyrimidine base and the ribose. In addition to its anabolic role, this cytoplasmic enzyme catabolizes nucleotides that are critical to the 'salvage' of nucleic acids. In an attempt to explain this amphibolic role, Naguib et al. (1987b) proposed that the variables responsible for the catabolic or anabolic action of UrdPase include: (1) the activities of other enzymes which compete for the same substrate and (2) the intracellular concentration of substrates. UrdPase is relatively nonspecific, since it also cleaves pyrimidine 2'- and 5'-deoxyribose nucleosides (Niedzwicki et al., 1983). This enzyme is widely distributed in many rat organs, but is reported to have the highest activity in small intestine (Ardalan and Glazer, 1981).
Renal.. ~. excretion ~
y FU I ~
Liver and extrahepitlc tlaaue
/ eq,
1.3
Ftrl~a , ~ U r d
T..,L
-':Fd~MP
eCl'II eq. 10 eq. I~ FUD~ > FdUDP
"I
FUTP
RNA
.I
FdUTP DNA
FIG. 4. The metabolism of 5-fluorouracil in humans. Equations 1-3 = dihydropyrimidine dehydrogenase, dihydropyrimidinase, fl-alanine synthase respectively; eq. 4 = uridine phosphorylase; eq. 5=uridine kinase; eq. 6= orotate phosphoribosyltransferase; eq. 7 = thymidine phosphorylase; eq. 8 = thymidine kinase; eq. 9 = nucleoside monophosphate and diphosphate kinases; eq. 10 = ribonucleotide diphosphate reductase. (eq. corresponds to equation number in text.)
Pyrimidine analogues and their nucleosides
O
O
I1~ H
I H
195
H or IF)
H
(4)
In order to enhance the anabolism and effectiveness of fluoropyrimidine drugs, several UrdPase inhibitors have been synthesized. Recent evidence has shown that 5-ethyl-2,2'-anhydrouridine (ANEUR) is a potent inhibitor of UrdPase. When ANEUR (280 mg/kg for 3 days) or FUrd (5 mg/kg for 3 days) were administered as a single agent to mice bearing S-180 solid tumors, no decrease in tumor weight was observed. However, co-administration of FUrd with ANEUR (i.e. 5 mg/kg for 3 days and 280 mg/kg for 3 days, respectively) resulted in a 91% decrease in tumor weight vs untreated controls, suggesting that the potentiating effect of ANEUR resulted from inhibition of UrdPase (Verez et al., 1987). In addition, several benzylacyclouridine compounds have been shown to be potent inhibitors of UrdPase. These agents increased the level of plasma uridine as well as the salvage of uridine in various tissues resulting in decreased FUra toxicity (Darnowski and Handschumacher, 1985). Furthermore, other studies with fluoropyrimidines have shown that benzylacyclouridines increase efficacy and potentiate the selective toxicity of FdUrd (Chu et al., 1984). Of the various benzylacyclouridine compounds aminomethyibenzyloxybenzylacyluracil, hydroxymethylbenzyloxybenzylacyluracil, and aminomethylbenzylacyluridine were found to be the most potent inhibitors with a K~ of 18 nM, 70rim and 92riM, respectively. In contrast, the Km of UrdPase for uridine was found to be 242/~ M in human tissue and 143 pM in murine tissue (Naguib et al., 1987b).
O
H~
idine (FUrd) to 5-fluorouridine-5'-monophosphate (FUMP). As shown in eqn 5, uridine kinase transfers a phosphate group from ATP to the hydroxyl group on the 5' carbon of FdUrd. This reaction has been demonstrated to be rate-limiting in the pyrimidine salvage pathway (Anderson, 1973). In addition, phosphorylation of the ribose to form the nucleotide prevents its efflux through the cell membrane (Wohlhueter and Plagemann, 1980). Multiple forms of uridine kinase have been observed in normal and neoplastic tissues when crude or partially purified enzyme was further separated by gel filtration (Greenberg et al., 1977; Keefer et al., 1975; Krystal and Scholefield, 1973), by isoelectric focusing (Absil et al., 1980; Ahmed, 1982; Ullman et al., 1979), by ion exchange chromatography (Fulchignoni-Lataud et al., 1976; Sk61d, 1963) and by affinity chromatography (Vesely and Smrt, 1977). With the exception of one report (Krystal and Scholefield, 1973), it is generally believed that the different forms of the enzyme observed by these techniques represent isoenzymes. These isoenzymes have been shown to differ with respect to size, net charge and heat stability. The differential expression of such isoenzymes in rapidly proliferating tumors may be important in cancer chemotherapy. Subtle changes in the physical properties of uridine kinase in tumor cells may impair FUra anabolism (Richard et al., 1962) which may lead to the development of FUra resistance (Sk61d, 1963).
O
H or (F)
xr'F'
H
H
(5)
011011
1.4.2.2. Uridine k&ase. Uridine, the uridine phosphorylase, is anabolized uridine-5'-monophosphate by uridine 2.7.1.48). This enzyme also converts
O1-1Oit
product of further to kinase (EC 5-fluorour-
More recently, Payne et al. (1985) purified uridine kinase (~60,000-fold) from Ehrlich ascites tumor cells with phosphocellulose and adenosine5'-triphosphate agarose. With two-dimensional gel
196
G.C. DAI-'IERet al.
electrophoresis, this enzyme migrated as one band suggesting the presence of a single enzyme. Earlier studies by the same group had suggested that
O
I'~
sequence of the reaction. Since substrates are channeled from one active site to the next, side reactions are minimized.
O
,,.r,F,
H or (F) M~'+ H H
this enzyme may form a variety of aggregates in the presence of appropriate effectors such as substrates (e.g. ATP) and feedback inhibitors (e.g. UTP, CTP) to form different polymers rather than isoenzymes (Payne and Traut, 1982). Later, they (Cheng et al., 1986) proposed that uridine kinase activity is regulated by changing its polymeric state. Included in this model was an ailosteric regulatory site which governs the dissociation of the enzyme. Based on this model it was suggested that feedback inhibitors can specifically bind to the regulatory site producing conformational changes which lead to dissociation of the active polymeric uridine kinase into an inactive monomer. On the other hand, substrates such as ATP act as primary effectors by competing with CTP and binding specifically at the regulatory site. ATP may have a dual role: (1) phosphate donor, when binding to the active site and (2) stabilizing factor by binding the allosteric site and producing a positive change in polymerization and activity. While uridine kinase inhibitors may be of theoretical interest, no therapeutically effective inhibitors have been developed. 1.4.2.3. Orotate phosphoribosyltransferase. A more direct route for conversion of FUra to FUMP is catalyzed by the enzyme orotate-phosphoribosyltransferase (OPRTase; EC 2.4.2.10), an enzyme in the de novo pathway of nucleic acid biosynthesis. This enzyme catalyzes the condensation of FUra with PRPP to yield FUMP. In this reaction (eqn 6), pyrophosphate is hydrolized from PRPP resulting in sufficient energy to drive the reaction forward (Heidelberger, 1975; Reyes, 1969). OPRTase is associated with a bifunctional enzyme complex that also contains orotidylate decarboxylase (EC 4.1.1.23). Each of these enzymes comprise a particular domain of a single 55-60 kDa polypeptide (Reyes and Guganig, 1975). Clustering of enzymatic activity in the same protein moiety probably evolved by exon shuffling. The presence of multiple domains on a single protein may direct the
H
(6)
While it is clear that the anabolism of FUra to FUMP may proceed by either (1) OPRTase or (2) the sequential action of UrdPase and uridine kinase, there are conflicting reports in the literature as to which anabolic pathway is dominant. Many tumor cell lines (e.g. murine leukemia, murine colonic adenocarcinoma) have been shown to utilize primarily OPRTase (Ardalan et al., 1982; Kessel et al., 1972; Mulkins and Heidelberger, 1982), while other tumors have been demonstrated to utilize primarily uridine phosphorylase and uridine kinase (Ardalan et al., 1980; Houghton and Houghton, 1983; Schwartz et al., 1985). Recently, Naguib et al. (1987a) showed that OPRTase activity was higher in rapidly proliferating tumor cells than in normal lymphocytes or liver. They suggested that this enzyme should be considered as a marker for cell growth and maturation. Other studies with FU1-2 cells, a mouse T-lymphoma ($49) variant selected for its resistance to FUra, have shown diminished OPRTase activity (Levinson et al., 1979). In these clones, a three- to four-fold increase in the apparent Km of OPRTase for FUra was observed, which has been suggested to be due to a structural gene mutation. A variety of pyrimidine base analogues have been evaluated as inhibitors of OPRTase. 4,6-Dihydroxypyrimidine was observed to be a potent inhibitor (Niedzwicki et al., 1984) which can be used experimentally in elucidating the metabolic pathways of pyrimidine base analogues and assessing the dominance of these anabolic pathways. 1.4.2.4. Thymidine phosphorylase. In the presence of 2-deoxyribose-l-phosphate (dR-l-P), the conversion of uracil (Ura) or thymine to their respective nucleosides deoxyuridine and thymidine (eqn 7) are catalyzed by thymidine phosphorylase (dThdPase; EC 2.4.2.4). It should be noted however, that the conditions within most cells favor the reverse reaction, especially when appropriate amount of the pyrimidine deoxyribonucleoside donor is not provided.
Pyrimidine analogues and their nucleosides 0
197 0
I~
H or (F) I
H
H
(7)
While this step has been suggested to be an alternate route for the conversion of FUra to FdUrd (eqn 7), the quantitative importance of this step has been questioned since little evidence for FdUrd formation has been observed even after administration of large doses of FUra in vivo (Woodcock et al., 1980). However, co-administration of thymidine with FUra leads to significant FdUrd formation, suggesting that transferase activity is limited by availability of cosubstrate (the deoxyribose donor) and is zero order with respect to FUra. It was originally believed that the specificity of this enzyme was restricted to pyrimidine-2'-deoxyribonucleosides, as pyrimidine ribonucleosides are not cleaved by this enzyme (Niedzwicki et al., 1983). However, it has been shown that dThdPase is not only specific for pyrimidine-2'-deoxyribonucleosdies but is also capable of releasing the sugar from 5'-deoxyfluorouridine (Iltzsch et al., 1985; Kono et al., 1983). The activity of dThdPase has been reported to be generally higher in normal liver or lymphocytes than in tumors. This activity increased upon treatment of the tumor with maturational agents (Naguib et al., 1987a) and this activity was observed to increase during the course of maturation of rat intestinal cells and acute myeloid leukemia cells in vitro (Imondi et al., 1969; Palu, 1980). Higher activity has been detected in mature peripheral blood cells than in rapidly proliferating cells such as blast cells, bone marrow, acute and chronic lymphocytic leukemic cells (Fox et al., 1979; Gallo and Seymour, 1969b; Marsh and Perry, 1964; Rabinowitz and Wilhite, 1969). Palu (1980) suggested that dThdPase should be considered as a possible marker for cellular maturity. This enzyme has been purified from a variety of mammalian sources (Desgranges et al., 1981; Gallo and Seymour, 1969a; Krenitsky, 1968; Kubilus et al., 1978; Zimmerman, 1964). A detailed kinetic study was undertaken in mouse liver (Iltzsch et aL, 1985) to elucidate its mechanism of action. This study indicated that in the presence of thymine, both phosphate and thymidine 0
~ H
or (F)
have 'cooperative effects' on dThdPase. Thus, this enzyme may have multiple allosteric binding sites that interact cooperatively. Studies of the concentration-dependent degradation of thymidine by intact platelets suggested that dThdPase follows Michaelis-Menton kinetics with an apparent Km of 0.10-0.14 mM. This phosphorolysis of thymidine is competitively inhibited by various C-5or C-6-substituted uracils. 6-Amino-5-bromouracil, 6-aminothymine, 5-bromouracil, and thymine were the most active inhibitors with a Ki of 6, 11, 28 and 270/~M, respectively. In acellular extracts the K i of these inhibitors was 2, 8, 15, and 72 #M, respectively (Desgranges et al., 1982). Intracellular degradation of thymidine may decrease its availability for anabolism limiting the production of thymidine-5'-triphosphate (dTTP). An increase in the dTTP pool will inhibit ribonucleotide reductase and, thus the conversion of cytidine-5'-diphosphate to deoxycytidine-5'-diphosphate needed for DNA synthesis. The potential role of dThdPase in the regulation of thymidine metabolism has been suggested from both in vivo (Kufe et al., 1980a) and in vitro (Fox et al., 1979) studies in lymphoid cells. Resistance to thymidine in Blymphoblasts differed from sensitive myeloblasts and T-lymphoblasts in having a ten-fold increase in dThdPase activity as well as a ten-fold decrease in thymidine kinase activity (Kufe et al., 1980a). Therefore, resistance to thymidine-induced toxicity is more likely to be due to an increase in the ratio of dThdPase to thymidine kinase activities than to an increase in dThdPase activity alone. 1.4.2.5. Thymidine kinase. Thymidine kinase (dThd kinase; EC 2.7.1.75) catalyzes the phosphorylation of not only thymidine but also a number of other structurally similar compounds. As shown in eqn 8, FdUrd is also a substrate for dThd kinase and is anabolized to FdUMP. Similar to uridine kinase, this enzyme also requires Mg 2÷ to complex with ATP and drive the reaction through this rate-limiting step. 0
H~
H or (F)
(8)
198
G.C. DArmRet al.
In human tissue, dThd kinase is present as 2 isoenzymes termed dThd kinase-1 and dThd kinase-2 (Show et al., 1979). Gel electrophoresis of subcellular fractions from fetal liver have shown that dThd kinase-1, the 'fetal' form, migrates slowly on the gel and is associated with the cytoplasmic fraction. In contrast, dThd kinase-2, the 'adult' form, migrates rapidly on the same gel and is associated with the mitochondrial fraction (Kit and Leung, 1974; Taylor et al., 1972). These two isoenzymes also differ in sedimentation coefficient, pH optimum, inhibition by dCTP and phosphate donor specificity (Berk and Clayton, 1973; Kit, 1976; Kit and Leung, 1974; Taylor et al., 1972). It is interesting to note that during cellular proliferation, the increase in DNA synthesis is accompanied by an increase in dThd kinase activity, thus indicating that this enzyme may be useful as a marker of the transition from the quiescent to the replicative phase. The activity of the dThd kinase- i isoenzyme increases when G] or G Ophase cells are stimulated or infected with oncogenic viruses (Kit, 1976). Variation in dThd kinase activity also occurs in response to hormonal influence (Bourtourault et al., 1984; Masui and Garren, 1971). It has been shown that human dThd kinase from both normal and leukemic leukocytes (chronic mylogeneous, chronic lymphocytic and monocytic leukemia) is competitively inhibited by dTTP, a substrate for DNA polymerase (Breznick and Karjala, 1964). In this study, the apparent Km for thymidine was 3.6 x l0 5M, while the Ki for dTTP was 1.5 × 10 -6 M. In addition, other studies (Breitman, 1963; Fiala et al., 1962; Ives et al., 1963) have also shown that dTTP regulates the enzymatic activity of thymidine kinase by feedback inhibition. These studies suggest that dTTP can inhibit dThd kinase either allosterically or isosterically (Monod et al., 1963). Inhibition of dThd kinase activity, regardless of the
type, may be detrimental therapeutically. The resulting decrease in anabolism of nucleoside analogues can impair the chemotherapeutic effectiveness of these drugs. Recently, Fisher et al. (Fisher and Baxter, 1981; Fisher et al., 1983, 1988; Fisher and Phillips, 1984) demonstrated that 5'-amino derivatives (e.g. 5'-aminothymidine) could reverse the inhibition of dThd kinase produced by dTTP, thereby stimulating nucleoside phosphorylation. These findings are particularly relevant to the design of new nucleoside analogues and anatagonists of allosteric inhibition. In addition, other inhibitors with different sites of action have been studied. Celiptium ~ (N-2-methyl-9hydroxyellipticinium acetate) is an ellipticine derivative used in the therapy of breast cancers. This drug is believed to exert its action on the dThd kinase- 1 gene instead of the active or regulatory sites of the enzyme (El Hiyani et al., 1987). It has been suggested that Celiptium "~may interfere with the nuclear binding site of the estrogen receptor and, consequently, suppress the induction of dThd kinase synthesis. Therefore, stimulation of dThd kinase-1 activity by estradiol (Bourtourault et al., 1984) will be inhibited. 1.4.2.6. Nucleoside monophosphate and diphosphate Nucleoside monophosphate kinase (NMP kinase; EC 2.7.4.4) and nucleoside diphosphate kinase (NDP kinase; EC 2.7.4.6) occupy pivotal positions in biosynthesis of pyrimidine nucleotides, since their substrates are products of both de noro and salvage pathways. Like other phosphotransferases in subgroup 2.7.4, ATP is utilized as the phosphate donor for the conversion of each nucleoside 5'-monophosphate to its corresponding di- and triphosphate. As shown in eqn 9, these two enzymes participate in both ribosyl and deoxyribosyl nucleotide formation catalyzing the generation of 5-fluoro(deoxy)uridine diphosphate [F(d)UDP] and 5-fluoro(deoxy)uridine triphoshate [F(d)UTP].
kinase.
O
O
(eq.9)
OH
H or (OH)
H or (OH)
(9)
I~ JL ~or(F) 0 ~
OH
H or (OH)
(ADPM ' g+~
199
Pyrimidine analogues and their nucleosides Partial purification and characterization of these two enzymes (Imazawa and Eckstein, 1979; Nakamura and Sugino, 1966) have shown that both enzymes lack specificity for either the base or sugar moiety of nucleotides. Neither the 2'- nor the 3'-hydroxyl moiety of natural nucleotides appear to be necessary for pyrimidine nucleoside mono (or di) phosphate kinase activity. This broad specificity for different congeners is appealing for the rational synthesis of nucleotide analogues that may be phosphorylated to nucleotide precursors of nucleic acids. In comparison to the activity of NMP kinase, the activity of NDP kinase is greater (Sugino et al., 1966). An elevated level of NDP kinase activity has been shown in normal, nonproliferating rat liver tissues (Bianchi et al., 1964; Chiga et al., 1963; Nakamura and Sugino, 1966) and rat or human erythrocytes (Bianchi et al., 1964; Mourad and Parks, 1966) as well as growing tissues such as Novikoff hepatoma cells (Ives, 1965), regenerating rat liver (Chiga et al., 1963), rat ascites tumor cells (Nakamura and Sugino, 1966), and Landschutz-ascites tumor cells (Grav and Smellie, 1963). By contrast, NMP kinase has been shown to have a significantly lower specific activity in a resistant murine leukemia (P388/FUra) cell line (Ardalan et al., 1980). Thus, it is conceivable that phosphorylation of nucleoside monophosphate to nucleoside diphosphate is the rate-limiting step in the formation of nucleoside triphosphates. 1.4.2.7. Ribonucleotide reductase. Ribonucleotide reductase (EC 1.17.4.1) is also a key enzyme in the synthesis of DNA. It is highly regulated and solely responsible for the de novo synthesis of deoxyribonucleoside diphosphates from their corresponding ribonucleoside diphosphates. In addition it also converts the fluorinated nucleotide FUDP to FdUDP and thus it is important in fluoropyrimidine metabolism. The mechanism of this enzyme is more complex than depicted in eqn 10. The mammalian enzyme consists of two dissimilar subunits called protein M1 and M2. Protein MI is a 170 kDa dimer that contains the allosteric binding sites (Thelander et al., 1980). Protein M2 is an 88 kDa dimer. Each monomer of M2 contains a nonheme iron center and a stable tyrosyl radical cation. The presence of this radical is critical for its catalytic activity, since the addition of hydroxyurea, a quencher of free radicals, destroys the enzymatic activity of the holoenzyme. However, the tyrosyl radical can be readily regenerated upon incubation of the radical-free protein with iron-dithiothreitol in the presence of air (Thelander et al., 1985). o
Ribonucleotide reductase is a cell-cycle dependent enzyme with maximal enzymatic activity during Sphase (Thelander and Reichard, 1979). Using synchronized cell populations, variation in enzymatic activity has been shown to be regulated by both de novo synthesis and degradation of the M2 subunit (Eriksson et al., 1984). In contrast, the M1 protein is expressed at a constant level and is present in excess throughout the cell cycle (Mann et al., 1987, 1988). Other studies utilizing monoclonal and polyclonal antibodies directed against M I and M2 subunits demonstrated localization of these two proteins in the cytoplasm (EngstrSm and Rozell, 1988; Engstr6m et al., 1984). Furthermore these immunocytochemical studies (Engstr6m et al., 1984) demonstrated that the M1 protein is only present in actively proliferating cells and is absent in fully differentiated cells. Hence, immunofluorescent studies of the M1 subunit may be promising as a marker for cell proliferation in normal, inflammatory, or neoplastic tissues. Additional biochemical studies from mouse T-lymphoma cells demonstrated the existence of two independent regulatory domains that can be genetically altered on the M1 protein. As shown in Fig. 5, one regulatory site is responsible for the overall activity of the enzyme through binding of ATP (activator) or dATP (inhibitor), and the other is responsible for substrate specificity through binding of ATP, dGTP and dTTP (Wright, 1983). It has also been shown that dATP can bind the substrate specificity site of M 1 and mimic the effects observed with ATP (Thelander et al., 1980). The antitumor agent, hydroxyurea, can passively diffuse into mammalian cells (Tagger et aL, 1987) where it specifically inhibits ribonucleotide reductase by destroying the M2 tyrosyl radical (McClarty et al., 1987; Thelander, et al., 1985). However, resistance to hydroxyurea may develop. This has proven useful as a selective agent in tissue culture to isolate drug-resistant cell lines with altered ribonucleotide reductase activity. Recent molecular and cellular characterization studies of two chinese hamster ovary cell lines have shown that resistance to cytotoxic concentrations of hydroxyurea was correlated with increased levels of ribonucleotide reductase (Tagger and Wright, 1988). Electron paramagnetic resonance measurements of free radical levels and studies with M l-specific antibodies suggested that the elevation in enzyme activity was entirely due to an increase in the presence of the M2 component. Furthermore, studies with M2 cDNA from both low and high resistant cell lines indicated that elevation in the M2 message could o
(10)
200
G.C. DAHERet al.
M1
~>
H
Substrate binding sites R" Tyrosyl free radical
FIG. 5. Model of ribonucleotide diphosphate reductase depicting two subunits, M~ and M2. also explain the observed increase in synthesis of the M2 subunit. Elevation of M2 mRNA in the most resistant cell line was shown to result from gene amplification (Tagger and Wright, 1988). Patterns of cross-resistance in hydroxyurea-resistant L1210 cells were examined for sensitivity to a variety of inhibitors specifically directed at the individual subunits of ribonucleotide reductase (Carter and Cory, 1988). HU-7-S7 L1210 cells were cross-resistant to 2,3-dihydro-IH-pyrazole[2,3-a]imidazole. However, it is of interest that they were sensitive to inhibitors (e.g. 4-methyl-5-amino-l-formyl-isoquinoline thiosemicarbazone and 1-isoquinolylmethyleneN-hydroxy-N'-aminoguanidine tosylate) that are specifically directed at the same subunit as hydroxyurea (Cory and Fleisher, 1979; Weckbecker et al., 1988). These cells retained their sensitivity to deoxyadenosine/erythro-9-(2-hydroxy-3-nonyl)adenine and deoxyguanosine/8-aminoguanosine through intracellular accumulation of dATP and dGTP, respectively (by their subsequent allosteric inhibition of ribonucleotide reductase) (Carter and Cory, 1988). It has been shown previously (Cory and Carter, 1988) that L1210 cells selected for resistance to deoxyadenosine/erythro-9-(2-hydroxy-3-nonyl)adenine were cross-resistant to deoxyguanosine/8-aminoguanosine, 2-fluorodeoxyadenosine and 2-fluoro-9-flD-arabinofuranosyladenine, but retained their sensitivity to hydroxyurea, 4-methyl-5-amino-l-formylisoquinoline thiosemicarbazone and 1-isoquinolylmethylene-N-hydroxy-N'-aminoguanidine tosylate. These studies suggest that development of resistance at the ribonucleotide reductase site is limited to the type of agent used and the subunit of reductase interacting with the inhibitor. Inhibition of ribonucleotide reductase with these agents can affect the metabolism of fluoropyrimidine and hence, modify its anticancer activity. 1.5. METABOLIC SITE OF ACTION
1.5.1. Thymidylate Synthase Thymidylate synthase (TS; EC 2.1.1.45) is crucial for the de novo synthesis of thymidylate (dTMP) needed for DNA synthesis. Specifically, this enzyme
is responsible for the methylation of dUMP to dTMP, with the concomitant oxidation of NS,N ~°methylenetetrahydrofolate (NS,N~°-CH:-H4 PteGlu) to dihydrofolate (H2PteGlu). NS,NI°-CH2-H4PteGIu serves both as a one-carbon donor and as an electron donor. Through this reaction a methyl group is added to the fifth position of dUMP. During this reduction step, two electrons are donated from the ring of the tetrahydrofolate moiety itself as a hydride ion (H:-). Recently, the molecular basis of TS-fluoropyrimidine interaction has been thoroughly reviewed (Cisneros et al., 1988; Heidelberger et al., 1983). The catalytic mechanism consists of a sequence of three different steps (Fig. 6). The first step initials the formation of a covalent bond between the sixth position of dUMP (or FdUMP) and a sulfhydryl group in the catalytic site of the enzyme (Hardy et al., 1987). Formation of this covalent adduct activates the vinylic fifth carbon of the pyrimidine ring for condensation with the methylene group of NS,N ~°CH2-H4PteGIu. In the second step there is an electrophilic attack of the NS,N~°-methylene moiety on the activated fifth position of the pyrimidine ring. However, in the presence of FdUMP, the ternary complex (i.e. the active site of TS, FdUMP, and the folate cofactor; Fig. 6) does not usually proceed past this point resulting in a 'suicide-like' inhibition. The third step is a reduction reaction that involves /3 elimination and hydride ion transfer to an intermediate that acts as a hydride ion acceptor (Danenberg and Lockshin, 1981). In this step, the electron withdrawing effects of fluorine on the fifth position of the pyrimidine ring of FdUMP, not only serves to activate the C-6 position for attack by the enzyme, but also stabilizes the above intermediate, and thereby, prevents the complex from advancing any further (Cisneros et al., 1988). The bonding in this ternary complex containing FdUMP (Fig. 7), is extremely 'tight' with a Kd of approximately 10 ~2M, suggesting that only one molecule of the complex will dissociate for every trillion molecules of complex formed (Danenberg and Danenberg, 1978; Lockshin and Danenberg, 1981; Santi and Danenberg, 1984). Under certain circumstances, however, TS will spontaneously promote the dissociation of this complex, demonstrating that ternary complex formation is reversible and FdUMP is not a 'suicide' inhibitor.
Pyrimidine analogues and their nucleosides
201
~CH iH~ PteGlu) H ~
or L
-H °r (F)
~'j~"
~),~-i---~
Le-I'o"th*c"po°l'lOno
FIG. 6. The catalytic mechanism of thymidylate synthase. (1) Initial formation of a covalent bond between substrate and enzyme active site; (2) electrophilic attack of methylene group of NS,N~°-methylenetetrahydrofolate (CH2H4PteGIu) on the 5th position of the pyrimidine ring; (3) ternary complex formation of 5-FdUMP, thymidylate synthase and the folate cofactor; H2PteGlu = dihydrofolate. Investigations into the physical characteristics of TS and its interaction with the cofactor and substrates have delineated its mechanism of action. Mammalian TS is actually a dimeric protein (MW = 68 kDa) with an active site on each monomer (Moran, 1988). As shown in Fig. 8, during the
formation of ternary complex, TS must first bind its substrate (i.e. dUMP or FdUMP) before its contact with the cofactor. Once the substrate is bound, a binding site for the cofactor is created, and the interaction between all three components may then occur.
5,10-METHYLENETETRAHYDROFOLATE I
i
I
PTERIN
H I
~l~
"N"
~ . FdUMP
PABA
I
II
OH2 ~
I
.N.~ O
GLUTAMATE
II
~
/
\
O
,
I
(CH2)2
i
//~C-NH-CH COOH
I
O
II HO--P-O--~
I OH
OH
FIG. 7. Ternary complex of N 5, Nl°-methylenetetrahydrofolate, 5-fluoro-2'-deoxyuridinemonophosphate (FdUMP) and thymidylate synthase.
202
G.C. DAHERet al.
"x FIG. 8. The ordered sequential addition of substrates and cofactors to mammalian thymidylate synthase: 1 = binding of dUMP to active site of thymidylate synthase; 2 = creation of binding site for cofactor; 3 and 4 = covalent binding of enzyme, substrate, and cofactor; 5, 6 and 7 = release of product, free enzyme, and oxidized cofactor dUMP = 2'-deoxyuridine-5'-monophosphate; TMP = thymidine-5'-monophosphate; H2PteGlu = dihydrofolate; H4PteGlu = tetrahydrofolate; ser = serine; gly = glycine; CH2H4PteGlu = NS,N l°-methylenetetrahydrofolate. The affinity of TS for either substrates (i.e. dUMP and FdUMP) is nearly identical. When FdUMP is bound TS will temporarily be inactivated. During this time, dUMP synthesis will continue, leading to an accumulation of dUMP. When the FdUMP-ternary complex subsequently dissociates, the accumulated dUMP will then compete for binding to the newly-released TS, thus replacing FdUMP at the active site, permitting dTMP synthesis to resume. Recent studies have shown that FdUMP bound to TS has a half-life of approximately 2 hr in LI210 leukemia cells. However, addition of leucovorin (5-formyltetrahydrofolate) prior to or simultaneously with fluoropyrimidines, results in an increased half-life of complex formation (tl,, 2 >35 hr) with a concomitant decrease in thymidine nucleotide pools and increased cell death (Keyomarsi and Moran, 1988). Intracellularly, leucovorin is reduced to NS,N]°-CH2 H4PteGlu, thereby providing the optimal reduced folate concentration necessary for stable ternary complex formation (Bleyer, 1989). Recent evidence has shown that the intracellular level of NS,NI°-CH2 H4PteGlu in human tumors is insufficient for complete inhibition of TS in the presence of adequate FdUMP (Bleyer, 1989; Houghton et al., 1981; Ullman et al., 1978). However, several studies (Garmont et al., 1988; Laufman et al., 1987; Petrelli et al., 1987) have shown that the response rates were consistently higher with concomitant administration of FUra and leucovorin. Such a regimen has been suggested to be beneficial in patients with metastatic colorectal adenocarcinoma (Petrelli et al., 1987).
1.5.2. R N A Incorporation Shortly after FUra was introduced, additional biochemical studies demonstrated the incorporation of this agent into RNA (Chaudhuri et al., 1958). Over the years it has become apparent that FUra inhibits mRNA processing or its nuclear-cytoplasmic transport (Armstrong et al., 1986). Other studies have also shown that FUra causes an irreversible inactivation of tRNA methyltransferase. This enzyme is responsible for the transfer of a methyl group from Sadenosyl-methionine (SAM) to the 5-carbon of a specific Urd residue to form the m5U (ribosylthymine) moiety found in TIp C loop (loop IV) in tRNA (Santi and Hardy, 1987). Additional studies on RNA from other species have shown that in vitro exposure of mouse LI210 cells to 10 6M FUra inhibited the maturation process of rRNA precursors (Kanamaru et al., 1986). This is consistent with earlier studies where rRNA maturation was also inhibited at the 45S and 32S regions causing accumulation of preribosomal RNA (Wilkinson and Crumley, 1977; Wilkinson et al., 1975; Wilkinson and Pitot, 1973). Recently, a study on the action of FUra on L1210 cells concluded that FUra inhibits: (1) the processing of pre-rRNA to rRNA; (2) the methylation of pre-rRNA and tRNA; and (3) the synthesis of poly(A)RNA of mRNA. Furthermore, it impairs the synthesis of snRNA and enhances the translation of poly(A)RNA (Kanamaru and Wakui, 1988). In addition, a recent study by Danenberg et al. (1990) suggested that FUra destabilizes the active
Pyrimidine analogues and their nucleosides conformation of RNA by substitution, thereby forming a weaker base pairing between FUra and adenine due to partial ionization of FUra residues. In vitro and in vivo studies utilizing combinations of fluoropyrimidines with 'biochemical modulators' have been carried out in order to differentiate between the DNA and RNA toxicity and to determine the contribution of specific RNA targets to the mechanism of action of these drugs, dThd is one such modulator that has been studied extensively as an adjuvant of fluoropyrimidines (Au et al., 1982; Danhauser and Rustum, 1979; Speigelman et al., 1980a,b; Sternberg et al., 1984; Takimoto et al., 1987). It is especially important, since RNA-mediated cytotoxicity is not reversed by dThd such that the block at the ternary complex (i.e. fluoronucleotide-[NS,Nl°-CH2-H4PteGlu]-TS) may be bypassed, providing an alternate source of dTTP for DNA synthesis. Characterization of the effects of fluoropyrimidines on RNA and/or RNA processing is perhaps very important clinically. However, the therapeutic relevance of such a 'modulator' is questionable since there have been many conflicting results. Several studies have demonstrated that modulation of FUra cytotoxicity by dThd could improve the therapeutic efficacy of the antimetabolites in certain murine tumors (CD2F1 colon tumor 26 and CD8F1 mammary carcinoma) (Speigelman et al., 1980a,b). In addition, the incorporation of FUra into RNA can also be augmented by inhibiting the de novo pyrimidine synthesis and lowering UTP pools with agents such as phosphonacetyl-L-aspartic acid (PALA). This drug causes a 60-80% decrease in UTP levels in tissue culture cells (Johnson et al., 1978; Moyer and Handschumacher, 1979) and four-fold increase of FUra incorporation into RNA in vivo (Spiegelman et al., 1980b). In addition, it acts synergistically to yield a greater than ten-fold increase in FUra incorporation when administered in combination with dThd. However, other studies where the combination of dThd and FUra was investigated in tumor models (Danhauser and Rustum, 1979, 1984) and in patients with advanced colorectal carcinoma (Au et al., 1982; Sternberg et al., 1984) showed no significant increase in the therapeutic index. These studies provide insights into the mechanism of action of FUra in various tumor models and cell lines. In colon carcinoma (Au et al., 1982; Sternberg et al., 1984), the combination of FUra with dThd failed to produce a significant improvement in the therapeutic efficacy of FUra. These results collectively suggest that incorporation of FUra into RNA may be more important to antitumor efficacy.
1.5.3. D N A Incorporation Throughout the 1970s, it was widely believed that the fluoropyrimidines, mainly FUra and FdUrd, act through the mechanisms above, (i.e. inhibition of thymidylate synthase by FdUMP and incorporation into RNA) (Heidelberger, 1975; Speigelman et al., 1980a,b). Later the isolation of (FUra)-DNA was demonstrated in both tumor and normal cells treated with FdUrd or FUra in tissue culture (Danenberg et
203
al., 1981; Herrick et al., 1982; Ingraham et al., 1982; Kufe et al., 1981; Schuetz et al., 1986). During this
time, other studies have shown that pretreatment with dThd in vivo decreases the amount of FUra incorporated into DNA, suggesting that FdUTP is a substrate of DNA polymerase (Sawyer et al., 1984). The apparent absence of intracellular FdUTP and the minimal amount of FUra in DNA have been explained by the recognition that FdUTP is a substrate for the enzyme deoxyuridinetriphosphate nucleotide hydrolase (dUTPase) and by the demonstration that uracil glycosylase is able to cleave FUra from DNA respectively (Caradonna and Cheng, 1980a,b; Ingraham et al., 1980). However, it should be noted that uracil glycosylase appears to be extremely specific for uracil compared to FUra (Caradonna and Cheng, 1980b). This implies that incorporation of FUra into DNA should occur more frequently than its naturally occurring analogue (i.e. uracil). Since the cleavage of fluoropyrimidine-DNA derivatives is much slower than their natural counterparts, it is to be expected that the former is more stable than the latter. Further studies on DNA as a site of action of fluoropyrimidines have demonstrated that under certain experimental conditions, FdUrd can be incorporated into DNA and may be partially responsible for its cytotoxic effects (Kufe et al., 1983). It is probable that such toxicity may result from misincorporation of FUra into DNA or from the excision-repair of these adducts by uracil glycosylase which can potentially lead to mutagenesis and eventual cytotoxicity. In our laboratory, we have demonstrated that DNA repair occurs subsequent to incorporation of FUra into DNA (Schuetz et al., 1988). However, such repair may not be fully effective, leading to inhibition of DNA elongation (Schuetz and Diasio, 1985) and DNA fragmentation (Schuetz et al., 1986). In this latter study, we demonstrated that FUra resulted in time-dependent fragmentation of bone marrow DNA with an associated time-dependent excision of FUra from DNA. This relationship between FUra removal and DNA chain degradation is consistent with the mechanism proposed in which FUra is excised from DNA by uracil glycosylase with subsequent repair of the apyrimidinic site by apyrimidine endonuclease (Caradonna and Cheng, 1980a,b). Other studies have also shown that fluoropyrimidine, specifically 5fluorodeoxycytidine, can incorporate into DNA and is associated with inhibition of DNA methylation and decrease in clonogenic survival (Kaysen et al., 1986).
1.5.4. Incorporation into Cell Membranes In addition to inhibition of TS and incorporation into DNA and RNA, it has also been reported that FUra can produce cell surface modifications (Kessel, 1980). Such alteration in cellular membranes is suggested to be due to FUDP-hexoses which may alter membrane structure by imparing glycoprotein biosynthesis (Kessel, 1980; Peters et al., 1984). Another study utilizing HeLa cells treated with FUra revealed a dampened oscillatory response in the transmembrane potential. In contrast, the 6-potential or surface charge of these cells were altered to a lesser extent by FUra exposure (Walliser and Redmann,
204
G.C. DAHERet al.
1978). Based on the hypothesis that the electrical potential of the cell membrane exerts an effect on mitotic activity of the cell, the authors suggested that the effect of FUra on the cell membrane is related to inhibition of cell growth. However, the relative importance of this cell membrane effect on cell growth has not been well studied and its contribution to toxicity remains to be established. 1.6. MISCELLANEOUSFLUOROPYRIMIDINEDRUGS
In addition to the fluoropyrimidines discussed above (i.e. FUra and FdUrd), several other fluoropyrimidine analogues have been investigated. These include compounds such as 1-(tetrahydro-2-furanyl)5-fluorouracil (i.e. ftorafur) and 5'-deoxy-5fluorouridine (5'dFUrd). Ftorafur is believed to function as a depot form of FUra and is converted to FUra within target cells (Au et al., 1979; Benvenuto et al., 1978). Initial studies with ftorafur (Hall et al., 1977) revealed gastrointestinal symptoms and neurotoxicity as the major clinical toxicities without the myelosuppression typically observed with FUra administration. Although utilized in many Eastern countries, its clinical activity compared to FUra has not been thoroughly evaluated and its use is still experimental in the United States. 5'dFUrd has shown promising antitumor activity and increased specificity for tumor cells as compared to normal tissues (Bollag and Hartmann, 1980). The substitution of an OH group at the 5' position for the CH2OH present in ribose sugars prevents further activation to a nucleotide. Instead, it must be converted to FUra by a nucleoside phosphorylase before it can be further anabolized to a nucleotide (Armstrong and Diasio, 1980). An additional hypothetical advantage is that 5'dFUrd may be active as an oral agent (Chabner, 1982). Clinical trials with 5'dFUrd are in progress in Europe and Japan.
2. ARABINOSYLCYTOSINE Arabinosylcytosine (araC; l-fl-D-arabinofuranosylcytosine; cytosar; cytarabine), an arabinose nucleoside, is primarily used in combination with anthracyclines to induce remission in patients with acute myeloblastic leukemia (Ellison et al., 1968). AraC has also been shown to have activity in other human tumors including chronic granulocytic leukemia (particularly blast crisis), acute lymphocytic leukemia, and histiocytic lymphoma (Bryan et al., 1974; Chabner and Myers, 1985). The metabolism of araC in tumor cells and its selective activity against rapidly growing tumors has limited its usefulness in the treatment of most solid malignancies (Chabner and Myers, 1985). AraC is profoundly affected by pharmacologic parameters such as dose, schedule of treatment, and route of administration (Ho and Freireich, 1975). These affects, in turn, can be attributed to araC metabolism in host and tumor cells. In addition to its activity against hematological leukemias, araC has been found to be useful in the treatment of leukemic or carcinomatous meningitis when administered intrathecally (Band et al., 1973; Spiers and Firth, 1972). Lastly, araC has been shown
to have activity against several DNA viruses (Shouval et al., 1986).
2.1. HISTORICALPERSPECTIVE Arabinosyl derivatives of pyrimidines were first isolated by Bergmann and Feeney from a Caribbean sponge, Cryptotethya crypta (Bergmann and Feeney, 1950, 1951). The isolated compounds were the 1-fl-Darabinofuranosyl derivatives of thymine and uracil (i.e. araT and araU). In contrast, araC has not been detected as a natural substance (Cohen, 1966). Numerous studies have focused on the antitumor activity of this unique class of compounds. The first report suggesting antitumor activity reported that araC inhibited growth in Escherichia coli and that this growth inhibition was reversed by pyrimidine nucleosides (Slechta, 1961). AraC was first shown to have antitumor activity in a variety of mouse tumors including leukemia L1210, Sarcoma 180, Ehrlich ascites, L5178Y lymphoma, and T 4 lymphoma (Evans et al., 1961, 1964). In another report araC inhibited growth in L1210, P288, P388, and K1964 mouse tumours (Dixon and Adamson, 1965). Thus, a large spectrum of mouse tumours was initially tested and a total of 38 were sensitive to araC. 2.2. CHEMISTRY
AraC is a synthetic nucleoside which differs from the natural nucleosides cytidine and deoxycytidine in that the sugar moiety is an arabinose rather than a ribose or deoxyribose, respectively (Fig. 9). AraC has a molecular weight identical to that of cytidine and the arabinose sugar is a pentose sugar with the same side groups as cytidine. The arabinose sugar moiety differs from ribose in that its 2' hydroxyl group is
OHOH Cytldlne
OH Deoxy©ytldlne
I IH 2
S) OH Arablnosyloytollne
Azloytldlne
FIG. 9. Structures of cytidine, deoxycytidine and cytidine analogues, arabinosylcytosine and azacytidine.
Pyrimidine analogues and their nucleosides
// Ar I 1 - ~
f•
205
dATP"I '--.I.-.-O.A
Ara-C 1-~-D- ArB-CMP 4---~-ArI.CDP-~.-e- Arlg-CTP
/1[ lrlI-U 'l.
l' Ara-UMP
FIG. 10. Transport and metabolism of arabinosylcytosine in humans. (1) Deoxycytidine kinase; (2) cytidine deaminase; (3) deoxycytidylatedeaminase; (4) deoxycytidylate kinase; (5) nucleoside diphosphate kinase; (6) DNA polymerase ct. oriented in the beta (fl) position. The inverted position of the 2' hydroxyl group causes the arabinose to 'act like' a deoxyribose sugar and, thus, its metabolism is more like deoxycytidine than cytidine. 2.3. UPTAKE INTO CELLS A prerequisite for araC-induced cellular toxicity is the transport of araC into the target cell (Fig. 10). Early studies of nucleoside transport were hindered by rapid metabolism within the cell. Equilibrium between intracellular and extracellular compartments is thought to occur within 90 sec with the intracellular nucleoside undergoing rapid phosphorylation to nucleotide analogues. Thus, it is important to separate the intracellular nucleoside from other metabolites, particularly nucleosides, in order to quantitate transport. In 1968, Kessel and Shurin (Kessel and Shurin, 1968) isolated a subline of L1210 murine leukemia cells which lacked the ability to phosphorylate either deoxycytidine or araC. This subline was utilized to study the transport of these two nucleosides. Saturation kinetics and structural specificity were clearly demonstrated with inhibition by other nucleosides suggesting that a mediated process was involved in nucleoside entry. The efflux of labeled nucleotides was determined to be a two-phase process with an initial rapid phase followed by a much slower phase (Kessel and Shurin, 1968). The rapid phase was inhibited by high intercellular levels of other nucleosides (e.g. thymidine, uridine, etc.) indicating a process with structural specificity and saturability (Kessel and Shurin, 1968). Subsequent studies have confirmed that nucleosides, as well as araC and other nucleoside analogues, enter the cell via a high Kin, facilitated diffusion system that seems to transport all ribonucleosides and deoxyribonucleosides, but with somewhat different efficiencies (Wohlhueter and Plagemann, 1980). Competitive kinetics were observed between araC and deoxycytidine for entry into leukemic blasts (Wiley et al., 1982, 1983, 1985) and cultured Novikoff rat hepatoma cells (Marz et al., 1977; Plagemann et al., 1978b). In addition, influx of araC into human leukemic blasts was blocked by phloretin, a broad-spectrum inhibitor of facilitated transport systems (Wiley et al., 1983).
The transport kinetics of araC have been studied in detail in a variety of malignant cells such as Novikoff hepatoma cells (Marz et al., 1977; Plagemann et al., 1978b), Yoshida sarcoma cells (Mulder and Harrap, 1975), and human leukemic blast cells (Wiley et al., 1982, 1983, 1985) and cultured Novikoff rat hepatoma ceils (Marz et al., 1977; Plagemann et al., 1978b) In addition, influx of araC into human leukemic blasts was blocked by phloretin, a broadspectrum inhibitor of facilitated transport systems (Wiley et al., 1983). The transport kinetics of araC have been studied in detail in a variety of malignant cells such as Novikoff hepatoma cells (Marz et al., 1977; Plagemann et al., 1978b), Yoshida sarcoma cells (Mulder and Harrap, 1975), and human leukemic blast cells (Wiley et al., 1982, 1983, 1985). Plagemann et al. (1978b) used a subline of wild-type Novikoff rat hepatoma cells that lacked nucleoside kinase activities. With the appropriate experimental conditions, the ATP level in these cells was maintained at less than 1% of the concentration typically observed and no substrate phosphorylation occurred. Lineweaver-Burke analysis showed that araC transport followed normal Michaelis-Menten kinetics with an apparent Km of 450#M and an apparent Vmax of 30pmol//~l cell H : O x sec. Mulder and Harrap (Mulder and Harrap, 1975) had similar findings in Yoshida sarcoma ceils but suggested that in these cells the facilitated diffusion process operated at two concentration ranges with a Km of 400/~M and 2000 #M. Passive diffusion only became a significant factor in the uptake of araC at extracellular concentrations above 1 mM. In human leukemic blasts, kinetic analysis of araC uptake yielded a Km of approximately 225/~M which was significantly higher than their normal counterparts (lymphocytes, K m = 50/~M; polymorphonuclear cells, K m = 140/IM) and positively correlated to the Vmax (Wiley et al., 1983). Based on these observations, it appears that the rate of transport of araC into the cell is 10 to 100 times faster than the rate of intracellular phosphorylation suggesting that phosphorylation rather than transport is the rate-determining step (Plagemann et al., 1978b; Wiley et al., 1985). However, there is some evidence that membrane transport may be a limiting factor in araC
206
G.C. DAHERet al.
chemotherapy based on studies that demonstrate that the number of cell nucleoside transport sites correlates closely with araC influx (Wiley et al., 1983, 1982) as well as clinical responsiveness to araC therapy (Wiley et al., 1982). The number of nucleoside transport sites on cells may be estimated by measuring the equilibrium binding of [3H]-nitrobenzylthioinosine (NBMPR). This compound is a nucleoside analogue known to have a high affinity for the nucleoside transport protein but is not transported into the cell. At extracellular concentrations below 1 #M, a close correlation was demonstrated between the maximal number of nucleoside binding sites (i.e. NBMPR binding sites) per cell in T lymphoblastic lymphoma blasts, myeloblasts, and lymphoblasts (Wiley et al., 1985) and the level of araCTP accumulation in these cells suggesting that membrane transport may be one of the major rate-limiting steps for araCTP accumulation at low extracellular concentrations (Heichal et al., 1979; Wiley et al., 1985). Also, Tanaka and Yoshida (Tanaka and Yoshida, 1987) examined araCTP accumulation, metabolic enzyme activities, and the nucleoside transport capacity in eleven human leukemic cell lines with different phenotypes. The sensitivity of the leukemic cell lines to araC (measured by clonogenic survival) correlated with the amount of araCTP formed within the cell. The accumulation of araCTP correlated with the nucleoside transport capacity in the leukemic cells, while no correlation was observed with the levels of enzymes involved in araC metabolism (deoxycytidine kinase, pyrimidine monophosphate kinase, and deoxycytidylate deaminase). An alteration in araC transport is one possible mechanism for acquired resistance to araC during chemotherapy and is supported by the isolation of murine lymphoma cells with defective transport
metabolized by the enzymes involved in the natural processing of these nucleotide precursors. The balance between activating (i.e. anabolic) and inactivating (i.e. catabolic) enzymes is crucial in determining the quantity of drug converted to the active intermediate, araCTP (Fig. 10). 2.4.1. Catabol&m Opposing the activation of araC to araCTP are two enzymes: cytidine deaminase (EC 3.5.4.5) and deoxycytidylate deaminase (EC 3.5.4.12). The respective contribution of these enzymes, either collectively or independently, in the regulation of the catabolism and intracellular retention of araC in intact cells has not been thoroughly investigated. In addition, nucleoside tri- and diphosphatases are assumed to be involved in the conversion of araCTP to araCMP. However, these pathways are not well defined and will not be discussed in this paper. The conversion of araCMP to araC by 5'-nucleotidase is another plausible pathway for dephosphorylation but will not be discussed. 2.4.1.1. Cytidine deaminase. Cytidine deaminase (EC 3.5.4.5) catalyzes the deamination of cytidine and deoxycytidine. Initial studies with intravenously administered araC in human cancer patients (Smith et al., 1959) demonstrated that araC was cleared very rapidly from the bloodstream. Since the rate of disappearance could not be accounted for by urinary excretion, it was postulated that araC must be metabolized to an inactive product or products. Subsequent studies with human liver homogenates (Camiener and Smith, 1965) demonstrated that araC was deaminated to an inactive product, uracil arabinoside (araU; eqn 11) by cytidine deaminase.
(11)
OH
systems for purine or pyrimidine nucleosides (Cass et al., 1981; Cohen et al., 1979). However, in a variety of lymphoblasts known to be resistant to araC, no significant changes in nucleoside transport characteristics were noted (Young et al., 1985) and the frequency of altered nucleoside transport in cells resistant to araC has not been determined. 2.4. METABOLISM As with most antimetabolites, metabolism has a major role in determining the therapeutic effectiveness of araC. The close structural similarity of araC to cytidine and deoxycytidine (Fig. 9) permits it to be
OI4
Cytidine deaminase is widely distributed in mammalian tissues including liver, kidney, heart, intestinal mucosa, and granulocytes (Camiener and Smith, 1965; Chabner et al., 1974; Chou et al., 1975) as well as various tumors (Camiener and Smith, 1965; Chabner et al., 1973; Hart et al., 1972; Ho and Frei, 1971). High levels of enzyme activity have been reported in normal liver and kidney (Camiener, 1967; Camiener and Smith, 1965) and in some human solid tumor samples (Cheng and Capizzi, 1982). The relatively high specific activity of cytidine deaminase found in most solid tumors may account for the poor response of such tumors to conventional doses of araC. In contrast, acute and chronic myelocytic leukemia cells
Pyrimidine analogues and their nucleosides contained significantly less cytidine deaminase than normal circulating granulocytes (Chabner et al., 1974) which may explain the increased cytotoxic response of these cells to araC. Cytidine deaminase has been partially purified and characterized from a number of sources and cells including bacteria (Ashley and Bartlett, 1984; Vita et al., 1985), mouse kidney (Creasey, 1963; Tomchick et al., 1968), mouse spleen (Malathi and Silber, 1971), human, canine, chicken, and sheep liver (Fanucchi et al., 1986; Vita et al., 1987; Wisdom and Orsi, 1969), and human normal and leukemic granulocytes (Chabner et al,, 1974). In humans, cytidine deaminase activity is highest in the liver. Kinetic analysis of human liver prepared from freshly frozen autopsy material yielded Km values of 12#M, 19pM, and 87/~M for cytidine, deoxycytidine, and araC, respectively (Chabot et al., 1983). Utilizing the same substrates, Fanucchi et al. (Fanucchi et al., 1986) reported Km values of 49pM, 55 #M, and 270#M, respectively, for cytidine deaminase isolated from human liver. Cytidine deaminase purified from normal and leukemic granulocytes (Chabner et al., 1974) had similar Michaelis constants with Km values of 11/~M, 26/~M, and 88 pM for cytidine, deoxycytidine, and araC, respectively. Comparison of cytidine deaminase from normal, acute myelocytic leukemic, and chronic myelocytic leukemic granulocytes yielded no biochemical or kinetic differences among the different cell types but showed significantly lower levels of enzyme in leukemic cells as opposed to normal granulocytes (Chabner et al., 1974). Tetrahydrouridine (THU) and araU are both potent inhibitors of cytidine deaminase (Capizzi et al., 1983; Ho et al., 1975; Yang et al., 1985). The K~ for THU was reported to be 3 x 10-sM (Stoller et al., 1978). Since cytidine deaminase is the primary catabolic enzyme responsible for the inactivation of
207
is an inhibitor of cytidine deaminase (Ki = 1 x 10-5 M) and decreases the inactivation of araC in both normal and malignant tissues that contain cytidine deaminase (Drake et al., 1980). The emergence or expansion of a population of araC resisant cells is probably the major cause of failure in araC chemotherapy. Alteration in the activity of cytidine deaminase has been suggested to have a role in either de novo or acquired resistance (Steuart and Burke, 1971), but the clinical relevance of this data has been questioned (Harris et al., 1981; Smyth et al., 1976). In a study with myeloid leukemic cells, Harris et al. (1981) suggested that araC in the medium was freely accessible to both metabolic pathways (anabolic and catabolic) and that changes in deaminase activity resulted in a decrease in the concentration of araC in the medium and not a decrease in intracellular araC. Although cells with a high deaminase/kinase ratio were apparently resistant to araC, when the concentration of araC remaining in the medium was plotted against the concomitant inhibition of DNA synthesis all cells had similar sensitivity to araC. Therefore, provided an adequate extracellular concentration of araC was maintained, cells with high deaminase activity would be as sensitive as those with much less deaminase activity. 2.4.1.2. Deoxycytidylate deaminase. Deoxycytidylate deaminase (EC 3.5.4.12) is the enzyme responsible for the deamination of CMP to UMP and provides an additional route for the catabolism of araC. l-fl-D-arabinofuranosylcytosine 5"-monophosphate (araCMP) may be deaminated to 1-fl-D-arabinofuranosyluridine 5'-monophosphate (araUMP) by deoxycytidylate deaminase (Fig. 7) with the subsequent release of l-~-D-arabinofuranosyluracil (araU) (E1lims et al., 1981, 1983). The reaction catalyzed by deoxycytidylate deaminase is given in eqn 12.
(12)
OH araC, inhibition of this enzyme may lead to increased intracellular levels of araC and, subsequently, increased araCTP. Recently, Avramis and Powell (Avramis and Powell, 1987) designed a study to determine if inhibition of cytidine deaminase would enhance the tumor cellular anabolism of araC to araCTP in tumor-bearing mice. In their study, pretreatment with THU and/or araU increased plasma araC concentrations but did not significantly affect the intracellular concentration of araCTP in the tumor cells nor did it affect the antitumor activity of araC. In addition to THU and araU, 3-deazauridine JPT 48/2--G
Deoxycytidylate deaminase has been highly purified from a number of sources including human leukemia cells (Ellims et al., 1983), human spleen (Ellims et al., 1981), donkey spleen (Geraci et aL, 1967), chick embryo extracts (Maley and Maley, 1964, 1968) and bacteria (MoUgaard and Neuhard, 1978; Sergott et al., 1971; Socca et al., 1969). The characterization of deoxycytidylate deaminase isolated from this variety of sources has revealed variations in the physical and kinetic properties of this enzyme relative to either species specificity or the unstable nature of the enzyme. Deoxycytidylate
208
G.C. DArmR et al.
deaminase purified from human leukemic CCRFCEM cells exists as a dimer with a native MW of 108 kDa and subunits of 51 kDa each (Ellims et al., 1983). Deoxycytidylate deaminase had the greatest affinity for dCMP, with less affinity for araCMP, and least affinity for cytidine monophosphate (CMP) (Ellims et al., 1983). The enzyme is regulated primarily by two allosteric effects, an inhibitory effect by deoxycytidine triphosphate (dCTP) and an activating effect by thymidine triphosphate (TTP) and araCTP. A possible effect of administration of araC would be to stimulate deoxycytidylate deaminase activity which may lead to an increase in dUMP pools and, in turn, may increase TMP and TTP pools. TTP has been shown to activate GDP reductase and inhibit CDP reductase and thymidine kinase (George and Cory, 1979). Therefore, one of the possible effects of araC may be alteration of deoxyribonucleotide pools leading to inhibition of DNA synthesis. Reports have shown that araCMP is an effective substrate of deoxycytidylate deaminase isolated from cultured CCRF-CEM human lymphoblastic leukemia cells (Drake et al., 1980; Ellims et al., 1983) and human spleen (Ellims et al., 1981), while araCMP is neither an inhibitor nor a substrate for enzyme isolated from acute myelocytic leukemia (Mancini and Cheng, 1983). Such differences in the characteristics of this important nucleotide-metabolizing enzyme add to the complexity of the antitumor activity of araC in human leukemic cells. Similar to the inhibition of cytidine deaminase by THU, deoxycytidylate deaminase is also inhibited by deoxytetrahydrouridine (dTHU). While, inhibition of cytidine deaminase by THU did not result in an increase in the accumulation of araCTP, inhibition of deoxycytidylate deaminase by dTHU resulted in an increased accumulation of araCTP from araC in CCRF-CEM human leukemia cells of approximately 50% over that of araC alone (Fridland and Verhoef, 1987). These studies also indicated that deoxycytidylate deaminase functions as the major pathway for araC nucleotide catabolism in these cells. Interestingly, deoxycytidylate deaminase did not contribute significantly to araC nucleotide catabolism in PF-2S lymphoblasts despite high levels of dCMP and araCMP deamination in extracts of these cells (Fridland and Verhoef, 1987) suggesting that another pathway (e.g. 5'-nucleotidase) was operating in these cells and at a much higher rate than in the CEM lymphoblasts. In addition to dTHU, several purine and pyrimidine nucleotides are competitive inhibitors of deoxycytidylate deaminase such as dGMP (Ki= 1.8 × 10-4M), dUMP (Ki= 1.2 x 10-3M) and TMP (Ki = 1.4 × 10 -3 M) (Ellims et al., 1981). A potentially important mechanism of resistance to araC chemotherapy is a marked expansion of dCTP pools due to increased CTP synthetase activity or deficiency of deoxycytidylate deaminase activity (Momparler et al., 1968). The increased intracellular concentration of dCTP may contribute to araC resistance by feedback inhibition of deoxycytidine kinase and/or competition with araCTP for insertion into DNA and subsequent inhibition of DNA synthesis. In addition, increased activity of
deoxycytidylate deaminase may provide a mechanism of acquired resistance by depletion of arabinosyl nucleotides and increased inactivation of araCMP to araUMP. 2.4.2. Anabolism Following entry into the cell, araC must be anabolized to araCTP in order for it to be effective as an anticancer agent. Because of the similarity of araC to the naturally-occurring nucleoside, deoxycytidine, it is anabolized by the same anabolic pathway. AraC is initially converted to araCMP by deoxycytidine kinase, than araCMP is converted to araCDP by deoxycytidylate kinase, and, finally, araCDP is converted to araCTP by nucleoside diphosphate kinase (Fig. 10). Formation of araCTP and maintenance of the intracellular pool of the araCTP are two factors that are clearly important in determining the antitumor effectiveness of araC (Preisler et al., 1985; Rustum and Preisler, 1979). 2.4.2.1. D e o x y c y t i d i n e kinase. Deoxycytidine kinase (EC 2.7.1.74) is the initial enzyme in the anabolic pathway and is responsible for the conversion of deoxycytidine to deoxycytidylate in the presence of ATP. Upon exposure of the cell to araC, this enzyme also catalyzes the conversion of araC to araCMP (eqn 13). Deoxycytidine kinase isolated from cultured human T-lymphoblasts has been shown to have a MW of 60 kDa with a requirement for divalent ions (particularly, Mg 2+ or Mn 2+) (Datta et al., 1989). Of the three enzymatic steps in the formation of araCTP, deoxycytidine kinase is believed to be rate-limiting due to its relatively low intracellular concentration. The Km for the naturally occurring substrate, deoxycytidine, has been estimated to be 7.8/~M, while the Km for araC is thought to be approximately 20 # u (Coleman et al., 1975; Hande and Chabner, 1978). Deoxycytidine kinase is inhibited strongly by deoxycytidine (Ki = 1.7 x 10 -7 M) and dCTP (Ki = 7.3 × 10 -6 M) and weakly by araCTP (K~ = 1.3 × 10 -7 M). As the levels of dCTP rise, deoxycytidine kinase is allosterically inhibited. High levels of dCTP can compete with araCTP for incorporation into DNA (Yang et al., 1985). A decrease in the dCTP pool by pretreatment with hydroxyurea (Walsh et al., 1980), deazauridine (Mills-Yamamoto et al., 1978), or high doses of thymidine (Danhauser and Rustum, 1980) has been shown to potentiate the cytotoxicity of araC. Deoxycytidine kinase is an important site of metabolic resistance to araC chemotherapy. In murine cell lines, deficiency of deoxycytidine kinase is the most frequently observed cause of drug resistance (Chu and Fischer, 1965; Drahovsky and Kreis, 1970; Schrecker and Urshel, 1968). Also, human tumors deficient in deoxycytidine kinase have been shown to be resistant to araC (Tattersall et al., 1974). However, reduced deoxycytidine kinase activity is not always demonstrated in araC-resistant cells and it is unclear how frequently deoxycytidine kinase deficiency occurs in man. In a study of araC-resistant and araCsensitive murine leukemia cells, Momparler et al. (1968) failed to find significant differences in araC
209
Pyrimidine analogues and their nucleosides
(13)
OH
OH
phosphorylation between these two cell lines. The resistant cell line had an increased dCTP pool which may contribute to araC resistance by feedback inhibition on deoxycytidine kinase and/or competition with araCTP for insertion into DNA. 2.4.2.2. Deoxycytidylate kinase. The second step in the anabolism of araC is the conversion of araCMP to araCDP (eqn 14) catalyzed by deoxycytidylate kinase (EC 2.7.4.14). Deoxycytidylate kinase has been purified from rat Novikoff ascites hepatoma (Maness and Orengo, 1976), rat liver (Maness and Orengo, 1975), calf thymus (Sugino et al., 1966), Yoshida sarcoma cells (Arima et al., 1977) and human leukemic blast cells (Hande and Chabner, 1978). A MW of 28 kDa was reported for deoxycytidylate kinase from leukemic blasts (Hande and Chabner, 1978), while 17 kDa has been reported for enzyme isolated from rat liver (Maness and Orengo, 1975). Studies with various pyrimidine nucleoside monophosphates and analogues have also shown that
NH~
a single enzyme is responsible for the phosphorylation of CMP, dCMP, UMP, dUMP, araCMP, FUMP, FdUMP, and araUMP. Variations in enzyme levels and substrate affinity have been shown in cells from different sources (Hande and Chabner, 1978). Blast cells isolated from normal human donors contained a mean level of deoxycytidylate kinase activity of 1.2/tmol/hr/mg protein (Hande and Chabnet, 1978). Higher levels were reported in normal human lymphocytes (18 #mol/hr/mg protein) but a different substrate was used in this study (Scholar and Calabresi, 1973). Both studies, however, reported a two-fold increase in leukemic cells compared to their normal counterparts. Levels of deoxycytidylate kinase were 100-fold higher in human leukemic blasts than deoxycytidine kinase (Coleman et al., 1975; Hande and Chabner, 1978). In addition, deoxycytidine kinase had considerably higher affinity for its substrates, deoxycytidine (Km= 7.8 × l0 -6) and araC (Kin = 2.6 x 10-5) than deoxycytidylate kinase had for its substrates,
NH2
(14)
Oil
210
G. C. DAHERet al.
dCMP (Km= 1.9 x 10 -3) and araCMP (Kin = 6.8 x 10-4). Therefore, deoxycytidylate kinase is probably not the rate-limiting step in the activation of araC. However, it has been suggested that under conditions of low araCMP concentration (i.e. less than 10 -5 M), deoxycytidylate kinase may become rate-limiting (Hande and Chabner, 1978). 2.4.2.3. Nucleoside diphosphate kinase. Nucleoside diphosphate kinase (NDP kinase; EC 2.7.4.6) utilizes ATP as a phosphate donor in the conversion of nucleoside 5'-diphosphates to their corresponding nucleoside 5'-triphosphates. NDP kinase is also responsible for the conversion of araCDP to araCTP, the active metabolite in araC chemotherapy (eqn 14). NDP kinase was discussed previously in Section 1.4.2.6. 2.5. METABOLICSITESOF ACTION Biochemical studies into the mechanisms by which araC exerts its cytotoxic effect on cells reveal that araC acts as an analogue of deoxycytidine and has multiple effects on DNA synthesis. Identification of the specific event which produces cellular toxicity is difficult because of the complexity of the biochemical and cellular processes responsible for cell replication and cell function. A correlation between the occurrence of a specific event and clonogenic cell survival is the initial step in the identification of the mechanism or mechanisms of drug-induced cytotoxicity. Studies with araC have focused on several specific area including inhibition of DNA biosynthesis, inhibition of DNA repair, incorporation into nucleic acids (i.e. RNA and DNA), and inhibition of membrane precursor synthesis. 2.5.1. Inhibition o f DNA Biosynthesis Following formation of araCTP from araC, araCTP has been shown to competitively inhibit the interaction of dCTP and DNA polymerase • in a variety of tissues including calf thymus (Furth and Cohen, 1968), murine tumors (Graham and Whitmore, 1970a; Kimball and Wilson, 1968), and human leukemic cells (Inagati et al., 1970). AraCTP and dCTP have relatively equal affinity for DNA polymerase c~ in the range of 1 x 10-sM (Momparler, 1972) and the inhibition is reversed by the addition of dCTP in cell-free systems or deoxycytidine in intact cells (Chu and Fischer, 1962; Kinahan et al., 1979). However, enzyme kinetic studies indicate that araCTP is a weak competitive inhibitor of DNA polymerase :¢(Graham and Whitmore, 1970b; Momparler, 1972). Since DNA synthesis can be inhibited to approximately 50% by araCTP levels 100-200-fold lower than that required for competitive inhibition of DNA polymerase ~, there must be additional mechanisms by which araC inhibits DNA synthesis. 2.5.2. Inhibition o f DNA Repair AraCTP has only a slight inhibitory effect on DNA repair. The K~ for inhibition of DNA polymerase fl (i.e. the DNA repair enzyme) is 26/~M (Dunn and Regan, 1979). The affinity of dCTP, the natural
substrate for the polymerase, is five times that of araCTP (Muller, 1977). Repair inhibition is reversed by 50% when araC is removed and by 9 5 o if deoxycytidine is added following araC exposure (Dunn and Regan, 1979). Therefore, inhibition of DNA excision repair by araCTP is significant only if dCTP pools are depleted by additional pharmacologic manipulation (Dunn and Regan, 1979; Hiss and Preston, 1978). Fram and Kufe (Fram and Kufe, 1982) showed that the inhibition of DNA repair leads to the accumulation of single strand breaks in the DNA that increased with both araC concentration and exposure time. This data supports a model that has been proposed relating inhibition of repair with cytotoxicity (Stenstrom et al., 1974). In addition to its effects on eukaryotic polymerases, araCTP is an extremely potent inhibitor of viral RNA-directed DNA polymerase with a Ki = 0.1 /~M (Muller, 1977). 2.5.3. Incorporation into Nucleic Acids The effects of araC on DNA include not only the inhibition of DNA elongation and repair at the polymerase step but also disruption of DNA function secondary to incorporation of araCTP into DNA (Kufe and Major, 1982). Although earlier studies had indicated that incorporation of araC into nucleic acids (DNA or RNA) did not correlate with araC-induced cytotoxicity (Graham and Whitmore, 1970a), more recent studies (Kufe et al., 1980b) revealed a relationship between the incorporation of araC into DNA and cell lethality in L1210 cells in culture. This relationship suggested that the probability of cell survival was about 50% when 0.66 pmol. araC was incorporated into DNA. This is equivalent to about 5 araC nucleotides per 104 bases in DNA. A similar relationship has also shown for a human promyeloblast cell line and myeloblasts isolated from a patient with acute myelogenous leukemia (Major et al., 1981). This process is very complex and it is difficult to understand the mechanisms of araC cytotoxicity based on these observations. Ross et al. (1987) noted that in studies of blast cells from patients with acute nonlymphocytic leukemia the intracellular araCTP concentration was not linearly correlated with the incorporation of araCTP (into DNA). This finding may be the result of increasing inhibition of DNA polymerase ~ by araCTP with an increase in the intracellular concentration of araCTP. 2.5.4. Inhibition o f Membrane Precursor Synthesis AraC has been shown to inhibit enzymes involved in the synthesis of glycoproteins and glycolipids in mammalian cells (Hawtrey et al., 1974a,b; MyersRobfogel and Spataro, 1980). Since these are essential components of the cell membrane, araC may alter the composition of the membrane and, subsequently, its function. AraC inhibits the incorporation of glucosamine into glycoproteins and glycolipids (Hawtrey et al., 1974a,b) and the transfer of galactose, Nacetyl-D-glucosamine, and sialic acid from their respective nucleotide carrier to glycoproteins (Hawtrey
Pyrimidine analogues and their nucleosides
NH2
NH2 0
211
N.~
2~r "NH HOC~ OH OH {A)
HOCH2
(B)
{C)
FIG. 11. Reversible spontaneous decomposition of azacytidine (A) to N-(formylamidino)-N'-fl-D-ribofuranosylurea (B) which may further decompose to 1-fl-D-ribofuranosyl-3-guanylurea (C).
et al., 1974a,b; Klohs et al., 1979). AraCMP and
araCTP also competitively inhibit sialytransferases which synthesize sialylglycoproteins (Myers-Robfogel and Spataro, 1980). Inhibition of membrane precursor synthesis appears to occur at high araC concentrations which also inhibit D N A synthesis and, thus, its relationship to and impact on cell survival are difficult to determine. In addition, araCTP serves as a substrate for the synthesis of araCDP-choline (Lauzon et al., 1978a,b) and, therfore, may serve as a substrate for membrane phospholipid synthesis. The rapid lysis of leukemic blasts noted in clinical trials with high-dose araC (Capizzi et al., 1984) suggests that such a mechanism may exist in addition to inhibition of DNA synthesis.
utilized this synthetic azaCyd to demonstrate that azaCyd produced growth inhibition in bacteria. Two years later, azaCyd was also isolated as an antibiotic from fungal cultures (Hanka et al., 1966). Based on the concept that initiated the synthesis of azaCyd several other nucleosides closely related to azaCyd have also been synthesized and investigated for antitumor activity. These include 2'-deoxy-5-azacytidine (Pliml and Sorm, 1964), 5,6-dihydro-5-azacytidine (Beisler et al., 1976), and the arabinosyl nucleoside of azaCyd, arabinosyl-5-azacytidine (Beisler et al., 1977). At present, the metabolism, mechanism of action, or therapeutic efficacy of these compounds relative to azaCyd has not been thoroughly evaluated. 3.2. CHEMISTRY
3. AZACYTIDINE The success of araC as an antileukemic drug stimulated the research for other cytidine analogues that may have antitumor activity. Of particular interest were analogues that did not require activation by deoxycytidine kinase since its activity is decreased in many of the araC-resistant tumors. Therefore, cytidine analogues were examined with substitutions in the pyrimidine ring rather than in the ribose moiety since these analogues would most likely be activated by uridine/cytidine kinase instead of deoxycytidine kinase. This rationale led to the development of azacytidine (azaCyd; 5-azacytidine). Although azaCyd was shown to have antitumor activity in initial tumor screening, clinical trials have been less impressive limiting enthusiasm for this drug as an antitumor agent (Karon et al., 1973; McCredie et al., 1973; Vogler et al., 1976). Occasional responses have been reported in patients with solid tumors (e.g. breast, colon, and malignant melanoma) but the therapeutic efficacy was very low and further clinical trials were not warranted (Von Holt et al., 1976). The only therapeutic role for azaCyd today is in the treatment of acute myleoblastic leukemia. 3.1. HISTORICALPERSPECTIVE AzaCyd was first synthesized in 1964 by Sorm and his colleagues (Sorm et al., 1964). Sorm et al. (1964)
AzaCyd is an analogue of cytidine and differs from the de novo nucleoside by the presence of a nitrogen in the pyrimidine ring (Fig. 9). This substitution destabilizes the heterocyclic ring and leads to spontaneous decomposition of azaCyd in neutral or alkaline solutions (Fig. 11) with a half-life of approximately 4 hr (Beisler, 1978). At acidic pH or in buffered solutions azaCyd is much more stable with a half-life of 65 hr at 25°C and 94 hr at 20°C (Israili et al., 1976). 3.3. UPTAKEINTOCELLS AzaCyd readily enters all mammalian cells by the same facilitated nucleoside transporter as the de novo nucleosides, uridine and cytidine (Notari and DeYoung, 1975). This transport system was discussed in detail previously in Sections 1.3 and 2.3. 3.4. METABOLISM As discussed previously for both the fluoropyrimidines (Section 1.4) and araC (Section 2.4), the metabolism of azaCyd by host and tumor cells is an important aspect of its usefulness as an antitumor agent. As with the previous antimetabolites, the close structural similarity of azaCyd to physiological nucleosides (Fig. 9) results in its metabolism by the same enzymes that metabolize cytidine.
212
G.C. DAHERet al. .HH2
.NH2
(~'~N~1 0 ~ j 3
I 4 I
.NH2
.
¢.J
/\ RNA
DNA
¢.2
FIG. 12. Metabolism of azacytidine (azaCyd) in humans. Anabolism of azaCyd is catalyzed initially by uridine/cytidine kinase (I) to azacytidine monophosphate (azaCMP) followed by nucleotide kinases (2) which ultimately form azacytidine triphosphate (azaCTP). AzaCTP may be incorporated into DNA or RNA leading to inhibition of nucleic acid synthesis. Catabolism of azaCyd primarily occurs via cytidine deaminase (3) to form azauridine. Deamination of azaCMP to azauridine monophosphate by deoxycytidylate deaminase (4) is also suspected. 3.4.1. Catabolism
3.4.2. Anabolism
In addition to spontaneous decomposition in neutral or alkaline solutions, azaCyd may be catabolized to inactive products (Fig. 12) via the enzymes, cytidine deaminase (EC 3.5.4.5) and deoxycytidylate deaminase (EC 3.5.4.12). These enzymes were discussed in the sections on araC catabolism (see Sections 2.4.1.1 and 2.4.1.2). The importance of deamination in azaCyd pharmacokinetics has been demonstrated by studies with THU (Neil et al., 1975). As noted earlier, THU is a potent cytidine deaminase inhibitor. These studies demonstrate that THU increases the plasma concentration of azaCyd as well as the toxicity and therapeutic activity of orally administered azaCyd. The role of cytidine deaminase in the development of resistance to azaCyd has not been determined.
The initial step in the activation of azaCyd is its conversion to a monophosphate (azaCMP) by uridine/cytidine kinase with subsequent conversion to azaCyd triphosphate (azaCTP) by nucleotide kinases (Fig. 12).
NH2
3.4.2.1. Uridine/cytidine kinase. Uridine/cytidine kinase (EC 2.7.1.48) is responsible for the conversion of either uridine or cytidine to their respective monophosphates, uridine monophosphate (UMP) or cytidine monophosphate (CMP). In addition, when cells are exposed to azaCyd, uridine/cytidine kinase catalyzes the conversion of azaCyd to azaCMP (eqn 15). Uridine/cytidine kinase has a lower affinity for azaCyd with a Km between 0.2 and 11 mM than for NH 2
H
OHOH
H
OHOH
(15)
213
Pyrimidine analogues and their nucleosides uridine or cytidine with a Km of 0.05 M (Drake et al., 1977; Lee et al., 1974) and is the rate-limiting step in azaCyd anabolism. Both uridine (Vadlamudi et al., 1970b) and cytidine competitively inhibit the anabolism of azaCyd and have been shown to prevent azaCyd toxicity in whole animals and tissue culture (Vadlamudi et al., 1970a). Deletion of uridine/ cytidine kinase has been observed in mutant Novikoff hepatoma cells that were resistant to azaCyd (Plagemann et al., 1978a). 3.4.2.2. Nucleotide kinases. Further activation of azaCyd involves the conversion of azaCMP to azaCDP and, subsequently, azaCTP. These reactions are most likely catalyzed by the enzymes deoxcytidylate kinase and N D P kinase, respectively. Deoxycytidylate kinase was previously discussed in regard to araC metabolism in Section 2.4.2.2 and NDP kinase was previously discussed in Sections 1.4.2.6 and 2.4.2.3.
3.5. METABOLICSITESOF ACTION Over the past 25 years there have been several studies on the cytotoxic effects of azaCyd. Both drug concentration and duration of exposure to azaCyd greatly influence the cytotoxicity observed in cultured cells (Chabner, 1982). AzaCyd is primarily cytotoxic to cells in the S phase of the cell cycle with little, if any, effect against nondividing cells (Li et aL, 1970; Lloyd et al., 1972). These findings would suggest that one of the effects of azaCyd is on D N A synthesis (Li et al,, 1970). Dose-response curves in both normal and tumor cell cultures are biphasic indicating more than one mechanism is probably responsible for the cytotoxicity of azaCyd (Chabner, 1982). The incorporation of azaCyd (or more specifically, azaCTP) into R N A and D N A was demonstrated in both cultured rhesus monkey kidney cells (Sorm et al., 1964) and L1210 cells (Li et al., 1970). In both cases, inhibition of D N A synthesis was more pronounced than R N A synthesis. While incorporation of azaCTP into D N A is one possible mechanism for the inhibition of D N A synthesis, another is by the competition between azaCTP and dCTP for D N A polymerase as was shown to occur with araCTP. However, if this were one of the major mechanisms for the inhibition of D N A synthesis deoxycytidine would be expected to alleviate azaCyd inhibition of D N A synthesis which is not the case (Li et al., 1970). Therefore, interference with the synthesis of de novo pyrimidine nucleotides and the inhibition of D N A synthesis due to incorporation of azaCTP appear to be the major mechanisms of cytotoxicity following exposure to azaCyd. In addition to its cytotoxic effects, azaCyd has other biological actions that may be of clinical importance. AzaCyd stimulates the activity of various hepatic enzymes including tyrosine aminotransferase (Cihak et al., 1973) and uridine/cytidine kinase (Cihak and Vesely, 1973). It also stimulates differentiation in certain mouse embryo cells in culture (Constantinides et al., 1977). This 'differentiating' effect of azaCyd has not been thoroughly examined
in tumor cells. AzaCyd also has mutagenic and teratogenic effects. Chromatid breaks have been observed in cultured hamster fibroblasts (Karon and Benedict, 1972) and LI210 cells (Li et al., 1970). However, no secondary malignancies related to azaCyd treatment have been described in animals or humans. REFERENCES ABSIL,J., TUILIE,M. and Roux, J. M. (1980) Electrophoretitally distinct forms of uridine kinase in the rat. Tissue distribution and age-dependence. Biochem. J. 185: 273-276. ArtM~D, N. K. (1982) Multiple forms and inhibitors of uridine-cytidine kinase in neoplastic cells. Int. J. Biochem. 14: 259-262. ANDERSON,E. P. (1973) Nucleoside and nucleotide kinases. In: The Enzymes, pp. 49-96, BOYER,P. D. (ed.) Academic Press, New York. ARDALAN, B. and GLAZER,R. (1981) An update on the biochemistry of 5-fluorouracil. Cancer Treat. Rev. 8: 157-167. ARDALAN,B., COONEY,D. A., JAYARAM,H. N,, CARRICO,C. K., GLAZER, R. I., MACDONALD, J. and SCHEIN, P. S. (1980) Mechanism of sensitivity and resistance of murine tumors to 5-fluorouracil. Cancer Res. 40: 1431-1437. ARDALAN, B., VILLACORTE,O., HECK, D. and CORBETT,T. (1982) Phosphoribosyl pyrophosphate, pool size and tissue levels as a determinant of 5-fluorouracil response in murine colonic adenocarcinomas. Biochem. Pharmac. 31: 1989-1992. ARIMA, T., AKIYOSHI,H. and FUJII, S. (1977) Characterization of pyrimidine monophosphokinase in normal and malignant tissues. Cancer Res. 37:1593-1597. ARMSTRONG,R. D. and DIASIO,R. B. (1980) Metabolism and biological activity of 5'-deoxy-5-fluorouridine, a novel fluoropyrimidine. Cancer Res. 40: 3333-3338. ARMSTRONG,R. D., LEWIS,M., STERN,S. G. and CADMAN, E. C. (1986) Acute effect of 5-fluorouracil on cytoplasmic and nuclear dihydrofolate reductase messenger RNA metabolism. J. biol. Chem. 261: 7366-7371. ASHLEY,G. W. and BARTLETT,P. A. 0984) Purification and properties of cytidine deaminase from Escherichia coil J. biol. Chem. 259: 13615-13620. Au, J. L., Wu, A. T., FRIEDMAN,M. A. and SADEE,W. (1979) Pharmacokinetics and metabolism of ftorafur in man. Cancer Treat. Rep. 63: 343-350. AU, J. L., RUSTUM,Y. M., LEDESMA,E. J., MITTELMAN,A. and CR~.AVEN,P. J. (1982) Clinical pharmacological studies of concurrent infusion of 5-fluorouracil and thymidine in treatment of colorectal carcinoma. Cancer Res. 42: 2930-2937. AVRAMIS,V. I. and POWELL,W. (1987) Pharmacology of combination chemotherapy of cytosine arabinoside (araC) and uracil arabinoside (ara-U) or tetrahydrouridine (THU) against murine leukemia LI210/0 in tumorbearing mice. Cancer Invest. 5: 293-299. BAND, P. R., HOLLAND, J. F., BERNARD, J., WEIL, M., WALKER, M. and RALL, D. (1973) Treatment of central nervous system leukemia with intrathecal cytosine arabinoside. Cancer 32: 744-748. BEISLER,J. A. (1978) Isolation, characterization, and properties of a labile hydrolysis product of the antitumor nucleoside, 5-azacytidine. J. reed. Chem. 21: 204-208. BEISLER, J. A., ABBASI,M. M. and DR1SCOLL,J. S. (1976) Dihydro-5-azacytidine hydrochloride, a biologically active and chemically stable analog of 5-azacytidine. Cancer Treat Rep. 60: 1671-1674. BEISLER,J. A., ABBASI,M. M. and DRISCOLL,J.S. (1977) The synthesis and antitumor activity of arabinosyl-5-azacytosine. Biochem. Pharmac. 26: 2469-2472.
214
G.C. DAI~R et al.
BENVENUTO, J., LU, K., HALL, S. W., BENJAMIN, R. S. and Loo, T. L. (1978) Metabolism of 1-(tetrahydro-2-furanyl)5-fluorouracil (ftorafur). Cancer Res. 38: 3867-3870. BERENS, K. and SHUGAR,D. (1963) Ultraviolet absorption spectra and structure of halogenated uracils and their glycosides. Acta biochimica polonica 10: 25-47. BERGMANN,W. and FEENEY, R. J. (1950) The isolation of a new thymine pentoside from sponges. J. Am. chem. Soc. 72: 2809-2810. BERGMANN, W. and FEENEY, R. J. (1951) Contributions to the study of marine products. XXXII. The nucleosides of sponges. J. org. Chem. 16: 981. BERg, A. J. and CLAYTON,D. A. (1973) A genetically distinct thymidine kinase in mammalian mitochondria. Exclusive labelling of mitochondrial deoxyribonucleic acid. J. biol. Chem. 248: 2722-2729. BIANCHI, P. A., FARINA, M. V. and POLLI, E. (1964) Phosphorylation of thymidine diphosphate in resting and proliferating mammalian cells. Biochim. biophys. Acta 91: 323-325. BLEYER, W. A. (1989) New vistas for leukovorin in cancer chemotherapy. Cancer 63: 995-1007. BOLLAG, W. and HARTMANN,H. R. (1980) Tumour inhibitory effects of a new fluorouracil derivative: 5'-deoxy-5fluorouridine. Eur. J. Cancer 16: 427-432. BOURTOURAULT,M., MAHOUDO, H., HARAS, D., SAMPEREZ, S. and JOUAN, P. (1984) Stimulation by estradiol-17fl of thymidine kinase activity in the rat uterus. J. Steroid Biochem. 21: 613~20. BOWEN, D., WHITE, J. C. and GOLDMAN,I. D. (1978) A basis for fluoropyrimidine-induced antagonism to methotrexate in Ehrlich ascites tumor cells in vitro. Cancer Res. 38: 219-222. BREITMAN, T. R. (1963) The feedback inhibition of thymidine kinase. Biochim. biophys. Acta 67: 153-155. BREZNICK, E, and KARJALA, R. J. (1964) End-product inhibition of thymidine kinase activity in normal and leukemic human leukocytes. Cancer Res. 24: 841-846. BROOKS, K. P., KIM, B. D. and SANDER, E. G. (1979) Dihydropyrimidine amidohydrolase is a zinc metalloenzyme. Biochim. biophys. Acta 570: 213-214. BROOKS, K. P., JONES, E. A., KIM, B. D. and SANDER,E. G. (1983) Bovine liver dihydropyrimidine amidohydrolase: Purification, properties, and characterization as a zinc metalloenzyme. Archs Biochem, Biophys. 226: 469-483. BRYAN,J. H., HENDERSON,E. S. and LEVENTHAL,B. G. (1974) Cytosine arabinoside and 6-thioguanine in refractory acute lymphocytic leukemia. Cancer 33: 539-544. CAMIENER, G. W. (1967) Studies on the enzymatic deamination of cytosine arabinoside. II. Properties of the deaminase of human liver. Biochem. Pharmac. 16: 168 I-1689.
CAMIENER,G. W. and SMITH, C. G. (1965) Studies of the enzymatic deamination of cytosine arabinoside. I. Enzyme distribution and species specificity. Biochem. Pharmac. 14: 1405-1416. CANELLAKIS, E. S. (1956) Pyrimidine metabolism. I. Enzymatic pathways of uracil and thymine degradation. J. biol. Chem. 221: 315-321. CAPIZZI, R. L., YANG, J. L., CHENG, E., BJORNSSON, T., SAHASRABUDHE,D., TAN, R.-S. and CHENG, Y.-C. (1983) Alteration in the pharmacokinetics of high dose ara-C by its metabolite, ara-U, in patients with acute leukemia. J. c/in. Oncol. l: 763-771. CAPIZZI,R. L., POOLE, M., COOPER,M. R., R1CHARDS,F., II, STUART, J. J., JACKSON, D. V., JR, WHITE, D. R., SPURR, C. L., HOPKINS, J. O., Muss, M. B., RUDNICK, S. A., WELLS,R., GABRIEL,D. and Ross, D. (1984) Treatment of poor risk acute leukemia with sequential high dose araC and asparaginase. Blood 63: 694-700. CARADONNA,S. J. and CnENG, Y.-C. (1980a) The role of deoxyuridine triphosphate nucleotidohydrolase, uracilDNA glycosylase, and DNA glycosylase, and DNA
polymerase ct in the metabolism of FUdR in human tumor cells. Molee. Pharmac. 18: 513-520. CARADONNA, S. J. and CnENG, Y.-C. (1980b) Uracil DNAglycosylase. Purification and properties of this enzyme isolated from blast cells of acute myelocytic leukemia patients. J. biol. Chem. 255: 2293-2300. CARTER, G. L. and CoRv, J. G. (1988) Cross-resistance patterns in hydroxyurea-resistant leukemia LI210 cells. Cancer Res. 48: 5796-5799. CASS, C. E., KOLASSA,N., UEHARA,Y., DAHLING-HARLEY,E., HARLEY, E. and PATERSON, A. R. P. (1981) Absence of binding sites for the transport inhibitor nitrobenzylthioinosine on nucleoside transport deficient mouse lymphoma cells. Biochem. biophys. Acta 649: 769-777. CHABNER, B. A. (1982) Pyrimidine antagonists. In: Pharmacologic Principles of Cancer Treatment, pp. 183-212, CHABNER, B. A. (ed.) W. B. Saunders Co., Philadelphia. CHAaNER, B. A. and MYERS, C. E. (1985) Clinical pharmacology of cancer chemotherapy. In: Cancer--Principles and Practice of Oncology (2nd edn), pp. 287-328, DEVITA, V. T., JR, HELLMAN,S. and ROSENaERG,S. A. (eds) Lippincott, Philadelphia. CHABNER, B. A., DRAKE, J. C. and JOHNS, D. G. (1973) Deamination of 5-azacytidine by a human leukemia cell cytidine deaminase. Biochem. Pharmac. 22: 27632765. CHABNER, I . A., JOHNS, D. G., COLEMAN,C. N., DRAKE,J. C. and EVANS, W. H. (1974) Purification and properties of cytidine deaminase from normal and leukemic granulocytes. J. clin. Invest. 53:922-93 I. CHABOT, G. G., BOUCHARD,J. and MOMPARLER,R. L. (1983) Kinetics of deamination of 5-aza-2'-deoxycytidine and cytosine arabinoside by human liver cytidine deaminase and its inhibition by 3-deazauridine, thymidine, or uracil arabinoside. Biochem. Pharmac. 32:1327-1328. CHAUDHURI, N. K., MONTAG, B. J. and HEIDELBERGER, C. (1958) Studies on fuorinated pyrimidines III. The metabolism of 5-fuorouracil-2-C14 acid in vivo. Cancer Res. 18: 318-328. CI-IENG, N., PAYNE, R. C. and TRAUT, T. W. (1986) Regulation of uridine kinase: Evidence for a regulatory site. J. biol. Chem. 261: 13006-13012. CHENG, Y.-C. and CAPIZZI, R. L. (1982) Enzymology of cytosine arabinoside. Med. Pediat. Oncol. 10 (Suppl. I): 27-31. CHIGA, M., ODA, A. and HOLTZER,R. L. (1963) The activities of certain nucleoside diphosphokinases of normal and regenerating rat liver. Archs Biochem. Biophys. 103: 366-370. CHOU, T.-C., HUTCHISON, D. J., SCHMID, F. A. and PHILIPS, F. S. (1975) Metabolism and selective effects of 1-fl-D-arabinofuranosylcytosine in LI210 and host tissues in vivo. Cancer Res. 35: 225-236. CHU, M. Y. and FISCHER, G. A. (1962) A proposed mechanism of action of l-fl-D-arabinofuranosylcytosine as an inhibitor of the growth of leukemic cells. Biochem. Pharmac. 11: 423-430. CHU, M. Y. and FISCH/~R,G. A. (1965) Comparative studies of leukemic cells sensitive and resistant to cytosine arabinoside. Biochem. Pharmac. 14: 333-341. CHU, M. Y., NAGUIB, N. M., ILTZSCH, L. H., EL KOUNI, M. H., CHU, S. H. and CALABRESI,P. (1984) Potentiation of 5-fluoro-2'-deoxyuridine antineoplastic activity by the uridine phosphorylase inhibitors benzylacyclouridine and benzyloxybenzylacyclouridine. Cancer Res. 44:1852-1856. CIHAK,A. and VESELY,J. (1973) Enhanced uridine kinase in rat liver following 5-azacytidine administration. J. biol. Chem. 248: 1307-1313. CIHAK, A., LAMAR,C. and PITOT, H. C. (1973) Studies on the mechanism of the stimulation of tyrosine aminotransferase activity in vivo by pyrimidine analogs: The role of enzyme synthesis and degradation. Archs Biochem. BiDphys. 156: 176-187.
Pyrimidine analogues and their nucleosides
215
CISNEROS, R. J., SILKS, L. A. and DUNLAP, R. B. (1988) DESGRANGES, C., RAZAKA, G., RABAUD, i . , PICARD, P., DUPUCH, F. and BRICAUD,H. (1982) The human blood Mechanistic aspects of thymidylate synthase: Molecular platelet: A cellular model to study the degradation of basis for drug design. Drugs of the Future 13: 859-881. thymidine and its inhibition. Biochem. Pharmac. 31: COl-raN, A., ULLMAN,B. and MARTIN,D. W. (1979) Charac2755-2759. terization of a mutant mouse lymphoma cell line with DESGRANGES,C., RAZAKA,G., DE CLERCQ,E., HERDEWIJN, deficient transport of purine and pyrimidine nucleosides. P., BALZARINI,J., DROUILLET,F. and BRICAUD,H. (1986) J. biol. Chem. 254: 110-116. Effect of (E)-5-(2-bromovinyl)uracil on the catabolism COHEN, S. S. (1966) Introduction to the biochemistry of and antitumor activity of 5-fluorouracil in rats and D-arabinosyl nucleosides. In: Progress in Nucleic Acid leukemic mice. Cancer Res. 46:1094-1101. Research and Molecular Biology, pp. 1-88, DAV1DSON, DIASIO, R. B., BEAVERS,T. L. and CARPENTER,J. T. (1988) J. N. and COHN,W. E. (eds) Academic Press, New York. Familial deficiency of dihydropyrimidine dehydroCOLEMAN,C. N., STOLLER,R. G., DRAKE,J. C. and CHABNER, genase. Biochemical basis for familial pyrimidinemia B. A. (1975) Deoxycytidine kinase: properties of the and severe 5-fluorouracil-induced toxicity. J. clin. Invest. enzyme from human leukemic granulocytes. Blood 46: 81: 47-51. 791-803. DIXON, R. L. and ADAMSON,R. H. (1965) Antitumor activity CONSTANTINIDES,P. G., JONES,P. A. and GEVERS,W. (1977) and pharmacologic disposition of cytosine arabinoside Functional striated muscle cells from non-myoblast pre(NSC 63878). Cancer Chemother. Rep. 48: 11-16. cursors following 5-azacytidine treatment. Nature 267: DRAHOVSKY, D. and KKEIS, W. (1970) Studies on drug 364-366. resistance. II. Kinase patterns in P815 neoplasms sensitive COOPER, G. M., DUNNING,W. F. and GREER,S. (1972) Role and resistant to l-fl-o-arabinofuranosylcytosine. BiDof catabolism in pyrimidine utilization for nucleic acid chem. Pharmac. 19: 940-944. synthesis in vivo. Cancer Res. 32: 390-397. CORY, J. G. and CARTER,G. L. (1988) Leukemia L1210 cell DRAKE, J. C., STOLLER,R. G. and CHAnNER, B. A. (1977) Characteristics of the enzyme ufidine-cytidine kinase lines resistant to ribonucleotide reductase inhibitors. isolated from a cultured human cell line. Biochem. PharCancer Res. 48: 839-843. mac. 26: 64-66. CORY, J. G. and FLEISHER,A. E. (1979) Specific inhibitors directed at the individual components of ribonucleotide DRAKE, J. C., HANDE,K. R., FULLER,R. W. and CHABNER, B. A. (1980) Cytidine and deoxycytidylate deaminase reductase as an approach to combination chemotherapy. inhibition by uridine analogs. Biochem. Pharmac. 29: Cancer Res. 39: 4600-5604. 807-811. CREAS~Y,W. A. (1963) Studies on the metabolism of 5-iodoDUDLEY, K. H., BUTLER,T. C. and BIDS,D. L. (1974) The 2'-deoxycytidine in vitro. Purification of nucleoside deamrole of dihydropyrimidinase in the metabolism of some inase from mouse kidney. J. biol. Chem. 238: 1772-1776. hydantoin and succinimide drugs. Drug Metab. Dispos. 6: DANENBERG,P. V. and DANENBERG,K. D. (1978) Effects of 601~505. 5,10-methylenetetrahydrofolate on the dissociation of 5fluoro-2'-deoxyuridylate from thymidylate synthase: Evi- DUNN, W. C. and REGAN,J. D. (1979) Inhibition of DNA excision repair in human cells by arabinofuranosyl cydence for an ordered mechanism. Biochemistry 17: tosine: Effect on normal and xeroderma pigmentosa cells. 4018-4024. Molec. Pharmac. 15: 367-374. DANENBERG,P. V. and LOCKSH1N,A. (1981) Fluorinated pyrimidines as tight-binding inhibitors of thymidylate DUSCHINSKY,R., PLEVEN,E. and HEIDELBERGER,C. (1957) synthase. Pharmac. Ther. 13: 69-90. The synthesis of 5-fluoropyrimidines. J. Am. chem. Soc. 79: 4559-4560. DANENBERG,P. V., HEIDELBERGER,C., MULKINS,M. k. and PETERSON,A. R. (1981) The incorporation of 5-fluoro-2'- EL HIYANI,L., SAMPEREZ,S. and JOUAN,P. (1987) Inhibition deoxyuridine into DNA of mammalian tumor cells. by Celeptium~ of the fetal thymidine kinase synthesis induced by estrogens in the rat uterus. Chem.-Biol. Biomed. biophys. Res. Commun. 102: 654q558. DANENBERG,P. V,, SHEA,L. C. C. and DANENBERG,K. (1990) Interact. 62: 167-178. ELLIMS, P. H., KAO, A. Y. and CHABNER,B. A. (1981) Effect of 5-fluorouracil substitution on the self-splicing activity of tetrahymena ribosomal RNA. Cancer Res. 50: Deoxycytidylate deaminase: Purification and some properties of the enzyme isolated from human spleen. J. biol. 1757-1763. DANHAUSER,L. L. and RUSTUM,Y. M. (1979) A method for Chem. 256: 6335~5340. continuous drug infusion in unrestrained rats: Its appli- ELLIMS,P. H., KAO,A. Y. and CHABNER,B. A. (1983) Kinetic cation in evaluating the toxicity of 5-fluorouracil/ behaviour and allosteric regulation of human deoxythymidine combination. J. Lab. clin. Med. 93: 1047-1053. cytidylate deaminase derived from leukemic cells. Molec. DANHAUSER, L. L. and RUSTUM,Y. M. (1980) Effect of cell. Biochem. 57: 185-190. thymidine on the toxicity, antitumor activity and metabELLISON, R. R., HOLLAND,J. F., WELL,M., JACQUILLAT,C., olism of l-fl-o-arabinofuranosylcytosine in rats bearing a BOIRON,M., BERNARD,J., SAWITSKY,A., ROSNER,F., GUSchemically induced colon carcinoma. Cancer Res. 40: SOFF,B., SILVER,R. m., KARANAS,k., CUTTNER,J., SPURR, 1274-1280. C., HAYES, D. M., BLOM, J., LEONE, L. A., HAUVANI,F., DANHAUSER,L. L. and RUSTUM,Y. M. (1984) ChemotheraKYLE, R., HUTCmSON, J. U, FORClER, R. J. and MOON, peutic efficacy of 5-fluorouracil with concurrent J. H. (1968) Arabinosyl cytosine: a useful agent in the thymidine infusion against transplantable colon tumor in treatment of acute leukemia in adults. Blood 32: 507-523. rodents. Cancer Drug Delivery 1: 269-282. ENGSTRrM, Y. and ROZELL,B. (1988) Immunocytochemical DARNOWSKI, J. W. and HANDSCHUMACHER,R. E. (1985) evidence for cytoplasmic localization and differential exTissue-specific enhancement of uridine utilisation and pression during the cell cycle of M 1 and M2 subunits of 5-fluorouracil therapy in mice by benzylacyclouridine. mammalian ribonucleotide reductase. EMBO J. 7: Cancer Res. 44: 5364-5368. 1615-1620. DATTA, N. S., SHEWACH,D. S., HURLEY,M. C., MITCHELL, ENGSTROM, Y., ROZELL, B., HANSSON,H., STEMME,S. and B. S. and Fox, I. H. (1989) Human T-lymphoblast deoxyTHELANDER,L. (1984) Localization of ribonucleotide recytidine kinase: Purification and properties. Biochemistry ductase in mammalian cells. EMBO J. 3: 863-867. 28:114-123. ERIKSSON,S., GR.~SLUND,A., SKOG, S., THELANDER,L. and DESGRANGES,C., RAZAKA,G., RABAUD,M. and BRICAUD,H. TRIBUKAIT,B. (1984) Cell cycle-dependent regulation of (1981) Catabolism of thymidine in human blood platelets: mammalian ribonucleotide reductase. The S phase-correPurification and properties of thymidine phosphorylase. lated increase in subunit M2 is regulated by de novo Biochem. biophys. A cta 654:211-218. protein synthesis. J. biol. Chem. 259:11695-11700.
216
G.C. DAHER et al.
ERLICHMAN, C., FINE, S. and ELHAKIM,T. (1986) Plasma pharmacokinetics of 5-fluorouracil by continuous infusion with allopurinol. Cancer Treat. Rep. 70: 903-904. EVANS,J. S., MUSSER,E. A., MENGEL,G. D., FORSBLAD,K. R. and HUNTER,J. H. (1961) Antitumor activity of 1-fl-Darabinofuranosylcytosine hydrochloride. Proc. Soc. exp. Biol. Med. 106: 350-353. EVANS, J. S., MUSSER, E. A., BOSTWICK, L. and MENGEL, G. D. (1964) The effect of l-fl-D-arabinofuranosylcytosine hydrochloride on murine neoplasms. Cancer Res. 24: 1285-1293. FANUCCHI,M. P., WATANABE,K. A., Fox, J. J. and CHOU, T.-C. (1986) Kinetics and substrate specificity of human and canine cytidine deaminase. Biochem. Pharmac. 35: 1199-1201. FIALA, S., F1ALA,A., TOBAR,G. and McQUILLA, H. (1962) Deoxynucleotidase activity in rat liver and certain tumors. J. natn. Cancer Inst. 28: 1269-1289. FISHER, P. H. and BAXTER,D. (1981) Enzyme regulatory site-directed drugs. Modulation of thymidine triphosphate inhibition of thymidine kinase by 5'-amino-5'deoxythymidine. Molec. Pharmac. 22: 231-234. FISHER, P. H. and PHILLIPS,A. W. (1984) Antagonism of feedback inhibition. Stimulation of the phosphorylation of thymidine and 5-iodo-2'-deoxyuridine by 5-iodo-5'amino-2',5'-dideoxyuridine. Molec. Pharmac. 25: 446-451. FISHER, P. H., WEDDLE, M. A. and MOSSIE, R. D, (1983) Modulation of the metabolism and cytotoxicity of iododeoxyuridine by 5'-amino-5'-deoxythymidine. Molec. Pharmac. 23: 709-716. FISHER, P. H., FANG, T.-T., LIN, T.-S., HAMPTON, A. and BRUGGINK,J. (1988) Structure-activity analysis of antagonism of the feedback inhibition of thymidine kinase. Biochem. Pharmac. 37: 1293-1298. Fox, R. M., PIDOINGTON,S. K., TRIPP, E. H., DUNMAN,N. P. and TATTERSALL,M. H. N. (1979) Thymidine sensitivity of cultured leukaemic lymphocytes. Lancet 2: 391-393. FRAM, R. J. and KUFE, O. W. (1982) DNA strand breaks caused by inhibitors of DNA synthesis: l-fl-a-arabinofuranosylcytosine and aphidicolin. Cancer Res. 42: 4050-4053. FRIDLAND,A. and VERHOEF,V. (1987) Mechanism for araCTP catabolism in human leukemic cells and effect of deaminase inhibitors on this process. Sem. Oncol. 14 (Suppl. 1): 262-268. FRITZSON, P. (1960) Properties and assay of dihydrouracil dehydrogenase of rat liver. J. biol. Chem. 235: 719-725. FRITZSON,P. (1962) The relation between uracil-catabolizing enzymes and rate of rat liver regeneration. J. biol. Chem. 237: 150-156, FULCH1GNONI-LATAUD,M.-C., TUILIE,M. and Roux, J.-M. (1976) Uridine-cytidine kinase from foetal and adult rat liver: Purification and study of some properties. Fur. J. Biochem. 69: 217-222. FURTH, J. J. and COHEN, S. S, (1968) Inhibition of mammalian DNA polymerase by the 5'-triphosphate of 1-fl-n-arabinofuranosylcytosine and the 5'-triphosphate of 9-/~-o-arabinofuranosyladenine. Cancer Res. 28: 2061-2067. GALLO, R. C. and SEYMOUR,P. (1969a) The enzymatic mechanisms for deoxythymidine synthesis in human leukocytes. IV. Comparisons between normal and leukemic leukocytes. J. clin. Invest. 48: 105-116. GALLO, R. C. and SEYMOUR,P. (1969b) Enzyme abnormality in human leukaemia. Nature 218: 465-466. GARMONT, A. D., KRULIK, M., CADY, J., LAGADEC, B., MAISANI, J.-E., LOISEAU,J.-P., GRANGE,J.-D., GUSTAVO, G.-C., BENEDICTE,D., LOUVET,C., SEROKA,J., DRAY, C. and DEBRAY,J. (1988) High-dose folonic acid and 5fluorouracil bolus and continuous infusion in advanced colorectal cancer. Eur. J. din. Onco/. 9: 1499-1503.
GEORGE,C. B. and CORY,J. G. (1979) Activation of deoxycytidylate deaminase by 1-fl-o-arabinofuranosylcytosine5'-triphosphate. Biochem. Pharmac. 28: 1699-1701. GERACI,G., ROSSI,M. and SCARANO,E. (1967) Deoxycytidylate aminohydrolase. I. Preparation and properties of the homogeneous enzyme. Biochemistry 6: 183-191. GRAHAM, F. L. and WHITMORE,G. F. (1970a) The effect of 1-//-arabinofuranosylcytosine on growth, viability, and DNA synthesis of mouse L-cells. Cancer Res. 30: 2627-2635. GRAHAM,F. L. and WHITMORE,G. F. (1970b) Studies in the mouse L-cells on the incorporation of 1-fl-D-arabinofuranosylcytosine into DNA and on inhibition of DNA polymerase by 1-//-o-arabinofuranosylcytosine-5'-triphosphate. Cancer Res. 30: 2636-2644. GRAY, H. J. and SMELLIE,R. M. S. (1963) The mechanism of formation of thymidine 5'-triphosphate by enzymes from Landschutz ascites-tumor cells. Biochem. J. 89: 486-491, GREENBERG,N., SCHUMM,D. E. and WEBa, T. (1977) Uridine kinase activities and pyrimidine nucleoside phosphorylation in fluoropyrimidine-sensitive and resistant cell lines of the Novikoff hepatoma. Biochem. J. 164: 379-387. HALL, S. W., VALDIVIESO,M. and BENJAMIN,R. S. (1977) Intermittent high single dose ftorafur. A phase I clinical trial with pharmacologic-toxicity correlations. Cancer Treat. Rep. 61: 1495-1498. HANDE, K. R. and CHABNER,B. A. 0978) Pyrimidine nucleoside monophosphate kinase from human leukemic blast cells. Cancer Res. 38: 579-585. HANKA, L. J., EVANS,J. S., MASON, D. J. and DIETZ, A. (1966) Microbiological production of 5-azacytidine. 1. Production and biological activity. Antimicrob. Ag. Chemother. 619-624. HARDY, L. W., FINER-MOORE, J. S., MONTFORT, W. R., JONES, M. O., SANTI, D. V. and STROUD,R. M. (1987) Atomic structure of thymidylate synthase: Target for rational drug design. Science 235: 448-455. HARRIS, A. L., GRAHAME-SMITH,D. G., POTTER, C. G. and BUNCH, C. (1981) Cytosine arabinoside deamination in human leukaemic myeloblasts and resistance to cytosine arabinoside therapy. Clin. Sci. 60: 191-198. HARRIS, B. E., SONG, R., HE, Y.-J., SOONG,S.-J. and DIASIO, R. B. (1988) Circadian rhythm of rat liver dihydropyrimidine dehydrogenase. Possible relevance to fluoropyrimidine chemotherapy. Biochem. Pharmac. 37: 4759-4762. HARRIS, B. E., SONG, R., SOONG, S.-J. and DIAS10, R. B. (1990) Relationship between dihydropyrimidine dehydrogenase activity and plasma 5-fluorouracil levels: Evidence for circadian variation of plasma drug levels in cancer patients receiving 5-fluorouracil by protracted continuous infusion. Cancer Res. 50: 197-201. HART, J. S., HO, D. H., GEORGE,S. L., SALEM,P., GOTTLIEB, J. A. and FREI, E. I. (1972) Cytokinetic and molecular pharmacology studies of arabinosylcytosine in metastatic melanoma. Cancer Res. 22: 2763-2765. HARTMANN,K. V. and HEIDELBERGER,C. (1961) Studies on fluorinated pyrimidines XIII. Inhibition of thymidylate synthetase. J. biol. Chem. 236: 3006-3013. HAWTREY,A. O., SCOTT-BURDEN,T., JONES,P. and ROBERTSON, G. (1974a) Inhibition by l-fl-D-arabinofuranosylcytosine of the incorporation of N-acetylneuraminic acid into glycolipids and glycoproteins of hamster embryo cells. Biochem. biophys. Res. Commun. 54: 1282-1287. HAWTP~Y, A. O., SCOTT-BUROEN,T. and ROBERTSON,G. (1974b) Inhibition of glycoprotein and glycolipid synthesis in hamster embryo cells by cytosine arabinoside and hydroxyurea. Nature 252: 58-60. HEGGIE, G. D., SAMMADOSSI,J. P., CROSS, D. S., HUSTER, W. J. and DIASlO,R. B. (1987) Clinical pharmacokinetics of 5-fluorouracil and its metabolites in plasma, urine, and bile. Cancer Res. 47: 2203-2206. HEICHAL,O., IsH-SHALOM,D., KOREN, R. and STEIN,W. D. (1979) The kinetic dissection of transport from metabolic
Pyrimidine analogues and their nucleosides trapping during substrate uptake by intact cells. Uridine uptake by quiescent and serum-activated Nil 8 hamster cells and their murine sarcoma virus-transformed counterparts. Biochim. biophys. Acta 551: 169-186. HEIDELBERGER, C. (1975) Fluorinated pyrimidines and their nucleosides. In: Antineoplastic and Immunosuppressive Agents Part 11, pp. 193-231, SARTORELLi,A. C. and JOHNS, D. G. (eds) Springer-Verlag, New York. HEIDELBERGER, C., DANENBERG, P. V. and MORAN, R. G. (1983) Fluorinated pyrimidines and their nucleosides. Adv. Enzymol. 54:57-119. HERRICK, n . J., MAJOR, P. P. and KUFE, D. W. (1982) Effect of methotrexate on incorporation and excision of 5fluorouracil residues in human breast carcinoma DNA. Cancer Res. 42: 5015-5017. Hiss, E. A. and PRESTON, R. J. (1978) The effect of cytosine arabinoside on the presence of single strand breaks in DNA of mammalian cells following irradiation or chemical treatment. Biochem. biophys. Acta 478: 1-8. Ho, D. H. and FREI, E. I. (1971) Clinical pharmacology of l-fl-D-arabinofuranosyl cytosine. Clin. Pharmac. Ther. 12: 944-954. Ho, D. H. W. and FREIREICH, E. J. (1975) Clinical pharmacology of arabinosylcytosine. In: Handbook of Experimental Pharmacology: Antineoplastic and lmmunosuppressive Agents (Part H), pp. 232-256, SARTORELLI,A. C. and JOHNS, D. G. (eds) Springer-Verlag, New York. Ho, D. H. W., CARTER, C. J. and Loo, T. L. (1975) Effects of tetrahydrouridine (THU) on the uptake and metabolism of arabinosylcytosine by human acute myelogenous leukemia (AML) cells. Proc. Am. Ass. Cancer Res. 16: 57. HOUGHTON, J. A. and HOUGHTON,P. J. (1983) Elucidation of pathways of 5-fluorouracil metabolism in xenografts of human coiorectal adenocarcinoma. Fur. J. Cancer. clin. Oncol. 19:807-815. HOUGHTON, J. A., MARODA, S. J., PHILIPS, J. D. and HOUGHTON, P. J. (1981) Biochemical determinants of responsiveness to 5-fluorouracil and its derivatives in xenografts of human colorectal adenocarcinomas in mice. Cancer Res. 41: 144-149. IIGO, M., ARAKI, E., NAKAJ1MA, Y., HOSHI, A. and DE CLERCQ, E. (1988) Enhancing effect of bromovinyldeoxyuridine on antitumor activity of 5-fluorouracil against adenocarcinoma 755 in mice: Increased therapeutic index and correlation with increased plasma 5fluorouracil level. Biochem. Pharmac. 37: 1609-1613. ILTZSCH, M. H., EL KOUNL M. H. and CHA, S. (1985) Kinetic studies of thymidine phosphorylase from mouse liver. Biochemistry 24: 6799-6807. IMAZAWA,M. and ECKSTEIN, F. (1979) Synthesis of sugarmodified nucleoside 5'-triphosphates with partially purified nucleotide kinases from calf thymus. Biochim. biophys. Acta 570: 284-290. IMONDL A. R., BALLS,M. E. and LIPKIN, M. (1969) Changes in enzyme levels accompanying differentiation of intestinal epithelial cells. Exp Cell Res. 58: 323-330. INAGATI, A., NAKAMERA, T. and WAKINSOKA,G. (1970) Studies on the mechanism of action of 1-beta-o-arabinofuranosylcytosine as an inhibitor of DNA synthesis in human leukemic leukocytes. Cancer Res. 29: 2169-2176. INGRAHAM, H. A., TSENG, B. Y. and GOULIAN, M. (1980) Mechanism for exclusion of 5-fluorouracil from DNA. Cancer Res. 40: 998-1001. INGRAHAM, H. A., TSENG, B. Y. and GOULIAN, M. (1982) Nucleotide levels and incorporation of 5-fluorouracil and uracil into DNA of cells treated with 5-fluorodeoxyuridine. Molec. Pharmac. 21: 211-216.
ISRAILI,Z. H., VOGLER,W. R., MINGIOLI,E. S., PIRKLE,J. L., SMITHWICK, R. W. and GOLDSTEIN, J. H. (1976) The disposition of pharmacokinetics in humans of 5-azacytidine administered intravenously as a bolus or by continuous infusion. Cancer Res. 36: 1453-1461.
217
IvEs, D. H. (1965) Evidence for thymidine diphosphate as the precursor of thymidine triphosphate in tumor. J. biol. Chem. 240: 819-824. IRES, D. H., MORSE, P. A. J. and PORTER, V. R. (1963) Feedback inhibition of thymidine kinase by thymidine triphosphate. J. biol. Chem. 238: 1467-1474. JAcQtmZ, J. A. (1962) Permeability of Ehrlich cells to uracil, thymine, and fluorouracil. Proc. Soc. exp. Biol. Med. 109: 132-135. JOHNSON, R. K., SWYRYD, E. A. and STARK, G. R. (1978) Effects of N-(phosphonacetlyl)-L-aspartate on murine tumors and normal tissues in vivo and in vitro and the relationship of sensitivity to rate of proliferation and level of aspartate transcarbamylase. Cancer Res. 38: 371-378. KANAMARU,R. and WAKUI,A. (1988) Mechanism of action of anti-cancer drugs from the viewpoint of RNA metabolism. Jap. J. Cancer Chemother. 15:1011-1018. KANAMARO, R., KAKUTA,H., SATO, T., ISmOKA, C. and WAKUI,A. (1986) The inhibitory effects of 5-fluorouracil on the metabolism of preribosomal and ribosomal RNA in L-1210 cells in vitro. Cancer Chemother. Pharmac. 17: 43-46. KARON, M. and BENEDICT,W. (1972) Chromatid breakage: differential effect of inhibitors of DNA synthesis during G 2 phase. Science 178: 62.
KARON, i . , SIEGER,L., LEIMBROCK,S., FINKLESTEIN,J. Z., NESmT, M. E. and SWANEY,J. J. (1973) 5-Azacytidine: a new active agent for the treatment of acute leukemia. Blood 42: 359-365. KAWAI,i . , ROSENFELD,J., MCCULLOCH,P. and HILLCOAT, B. L. (1976) Blood levels of 5-fluorouracil during intravenous infusion. Br. J. Cancer 36: 346-347. KAYSEN, J., SPRIGGS,D. and KUFE, D. (1986) Incorporation of 5-fluorodeoxycytidine and metabolites into nucleic acids of human MCF-7 breast carcinoma cells. Cancer Res. 46: 4534-4538. KEE~R, R. C., MCNAMARA, D. J. and WEaa, T. E. (1975) The separation of two forms of uridine kinase from the Novikoff hepatoma. Cancer Biochem. Biophys. 1: 107-110. KESSEL, D. (1980) Cell surface alterations associated with exposure of leukemia LI 210 cells to fluorouracil. Cancer Res. 40: 322-324. KESSEL, D. and HALL, T. C. (1967) Studies on drug transport by normal human leukocytes. Biochem. Pharmac. 16: 2395-2403. KESSEL, D. and SHUmN, S. B. (1968) Transport of two non-metabolized nucleosides, deoxycytidine and cytosine arabinoside, in a sub-line of the L1210 murine leukemia. Biochim. biophys. Acta 163: 179-187. KESSEL, D., DEACON, J., COFFEY, B. and BAKAMAJIAN,A. (1972) Some properties of a pyrimidine phosphoribosyltransferase from murine leukemia cells. Molec. Pharmac. g: 731-739. KEYOMARSI, K. and MORAN, R. G. (1988) Mechanism of cytotoxic synergism of fluoropyrimidines and folinic acid in mouse leukemia cells. J. biol. Chem. 263: 14402-14409.
KIKUGAWA,M., FUJIMOTO,S., MIZOTA,C. and TAMAKI,N. (1988) Bisfunction of proprionic acid on purified rat liver fl-ureidopropionase. FEBS Lett. 229: 345-348. KIM, B. D., KEENAN, S., BODNAR, J. K. and SANDER,E. G. (1976) Role of enzymatically catalyzed 5-iodo-5,6-dihydrouracil ring hydrolysis on the dehalogenation of 5iodouracil. J. biol. Chem. 251: 6909-6914. KIMBALL,A. P. and WILSON, M. J. (1968) Inhibition of DNA polymerase by fl-D-arabinosylcytosine and reversal of inhibition of deoxycytidine-5'-triphosphate. Proc. Soc. exp. Biol. Med. 127: 429432. KINAHAN, J. J., OTTEN, M. and GRINDEY, G. B. (1979) Evaluation of ribonucleoside and deoxyribonucleoside triphosphate pools in cultured leukemia cells during exposure to methotrexate or methotrexate plus thymidine. Cancer Res. 39: 3531-3539.
218
G.C. DAI-'IERet al.
KIT, S. (1976) Thymidine kinase, DNA synthesis and cancer. Molec. cell. Biochem. 11: 161-182. KIT, S. and LEUNO,W.-C. (1974) Submitochondrial localization and characteristics of thymidine kinase molecular forms in parental and kinase-deficient HeLa cells. BiDchem. Genetics 11: 231-347. KLOHS, W. D., BERNACKI,R. J. and KORYTNYK,W. (1979) Effects of nucleotides and nucleotide-analogs on human serum sialytransferase. Cancer Res. 39: 1231-1238. KONO, A., HARA, Y., SUGATA,S., KAUBE,Y., MATSUSHIMA, Y. and ISH1TSUKA,H. (1983) Activation of 5'-deoxy-5fluorouridine by thymidine phosphorylase in human tumors. Chem. pharm. Bull. 31: 175-178. KRENITSKY,T. A. (1968) Pentosyl transfer mechanisms of the mammalian nucleoside phosphorylases. J. biol. Chem. 243: 2871-2875. KRYSTAL, G. and SCHOLEEmLD,P. G. (1973) The partial purification and properties of uridine kinases from Ehrlich ascites tumor cells. Can. J. Biochem. 51: 379-389. KUmLUS,J., LEE, L. D. and BADEN,H. P. (1978) Purification of thymidine phosphorylase from human amniochorion. Biochem. biophys. Acta 527: 221-228. KUEE, D. W. and MAJOR, P. P. (1982) Studies on the mechanism of action of cytosine arabinoside. Med. Pedlar. Oncol. 1: 49-67. KUFE, D. W., BEARDSLEY, P., KARP, D., PARKER, L., ROSOWSKY, A., CANELLOS,G. and FP,EI, E., III (1980a) High-dose thymidine infusions in patients with leukemia and lymphoma. Blood 55: 580°589. KUFE, D. W., MAJOR, P. P., EGAN, E. M. and BEARDSLEY, G. P. (1980b) Correlation of cytotoxicity with incorporation of araC into DNA. J. biol. Chem. 255: 8897-9000. KUFE, D. W., MAJOR,P. P., EGAN,E. M. and Lo,, E. (1981) 5-Fluoro-2'-deoxyuridine. Incorporation in L1012 DNA. J. biol. Chem. 256: 8885-8888. KUFE, D. W., SCOTT, P., FRAM, R. and MAJOR, P. (1983) Biological effect of 5-fluoro-2'-deoxyuridine incorporation in L1210 deoxyribonucleic acid. Biochem. Pharmac. 32: 1337-1340. LAUFMAN,L. R., KRZECZOWSKI,R. R. and SEGAL,M. (1987) Leucovorin plus 5-fluorouracil: An effective treatment for metastatic colon cancer. J. clin. Oncol. 5: 1394-1400. LAUZON,(3. J., PARAN,J. H. and PATERSON,A. R. P. (1978a) Formation of l-fl-n-arabinofuranosylcytosine diphosphate choline in cultured human leukemic RPMI 6410 cells. Cancer Res. 38: 1723-1729. LAUZON,(3. J., PATERSON,A. R. P. and BELCH,A. W. (1978b) Formation of 1-fl-n-arabinofuranosylcytosine diphosphate choline in neoplastic and normal cells. Cancer Res. 38:1730-1733. LEE, M. H., COWLING,R. A., SANDER,E. G. and PETTIGREW, D. W. (1986) Bovine liver dihydropyrimidine amidohydrolase: pH dependencies of inactivation by chelators and steady state kinetic properties. Archs Biochem. Biophys. 248: 368-378. LEE, M. H., PETTIGREW,D. W., SANDER,E. G. and NOWAK, T. (1987) Bovine liver dihydropyrimidine amidohydrolase: pH dependencies of the steady-state kinetic and proton relaxation rate properties of the Mn(lI)-containing enzyme. Archs Biochem. Biophys. 259: 597-604. LEE, T., KARON,M. and MOMPARLER,R. L. (1974) Kinetic studies on phosphorylation of 5-azacytidine with the purified uridine-cytidine kinase from calf thymus. Cancer Res. 34: 2481-2488. LEVINSON, B. S., ULLMAN, B. and MARTIN, D. W. (1979) Pyrimidine pathway variants of cultured mouse lymphoma cells with altered levels of both orotate phosphoribosyltransferase and orotidylate decarboxylase. J. biol. Chem. 254: 4396-4401. LI, L. H., OLIN, E. J., FRASER,T. J. and BHUYAN,B. K. (1970) Phase specificity of 5-azacytidine against mammalian cells in tissue culture. Cancer Res. 30: 277002775.
LLOYD, H. H., DALMADGE,E. A. and WIKOFF,L. J. (1972) Kinetics of the reduction in viability of cultured L1210 leukemic cells exposed to 5-azacytidine (NSC-102816). Cancer Chemother. Rep. 56: 585-591. LOCKSmN, A. and DANENBERG,P. V. (1981) Biochemical factors affecting the tightness of 5-fluorodeoxyuridylatc binding to human thymidylate synthetase. Biochem. Pharmac. 30: 247-257. LOZERON, H. A., GORDON, M. P., GABRIEL,T., TAUTZ, W. and DUSCHINSKY,R. (1964) The photochemistry of 5fluorouracil. Biochemistry 3: 1844-1850. MAEHERA,Y., NAGAYAMA,S., OKAZAKI,H., NAKAMURA,H., SHIRAZAKA, T. and FUJI, S. (1981) Metabolism of 5fluorouracil in various human normal and tumor tissues, Gann 72: 824-827. MAGUIRE, J. H. and DUDLEY,K. H. (1978) Partial purification and characterization of dihydropyrimidinases from calf and rat liver. Drug Metab. Dispos. 6: 601--605. MAJOR, P., EGAN,E. M., BEARDSLEY,G. P., MINDEN,M. D. and KUFE, D. W. (1981) Lethality of human myeloblasts correlates with the incorporation of araC into DNA. Proc. natn. Acad. Sci. U.S.A. 78: 3235-3239. MALATHI, V. G. and SILBER, R. (1971) Effects of viral leukemia on spleen nucleoside deaminase: Purification and properties of the enzyme from leukemic spleen. Biochem. biophys. Acta 238: 377-387. MALEY, G. F. and MALEY, F. (1964) The purification and properties of deoxycytidylate deaminase from chick embryo extracts. J. biol. Chem. 239: I168-I 176. MALEY,G. F. and MALEY,F. (1968) Regulatory properties and subunit structure of chick embryo deoxycytidylate deaminase. J. biol. Chem. 243: 4506-4512. MANCINI, W. R. and CrtENG, Y.-C. (1983) Human deoxycytidylate deaminase: Substrate and regulator specificities and their chemotherapeutic implications. Molec. Pharmac. 23: 159-164. MANE.KS,P. and ORENGO,A. (1975) A pyrimidine nucleoside monophosphate kinase from rat liver. Biochemistry 14: 1484-1489. MANESS, P. and ORENGO, A. (1976) Differences between pyrimidine nucleoside monophosphate kinase from rat Novikoff ascites hepatoma and rat liver. Cancer Res. 36: 2312-2316. MANN, G. J., DYNE, M. and MUSGROVE,E. A. (1987) Immunofluorescent quantification of ribonucleotide reductase Ml subunit and correlation with DNA content by flow cytometry. Cytometry 8: 509-517. MANN, G. J., MUSGROVE,E. A., FOX, R. M. and THELANDER, L. (1988) Ribonucleotide reductase M1 subunit in cellular proliferation, quiescence, and differentiation. Cancer Res. 48: 5151-5156. MARSH,J. C. and PERRY,S. (1964) Thymidine catabolism by normal and leukemic human leukocytes. J. clin. Invest. 43: 267-278. MARZ, R., WOHLHUETER,R. M. and PLAGEMANN,P. G. W. (1977) Relationship between thymidine transport and phosphorylation in Novikoff rat hepatoma cells as analyzed by a rapid sampling method. J. supramolec. Struct. 6: 433-440. MASAAK1,I., ARAKI, E., NAKAJIMA,Y. and DE CLERCQ, E. (1988) Enhancing effects of bromovinyldeoxyuridine on antitumor activity of 5-fluorouracil against adenocarcinoma 755 in mice. Increased therapeutic index and correlation with increased plasma 5-fluorouracil levels. Biochem. Pharmac. 37: 1609-1613. MASUI, H. and GARREN,L. D. (1971) On the mechanism of action of adrenocorticotropic hormone. The stimulation of thymidine kinase activity with altered properties and changed subcellular distribution. J. biol. Chem. 246: 5407-5413. MATTHES, E., BARWOLFF,D. and LANGEN,P. (1974) Inhibition by 6-aminothymine of the degradation of nucleosides (5-iododeoxyuridine, thymidine) and pyrimidine
Pyrimidine analogues and their nucleosides bases (5-iodouracil, uracil and 5-fluorouracil) in vivo. Acta biol. med. germ. 32: 483-502. MATTHEWS, M. M. and TRAUT, T. W. (1987) Regulation of N-carbamoyl-fl-alanine amidohydrolase, the terminal enzyme in pyrimidine catabolism, by ligand-induced change in polymerization. J. biol. Chem. 262: 72327237. MCCLARTY, G. A., CHAN, A. K., ENGSTROM,Y., WRIGHT, J. A. and THELANDER,L. (1987) Elevated expression and M1 and M2 components and drug-induced posttranscriptional modulation of ribonucleotide reductase in a hydroxyurea-resistant mouse cell line. Biochemistry 26: 8004-8011. McCREDIE, K. B., BODEY, G. P., BURGESS, M. A., GUTTERMAN,J. U., RODRIGUEZ,V., SULLIVAN,M. P. and FREIREICH,E. J. (1973) Treatment of acute leukemia with 5-azacytidine (NSC-102816). Cancer Chemother. Rep. 57: 319-323. MENTRE, F., STEIMER,J.-L., SOMMADOSSI,J.-P., DIASIO,R. B. and CANO, J. P. (1984) A mathematical model of the kinetics of 5-fluorouracil and its catabolites in freshly isolated rat hepatocytes. Biochem. Pharrnac. 33: 2727-2732. MILLS-YAMAMOTO,C., LAUZON,G. J. and PATERSON,A. R. P. (1978) Toxicity of combinations of arabinosylcytosine and 3-deazuridine toward neoplastic cells in culture. Biochem. Pharmac. 27: 181-186. MOLLGAARD,H. and NEUHARD,J. (1978) Deoxycytidylate deaminase from Bacillus subtilis. J. biol. Chem. 253: 3536-3542. MOMPARLER, R. (1972) Kinetic and template studies with l-fl-o-arabinofuranosylcytosine 5'-triphosphate and mammalian deoxyribonucleic acid polymerase. Molec. Pharmac. 8: 362-370. MOMPARLER,R. L., CHU, M. Y. and FISCHER,G. A. (1968) Studies on a new mechanism of resistance of L5178Y murine leukemia cells to cytosine arabinoside. Biochem. biophys. Acta 161: 481-493. MONOD, J., CHANGEAUX,J. and JACOB,J. (1963) Allosteric proteins and cellular control systems. J. molec. Biol. 6" 30(~329. MORAN, R. G. (1988) The biochemistry of leucovorin potentiation of 5-FU. Advances in Chemotherapy: Status of 5-fluorouracil / leucovorin in advanced malignancies, pp. 8-9, American Cancer Society, Cleveland, OH. MOURAD, N. and PARKS, R. E. J. (1966) Erythrocytic nucleoside diphosphokinase II. Isolation and kinetics. J. biol. Chem. 241: 271-278. MOVER,J. D. and HANDSCHUMACHER,R. E. (1979) Selective inhibition of pyrimidine synthesis and depletion of nucleotide pools by N-(phosphonacetyl)-L-aspartate. Cancer Res. 39: 3089-3094. MULDER, J. H. and HARRAP, K. R. (1975) Cytosine arabinoside uptake by tumor cells in vitro. Fur. J. Cancer ll: 373-379. MULKINS,M. A. and HEIDELBERGER,C. (1982) Biochemical characterization of fluoropyrimidine-resistant murine leukemic cell lines. Cancer Res. 42: 965-973. MULLER, W. E. G. (1977) Rational design of arabinosyl nucleosides as antitumor and antiviral agents. J. Antibiot., Tokyo 30 (Suppl.): 104-120. MYERS-ROBFOGEL,M. W. and SPATARO,A. C. (1980) 1-fl-Darabinofuranosylcytosine nucleotide inhibition of sialic acid metabolism in WI-38 cells. Cancer Res. 40: 1940-1943. NAGUIB, F. N. M., EL KOUNI, M. H. and CHA, S. (1985) Enzymes of uracil catabolism in normal and neoplastic human tissues. Cancer Res. 45: 5405-5412. NAGUIB, F. N. M., NIEDZWICKI, J. G., ILTZSCH, M. H., WmMANN,M. C., EL KOUNI,M. and SUNGMAN,C. (1987a) Effects of N,N-dimethylformamide and sodium butyrate on enzymes of pyrimidine metabolism in cultured human tumor cells. Leukemia Res. 11: 855-861.
219
NAGUIB,F. N. M., EL KOUNI,M. H., CHU, S. H. and CHA, S. (1987b) New analogues of benzylacylcycloufidines, specific and potent inhibitors of uridine phosphorylase from human and mouse livers. Biochem. Pharmac. 36: 2195-2201. NAGUIB, F. N. M., EL KOUNI, M. H. and CHA, S. (1989) Structure-activity relationship of ligands of dihydrouracil dehydrogenase from mouse liver. Biochem. Pharmac. 38: 1471-1480. NAKAMURA,H. and SUGINO,Y. (1966) Metabolism of deoxyribonucleotides. II. Enzymatic phosphorylation of deoxycytidylic acid in normal rat liver and rat ascites hepatoma cells. Cancer Res. 26 (Part 1): 1425-1429. NEIL, G. L., MOXLEY,T. E., KUENTZEL,S. L., MANAK,R. C. and HANKA, L. J. (1975) Enhancement by tetrahydrouridine (NSC-I 12907) of the oral activity of 5-azacytidine (NSC-102816) in L1210 leukemic mice. Cancer Chemother. Rep. 59: 459-465. NmDZWICKI,J. G., EL KOUNI,M. H., CHU, S. H. and C,A, S. (1983) Structure-activity relationship of ligands of the pyrimidines nucleoside phosphorylases. Biochem. Pharmac. 32: 399-415. NIEDZWICKi, J. G., ILTZSCH, M. H., EL KOUNI, M. H. and CHA, S. (1984) Structure-activity relationship of pyrimidine base analogs as ligands of orotate phosphoribosyltransferase. Biochem. Pharmac. 33: 2383-2395. NOTARI, R. E. and DEYOUNG, J. L. (1975) Kinetics and mechanisms of degradation of the antileukemic agent 5-azacytidine in aqueous solutions. J. pharm. Sci. 64: 1148-I157. O'DONOVAN,G. A. and NEUaARD,J. (1970) Pyrimidine metabolism in microorganisms. Bacteriol. Rev. 34: 278-343. PALU, G. (1980) Thymidine phosphorylase: A possible marker of cell maturation in acute myeloid leukaemia. Tumori 66: 35-42. PAYNE,R. C. and TRAUT,T. W. (1982) Regulation of uridine kinase quaternary structure. J. biol. Chem. 257: 12485-12488. PAYNE, R. C., CHENG,N. and TRAUT,T. W. (1985) Uridine kinase from Ehrlich ascites carcinoma: Purification and properties of homogeneous enzyme. J. biol. Chem. 260: 10242-10247. PETERS,G. J., LAURENSSE,E., LANKELMA,J., LEYVA,A. and PINEOO,H. M. (1984) Separation of several 5-fluorouracil metabolites in various melanoma cell lines. Evidence for the synthesis of 5-fluorouracil-nucleotide sugars. Eur. J. Cancer clin. Oncol. 20: 1425-1431. PETIT, E., MILANO,G., LEVI,F., THYSS,A., BA1LLEUL,F. and SCHNEIDER,M. (1988) Circadian rhythm-varying plasma concentration of 5-fluorouracil during a five day continuous venous infusion at a constant rate in cancer patients. Cancer Res. 48: 1676-1679. PETRELLI, N., HERR.ERA, L., RUSTUM, Y., BURKE, P., CREAVEN,P., STULC,J., EMRICH,L, J. and MITTELMAN,A. (1987) A prospective randomized trial of 5-fluorouracil versus 5-fluorouracil and high-dose leucovorin versus 5-fluorouracil and methotrexate in previously untreated patients with advanced colorectal carcinoma. J. clin. Oncol. 5: 1559-1565. PLAGEMANN, P. G. W., BEHRENS, M. and ABRAHAM,D. (1978a) Metabolism and cytotoxicity of 5-azacytidine in cultured Novikoff rat hepatoma and P388 mouse leukemia cells and their enhancement by preincubation with pyrazofurin. Cancer Res. 38: 2458-2466. PLAGEMANN,P. G. W., MARZ, R. and WOHLHUETER,R. M. (1978b) Transport and metabolism of deoxycytidine and I-~-D-arabinofuranosylcytosine into cultured Novikoff rat hepatoma cells, relationship to phosphorylation, and regulation of triphosphate synthesis. Cancer Res. 38: 978-989. PLIML, J. and SORM,F. (1964) Synthesis of 2'-deoxy-D-furanosyl-5-azacytosine. Colin Czech. chem. Commun. 29: 2576-2578.
220
G.C. DArtER et al.
PRE1SLER, H. D., RUSTUM, Y. and PRIORE, R. L. (1985) Relationship between leukemic cell retention of cytosine arabinoside triphosphate and the duration of remission in patients with acute non-lymphocytic leukemia. Eur. J. Cancer din. Oncol. 21: 23-30. QUEENER, S. F., MORIS, H. P. and WEBER, G. (1971) Dihydrouracil dehydrogenase activity in normal, differentiating, and regenerating liver and in hepatomas. Cancer Res. 31: 1004-1009. RABINOW1TZ,Y. and WILHITE,B. A. (1969) Thymidine salvage pathway in normal and leukemic leukocytes with effects of ATP on enzyme control. Blood 33: 759-771. REYES, P. (1969) The synthesis of 5-fluorouridine 5'-phosphate by a pyrimidine phosphoribosyltransferase of mammalian origin. I. Some properties of the enzyme from P1534J mouse leukemic cells. Biochemistry g: 2057-2062. REYES, P. and GU~ANIG, M. E. (1975) Studies on a pyrimidine phosphoribosyltransferase from murine leukemia PI534J. J. biol. Chem. 250: 5097-5108. RICHARD, P., SKOLD, O., KLIEN, G., R&v~sz, U and MAGNUSSON, P.-H. (1962) Studies on resistance against 5fluorouracil I. Enzymes of the uracil pathway during development of resistance. Cancer Res. 22: 235-243. Ross, D. D., THOMPSON, B. W., JONECKIS, C. C., AKMAN, S. A. and SCmFFER, C. A. (1987) Studies of araC metabolism in acute leukemia. Sem. Oncol. 14 (Suppl. 1): 182-191. RUSTUM, Y. M. and PREISLER, H. D. (1979) Correlation between leukemic cell retention of 1-fl-D-arabinofuranosylcytosine 5'-triphosphate and response to therapy. Cancer Res. 39: 42~,9. SANNO, Y., HOLZER, M. and SCHIMKE, R. T. (1970) Studies of a mutation affecting pyrimidine degradation in inbred mice. J. biol. Chem. 245: 5668-5676. SANTI, D. V. and DANENBERG,P. V. (1984) Folates in pyrimidine nucleotide biosynthesis. In: Folates and Pterins, pp. 345-398, BLAKLEY, R. L. and BENKOVIC, S. J. (eds) John Wiley & Sons, New York. SANTI, D. V. and HARDY, L. W. (1987) Catalytic mechanism and inhibition of tRNA (uracil-5-)methyltransferase: Evidence for covalent catalysis. Biochemistry 26: 85998606. SAWYER,R. C., STOLFI,R. L., MARTIN, D. S. and SPIEGELMAN, S. (1984) Incorporation of 5-fluorouracil into murine bone marrow DNA in vivo. Cancer Res. 44: 1847 1851. SCHOLAR,E. and CALABRESI,P. (1973) Identification of the enzymatic pathways of nucleoside metabolism in human lymphocytes and leukemic ceils. Cancer Res. 33: 94-103. SCHRECKER, A. W. and URSHEL, M. J. (1968) Metabolism of 1-fl-D-arabinofuranosylcytosine nucleoside inhibition of sialic acid metabolism of WI-38 cells. Cancer Res. 28: 793-810. SCHUETZ, J. D. and DIASIO, R. B. (1985) The effect of 5-fluorouracil on DNA chain elongation in intact bone marrow ceils. Biochem. biophys. Res. Commun. 133: 361-367. SCHUETZ, J. D., COLLINS, J. M., WALLACE, H..l. and DIASlO, R. B. (1986) Alteration of the secondary structure of newly synthesized DNA from murine bone marrow cells by 5-fluorouracil. Cancer Res. 46:119 123. SCHUETZ, J. D., WALLACE,H. J. and D1ASlO, R. B. (1988) DNA repair following incorporation of 5-fluorouracil into DNA of mouse bone marrow ceils. Cancer Chemother. Pharmac. 21: 208-210. SCHWARTZ, P. M., MOIR, R. D., HYDE, C. M., TUREK, P. J. and HANDSCHUMACHER, R. E. (1985) Role of uridine phosphorylase in the anabolism of 5-fluorouracil. Biochem. Pharmac. 34: 3585-3589. SERGOTT, R. C., DEBEER, L. J. and BESSMAN,M. J. (1971) On the regulation of bacterial deoxycytidylate deaminase. J. biol. Chem. 246: 7755-7758.
SHIOTANI,T. and WEBER,G. (1981) Purification and properties of dihydrothymine dehydrogenase from rat liver. J. biol. Chem. 256: 219-224.
SHOUVAL, D., ADLER, R., WANDS, J. R. and HURWITZ, E. (1986) Conjugates between monoclonal antibodies to HBsAg and cytosine arabinoside. J. Hepatol. 3 (Suppl. 2): $87-95. Snow, T. B., ALPER, C. A., BOOTSMA, D., DORF, M., DOUGLAS,T., HUISMAN, T., KIT, S., KLINGER, H. P., KOZAK, P. A., LALLEY, P. A., LINDLEY, D., McALPINE, P. J., McDOUGALL, J. K., MEERAKHAN, P., MEISLER, M., MORTON, N. E., OPlTZ, C. W., PARTRIDGE,C. W., PAYNE, R., RODERICK, T. H., RUBINSTEIN, P., RUDDLE, F. H., SHAW, M., SPRANGER, J. W. and WEISS, K. (1979) International system for human gene nomenclature. Cytogenet. Cell Genet. 25:96-116. SKrLD, O. (1958) Enzymic ribosidation and ribotidation of 5-fluorouracil by extracts of the Ehrlich-ascites tumor. Biochim. biophys. Acta 29: 651. SKOLD, O. (1963) Studies on resistance against 5-fluorouracil IV. Evidence for an altered uridine kinase in resistant cells. Biochim. biophys. Acta 76: 160-162. SLECHTA, L. (1961) Effect of arabinonucleotides on growth and metabolism of E. coll. Fedn Proc. 20: 357. SMITH, C. G., LUMMIS,W. L. and GRADY, J. E. (1959) An improved tissue culture assay. II. Cytotoxicity studies with antibiotics, chemicals and solvents. Cancer Res. 19: 847-852. SMYTH, J. F., ROmNS, A. B. and LEESE, L. L. (1976) The metabolism of cytosine arabinoside as a predictive test for clinical response to the drug in acute myeloid leukaemia. Eur. J. Cancer 12: 567-575. SOCCA, J. J., PANNY, S. A. and BESSMAN,M. J. (1969) Studies of deoxycytidylate deaminase from T4-infected Escherichia coli. J. biol. Chem. 244: 3698-3706. SOMMADOSSI,J.-P., GEWIRTZ, D. A., DIASIO, R. B., AUaERT, C., CANO, J. P. and GOLDMAN, I. D. (1982) Rapid catabolism of 5-fluorouracil in freshly isolated hepatocytes as analyzed by high performance liquid chromatography. J. biol. Chem. 257: 8171-8176. SORM, F., PISKALA, A., CIHAK, A. and VESELY, J. (1964) 5-Azacytidine, a new highly effective cancerostatic. Experientia 20: 202-203. SPIEGELMAN, S., NAYAK, R., SAWYER, R., STOLFI, R. and MARTIN, D. (1980a) Potentiation of the anti-tumor activity of 5-FU by thymidine and its correlation with the formation of (5FU) RNA. Cancer 45:1129-1134. SPIEGELMAN, S., SAWYER, R., NAYAK, R., RITZ1, E., STOLFI, R. and MARTIN, D. (1980b) Improving the anti-tumor activity of 5-fluorouracil by increasing its incorporation into RNA via metabolic modulation. Proc. natn. Acad. Sci. U.S.A. 77: 4966-4970. SPlERS, A. S. D. and FIRTH, J. L. (1972) Treating the nervous system in acute leukemia. Lancet 2: 1433. STENSTROM, M. L., EDELSTEIN, M. and GRISHAM, J. W. (1974) Effect of ara-CTP on DNA replication and repair in isolated hepatocyte nuclei. Expl Cell Res. 89: 439-442. STERNBERG, A., PETRELLL N., AU, J. L.-S., RUSTUM, Y. M., MITTELMAN, A. and CREAVEN,P. (1984) A combination of 5-fluorouracil and thymidine in advanced colorectal carcinoma. Cancer Chemother. Pharmac. 13: 218-222. STEUART,C. D. and BURKE,P. J. (1971) Cytidine deaminase and the development of resistance to arabinosyl cytosine. Nature New Biol. 233: 109-110. STOLLER, R. G., MYERS, C. E. and CHABNER, B. A. (1978) Analysis of cytidine deaminase and tetrahydrouridine interaction by use of ligand techniques. Biochem. Pharmac. 27:53 59. SUGINO, Y., TERAOKA, H. and SHIMONO, H. (1966) Metabolism of deoxyribonucleotides. I. Purification and properties of deoxycytidine monophosphokinase of calf thymus. J. biol. Chem. 241: 961-969.
Pyrimidine analogues and their nucleosides SWEENY, D. J., BARNES,S., HEGGIE, G. H. and DIASIO, R. B. (1987) Metabolism of 5-fluorouracil to an N-cholyl-2fluoro-b-alanine conjugate: Previously unrecognized role for bile acids in drug conjugation. Proe. natn. Acad. Sci., U.S.A. 84: 5439-5443. SWEENY, n . J., MARTIN, M. and D1ASIO, R. B. (1988) Nchenodeoxycholyl-2-fluoro-b-alanine: A biliary metabolite of 5-fluorouracil in humans. Drug Metab. Dispos. 16: 892-893. TAGGER, A. Y. and WRIGHT, J. A. (1988) Molecular and cellular characterization of drug resistant hamster cell lines with alterations in ribonucleoside reductase. Int. J. Cancer 42: 760-766. TAGGER, A. Y., Soux, J. and WRIGHT, J. A. (1987) Hydroxy[t4C]urea uptake by normal and transformed human cells: Evidence for a mechanism of passive diffusion. Biochem. Cell Biol. 65: 925-929. TAKIMOTO, C. H., TAN, Y. Y., CADMAN, E. C. and ARMSTRONG, R. D. (1987) Correlation between ribosomal RNA production and RNA-directed fluoropyrimidinecytotoxicity. Biochem. Pharmac. 36: 3243-3248. TAMAKI, N., MIZUTANI, N., KIKUGAWA, M., FUJIMOTO, S. and MIZOTA, C. (1987) Purification and properties of b-ureidopropionase from the rat liver. Eur. J. Biochem. 169: 21-26. TANAKA, M. and YOSH1DA,S. (1987) Formation of cytosine arabinoside-5'-triphosphate in cultured human leukemic cell lines correlates with nucleoside transport capacity. Jap. J. Cancer Res. 78: 851-857. TATTERSALL,M. N. H., GANESHAGURU,K. and HOFFBRAND, A. V. (1974) Mechanisms of resistance of human acute leukaemia cells to cytosine arabinoside. Br. J. Haemat. 27: 39-46. TAYLOR, A. T., STAFFORD, M. A. and JONES, O. W. (1972) Properties of thymidine kinase partially purified from human fetal and adult tissue. J. biol. Chem. 247: 1930-1935. THELANDER, L. and REICHARD, P. (1979) Reduction of ribonucleotides. A. Rev. Biochem. 48: 133-158. THELANDER, L., ERIKSSON,S. and ,~KERMAN,M. (1980) Ribonucleoside reductase from calf thymus. Separation of the enzyme into two nonidentical subunits, proteins MI and M2. J. biol. Chem. 255: 7426-7432. THELANDER, M., GR.~SLUND, A. and THELANDER, L. (1985) Subunits M2 of mammalian ribonucleoside reductase. Characterization of a homogeneous protein isolated from M2-overproducing mouse cells. J. biol. Chem. 260: 2737-2741. TOMCHICK, R., SASLAW, L. D. and WARAVDEKAR, V. S. (1968) Mouse kidney cytidine deaminase. Purification and properties. J. biol. Chem. 243: 2534-2537. TRAUT, T. W. and LOECnEL, S. (1984) Pyrimidine catabolism: Individual characterization of three sequential enzymes with a new assay. Biochemistry 23: 2533-2539. TUCHMAN, M., STOECKELER, J. S., KIANG, D. T., O'DEA, R. F., RAMNARAINE, M. L. and MIRKIN, B. L. (1985) Familial pyrimidinemia and pyrimidinuria associated with severe ttuorouracil toxicity. New Engl. J. Med. 313: 245 249. ULLMAN, B., LEE, M., MARTIN, D. W. and SANTI,D. V. (1978) Cytotoxicity of 5-fluoro-2'-deoxyuridine: Requirement for reduced folate cofactors and antagonism by methotrexate. Proc. natn. Acad. Sci. U.S.A. 75: 980-983. ULLMAN, B., LEVINSON, B. B., ULLMAN, D. H. and MARTIN, D. W. J. (1979) Isolation and characterisation of cultured mouse T-lymphoma deficient in uridine-cytidine kinase. J. biol. Chem. 254: 8736-8739. VADLAMUD1,S., CHOUDRY,J. N., WARAVDEKAR,V. S., KLINE, I. and GOLDIN, A. (1970a) Effect of combination treatment with 5-azacytidine and cytidine on the life span and spleen and bone marrow cells of leukemic (L1210) and non-leukemic mice. Cancer Res. 30: 362-369.
221
VADLAMUDI, S., PADARATHSINGH, M., BONMASSAR,E. and GOLDIN, A. (1970b) Reduction of antileukemic and immunosuppressive activities of 5-azacytidine in mice by concurrent treatment with uridine. Proc. Soe. exp. Biol. Med. 133: 1232-1238. VEREZ,Z., SZINAI,I., SZABOLCS,A., UJSZASZY,K. and DENES, G. (1987) Biological activity of the potent uridine phosphorylase inhibitor 5-ethyl-2,2'-anhydrouridine. Drugs under exp. clin. Res. 13: 615-621. VESELY,J. and SMRT, J. (1977) Enzymes relevant to cancer chemotherapy. II. Heterogeneity of rat kidney uridine kinase following chromatography on sepharose 6B coupled to N-(5-amino-pentyl)-cytidine. Neoplasma (Bristol) 24: 461-467. VESSEY,D. A., CRISSEY, M. H. and ZAKIM,D. (1977) Kinetic studies on the enzymes conjugating bile acids with taurine and glycine in bovine liver. Biochem. J. 163: 181-183. VITA, A., AM1CI, A., CACCIAMANI, T., LANCIOTTI, M. and MAGNI, G. (1985) Cytidine deaminase from Escherichia coil B. Purification and enzymatic and molecular properties. Biochemistry 24: 6020-6024. VITA, A., CACCIAMANI, T., NATALINI, P., RUGGIERI, S., SANTARELLI, I. and MAGNI, G. (1987) Purification of cytidine deaminase from chicken liver. Ital. J. Biochem. 36: 275-282. VOGLER, W. R., MILLER, D. S. and KELLER, J. W. (1976) 5-Azacytidine (NSC-I02816): a new drug for the treatment of myeloblastic leukemia. Blood 48: 331-337. VON HOFF, D. D., SLAVIK,M. and MUGGIA,F. M. (1976) A new anticancer drug with effectiveness in acute myelogenous leukemia. Ann. intern. Med. 85: 237-245. WALLACH, O. P. and SANTIAGO,G. (1957) The purification and properties of hydropyrimidine hydrase. J. biol. Chem. 251: 6909-6914. WALLISER,S. and REDMANN,K. (1978) Effect of 5-fluorouracil and thymidine on the transmembrane potential and 6 potential of HeLa cells. Cancer Res. 38: 3555-3559. WALSH, C. Z., CRAIG, R. W. and AGARWAL, R. P. (1980) Increased activation of 1-fl-arabinofuranosylcytosine by hydroxyurea in L1210 cells. Cancer Res. 40: 3286-3292. WEBER, G. (1980) Recent advances in the design of anticancer chemotherapy. Oneology (Basel)(Suppl. l): 19-24. WECKBECKER,G., LIEN, E. J. and CORY, J. G. (1988) Properties of N-hydroxy-N'-aminoguanidine derivatives as inhibitors of mammalian ribonucleotide reductase. Biochem. Pharmac. 37: 529-534. WILEY, J. S., JONES, P. E., SAWYER, W. H. and PATERSON, A. R. P. (1982) Cytosine arabinoside influx and nucleoside transport sites in acute leukemia. J. clin. Invest. 69: 479-489. WILEY, J. S., JONES,P. E. and SAWYER,W. H. (1983) Cytosine transport by human leukemic cells. Fur. J. Cancer clin. Oncol. 19: 1067-I074. WILEY, J. S., TAUPIN, J., JAMIESON, G. P., SNOOK, M., SAWYER,W. H. and FINCH, L. R. (1985) Cytosine arabinoside transport and metabolism in acute leukemias and T cell lymphoblastic lymphoma. J. clin. Invest. 75: 632-642. WILKINSON, D. S. and CRUMLEY, J. (1977) Metabolism of 5-fluorouracil in sensitive and resistant Novikoff hepatoma cells. J. biol. Chem. 252: 1051-1056. WILKINSON, L. S. and PITOT, H. C. (1973) Inhibition of ribosomal ribonucleic acid maturation on Novikoff hepatoma cells by 5-fluorouracil and 5-fluorouridine. J. biol. Chem. 248: 63-68. WILKINSON, D. S., TlSTY, T. D. and HANAS, R. J. (1975) The inhibition of ribosomal RNA synthesis and maturation in Novikoff hepatoma cells by 5-fluorouridine. Cancer Res. 35: 3014-3020. WISDOM, G. B. and ORSl, B. A. (1969) The purification and properties of cytidine aminohydrolase from sheep liver. Fur. J. Biochem. 7: 223-230.
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WOHLHUETER, R. M. and PLAGEMANN, P. G. W. (1980) Role of transport and phosphorylation in nutrient uptake in cultured animal cells. Int. Rev. Cytol. 64: 171-240. WOHLHUETER, R. M., MCIVAR, R. S. and PLAGEMAN, P. G. W. (1980) Facilitated transport of uracil and 5fluorouracil, and permeation of orotic acid into cultured mammalian cells. J. cell. Physiol. 194: 309-319. WOODCOCK, T. M., MARTIN, D. S., DAMIN, A. E. M., KEMENY, N. E. and YOUNG, C. W. (1980) Combination clinical trials with thymidine and fluorouracil: A phase 1 and clinical pharmacologic evaluation. Cancer 45: 1135-1143.
WRIGHT, J. A. (1983) Altered forms of mammalian nu-
cleoside diphosphate reductase from mutant cell lines. Pharmac. Ther. 22: 81-102. YANG, J. L., CHENG, E. H., CAPIZZl, R. L., CHENG, Y.-C. and KUTE, T. (1985) Effect of uracil arabinoside on metabolism and cytotoxicity of cytosine arabinoside in L5178Y murine leukemia. J. clin. Invest. 75: 141-146. YOUNG, I., YOUNG, G. J., WILEY, J. S. and VANDER WEYDEN, M. B. (1985) Nucleoside transport and cytosine arabinoside (araC) metabolism in human T lymphoblasts resistant to araC, thymidine and 6-methylmercaptopurine riboside. Eur. d. Cancer clin. OncoL 21: 1077-1082. ZIMMERMAN,M. (1964) II. Nucleoside:pyrimidine deoxyribosyltransferase activity of three partially purified thymidine phosphorylases. J. biol. Chem. 239: 2622-2627.
0163-7258/90 $0.00+ 0.50 © 1990 Pergamon Press pie
Specialist Subject Editor: G. Powls
METABOLISM OF PYRIMIDINE ANALOGUES A N D THEIR NUCLEOSIDES GEORGE C. DAHER, BARRY E. HARRIS and ROBERTB. DIASIO Department of Pharmacology, Division of Clinical Pharmacology, University of Alabama at Birmingham, Birmingham, AL 35294, U.S.A. Abstract--The pyrimidine antimetabolite drugs consist of base and nucleoside analogues of the naturally occurring pyrimidines uracil, thymine and cytosine. As is typical of antimetabolites, these drugs have a strong structural similarity to endogenous nucleic acid precursors. The structural differences are usually substitutions at one of the carbons in the pyrimidine ring itself or substitutions at one of the hydrogens attached to the ring of the pyrimidine or sugar (ribose or deoxyribose). Despite the differences noted above, these analogues, can still be taken up into cells and then metabolized via anabolic or catabolic pathways used by endogenous pyrimidines. Cytotoxicity results when the antimetabolite either is incorporated in place of the naturally occurring pyrimidine metabolite into a key molecule (such as RNA or DNA) or competes with the naturally occurring pyrimidine metabolite for a critical enzyme. There are four pyrimidine antimetabolites that are currently used extensively in clinical oncology. These include the fluoropyrimidines, fiuorouracil and fluorodeoxyuridine, and the cytosine analogues, cytosine arabinoside and azacytidine.
CONTENTS 1. Fluoropyrimidines 1.1. Historical perspective 1.2. Chemistry 1.3. Uptake into cells 1.4. Metabolism 1.4.1. Catabolism 1.4.2. Anabolism 1.5. Metabolic site of action 1.5.1. Thymidylate synthase 1.5.2. RNA incorporation 1.5.3. DNA incorporation 1.5.4. Incorporation into cell membranes 1.6. Miscellaneous fluoropyrimidine drugs 2. Arabinosylcytosine 2.1. Historical perspective 2.2. Chemistry 2.3. Uptake into cells 2.4. Metabolism 2.4.1. Catabolism 2.4.2. Anabolism 2.5. Metabolic sites of action 2.5.1. Inhibition of DNA biosynthesis 2.5.2. Inhibition of DNA repair 2.5.3. Incorporation into nucleic acids 2.5.4. Inhibition of membrane precursor synthesis 3. Azacytidine 3.1. Historical perspective 3.2. Chemistry 3.3. Uptake into cells 3.4. Metabolism 3.4.1. Catabolism 3.4.2. Anabolism 3.5. Metabolic sites of action References 189
190 190 190 190 191 192 194 200 200 202 203 203 204 204 204 204 205 206 206 208 210 210 210 210 210 211 211 211 211 211 212 212 213 213
190
G.C. DAHERet al. 1. F L U O R O P Y R I M I D I N E S
Over the past few decades, much information has been accumulated on the metabolism and clinical pharmacology of fluoropyrimidines. 5-Fluorouracil (FUra) and 5-fluoro-2'-deoxyuridine (FdUrd) have been shown to be effective antimetabolites with antitumor activity in a number of different tumors including carcinomas of the gastrointestinal tract, breast, ovary, and skin. Extensive studies have produced a large volume of information on the metabolism of fluoropyrimidines but the relative importance of various metabolites in the antitumor activity of these drugs is still unclear. Also, despite many pharmacokinetic studies the best route or schedule of administration for fluoropyrimidines has not been determined. Several routes of administration are currently used including continuous infusion, bolus intravenous, intraperitoneal, and hepatic arterial infusion.
chemistry especially after it became apparent that FUra and its derivatives showed antitumor activity. In this review, our discussion of the chemistry of FUra will be narrowed to what is pertinent to its role as an anticancer agent. FUra differs from the naturally-occurring nucleic acid precursor, uracil, by the presence of a fluorine on the fifth carbon. In addition, the van der Waal's radius of the fluorine atom (1.35 A) is similar to that of hydrogen (1.2 A), thus minimizing alteration of the structural conformation and insuring that F U r a can be metabolized by the enzymes that metabolize uracil. The consequences of the substitution of the fluorine for the hydrogen at the fifth position of the pyrimidine ring will be discussed in detail in Section 1.5.1. The electronegativity of the fluorine atom on the ring increases the acidity of FUra, which is reflected by a lower pK a as compared to its natural analogue uracil (Berens and Shugar, 1963).
1.1. HISTORICAL PERSPECTIVE The fluoropyrimidine drugs (Fig. 1), represented primarily by the pyrimidine base FUra and its nucleoside FdUrd, were introduced over thirty years ago. In contrast to most chemotherapy agents, these drugs were rationally synthesized. The motivation for the design of these drugs came from an earlier observation that certain tumors (e.g. rat hepatomas) required exogenous uracil to sustain nucleic acid synthesis and, hence, tumor growth. This, in turn, led to the suggestion that pyrimidine analogues could be synthesized with physicochemical properties similar to uracil. Because of their structural similarity, these analogues can be taken up by cells and metabolized. However, the minor structural alteration may interfere with a critical enzymatic step or may replace the naturally-occurring pyrimidine nucleotides. Since most tumors are rapidly dividing these drugs may be selective for tumor cells in comparison to their normal counterparts. 1.2. CHEMISTRY
Since FUra was first synthesized by Duschinsky from acyclic precursors (Duschinsky et al., 1957), considerable attention has been focused on its bio-
0
Uracil 5-Fluorouracil
pKl 9.50 8.00
F U r a is commercially available in solution buffered with NaOH to yield an alkaline solution with a pH of approximately 9.0. Upon ultraviolet exposure, there is no evidence of dimer formation. However, in aqueous solution, FUra undergoes a photochemically induced reaction to form an alkalilabile, acid-soluble compound that has been identified as 5-fluoro-6-hydroxy-5,6-dihydrouracil (Lozeron et al., 1964). Exposure to wavelengths less than 270 nm causes secondary reactions of this initial product releasing the fluorine from the fifth carbon to form barbituric acid.
1.3. UPTAKE INTO CELLS
The pyrimidine base F U r a had long been thought to enter cells through a simple diffusion process achieving equilibrium rapidly between intracellular and extracellular compartments (Jacquez, 1962; Kessel and Hall, 1967). Subsequent studies (Wohlhueter et al., 1980) using rapid sampling techniques suggested that both F U r a and uracil enter the cell via a carrier-mediated transport process which
0
O H
H Uracil
pK2 > 13.0 ~ 13.0
5-Fluorouracll (FUrs)
OH 5 - F l u o r o - 2 ' - d e o x y u rldlne (FdUrd)
FIG. |. Structures of uracil, 5-fluorouracil and 5-fluoro-2'-deoxyuridine.
Pyrimidine analogues and their nucleosides appeared to be saturable and nonconcentrative. Concentrations which resulted in 'half saturation' were relatively high such that entry of either pyrimidine at pharmacological concentrations was first order. At relatively low concentrations of 5-fluorouracil, transport was not rate-limiting to intracellular metabolism of either pyrimidine. The nucleoside F d U r d is believed to enter cells via the same mechanism used by the naturally occurring nucleosides uridine and deoxyuridine. As with studies examining uptake of pyrimidine bases, these studies have been complicated by technical limitations, particularly the need for rapid sampling techniques to discriminate between transport and metabolism. This is due to the fact that the nucleoside is rapidly phosphorylated within the cells and, thus, cannot pass back out of the cell. The studies of Bowen et al. (1978) demonstrated that under pharmacologic conditions, transport of F d U r d was rapid and not ratelimiting. In contrast, metabolism of FdUrd appeared to be the rate-limiting step.
Liver end ..~ extrshepetlc tissues
191
-8O'/,
FUrs
1-3%
Anebollsm
1s-2o%) Urinary sx¢retlon
FIG. 2. Metabolic fate of 5-fluorouracil in humans. 1.4. METABOLISM
Fluoropyrimidines must be initially metabolized before they are clinically effective. Biotransformation may result in the formation of either less toxic catabolites or more toxic anabolites. In this review we will discuss the metabolism of pyrimidine analogues, their nucleosides, as well as, the biochemical modulation of their metabolism. In addition, we will review their sites of action.
H o r (F)
A
NAD(P)H'~
NAD(P)H
NAD(p),dk" ~ [ "~"NAD(P)
q~°'~o O
N.,C~.
FO
© 9
FIG. 3. Catabolism of 5-fluorouracil in humans. (1) Dihydropyrimidine dehydrogenase (DPD); (2) dihydropyrimidinase; (3) fl-alanine synthase; (4) bile acid n-acyl CoA transferase. FUPA = 5-fluoroureidopropionic acid; FBAL = 5-fluoro-fl-alanine; N-cholyI-FBAL = N-cholyl-5-fluoro-fl-alanine. JPT 48~2--F
192
G.C. DAHERet al.
The metabolic fate of administered FUra is given in Fig. 2. The level of FUra available for anabolism is primarily determined by catabolism (Heggie et al., 1987). The importance of catabolism has been demonstrated in clinical studies in which FUra was co-administered with other pyrimidines such as thymine or uracil resulting in increased availability of FUra and, in turn, increased anabolism (Au et al., 1982). Therefore, the balance between catabolism and anabolism is determined by several factors including: (1) the intracellular environment (e.g. presence of NADPH, ATP, PRPP) of the exposed cell; (2) the intracellular concentration of the substrate and/or its analogue; and (3) the activities of anabolic vs catabolic enzymes which are present at variable levels in different cell types. 1.4.1. Catabolbm
Catabolism has a major role in regulating the variability of fluoropyrmidines. This degradative pathway is identical to the reductive pathway of the naturally occurring pyrmidines, uracil and thymine. The presence of a halogen on the fifth carbon of the pyrimidine ring does not impede its catabolism and,
~ 0
H or (F)
surprisingly, FUra is more efficiently degraded than is uracil (Naguib et al., 1985). In this catabolic pathway (Fig. 3), the double bond between C-5 and C-6 of the pyrimidine ring is first reduced in the presence of NADPH. The ring is then hydrolyzed between N-3 and C-4 and the resulting ~-fluoro-flureidopropionate (FUPA) is subsequently cleaved between N-I and C-2 to yield ~-fluoro-fl-alanine (FBAL), carbon dioxide, and ammonia. The degradative pathway consists of three enzymes: (1) dihydropyrimidine dehydrogenase (DPD), (2) dihydropyrimidinase (DHPase); and (3) fl-alanine synthase. Each of these enzymes will be discussed separately. Halogenated pyrimidine analogues are usually dehalogenated in the course of their degradation and excreted in urine. In contrast, catabolism of FUra does not result in defluorination. Our laboratory has examined the clinical pharmacokinetics of 3H.FUra and its metabolites in plasma, urine and bile (Heggie et al., 1987). Over 24 hr, FBAL represented the major metabolite in urine accounting for 50% of the administered dose. Biliary excretion of radioactivity over 24 hr represented 2-3% of the total dose. A significant percentage (i.e. 80-90%) of these biliary metabolites were previously unrecognized (Heggie et al., 1987). These novel metabolites were later identified as conjugates of FBAL and either cholic acid (Sweeny
et al., 1987) or N-chenodeoxycholic acid (Sweeny et al., 1988). This previously unrecognized reaction is
catalyzed by cholyl-CoA-glycine/taurine N-acyltransferase (EC 2.3.1.13). This enzyme is present in the soluble fraction of liver and requires the prior activation of bile acid to a CoA derivative (Vessey et al., 1977). The isolation and characterization of this enzyme are currently in progress in our laboratory. 1.4.1.1. Dihydropyrimidine dehydrogenase. DPD (EC
1.3.1.2) is the initial enzyme in the degradation of the pyrimidine bases uracil and thymine. Specifically, it catalyzes the reversible reduction of the pyrimidine base to its corresponding dihydropyrimidine. This enzyme (eqn 1) also catalyzes the conversion of FUra to dihydrofluorouracil (FUHz). In contrast, cytosine and its analogues are not substrates for DPD. These agents must be deaminated to uracil by cytosine deaminase before their subsequent degradation. It is important to note, however, that cytosine deaminase is restricted only to micro-organisms and is not present in mammalian cells (O'Donovan and Neuhard, 1970).
0
~~L~y or (F) (1)
DPD activity is confined primarily to cytosolic supernatant fraction (Canellakis, 1956; Fritzson, 1960; Shiotani and Weber, 1981). It has been purified to homogeneity from rat liver cytosol (Shiotani and Weber, 1981). This enzyme is composed of two identical subunits with an approximate MW of 110 _+ 3 kDa each. DPD has been detected in normal human liver, pancreas, lung, intestinal mucosa, lymphocytes and in various neoplastic and hematopoietic tumors (Naguib et al., 1985). DPD activity in most tumors has been shown to be equivalent or higher than that determined in normal tissues from which the tumors derive (Maehera et al., 1981; Naguib et al., 1985; Weber, 1980). The pH optimum for rat liver DPD has been shown to be 7.4. In the presence of NADPH, the apparent Km was 2.6 pM for thymine and 1.8 #M for uracil. The apparent Km for NADPH was 15 #M with thymine as a substrate and 11 p M with uracil. The specific activity of DPD at 20 #M FUra was reported to be 47.4#mol/mg protein/hr. In contrast, the specific activity of DPD at the same concentration of uracil was 34.9#mol/mg protein/hr (Shiotani and Weber, 1981). In studies with rat liver, it has been suggested that DPD is the rate-limiting enzyme for pyrimidine catabolism (Canellakis, 1956; Fritzson, 1960, 1962; Queener et al., 1971). Other studies have suggested
Pyrimidine analogues and their nucleosides that dihydropyrimidinase is the rate-limiting enzyme in rat hepatocytes (Mentre et al., 1984; Sommadossi et al., 1982) and in human extrahepatic tissues and hematopoietic neoplasms (Naguib et al., 1985). Still other studies have suggested that fl-alanine synthase is the rate-limiting enzyme in mouse liver (Sanno et al., 1970; Traut and Loechel, 1984). Although it is still unclear which enzyme is rate-limiting, the
193
catalyzes the hydrolysis of a variety of 5,6-dihydropyrimidines (Kim et al., 1976; Wallach and Santiago, 1957), including the reversible conversion of FUH2 to FUPA (eqn 2), as well as catalyzing the hydrolytic ring opening of 5-phenylhydantoin and ~-phenylsuccinamide (Dudley et al., 1974; Maguire and Dudley, 1978). It has also been implicated in the dehalogenation of other pyrimidines (Kim et al., 1976).
O
H~
I
H or (F)
H2N~ ( ~ "~1 % or IF) (2)
presence of DPD activity is critical since its deficiency results in severe neurologic toxicity (Diasio et al., 1988; Tuchman et al., 1985). This toxicity was suggested to be due to an increased anabolism of FUra (Diasio et al., 1988). Inhibition of DPD activity has been shown to potentiate the effect of FUra in vitro and in vivo (Cooper et al., 1972; Desgranges et al., 1986; Masaaki et al., 1988; Matthes et al., 1974). 5-Benzyloxybenzyluracil and 1-deazauracil appear to be the most potent inhibitors of DPD activity with apparent Ki values of 0.2 and 0.5 #M, respectively (Naguib et al., 1989). In contrast the Km value for uracil has been reported to be 9/a M. The potential therapeutic benefit of inhibitors of DPD has been suggested in studies using bromovinyideoxyuridine in the modulation of FUra metabolism (Iigo et al., 1988). However, in light of the severe toxicity in patients with DPD deficiency, extreme caution should be used before administration of these compounds in conjunction with FUra chemotherapy. Further studies on these inhibitors are needed to determine their in vivo metabolism and possible side effects. Varying plasma concentration of FUra has been demonstrated during continuous infusion of FUra (Erlichman et al., 1986; Kawai et al., 1976; Petit et al., 1988). Our laboratory has also demonstrated that DPD activity in rat liver varies over a 24 hr period in association with light~zlark cycle (Harris et al., 1988). This may provide a biochemical explanation for the periodic variation of the plasma FUra level. In patients receiving FUra by protracted continuous infusion (300 mg/m2/day), we demonstrated an apparent inverse relationship between DPD activity in human peripheral blood mononuclear cells and plasma FUra levels (Harris et al., 1990). This association suggests that DPD may be a major determinant of the circadian variation of FUra plasma concentration when this drug is administered by protracted continuous infusion. 1.4.1.2. Dihydropyrimidinase. Dihydropyrimidinase (EC 3.5.2.2) is the second enzyme in the degradative pathway of uracil and thymine. It catalyzes the reversible hydrolysis of dihydrouracil to fl-ureidopropionic acid (fl-UPA). Dihydropyrimidinase also
Dihydropyrimidinase has been partially purified from calf (Wallach and Santiago, 1957) and rat liver (Maguire and Dudley, 1978) and, more recently, the bovine liver enzyme has been purified to homogeneity (Brooks et aL, 1983, 1979; Lee et aL, 1986). It was shown to be a tetramer with a MW of 226 kDa, and subunit molecular weight of 56.5 kDa. This enzyme contains 4 gram atoms of tightly bound Zn 2+ per mole of active enzyme with a Ks >1.3 x 1 0 9 M -1 (Brooks et al., 1983; Lee et al., 1986), presumably one gram atom Zn 2÷ per subunit. Previous work suggested that dihydropyrimidinase is a true Zn 2÷ metalloenzyme, since there exists a linear relationship over an enrichment range of 0 to 4 gram atoms of Zn 2÷ per mole of enzyme (Brooks et al., 1979). Chelation of Zn 2÷ by 2,6-dipicolinic acid, 8-hydroxyquinoline-5-sulfonic acid, or o-phenanthroline will cause the loss of dihydropyrimidinase activity (Brooks et al., 1983). The reaction pathway for this inactivation process involves the formation of a ternary enzyme-metal-chelator complex which then dissociates to yield the apoenzyme and the Zn 2÷chelate binary complex (Lee et al., 1986). Enzymatic activity will only be restored by reconstituting the hoioenzyme with the reincubation of the apoenzyme with Zn 2÷, Co 2÷ or Mn 2÷ (Brooks et al., 1983). Electron paramagnetic resonance studies have shown that substitution of Mn 2÷ for Zn 2÷ increases the specific activity of the enzyme approximately six-fold but only increases the Km value for 5-bromo-5,6-dihydrouracil (BrUH2) two-fold (Lee et aL, 1987). Like other metalloenzymes (e.g. carbonic anhydrase, dihydroorotase), dihydropyrimidinase is inhibited by several substituted sulfonamides. With 4-nitrobenzene sulfonamide, the inhibition was found to be competitive with respect to BrUH2 with a K~ value of approximately 0.6 mM (Brooks et al., 1983). 1.4.1.3. fl-Alanine synthase (fl-ureidopropionase). flAlanine synthase (EC 3.5.1.6), the third enzyme in the catabolism of pyrimidines, irreversibly cleaves flUPA (N-carbamyl-fl-alanine) to fl-alanine and generates carbon dioxide from the second carbon of the pyrimidine ring and ammonia from N-3. In addition, fl-alanine synthase also splits FUPA into FBAL (eqn 3).
194
G. C. DAHERet al. F
FO
(3)
H
fl-Alanine synthase was recently purified to homogeneity from rat liver, and was shown to have a MW in the range of approximately 323 to 327 kDa with a pH optimum for enzyme activity of 7.0 and a pl of 6.4 (Tamaki et al., 1987). Kinetic studies with this enzyme demonstrated that propionic acid, the structural analogue of fl-UPA, acts as an allosteric activator as well as a competitive inhibitor of fl-alanine synthase (Kikugawa et al., 1988). This derivative has a K~ of approximately 0.3 mM at pH 7.0; whereas, the Km for fl-UPA was 0.06 mM. A recent report (Matthews and Traut, 1987) suggested that fl-alanine synthase may exist as three different molecular weight species: a native form in the absence of ligands with a MW of 235 kDa; an active, higher MW form in the presence of its substrate fl-UPA (or its analogues); and an inactive, lower MW form in the presence of its product fl-alanine (or its analogues). It is thought that flalanine synthase contains at least one catalytic and one regulatory site. More work is needed in order to elucidate the complete mechanism of action of this enzyme.
1.4.2. A n a b o l i s m The anabolism of FUra to nucleotides occurs through one of several pathways (Fig. 4). As with catabolism, these pathways are identical to the de nova pathways used by the naturally-occurring pyrimidine, uracil. These include:
(l) a direct conversion catalyzed by pyrimidine phosphoribosyl transferase of FUra to FUMP by the transfer of ribose phosphate from phosphoribosyl pyrophosphate (PRPP) (Reyes, 1969),
(2) a sequential, two-step reaction consisting of the initial addition of a ribose by uridine phosphorylase to yield FUrd, followed by phosphorylation to FUMP by uridine kinase (Skrld, 1958), and (3) the addition of deoxyribose-l-phosphate to FUra by thymidine phosphorylase to yield FdUrd followed by phosphorylation to FdUMP by thymidine kinase (Hartmann and Heidelberger, 1961). 1.4.2. I. Uridine phosphorylase. Uridine phosphorylase (UrdPase; EC 2.4.2.3) catalyzes the conversion of the naturally occurring pyrimidine, uracil, to its nucleoside, uridine. FUra has also been shown to be
a substrate for this enzyme. As shown in eqn 4, UrdPase cleaves inorganic phosphate (P~) from ribose-l-phosphate (R-I-P) and, subsequently, forms a nucleoside bond between the pyrimidine base and the ribose. In addition to its anabolic role, this cytoplasmic enzyme catabolizes nucleotides that are critical to the 'salvage' of nucleic acids. In an attempt to explain this amphibolic role, Naguib et al. (1987b) proposed that the variables responsible for the catabolic or anabolic action of UrdPase include: (1) the activities of other enzymes which compete for the same substrate and (2) the intracellular concentration of substrates. UrdPase is relatively nonspecific, since it also cleaves pyrimidine 2'- and 5'-deoxyribose nucleosides (Niedzwicki et al., 1983). This enzyme is widely distributed in many rat organs, but is reported to have the highest activity in small intestine (Ardalan and Glazer, 1981).
Renal.. ~. excretion ~
y FU I ~
Liver and extrahepitlc tlaaue
/ eq,
1.3
Ftrl~a , ~ U r d
T..,L
-':Fd~MP
eCl'II eq. 10 eq. I~ FUD~ > FdUDP
"I
FUTP
RNA
.I
FdUTP DNA
FIG. 4. The metabolism of 5-fluorouracil in humans. Equations 1-3 = dihydropyrimidine dehydrogenase, dihydropyrimidinase, fl-alanine synthase respectively; eq. 4 = uridine phosphorylase; eq. 5=uridine kinase; eq. 6= orotate phosphoribosyltransferase; eq. 7 = thymidine phosphorylase; eq. 8 = thymidine kinase; eq. 9 = nucleoside monophosphate and diphosphate kinases; eq. 10 = ribonucleotide diphosphate reductase. (eq. corresponds to equation number in text.)
Pyrimidine analogues and their nucleosides
O
O
I1~ H
I H
195
H or IF)
H
(4)
In order to enhance the anabolism and effectiveness of fluoropyrimidine drugs, several UrdPase inhibitors have been synthesized. Recent evidence has shown that 5-ethyl-2,2'-anhydrouridine (ANEUR) is a potent inhibitor of UrdPase. When ANEUR (280 mg/kg for 3 days) or FUrd (5 mg/kg for 3 days) were administered as a single agent to mice bearing S-180 solid tumors, no decrease in tumor weight was observed. However, co-administration of FUrd with ANEUR (i.e. 5 mg/kg for 3 days and 280 mg/kg for 3 days, respectively) resulted in a 91% decrease in tumor weight vs untreated controls, suggesting that the potentiating effect of ANEUR resulted from inhibition of UrdPase (Verez et al., 1987). In addition, several benzylacyclouridine compounds have been shown to be potent inhibitors of UrdPase. These agents increased the level of plasma uridine as well as the salvage of uridine in various tissues resulting in decreased FUra toxicity (Darnowski and Handschumacher, 1985). Furthermore, other studies with fluoropyrimidines have shown that benzylacyclouridines increase efficacy and potentiate the selective toxicity of FdUrd (Chu et al., 1984). Of the various benzylacyclouridine compounds aminomethyibenzyloxybenzylacyluracil, hydroxymethylbenzyloxybenzylacyluracil, and aminomethylbenzylacyluridine were found to be the most potent inhibitors with a K~ of 18 nM, 70rim and 92riM, respectively. In contrast, the Km of UrdPase for uridine was found to be 242/~ M in human tissue and 143 pM in murine tissue (Naguib et al., 1987b).
O
H~
idine (FUrd) to 5-fluorouridine-5'-monophosphate (FUMP). As shown in eqn 5, uridine kinase transfers a phosphate group from ATP to the hydroxyl group on the 5' carbon of FdUrd. This reaction has been demonstrated to be rate-limiting in the pyrimidine salvage pathway (Anderson, 1973). In addition, phosphorylation of the ribose to form the nucleotide prevents its efflux through the cell membrane (Wohlhueter and Plagemann, 1980). Multiple forms of uridine kinase have been observed in normal and neoplastic tissues when crude or partially purified enzyme was further separated by gel filtration (Greenberg et al., 1977; Keefer et al., 1975; Krystal and Scholefield, 1973), by isoelectric focusing (Absil et al., 1980; Ahmed, 1982; Ullman et al., 1979), by ion exchange chromatography (Fulchignoni-Lataud et al., 1976; Sk61d, 1963) and by affinity chromatography (Vesely and Smrt, 1977). With the exception of one report (Krystal and Scholefield, 1973), it is generally believed that the different forms of the enzyme observed by these techniques represent isoenzymes. These isoenzymes have been shown to differ with respect to size, net charge and heat stability. The differential expression of such isoenzymes in rapidly proliferating tumors may be important in cancer chemotherapy. Subtle changes in the physical properties of uridine kinase in tumor cells may impair FUra anabolism (Richard et al., 1962) which may lead to the development of FUra resistance (Sk61d, 1963).
O
H or (F)
xr'F'
H
H
(5)
011011
1.4.2.2. Uridine k&ase. Uridine, the uridine phosphorylase, is anabolized uridine-5'-monophosphate by uridine 2.7.1.48). This enzyme also converts
O1-1Oit
product of further to kinase (EC 5-fluorour-
More recently, Payne et al. (1985) purified uridine kinase (~60,000-fold) from Ehrlich ascites tumor cells with phosphocellulose and adenosine5'-triphosphate agarose. With two-dimensional gel
196
G.C. DAI-'IERet al.
electrophoresis, this enzyme migrated as one band suggesting the presence of a single enzyme. Earlier studies by the same group had suggested that
O
I'~
sequence of the reaction. Since substrates are channeled from one active site to the next, side reactions are minimized.
O
,,.r,F,
H or (F) M~'+ H H
this enzyme may form a variety of aggregates in the presence of appropriate effectors such as substrates (e.g. ATP) and feedback inhibitors (e.g. UTP, CTP) to form different polymers rather than isoenzymes (Payne and Traut, 1982). Later, they (Cheng et al., 1986) proposed that uridine kinase activity is regulated by changing its polymeric state. Included in this model was an ailosteric regulatory site which governs the dissociation of the enzyme. Based on this model it was suggested that feedback inhibitors can specifically bind to the regulatory site producing conformational changes which lead to dissociation of the active polymeric uridine kinase into an inactive monomer. On the other hand, substrates such as ATP act as primary effectors by competing with CTP and binding specifically at the regulatory site. ATP may have a dual role: (1) phosphate donor, when binding to the active site and (2) stabilizing factor by binding the allosteric site and producing a positive change in polymerization and activity. While uridine kinase inhibitors may be of theoretical interest, no therapeutically effective inhibitors have been developed. 1.4.2.3. Orotate phosphoribosyltransferase. A more direct route for conversion of FUra to FUMP is catalyzed by the enzyme orotate-phosphoribosyltransferase (OPRTase; EC 2.4.2.10), an enzyme in the de novo pathway of nucleic acid biosynthesis. This enzyme catalyzes the condensation of FUra with PRPP to yield FUMP. In this reaction (eqn 6), pyrophosphate is hydrolized from PRPP resulting in sufficient energy to drive the reaction forward (Heidelberger, 1975; Reyes, 1969). OPRTase is associated with a bifunctional enzyme complex that also contains orotidylate decarboxylase (EC 4.1.1.23). Each of these enzymes comprise a particular domain of a single 55-60 kDa polypeptide (Reyes and Guganig, 1975). Clustering of enzymatic activity in the same protein moiety probably evolved by exon shuffling. The presence of multiple domains on a single protein may direct the
H
(6)
While it is clear that the anabolism of FUra to FUMP may proceed by either (1) OPRTase or (2) the sequential action of UrdPase and uridine kinase, there are conflicting reports in the literature as to which anabolic pathway is dominant. Many tumor cell lines (e.g. murine leukemia, murine colonic adenocarcinoma) have been shown to utilize primarily OPRTase (Ardalan et al., 1982; Kessel et al., 1972; Mulkins and Heidelberger, 1982), while other tumors have been demonstrated to utilize primarily uridine phosphorylase and uridine kinase (Ardalan et al., 1980; Houghton and Houghton, 1983; Schwartz et al., 1985). Recently, Naguib et al. (1987a) showed that OPRTase activity was higher in rapidly proliferating tumor cells than in normal lymphocytes or liver. They suggested that this enzyme should be considered as a marker for cell growth and maturation. Other studies with FU1-2 cells, a mouse T-lymphoma ($49) variant selected for its resistance to FUra, have shown diminished OPRTase activity (Levinson et al., 1979). In these clones, a three- to four-fold increase in the apparent Km of OPRTase for FUra was observed, which has been suggested to be due to a structural gene mutation. A variety of pyrimidine base analogues have been evaluated as inhibitors of OPRTase. 4,6-Dihydroxypyrimidine was observed to be a potent inhibitor (Niedzwicki et al., 1984) which can be used experimentally in elucidating the metabolic pathways of pyrimidine base analogues and assessing the dominance of these anabolic pathways. 1.4.2.4. Thymidine phosphorylase. In the presence of 2-deoxyribose-l-phosphate (dR-l-P), the conversion of uracil (Ura) or thymine to their respective nucleosides deoxyuridine and thymidine (eqn 7) are catalyzed by thymidine phosphorylase (dThdPase; EC 2.4.2.4). It should be noted however, that the conditions within most cells favor the reverse reaction, especially when appropriate amount of the pyrimidine deoxyribonucleoside donor is not provided.
Pyrimidine analogues and their nucleosides 0
197 0
I~
H or (F) I
H
H
(7)
While this step has been suggested to be an alternate route for the conversion of FUra to FdUrd (eqn 7), the quantitative importance of this step has been questioned since little evidence for FdUrd formation has been observed even after administration of large doses of FUra in vivo (Woodcock et al., 1980). However, co-administration of thymidine with FUra leads to significant FdUrd formation, suggesting that transferase activity is limited by availability of cosubstrate (the deoxyribose donor) and is zero order with respect to FUra. It was originally believed that the specificity of this enzyme was restricted to pyrimidine-2'-deoxyribonucleosides, as pyrimidine ribonucleosides are not cleaved by this enzyme (Niedzwicki et al., 1983). However, it has been shown that dThdPase is not only specific for pyrimidine-2'-deoxyribonucleosdies but is also capable of releasing the sugar from 5'-deoxyfluorouridine (Iltzsch et al., 1985; Kono et al., 1983). The activity of dThdPase has been reported to be generally higher in normal liver or lymphocytes than in tumors. This activity increased upon treatment of the tumor with maturational agents (Naguib et al., 1987a) and this activity was observed to increase during the course of maturation of rat intestinal cells and acute myeloid leukemia cells in vitro (Imondi et al., 1969; Palu, 1980). Higher activity has been detected in mature peripheral blood cells than in rapidly proliferating cells such as blast cells, bone marrow, acute and chronic lymphocytic leukemic cells (Fox et al., 1979; Gallo and Seymour, 1969b; Marsh and Perry, 1964; Rabinowitz and Wilhite, 1969). Palu (1980) suggested that dThdPase should be considered as a possible marker for cellular maturity. This enzyme has been purified from a variety of mammalian sources (Desgranges et al., 1981; Gallo and Seymour, 1969a; Krenitsky, 1968; Kubilus et al., 1978; Zimmerman, 1964). A detailed kinetic study was undertaken in mouse liver (Iltzsch et aL, 1985) to elucidate its mechanism of action. This study indicated that in the presence of thymine, both phosphate and thymidine 0
~ H
or (F)
have 'cooperative effects' on dThdPase. Thus, this enzyme may have multiple allosteric binding sites that interact cooperatively. Studies of the concentration-dependent degradation of thymidine by intact platelets suggested that dThdPase follows Michaelis-Menton kinetics with an apparent Km of 0.10-0.14 mM. This phosphorolysis of thymidine is competitively inhibited by various C-5or C-6-substituted uracils. 6-Amino-5-bromouracil, 6-aminothymine, 5-bromouracil, and thymine were the most active inhibitors with a Ki of 6, 11, 28 and 270/~M, respectively. In acellular extracts the K i of these inhibitors was 2, 8, 15, and 72 #M, respectively (Desgranges et al., 1982). Intracellular degradation of thymidine may decrease its availability for anabolism limiting the production of thymidine-5'-triphosphate (dTTP). An increase in the dTTP pool will inhibit ribonucleotide reductase and, thus the conversion of cytidine-5'-diphosphate to deoxycytidine-5'-diphosphate needed for DNA synthesis. The potential role of dThdPase in the regulation of thymidine metabolism has been suggested from both in vivo (Kufe et al., 1980a) and in vitro (Fox et al., 1979) studies in lymphoid cells. Resistance to thymidine in Blymphoblasts differed from sensitive myeloblasts and T-lymphoblasts in having a ten-fold increase in dThdPase activity as well as a ten-fold decrease in thymidine kinase activity (Kufe et al., 1980a). Therefore, resistance to thymidine-induced toxicity is more likely to be due to an increase in the ratio of dThdPase to thymidine kinase activities than to an increase in dThdPase activity alone. 1.4.2.5. Thymidine kinase. Thymidine kinase (dThd kinase; EC 2.7.1.75) catalyzes the phosphorylation of not only thymidine but also a number of other structurally similar compounds. As shown in eqn 8, FdUrd is also a substrate for dThd kinase and is anabolized to FdUMP. Similar to uridine kinase, this enzyme also requires Mg 2÷ to complex with ATP and drive the reaction through this rate-limiting step. 0
H~
H or (F)
(8)
198
G.C. DArmRet al.
In human tissue, dThd kinase is present as 2 isoenzymes termed dThd kinase-1 and dThd kinase-2 (Show et al., 1979). Gel electrophoresis of subcellular fractions from fetal liver have shown that dThd kinase-1, the 'fetal' form, migrates slowly on the gel and is associated with the cytoplasmic fraction. In contrast, dThd kinase-2, the 'adult' form, migrates rapidly on the same gel and is associated with the mitochondrial fraction (Kit and Leung, 1974; Taylor et al., 1972). These two isoenzymes also differ in sedimentation coefficient, pH optimum, inhibition by dCTP and phosphate donor specificity (Berk and Clayton, 1973; Kit, 1976; Kit and Leung, 1974; Taylor et al., 1972). It is interesting to note that during cellular proliferation, the increase in DNA synthesis is accompanied by an increase in dThd kinase activity, thus indicating that this enzyme may be useful as a marker of the transition from the quiescent to the replicative phase. The activity of the dThd kinase- i isoenzyme increases when G] or G Ophase cells are stimulated or infected with oncogenic viruses (Kit, 1976). Variation in dThd kinase activity also occurs in response to hormonal influence (Bourtourault et al., 1984; Masui and Garren, 1971). It has been shown that human dThd kinase from both normal and leukemic leukocytes (chronic mylogeneous, chronic lymphocytic and monocytic leukemia) is competitively inhibited by dTTP, a substrate for DNA polymerase (Breznick and Karjala, 1964). In this study, the apparent Km for thymidine was 3.6 x l0 5M, while the Ki for dTTP was 1.5 × 10 -6 M. In addition, other studies (Breitman, 1963; Fiala et al., 1962; Ives et al., 1963) have also shown that dTTP regulates the enzymatic activity of thymidine kinase by feedback inhibition. These studies suggest that dTTP can inhibit dThd kinase either allosterically or isosterically (Monod et al., 1963). Inhibition of dThd kinase activity, regardless of the
type, may be detrimental therapeutically. The resulting decrease in anabolism of nucleoside analogues can impair the chemotherapeutic effectiveness of these drugs. Recently, Fisher et al. (Fisher and Baxter, 1981; Fisher et al., 1983, 1988; Fisher and Phillips, 1984) demonstrated that 5'-amino derivatives (e.g. 5'-aminothymidine) could reverse the inhibition of dThd kinase produced by dTTP, thereby stimulating nucleoside phosphorylation. These findings are particularly relevant to the design of new nucleoside analogues and anatagonists of allosteric inhibition. In addition, other inhibitors with different sites of action have been studied. Celiptium ~ (N-2-methyl-9hydroxyellipticinium acetate) is an ellipticine derivative used in the therapy of breast cancers. This drug is believed to exert its action on the dThd kinase- 1 gene instead of the active or regulatory sites of the enzyme (El Hiyani et al., 1987). It has been suggested that Celiptium "~may interfere with the nuclear binding site of the estrogen receptor and, consequently, suppress the induction of dThd kinase synthesis. Therefore, stimulation of dThd kinase-1 activity by estradiol (Bourtourault et al., 1984) will be inhibited. 1.4.2.6. Nucleoside monophosphate and diphosphate Nucleoside monophosphate kinase (NMP kinase; EC 2.7.4.4) and nucleoside diphosphate kinase (NDP kinase; EC 2.7.4.6) occupy pivotal positions in biosynthesis of pyrimidine nucleotides, since their substrates are products of both de noro and salvage pathways. Like other phosphotransferases in subgroup 2.7.4, ATP is utilized as the phosphate donor for the conversion of each nucleoside 5'-monophosphate to its corresponding di- and triphosphate. As shown in eqn 9, these two enzymes participate in both ribosyl and deoxyribosyl nucleotide formation catalyzing the generation of 5-fluoro(deoxy)uridine diphosphate [F(d)UDP] and 5-fluoro(deoxy)uridine triphoshate [F(d)UTP].
kinase.
O
O
(eq.9)
OH
H or (OH)
H or (OH)
(9)
I~ JL ~or(F) 0 ~
OH
H or (OH)
(ADPM ' g+~
199
Pyrimidine analogues and their nucleosides Partial purification and characterization of these two enzymes (Imazawa and Eckstein, 1979; Nakamura and Sugino, 1966) have shown that both enzymes lack specificity for either the base or sugar moiety of nucleotides. Neither the 2'- nor the 3'-hydroxyl moiety of natural nucleotides appear to be necessary for pyrimidine nucleoside mono (or di) phosphate kinase activity. This broad specificity for different congeners is appealing for the rational synthesis of nucleotide analogues that may be phosphorylated to nucleotide precursors of nucleic acids. In comparison to the activity of NMP kinase, the activity of NDP kinase is greater (Sugino et al., 1966). An elevated level of NDP kinase activity has been shown in normal, nonproliferating rat liver tissues (Bianchi et al., 1964; Chiga et al., 1963; Nakamura and Sugino, 1966) and rat or human erythrocytes (Bianchi et al., 1964; Mourad and Parks, 1966) as well as growing tissues such as Novikoff hepatoma cells (Ives, 1965), regenerating rat liver (Chiga et al., 1963), rat ascites tumor cells (Nakamura and Sugino, 1966), and Landschutz-ascites tumor cells (Grav and Smellie, 1963). By contrast, NMP kinase has been shown to have a significantly lower specific activity in a resistant murine leukemia (P388/FUra) cell line (Ardalan et al., 1980). Thus, it is conceivable that phosphorylation of nucleoside monophosphate to nucleoside diphosphate is the rate-limiting step in the formation of nucleoside triphosphates. 1.4.2.7. Ribonucleotide reductase. Ribonucleotide reductase (EC 1.17.4.1) is also a key enzyme in the synthesis of DNA. It is highly regulated and solely responsible for the de novo synthesis of deoxyribonucleoside diphosphates from their corresponding ribonucleoside diphosphates. In addition it also converts the fluorinated nucleotide FUDP to FdUDP and thus it is important in fluoropyrimidine metabolism. The mechanism of this enzyme is more complex than depicted in eqn 10. The mammalian enzyme consists of two dissimilar subunits called protein M1 and M2. Protein MI is a 170 kDa dimer that contains the allosteric binding sites (Thelander et al., 1980). Protein M2 is an 88 kDa dimer. Each monomer of M2 contains a nonheme iron center and a stable tyrosyl radical cation. The presence of this radical is critical for its catalytic activity, since the addition of hydroxyurea, a quencher of free radicals, destroys the enzymatic activity of the holoenzyme. However, the tyrosyl radical can be readily regenerated upon incubation of the radical-free protein with iron-dithiothreitol in the presence of air (Thelander et al., 1985). o
Ribonucleotide reductase is a cell-cycle dependent enzyme with maximal enzymatic activity during Sphase (Thelander and Reichard, 1979). Using synchronized cell populations, variation in enzymatic activity has been shown to be regulated by both de novo synthesis and degradation of the M2 subunit (Eriksson et al., 1984). In contrast, the M1 protein is expressed at a constant level and is present in excess throughout the cell cycle (Mann et al., 1987, 1988). Other studies utilizing monoclonal and polyclonal antibodies directed against M I and M2 subunits demonstrated localization of these two proteins in the cytoplasm (EngstrSm and Rozell, 1988; Engstr6m et al., 1984). Furthermore these immunocytochemical studies (Engstr6m et al., 1984) demonstrated that the M1 protein is only present in actively proliferating cells and is absent in fully differentiated cells. Hence, immunofluorescent studies of the M1 subunit may be promising as a marker for cell proliferation in normal, inflammatory, or neoplastic tissues. Additional biochemical studies from mouse T-lymphoma cells demonstrated the existence of two independent regulatory domains that can be genetically altered on the M1 protein. As shown in Fig. 5, one regulatory site is responsible for the overall activity of the enzyme through binding of ATP (activator) or dATP (inhibitor), and the other is responsible for substrate specificity through binding of ATP, dGTP and dTTP (Wright, 1983). It has also been shown that dATP can bind the substrate specificity site of M 1 and mimic the effects observed with ATP (Thelander et al., 1980). The antitumor agent, hydroxyurea, can passively diffuse into mammalian cells (Tagger et aL, 1987) where it specifically inhibits ribonucleotide reductase by destroying the M2 tyrosyl radical (McClarty et al., 1987; Thelander, et al., 1985). However, resistance to hydroxyurea may develop. This has proven useful as a selective agent in tissue culture to isolate drug-resistant cell lines with altered ribonucleotide reductase activity. Recent molecular and cellular characterization studies of two chinese hamster ovary cell lines have shown that resistance to cytotoxic concentrations of hydroxyurea was correlated with increased levels of ribonucleotide reductase (Tagger and Wright, 1988). Electron paramagnetic resonance measurements of free radical levels and studies with M l-specific antibodies suggested that the elevation in enzyme activity was entirely due to an increase in the presence of the M2 component. Furthermore, studies with M2 cDNA from both low and high resistant cell lines indicated that elevation in the M2 message could o
(10)
200
G.C. DAHERet al.
M1
~>
H
Substrate binding sites R" Tyrosyl free radical
FIG. 5. Model of ribonucleotide diphosphate reductase depicting two subunits, M~ and M2. also explain the observed increase in synthesis of the M2 subunit. Elevation of M2 mRNA in the most resistant cell line was shown to result from gene amplification (Tagger and Wright, 1988). Patterns of cross-resistance in hydroxyurea-resistant L1210 cells were examined for sensitivity to a variety of inhibitors specifically directed at the individual subunits of ribonucleotide reductase (Carter and Cory, 1988). HU-7-S7 L1210 cells were cross-resistant to 2,3-dihydro-IH-pyrazole[2,3-a]imidazole. However, it is of interest that they were sensitive to inhibitors (e.g. 4-methyl-5-amino-l-formyl-isoquinoline thiosemicarbazone and 1-isoquinolylmethyleneN-hydroxy-N'-aminoguanidine tosylate) that are specifically directed at the same subunit as hydroxyurea (Cory and Fleisher, 1979; Weckbecker et al., 1988). These cells retained their sensitivity to deoxyadenosine/erythro-9-(2-hydroxy-3-nonyl)adenine and deoxyguanosine/8-aminoguanosine through intracellular accumulation of dATP and dGTP, respectively (by their subsequent allosteric inhibition of ribonucleotide reductase) (Carter and Cory, 1988). It has been shown previously (Cory and Carter, 1988) that L1210 cells selected for resistance to deoxyadenosine/erythro-9-(2-hydroxy-3-nonyl)adenine were cross-resistant to deoxyguanosine/8-aminoguanosine, 2-fluorodeoxyadenosine and 2-fluoro-9-flD-arabinofuranosyladenine, but retained their sensitivity to hydroxyurea, 4-methyl-5-amino-l-formylisoquinoline thiosemicarbazone and 1-isoquinolylmethylene-N-hydroxy-N'-aminoguanidine tosylate. These studies suggest that development of resistance at the ribonucleotide reductase site is limited to the type of agent used and the subunit of reductase interacting with the inhibitor. Inhibition of ribonucleotide reductase with these agents can affect the metabolism of fluoropyrimidine and hence, modify its anticancer activity. 1.5. METABOLIC SITE OF ACTION
1.5.1. Thymidylate Synthase Thymidylate synthase (TS; EC 2.1.1.45) is crucial for the de novo synthesis of thymidylate (dTMP) needed for DNA synthesis. Specifically, this enzyme
is responsible for the methylation of dUMP to dTMP, with the concomitant oxidation of NS,N ~°methylenetetrahydrofolate (NS,N~°-CH:-H4 PteGlu) to dihydrofolate (H2PteGlu). NS,NI°-CH2-H4PteGIu serves both as a one-carbon donor and as an electron donor. Through this reaction a methyl group is added to the fifth position of dUMP. During this reduction step, two electrons are donated from the ring of the tetrahydrofolate moiety itself as a hydride ion (H:-). Recently, the molecular basis of TS-fluoropyrimidine interaction has been thoroughly reviewed (Cisneros et al., 1988; Heidelberger et al., 1983). The catalytic mechanism consists of a sequence of three different steps (Fig. 6). The first step initials the formation of a covalent bond between the sixth position of dUMP (or FdUMP) and a sulfhydryl group in the catalytic site of the enzyme (Hardy et al., 1987). Formation of this covalent adduct activates the vinylic fifth carbon of the pyrimidine ring for condensation with the methylene group of NS,N ~°CH2-H4PteGIu. In the second step there is an electrophilic attack of the NS,N~°-methylene moiety on the activated fifth position of the pyrimidine ring. However, in the presence of FdUMP, the ternary complex (i.e. the active site of TS, FdUMP, and the folate cofactor; Fig. 6) does not usually proceed past this point resulting in a 'suicide-like' inhibition. The third step is a reduction reaction that involves /3 elimination and hydride ion transfer to an intermediate that acts as a hydride ion acceptor (Danenberg and Lockshin, 1981). In this step, the electron withdrawing effects of fluorine on the fifth position of the pyrimidine ring of FdUMP, not only serves to activate the C-6 position for attack by the enzyme, but also stabilizes the above intermediate, and thereby, prevents the complex from advancing any further (Cisneros et al., 1988). The bonding in this ternary complex containing FdUMP (Fig. 7), is extremely 'tight' with a Kd of approximately 10 ~2M, suggesting that only one molecule of the complex will dissociate for every trillion molecules of complex formed (Danenberg and Danenberg, 1978; Lockshin and Danenberg, 1981; Santi and Danenberg, 1984). Under certain circumstances, however, TS will spontaneously promote the dissociation of this complex, demonstrating that ternary complex formation is reversible and FdUMP is not a 'suicide' inhibitor.
Pyrimidine analogues and their nucleosides
201
~CH iH~ PteGlu) H ~
or L
-H °r (F)
~'j~"
~),~-i---~
Le-I'o"th*c"po°l'lOno
FIG. 6. The catalytic mechanism of thymidylate synthase. (1) Initial formation of a covalent bond between substrate and enzyme active site; (2) electrophilic attack of methylene group of NS,N~°-methylenetetrahydrofolate (CH2H4PteGIu) on the 5th position of the pyrimidine ring; (3) ternary complex formation of 5-FdUMP, thymidylate synthase and the folate cofactor; H2PteGlu = dihydrofolate. Investigations into the physical characteristics of TS and its interaction with the cofactor and substrates have delineated its mechanism of action. Mammalian TS is actually a dimeric protein (MW = 68 kDa) with an active site on each monomer (Moran, 1988). As shown in Fig. 8, during the
formation of ternary complex, TS must first bind its substrate (i.e. dUMP or FdUMP) before its contact with the cofactor. Once the substrate is bound, a binding site for the cofactor is created, and the interaction between all three components may then occur.
5,10-METHYLENETETRAHYDROFOLATE I
i
I
PTERIN
H I
~l~
"N"
~ . FdUMP
PABA
I
II
OH2 ~
I
.N.~ O
GLUTAMATE
II
~
/
\
O
,
I
(CH2)2
i
//~C-NH-CH COOH
I
O
II HO--P-O--~
I OH
OH
FIG. 7. Ternary complex of N 5, Nl°-methylenetetrahydrofolate, 5-fluoro-2'-deoxyuridinemonophosphate (FdUMP) and thymidylate synthase.
202
G.C. DAHERet al.
"x FIG. 8. The ordered sequential addition of substrates and cofactors to mammalian thymidylate synthase: 1 = binding of dUMP to active site of thymidylate synthase; 2 = creation of binding site for cofactor; 3 and 4 = covalent binding of enzyme, substrate, and cofactor; 5, 6 and 7 = release of product, free enzyme, and oxidized cofactor dUMP = 2'-deoxyuridine-5'-monophosphate; TMP = thymidine-5'-monophosphate; H2PteGlu = dihydrofolate; H4PteGlu = tetrahydrofolate; ser = serine; gly = glycine; CH2H4PteGlu = NS,N l°-methylenetetrahydrofolate. The affinity of TS for either substrates (i.e. dUMP and FdUMP) is nearly identical. When FdUMP is bound TS will temporarily be inactivated. During this time, dUMP synthesis will continue, leading to an accumulation of dUMP. When the FdUMP-ternary complex subsequently dissociates, the accumulated dUMP will then compete for binding to the newly-released TS, thus replacing FdUMP at the active site, permitting dTMP synthesis to resume. Recent studies have shown that FdUMP bound to TS has a half-life of approximately 2 hr in LI210 leukemia cells. However, addition of leucovorin (5-formyltetrahydrofolate) prior to or simultaneously with fluoropyrimidines, results in an increased half-life of complex formation (tl,, 2 >35 hr) with a concomitant decrease in thymidine nucleotide pools and increased cell death (Keyomarsi and Moran, 1988). Intracellularly, leucovorin is reduced to NS,N]°-CH2 H4PteGlu, thereby providing the optimal reduced folate concentration necessary for stable ternary complex formation (Bleyer, 1989). Recent evidence has shown that the intracellular level of NS,NI°-CH2 H4PteGlu in human tumors is insufficient for complete inhibition of TS in the presence of adequate FdUMP (Bleyer, 1989; Houghton et al., 1981; Ullman et al., 1978). However, several studies (Garmont et al., 1988; Laufman et al., 1987; Petrelli et al., 1987) have shown that the response rates were consistently higher with concomitant administration of FUra and leucovorin. Such a regimen has been suggested to be beneficial in patients with metastatic colorectal adenocarcinoma (Petrelli et al., 1987).
1.5.2. R N A Incorporation Shortly after FUra was introduced, additional biochemical studies demonstrated the incorporation of this agent into RNA (Chaudhuri et al., 1958). Over the years it has become apparent that FUra inhibits mRNA processing or its nuclear-cytoplasmic transport (Armstrong et al., 1986). Other studies have also shown that FUra causes an irreversible inactivation of tRNA methyltransferase. This enzyme is responsible for the transfer of a methyl group from Sadenosyl-methionine (SAM) to the 5-carbon of a specific Urd residue to form the m5U (ribosylthymine) moiety found in TIp C loop (loop IV) in tRNA (Santi and Hardy, 1987). Additional studies on RNA from other species have shown that in vitro exposure of mouse LI210 cells to 10 6M FUra inhibited the maturation process of rRNA precursors (Kanamaru et al., 1986). This is consistent with earlier studies where rRNA maturation was also inhibited at the 45S and 32S regions causing accumulation of preribosomal RNA (Wilkinson and Crumley, 1977; Wilkinson et al., 1975; Wilkinson and Pitot, 1973). Recently, a study on the action of FUra on L1210 cells concluded that FUra inhibits: (1) the processing of pre-rRNA to rRNA; (2) the methylation of pre-rRNA and tRNA; and (3) the synthesis of poly(A)RNA of mRNA. Furthermore, it impairs the synthesis of snRNA and enhances the translation of poly(A)RNA (Kanamaru and Wakui, 1988). In addition, a recent study by Danenberg et al. (1990) suggested that FUra destabilizes the active
Pyrimidine analogues and their nucleosides conformation of RNA by substitution, thereby forming a weaker base pairing between FUra and adenine due to partial ionization of FUra residues. In vitro and in vivo studies utilizing combinations of fluoropyrimidines with 'biochemical modulators' have been carried out in order to differentiate between the DNA and RNA toxicity and to determine the contribution of specific RNA targets to the mechanism of action of these drugs, dThd is one such modulator that has been studied extensively as an adjuvant of fluoropyrimidines (Au et al., 1982; Danhauser and Rustum, 1979; Speigelman et al., 1980a,b; Sternberg et al., 1984; Takimoto et al., 1987). It is especially important, since RNA-mediated cytotoxicity is not reversed by dThd such that the block at the ternary complex (i.e. fluoronucleotide-[NS,Nl°-CH2-H4PteGlu]-TS) may be bypassed, providing an alternate source of dTTP for DNA synthesis. Characterization of the effects of fluoropyrimidines on RNA and/or RNA processing is perhaps very important clinically. However, the therapeutic relevance of such a 'modulator' is questionable since there have been many conflicting results. Several studies have demonstrated that modulation of FUra cytotoxicity by dThd could improve the therapeutic efficacy of the antimetabolites in certain murine tumors (CD2F1 colon tumor 26 and CD8F1 mammary carcinoma) (Speigelman et al., 1980a,b). In addition, the incorporation of FUra into RNA can also be augmented by inhibiting the de novo pyrimidine synthesis and lowering UTP pools with agents such as phosphonacetyl-L-aspartic acid (PALA). This drug causes a 60-80% decrease in UTP levels in tissue culture cells (Johnson et al., 1978; Moyer and Handschumacher, 1979) and four-fold increase of FUra incorporation into RNA in vivo (Spiegelman et al., 1980b). In addition, it acts synergistically to yield a greater than ten-fold increase in FUra incorporation when administered in combination with dThd. However, other studies where the combination of dThd and FUra was investigated in tumor models (Danhauser and Rustum, 1979, 1984) and in patients with advanced colorectal carcinoma (Au et al., 1982; Sternberg et al., 1984) showed no significant increase in the therapeutic index. These studies provide insights into the mechanism of action of FUra in various tumor models and cell lines. In colon carcinoma (Au et al., 1982; Sternberg et al., 1984), the combination of FUra with dThd failed to produce a significant improvement in the therapeutic efficacy of FUra. These results collectively suggest that incorporation of FUra into RNA may be more important to antitumor efficacy.
1.5.3. D N A Incorporation Throughout the 1970s, it was widely believed that the fluoropyrimidines, mainly FUra and FdUrd, act through the mechanisms above, (i.e. inhibition of thymidylate synthase by FdUMP and incorporation into RNA) (Heidelberger, 1975; Speigelman et al., 1980a,b). Later the isolation of (FUra)-DNA was demonstrated in both tumor and normal cells treated with FdUrd or FUra in tissue culture (Danenberg et
203
al., 1981; Herrick et al., 1982; Ingraham et al., 1982; Kufe et al., 1981; Schuetz et al., 1986). During this
time, other studies have shown that pretreatment with dThd in vivo decreases the amount of FUra incorporated into DNA, suggesting that FdUTP is a substrate of DNA polymerase (Sawyer et al., 1984). The apparent absence of intracellular FdUTP and the minimal amount of FUra in DNA have been explained by the recognition that FdUTP is a substrate for the enzyme deoxyuridinetriphosphate nucleotide hydrolase (dUTPase) and by the demonstration that uracil glycosylase is able to cleave FUra from DNA respectively (Caradonna and Cheng, 1980a,b; Ingraham et al., 1980). However, it should be noted that uracil glycosylase appears to be extremely specific for uracil compared to FUra (Caradonna and Cheng, 1980b). This implies that incorporation of FUra into DNA should occur more frequently than its naturally occurring analogue (i.e. uracil). Since the cleavage of fluoropyrimidine-DNA derivatives is much slower than their natural counterparts, it is to be expected that the former is more stable than the latter. Further studies on DNA as a site of action of fluoropyrimidines have demonstrated that under certain experimental conditions, FdUrd can be incorporated into DNA and may be partially responsible for its cytotoxic effects (Kufe et al., 1983). It is probable that such toxicity may result from misincorporation of FUra into DNA or from the excision-repair of these adducts by uracil glycosylase which can potentially lead to mutagenesis and eventual cytotoxicity. In our laboratory, we have demonstrated that DNA repair occurs subsequent to incorporation of FUra into DNA (Schuetz et al., 1988). However, such repair may not be fully effective, leading to inhibition of DNA elongation (Schuetz and Diasio, 1985) and DNA fragmentation (Schuetz et al., 1986). In this latter study, we demonstrated that FUra resulted in time-dependent fragmentation of bone marrow DNA with an associated time-dependent excision of FUra from DNA. This relationship between FUra removal and DNA chain degradation is consistent with the mechanism proposed in which FUra is excised from DNA by uracil glycosylase with subsequent repair of the apyrimidinic site by apyrimidine endonuclease (Caradonna and Cheng, 1980a,b). Other studies have also shown that fluoropyrimidine, specifically 5fluorodeoxycytidine, can incorporate into DNA and is associated with inhibition of DNA methylation and decrease in clonogenic survival (Kaysen et al., 1986).
1.5.4. Incorporation into Cell Membranes In addition to inhibition of TS and incorporation into DNA and RNA, it has also been reported that FUra can produce cell surface modifications (Kessel, 1980). Such alteration in cellular membranes is suggested to be due to FUDP-hexoses which may alter membrane structure by imparing glycoprotein biosynthesis (Kessel, 1980; Peters et al., 1984). Another study utilizing HeLa cells treated with FUra revealed a dampened oscillatory response in the transmembrane potential. In contrast, the 6-potential or surface charge of these cells were altered to a lesser extent by FUra exposure (Walliser and Redmann,
204
G.C. DAHERet al.
1978). Based on the hypothesis that the electrical potential of the cell membrane exerts an effect on mitotic activity of the cell, the authors suggested that the effect of FUra on the cell membrane is related to inhibition of cell growth. However, the relative importance of this cell membrane effect on cell growth has not been well studied and its contribution to toxicity remains to be established. 1.6. MISCELLANEOUSFLUOROPYRIMIDINEDRUGS
In addition to the fluoropyrimidines discussed above (i.e. FUra and FdUrd), several other fluoropyrimidine analogues have been investigated. These include compounds such as 1-(tetrahydro-2-furanyl)5-fluorouracil (i.e. ftorafur) and 5'-deoxy-5fluorouridine (5'dFUrd). Ftorafur is believed to function as a depot form of FUra and is converted to FUra within target cells (Au et al., 1979; Benvenuto et al., 1978). Initial studies with ftorafur (Hall et al., 1977) revealed gastrointestinal symptoms and neurotoxicity as the major clinical toxicities without the myelosuppression typically observed with FUra administration. Although utilized in many Eastern countries, its clinical activity compared to FUra has not been thoroughly evaluated and its use is still experimental in the United States. 5'dFUrd has shown promising antitumor activity and increased specificity for tumor cells as compared to normal tissues (Bollag and Hartmann, 1980). The substitution of an OH group at the 5' position for the CH2OH present in ribose sugars prevents further activation to a nucleotide. Instead, it must be converted to FUra by a nucleoside phosphorylase before it can be further anabolized to a nucleotide (Armstrong and Diasio, 1980). An additional hypothetical advantage is that 5'dFUrd may be active as an oral agent (Chabner, 1982). Clinical trials with 5'dFUrd are in progress in Europe and Japan.
2. ARABINOSYLCYTOSINE Arabinosylcytosine (araC; l-fl-D-arabinofuranosylcytosine; cytosar; cytarabine), an arabinose nucleoside, is primarily used in combination with anthracyclines to induce remission in patients with acute myeloblastic leukemia (Ellison et al., 1968). AraC has also been shown to have activity in other human tumors including chronic granulocytic leukemia (particularly blast crisis), acute lymphocytic leukemia, and histiocytic lymphoma (Bryan et al., 1974; Chabner and Myers, 1985). The metabolism of araC in tumor cells and its selective activity against rapidly growing tumors has limited its usefulness in the treatment of most solid malignancies (Chabner and Myers, 1985). AraC is profoundly affected by pharmacologic parameters such as dose, schedule of treatment, and route of administration (Ho and Freireich, 1975). These affects, in turn, can be attributed to araC metabolism in host and tumor cells. In addition to its activity against hematological leukemias, araC has been found to be useful in the treatment of leukemic or carcinomatous meningitis when administered intrathecally (Band et al., 1973; Spiers and Firth, 1972). Lastly, araC has been shown
to have activity against several DNA viruses (Shouval et al., 1986).
2.1. HISTORICALPERSPECTIVE Arabinosyl derivatives of pyrimidines were first isolated by Bergmann and Feeney from a Caribbean sponge, Cryptotethya crypta (Bergmann and Feeney, 1950, 1951). The isolated compounds were the 1-fl-Darabinofuranosyl derivatives of thymine and uracil (i.e. araT and araU). In contrast, araC has not been detected as a natural substance (Cohen, 1966). Numerous studies have focused on the antitumor activity of this unique class of compounds. The first report suggesting antitumor activity reported that araC inhibited growth in Escherichia coli and that this growth inhibition was reversed by pyrimidine nucleosides (Slechta, 1961). AraC was first shown to have antitumor activity in a variety of mouse tumors including leukemia L1210, Sarcoma 180, Ehrlich ascites, L5178Y lymphoma, and T 4 lymphoma (Evans et al., 1961, 1964). In another report araC inhibited growth in L1210, P288, P388, and K1964 mouse tumours (Dixon and Adamson, 1965). Thus, a large spectrum of mouse tumours was initially tested and a total of 38 were sensitive to araC. 2.2. CHEMISTRY
AraC is a synthetic nucleoside which differs from the natural nucleosides cytidine and deoxycytidine in that the sugar moiety is an arabinose rather than a ribose or deoxyribose, respectively (Fig. 9). AraC has a molecular weight identical to that of cytidine and the arabinose sugar is a pentose sugar with the same side groups as cytidine. The arabinose sugar moiety differs from ribose in that its 2' hydroxyl group is
OHOH Cytldlne
OH Deoxy©ytldlne
I IH 2
S) OH Arablnosyloytollne
Azloytldlne
FIG. 9. Structures of cytidine, deoxycytidine and cytidine analogues, arabinosylcytosine and azacytidine.
Pyrimidine analogues and their nucleosides
// Ar I 1 - ~
f•
205
dATP"I '--.I.-.-O.A
Ara-C 1-~-D- ArB-CMP 4---~-ArI.CDP-~.-e- Arlg-CTP
/1[ lrlI-U 'l.
l' Ara-UMP
FIG. 10. Transport and metabolism of arabinosylcytosine in humans. (1) Deoxycytidine kinase; (2) cytidine deaminase; (3) deoxycytidylatedeaminase; (4) deoxycytidylate kinase; (5) nucleoside diphosphate kinase; (6) DNA polymerase ct. oriented in the beta (fl) position. The inverted position of the 2' hydroxyl group causes the arabinose to 'act like' a deoxyribose sugar and, thus, its metabolism is more like deoxycytidine than cytidine. 2.3. UPTAKE INTO CELLS A prerequisite for araC-induced cellular toxicity is the transport of araC into the target cell (Fig. 10). Early studies of nucleoside transport were hindered by rapid metabolism within the cell. Equilibrium between intracellular and extracellular compartments is thought to occur within 90 sec with the intracellular nucleoside undergoing rapid phosphorylation to nucleotide analogues. Thus, it is important to separate the intracellular nucleoside from other metabolites, particularly nucleosides, in order to quantitate transport. In 1968, Kessel and Shurin (Kessel and Shurin, 1968) isolated a subline of L1210 murine leukemia cells which lacked the ability to phosphorylate either deoxycytidine or araC. This subline was utilized to study the transport of these two nucleosides. Saturation kinetics and structural specificity were clearly demonstrated with inhibition by other nucleosides suggesting that a mediated process was involved in nucleoside entry. The efflux of labeled nucleotides was determined to be a two-phase process with an initial rapid phase followed by a much slower phase (Kessel and Shurin, 1968). The rapid phase was inhibited by high intercellular levels of other nucleosides (e.g. thymidine, uridine, etc.) indicating a process with structural specificity and saturability (Kessel and Shurin, 1968). Subsequent studies have confirmed that nucleosides, as well as araC and other nucleoside analogues, enter the cell via a high Kin, facilitated diffusion system that seems to transport all ribonucleosides and deoxyribonucleosides, but with somewhat different efficiencies (Wohlhueter and Plagemann, 1980). Competitive kinetics were observed between araC and deoxycytidine for entry into leukemic blasts (Wiley et al., 1982, 1983, 1985) and cultured Novikoff rat hepatoma cells (Marz et al., 1977; Plagemann et al., 1978b). In addition, influx of araC into human leukemic blasts was blocked by phloretin, a broad-spectrum inhibitor of facilitated transport systems (Wiley et al., 1983).
The transport kinetics of araC have been studied in detail in a variety of malignant cells such as Novikoff hepatoma cells (Marz et al., 1977; Plagemann et al., 1978b), Yoshida sarcoma cells (Mulder and Harrap, 1975), and human leukemic blast cells (Wiley et al., 1982, 1983, 1985) and cultured Novikoff rat hepatoma ceils (Marz et al., 1977; Plagemann et al., 1978b) In addition, influx of araC into human leukemic blasts was blocked by phloretin, a broadspectrum inhibitor of facilitated transport systems (Wiley et al., 1983). The transport kinetics of araC have been studied in detail in a variety of malignant cells such as Novikoff hepatoma cells (Marz et al., 1977; Plagemann et al., 1978b), Yoshida sarcoma cells (Mulder and Harrap, 1975), and human leukemic blast cells (Wiley et al., 1982, 1983, 1985). Plagemann et al. (1978b) used a subline of wild-type Novikoff rat hepatoma cells that lacked nucleoside kinase activities. With the appropriate experimental conditions, the ATP level in these cells was maintained at less than 1% of the concentration typically observed and no substrate phosphorylation occurred. Lineweaver-Burke analysis showed that araC transport followed normal Michaelis-Menten kinetics with an apparent Km of 450#M and an apparent Vmax of 30pmol//~l cell H : O x sec. Mulder and Harrap (Mulder and Harrap, 1975) had similar findings in Yoshida sarcoma ceils but suggested that in these cells the facilitated diffusion process operated at two concentration ranges with a Km of 400/~M and 2000 #M. Passive diffusion only became a significant factor in the uptake of araC at extracellular concentrations above 1 mM. In human leukemic blasts, kinetic analysis of araC uptake yielded a Km of approximately 225/~M which was significantly higher than their normal counterparts (lymphocytes, K m = 50/~M; polymorphonuclear cells, K m = 140/IM) and positively correlated to the Vmax (Wiley et al., 1983). Based on these observations, it appears that the rate of transport of araC into the cell is 10 to 100 times faster than the rate of intracellular phosphorylation suggesting that phosphorylation rather than transport is the rate-determining step (Plagemann et al., 1978b; Wiley et al., 1985). However, there is some evidence that membrane transport may be a limiting factor in araC
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G.C. DAHERet al.
chemotherapy based on studies that demonstrate that the number of cell nucleoside transport sites correlates closely with araC influx (Wiley et al., 1983, 1982) as well as clinical responsiveness to araC therapy (Wiley et al., 1982). The number of nucleoside transport sites on cells may be estimated by measuring the equilibrium binding of [3H]-nitrobenzylthioinosine (NBMPR). This compound is a nucleoside analogue known to have a high affinity for the nucleoside transport protein but is not transported into the cell. At extracellular concentrations below 1 #M, a close correlation was demonstrated between the maximal number of nucleoside binding sites (i.e. NBMPR binding sites) per cell in T lymphoblastic lymphoma blasts, myeloblasts, and lymphoblasts (Wiley et al., 1985) and the level of araCTP accumulation in these cells suggesting that membrane transport may be one of the major rate-limiting steps for araCTP accumulation at low extracellular concentrations (Heichal et al., 1979; Wiley et al., 1985). Also, Tanaka and Yoshida (Tanaka and Yoshida, 1987) examined araCTP accumulation, metabolic enzyme activities, and the nucleoside transport capacity in eleven human leukemic cell lines with different phenotypes. The sensitivity of the leukemic cell lines to araC (measured by clonogenic survival) correlated with the amount of araCTP formed within the cell. The accumulation of araCTP correlated with the nucleoside transport capacity in the leukemic cells, while no correlation was observed with the levels of enzymes involved in araC metabolism (deoxycytidine kinase, pyrimidine monophosphate kinase, and deoxycytidylate deaminase). An alteration in araC transport is one possible mechanism for acquired resistance to araC during chemotherapy and is supported by the isolation of murine lymphoma cells with defective transport
metabolized by the enzymes involved in the natural processing of these nucleotide precursors. The balance between activating (i.e. anabolic) and inactivating (i.e. catabolic) enzymes is crucial in determining the quantity of drug converted to the active intermediate, araCTP (Fig. 10). 2.4.1. Catabol&m Opposing the activation of araC to araCTP are two enzymes: cytidine deaminase (EC 3.5.4.5) and deoxycytidylate deaminase (EC 3.5.4.12). The respective contribution of these enzymes, either collectively or independently, in the regulation of the catabolism and intracellular retention of araC in intact cells has not been thoroughly investigated. In addition, nucleoside tri- and diphosphatases are assumed to be involved in the conversion of araCTP to araCMP. However, these pathways are not well defined and will not be discussed in this paper. The conversion of araCMP to araC by 5'-nucleotidase is another plausible pathway for dephosphorylation but will not be discussed. 2.4.1.1. Cytidine deaminase. Cytidine deaminase (EC 3.5.4.5) catalyzes the deamination of cytidine and deoxycytidine. Initial studies with intravenously administered araC in human cancer patients (Smith et al., 1959) demonstrated that araC was cleared very rapidly from the bloodstream. Since the rate of disappearance could not be accounted for by urinary excretion, it was postulated that araC must be metabolized to an inactive product or products. Subsequent studies with human liver homogenates (Camiener and Smith, 1965) demonstrated that araC was deaminated to an inactive product, uracil arabinoside (araU; eqn 11) by cytidine deaminase.
(11)
OH
systems for purine or pyrimidine nucleosides (Cass et al., 1981; Cohen et al., 1979). However, in a variety of lymphoblasts known to be resistant to araC, no significant changes in nucleoside transport characteristics were noted (Young et al., 1985) and the frequency of altered nucleoside transport in cells resistant to araC has not been determined. 2.4. METABOLISM As with most antimetabolites, metabolism has a major role in determining the therapeutic effectiveness of araC. The close structural similarity of araC to cytidine and deoxycytidine (Fig. 9) permits it to be
OI4
Cytidine deaminase is widely distributed in mammalian tissues including liver, kidney, heart, intestinal mucosa, and granulocytes (Camiener and Smith, 1965; Chabner et al., 1974; Chou et al., 1975) as well as various tumors (Camiener and Smith, 1965; Chabner et al., 1973; Hart et al., 1972; Ho and Frei, 1971). High levels of enzyme activity have been reported in normal liver and kidney (Camiener, 1967; Camiener and Smith, 1965) and in some human solid tumor samples (Cheng and Capizzi, 1982). The relatively high specific activity of cytidine deaminase found in most solid tumors may account for the poor response of such tumors to conventional doses of araC. In contrast, acute and chronic myelocytic leukemia cells
Pyrimidine analogues and their nucleosides contained significantly less cytidine deaminase than normal circulating granulocytes (Chabner et al., 1974) which may explain the increased cytotoxic response of these cells to araC. Cytidine deaminase has been partially purified and characterized from a number of sources and cells including bacteria (Ashley and Bartlett, 1984; Vita et al., 1985), mouse kidney (Creasey, 1963; Tomchick et al., 1968), mouse spleen (Malathi and Silber, 1971), human, canine, chicken, and sheep liver (Fanucchi et al., 1986; Vita et al., 1987; Wisdom and Orsi, 1969), and human normal and leukemic granulocytes (Chabner et al,, 1974). In humans, cytidine deaminase activity is highest in the liver. Kinetic analysis of human liver prepared from freshly frozen autopsy material yielded Km values of 12#M, 19pM, and 87/~M for cytidine, deoxycytidine, and araC, respectively (Chabot et al., 1983). Utilizing the same substrates, Fanucchi et al. (Fanucchi et al., 1986) reported Km values of 49pM, 55 #M, and 270#M, respectively, for cytidine deaminase isolated from human liver. Cytidine deaminase purified from normal and leukemic granulocytes (Chabner et al., 1974) had similar Michaelis constants with Km values of 11/~M, 26/~M, and 88 pM for cytidine, deoxycytidine, and araC, respectively. Comparison of cytidine deaminase from normal, acute myelocytic leukemic, and chronic myelocytic leukemic granulocytes yielded no biochemical or kinetic differences among the different cell types but showed significantly lower levels of enzyme in leukemic cells as opposed to normal granulocytes (Chabner et al., 1974). Tetrahydrouridine (THU) and araU are both potent inhibitors of cytidine deaminase (Capizzi et al., 1983; Ho et al., 1975; Yang et al., 1985). The K~ for THU was reported to be 3 x 10-sM (Stoller et al., 1978). Since cytidine deaminase is the primary catabolic enzyme responsible for the inactivation of
207
is an inhibitor of cytidine deaminase (Ki = 1 x 10-5 M) and decreases the inactivation of araC in both normal and malignant tissues that contain cytidine deaminase (Drake et al., 1980). The emergence or expansion of a population of araC resisant cells is probably the major cause of failure in araC chemotherapy. Alteration in the activity of cytidine deaminase has been suggested to have a role in either de novo or acquired resistance (Steuart and Burke, 1971), but the clinical relevance of this data has been questioned (Harris et al., 1981; Smyth et al., 1976). In a study with myeloid leukemic cells, Harris et al. (1981) suggested that araC in the medium was freely accessible to both metabolic pathways (anabolic and catabolic) and that changes in deaminase activity resulted in a decrease in the concentration of araC in the medium and not a decrease in intracellular araC. Although cells with a high deaminase/kinase ratio were apparently resistant to araC, when the concentration of araC remaining in the medium was plotted against the concomitant inhibition of DNA synthesis all cells had similar sensitivity to araC. Therefore, provided an adequate extracellular concentration of araC was maintained, cells with high deaminase activity would be as sensitive as those with much less deaminase activity. 2.4.1.2. Deoxycytidylate deaminase. Deoxycytidylate deaminase (EC 3.5.4.12) is the enzyme responsible for the deamination of CMP to UMP and provides an additional route for the catabolism of araC. l-fl-D-arabinofuranosylcytosine 5"-monophosphate (araCMP) may be deaminated to 1-fl-D-arabinofuranosyluridine 5'-monophosphate (araUMP) by deoxycytidylate deaminase (Fig. 7) with the subsequent release of l-~-D-arabinofuranosyluracil (araU) (E1lims et al., 1981, 1983). The reaction catalyzed by deoxycytidylate deaminase is given in eqn 12.
(12)
OH araC, inhibition of this enzyme may lead to increased intracellular levels of araC and, subsequently, increased araCTP. Recently, Avramis and Powell (Avramis and Powell, 1987) designed a study to determine if inhibition of cytidine deaminase would enhance the tumor cellular anabolism of araC to araCTP in tumor-bearing mice. In their study, pretreatment with THU and/or araU increased plasma araC concentrations but did not significantly affect the intracellular concentration of araCTP in the tumor cells nor did it affect the antitumor activity of araC. In addition to THU and araU, 3-deazauridine JPT 48/2--G
Deoxycytidylate deaminase has been highly purified from a number of sources including human leukemia cells (Ellims et al., 1983), human spleen (Ellims et al., 1981), donkey spleen (Geraci et aL, 1967), chick embryo extracts (Maley and Maley, 1964, 1968) and bacteria (MoUgaard and Neuhard, 1978; Sergott et al., 1971; Socca et al., 1969). The characterization of deoxycytidylate deaminase isolated from this variety of sources has revealed variations in the physical and kinetic properties of this enzyme relative to either species specificity or the unstable nature of the enzyme. Deoxycytidylate
208
G.C. DArmR et al.
deaminase purified from human leukemic CCRFCEM cells exists as a dimer with a native MW of 108 kDa and subunits of 51 kDa each (Ellims et al., 1983). Deoxycytidylate deaminase had the greatest affinity for dCMP, with less affinity for araCMP, and least affinity for cytidine monophosphate (CMP) (Ellims et al., 1983). The enzyme is regulated primarily by two allosteric effects, an inhibitory effect by deoxycytidine triphosphate (dCTP) and an activating effect by thymidine triphosphate (TTP) and araCTP. A possible effect of administration of araC would be to stimulate deoxycytidylate deaminase activity which may lead to an increase in dUMP pools and, in turn, may increase TMP and TTP pools. TTP has been shown to activate GDP reductase and inhibit CDP reductase and thymidine kinase (George and Cory, 1979). Therefore, one of the possible effects of araC may be alteration of deoxyribonucleotide pools leading to inhibition of DNA synthesis. Reports have shown that araCMP is an effective substrate of deoxycytidylate deaminase isolated from cultured CCRF-CEM human lymphoblastic leukemia cells (Drake et al., 1980; Ellims et al., 1983) and human spleen (Ellims et al., 1981), while araCMP is neither an inhibitor nor a substrate for enzyme isolated from acute myelocytic leukemia (Mancini and Cheng, 1983). Such differences in the characteristics of this important nucleotide-metabolizing enzyme add to the complexity of the antitumor activity of araC in human leukemic cells. Similar to the inhibition of cytidine deaminase by THU, deoxycytidylate deaminase is also inhibited by deoxytetrahydrouridine (dTHU). While, inhibition of cytidine deaminase by THU did not result in an increase in the accumulation of araCTP, inhibition of deoxycytidylate deaminase by dTHU resulted in an increased accumulation of araCTP from araC in CCRF-CEM human leukemia cells of approximately 50% over that of araC alone (Fridland and Verhoef, 1987). These studies also indicated that deoxycytidylate deaminase functions as the major pathway for araC nucleotide catabolism in these cells. Interestingly, deoxycytidylate deaminase did not contribute significantly to araC nucleotide catabolism in PF-2S lymphoblasts despite high levels of dCMP and araCMP deamination in extracts of these cells (Fridland and Verhoef, 1987) suggesting that another pathway (e.g. 5'-nucleotidase) was operating in these cells and at a much higher rate than in the CEM lymphoblasts. In addition to dTHU, several purine and pyrimidine nucleotides are competitive inhibitors of deoxycytidylate deaminase such as dGMP (Ki= 1.8 × 10-4M), dUMP (Ki= 1.2 x 10-3M) and TMP (Ki = 1.4 × 10 -3 M) (Ellims et al., 1981). A potentially important mechanism of resistance to araC chemotherapy is a marked expansion of dCTP pools due to increased CTP synthetase activity or deficiency of deoxycytidylate deaminase activity (Momparler et al., 1968). The increased intracellular concentration of dCTP may contribute to araC resistance by feedback inhibition of deoxycytidine kinase and/or competition with araCTP for insertion into DNA and subsequent inhibition of DNA synthesis. In addition, increased activity of
deoxycytidylate deaminase may provide a mechanism of acquired resistance by depletion of arabinosyl nucleotides and increased inactivation of araCMP to araUMP. 2.4.2. Anabolism Following entry into the cell, araC must be anabolized to araCTP in order for it to be effective as an anticancer agent. Because of the similarity of araC to the naturally-occurring nucleoside, deoxycytidine, it is anabolized by the same anabolic pathway. AraC is initially converted to araCMP by deoxycytidine kinase, than araCMP is converted to araCDP by deoxycytidylate kinase, and, finally, araCDP is converted to araCTP by nucleoside diphosphate kinase (Fig. 10). Formation of araCTP and maintenance of the intracellular pool of the araCTP are two factors that are clearly important in determining the antitumor effectiveness of araC (Preisler et al., 1985; Rustum and Preisler, 1979). 2.4.2.1. D e o x y c y t i d i n e kinase. Deoxycytidine kinase (EC 2.7.1.74) is the initial enzyme in the anabolic pathway and is responsible for the conversion of deoxycytidine to deoxycytidylate in the presence of ATP. Upon exposure of the cell to araC, this enzyme also catalyzes the conversion of araC to araCMP (eqn 13). Deoxycytidine kinase isolated from cultured human T-lymphoblasts has been shown to have a MW of 60 kDa with a requirement for divalent ions (particularly, Mg 2+ or Mn 2+) (Datta et al., 1989). Of the three enzymatic steps in the formation of araCTP, deoxycytidine kinase is believed to be rate-limiting due to its relatively low intracellular concentration. The Km for the naturally occurring substrate, deoxycytidine, has been estimated to be 7.8/~M, while the Km for araC is thought to be approximately 20 # u (Coleman et al., 1975; Hande and Chabner, 1978). Deoxycytidine kinase is inhibited strongly by deoxycytidine (Ki = 1.7 x 10 -7 M) and dCTP (Ki = 7.3 × 10 -6 M) and weakly by araCTP (K~ = 1.3 × 10 -7 M). As the levels of dCTP rise, deoxycytidine kinase is allosterically inhibited. High levels of dCTP can compete with araCTP for incorporation into DNA (Yang et al., 1985). A decrease in the dCTP pool by pretreatment with hydroxyurea (Walsh et al., 1980), deazauridine (Mills-Yamamoto et al., 1978), or high doses of thymidine (Danhauser and Rustum, 1980) has been shown to potentiate the cytotoxicity of araC. Deoxycytidine kinase is an important site of metabolic resistance to araC chemotherapy. In murine cell lines, deficiency of deoxycytidine kinase is the most frequently observed cause of drug resistance (Chu and Fischer, 1965; Drahovsky and Kreis, 1970; Schrecker and Urshel, 1968). Also, human tumors deficient in deoxycytidine kinase have been shown to be resistant to araC (Tattersall et al., 1974). However, reduced deoxycytidine kinase activity is not always demonstrated in araC-resistant cells and it is unclear how frequently deoxycytidine kinase deficiency occurs in man. In a study of araC-resistant and araCsensitive murine leukemia cells, Momparler et al. (1968) failed to find significant differences in araC
209
Pyrimidine analogues and their nucleosides
(13)
OH
OH
phosphorylation between these two cell lines. The resistant cell line had an increased dCTP pool which may contribute to araC resistance by feedback inhibition on deoxycytidine kinase and/or competition with araCTP for insertion into DNA. 2.4.2.2. Deoxycytidylate kinase. The second step in the anabolism of araC is the conversion of araCMP to araCDP (eqn 14) catalyzed by deoxycytidylate kinase (EC 2.7.4.14). Deoxycytidylate kinase has been purified from rat Novikoff ascites hepatoma (Maness and Orengo, 1976), rat liver (Maness and Orengo, 1975), calf thymus (Sugino et al., 1966), Yoshida sarcoma cells (Arima et al., 1977) and human leukemic blast cells (Hande and Chabner, 1978). A MW of 28 kDa was reported for deoxycytidylate kinase from leukemic blasts (Hande and Chabner, 1978), while 17 kDa has been reported for enzyme isolated from rat liver (Maness and Orengo, 1975). Studies with various pyrimidine nucleoside monophosphates and analogues have also shown that
NH~
a single enzyme is responsible for the phosphorylation of CMP, dCMP, UMP, dUMP, araCMP, FUMP, FdUMP, and araUMP. Variations in enzyme levels and substrate affinity have been shown in cells from different sources (Hande and Chabner, 1978). Blast cells isolated from normal human donors contained a mean level of deoxycytidylate kinase activity of 1.2/tmol/hr/mg protein (Hande and Chabnet, 1978). Higher levels were reported in normal human lymphocytes (18 #mol/hr/mg protein) but a different substrate was used in this study (Scholar and Calabresi, 1973). Both studies, however, reported a two-fold increase in leukemic cells compared to their normal counterparts. Levels of deoxycytidylate kinase were 100-fold higher in human leukemic blasts than deoxycytidine kinase (Coleman et al., 1975; Hande and Chabner, 1978). In addition, deoxycytidine kinase had considerably higher affinity for its substrates, deoxycytidine (Km= 7.8 × l0 -6) and araC (Kin = 2.6 x 10-5) than deoxycytidylate kinase had for its substrates,
NH2
(14)
Oil
210
G. C. DAHERet al.
dCMP (Km= 1.9 x 10 -3) and araCMP (Kin = 6.8 x 10-4). Therefore, deoxycytidylate kinase is probably not the rate-limiting step in the activation of araC. However, it has been suggested that under conditions of low araCMP concentration (i.e. less than 10 -5 M), deoxycytidylate kinase may become rate-limiting (Hande and Chabner, 1978). 2.4.2.3. Nucleoside diphosphate kinase. Nucleoside diphosphate kinase (NDP kinase; EC 2.7.4.6) utilizes ATP as a phosphate donor in the conversion of nucleoside 5'-diphosphates to their corresponding nucleoside 5'-triphosphates. NDP kinase is also responsible for the conversion of araCDP to araCTP, the active metabolite in araC chemotherapy (eqn 14). NDP kinase was discussed previously in Section 1.4.2.6. 2.5. METABOLICSITESOF ACTION Biochemical studies into the mechanisms by which araC exerts its cytotoxic effect on cells reveal that araC acts as an analogue of deoxycytidine and has multiple effects on DNA synthesis. Identification of the specific event which produces cellular toxicity is difficult because of the complexity of the biochemical and cellular processes responsible for cell replication and cell function. A correlation between the occurrence of a specific event and clonogenic cell survival is the initial step in the identification of the mechanism or mechanisms of drug-induced cytotoxicity. Studies with araC have focused on several specific area including inhibition of DNA biosynthesis, inhibition of DNA repair, incorporation into nucleic acids (i.e. RNA and DNA), and inhibition of membrane precursor synthesis. 2.5.1. Inhibition o f DNA Biosynthesis Following formation of araCTP from araC, araCTP has been shown to competitively inhibit the interaction of dCTP and DNA polymerase • in a variety of tissues including calf thymus (Furth and Cohen, 1968), murine tumors (Graham and Whitmore, 1970a; Kimball and Wilson, 1968), and human leukemic cells (Inagati et al., 1970). AraCTP and dCTP have relatively equal affinity for DNA polymerase c~ in the range of 1 x 10-sM (Momparler, 1972) and the inhibition is reversed by the addition of dCTP in cell-free systems or deoxycytidine in intact cells (Chu and Fischer, 1962; Kinahan et al., 1979). However, enzyme kinetic studies indicate that araCTP is a weak competitive inhibitor of DNA polymerase :¢(Graham and Whitmore, 1970b; Momparler, 1972). Since DNA synthesis can be inhibited to approximately 50% by araCTP levels 100-200-fold lower than that required for competitive inhibition of DNA polymerase ~, there must be additional mechanisms by which araC inhibits DNA synthesis. 2.5.2. Inhibition o f DNA Repair AraCTP has only a slight inhibitory effect on DNA repair. The K~ for inhibition of DNA polymerase fl (i.e. the DNA repair enzyme) is 26/~M (Dunn and Regan, 1979). The affinity of dCTP, the natural
substrate for the polymerase, is five times that of araCTP (Muller, 1977). Repair inhibition is reversed by 50% when araC is removed and by 9 5 o if deoxycytidine is added following araC exposure (Dunn and Regan, 1979). Therefore, inhibition of DNA excision repair by araCTP is significant only if dCTP pools are depleted by additional pharmacologic manipulation (Dunn and Regan, 1979; Hiss and Preston, 1978). Fram and Kufe (Fram and Kufe, 1982) showed that the inhibition of DNA repair leads to the accumulation of single strand breaks in the DNA that increased with both araC concentration and exposure time. This data supports a model that has been proposed relating inhibition of repair with cytotoxicity (Stenstrom et al., 1974). In addition to its effects on eukaryotic polymerases, araCTP is an extremely potent inhibitor of viral RNA-directed DNA polymerase with a Ki = 0.1 /~M (Muller, 1977). 2.5.3. Incorporation into Nucleic Acids The effects of araC on DNA include not only the inhibition of DNA elongation and repair at the polymerase step but also disruption of DNA function secondary to incorporation of araCTP into DNA (Kufe and Major, 1982). Although earlier studies had indicated that incorporation of araC into nucleic acids (DNA or RNA) did not correlate with araC-induced cytotoxicity (Graham and Whitmore, 1970a), more recent studies (Kufe et al., 1980b) revealed a relationship between the incorporation of araC into DNA and cell lethality in L1210 cells in culture. This relationship suggested that the probability of cell survival was about 50% when 0.66 pmol. araC was incorporated into DNA. This is equivalent to about 5 araC nucleotides per 104 bases in DNA. A similar relationship has also shown for a human promyeloblast cell line and myeloblasts isolated from a patient with acute myelogenous leukemia (Major et al., 1981). This process is very complex and it is difficult to understand the mechanisms of araC cytotoxicity based on these observations. Ross et al. (1987) noted that in studies of blast cells from patients with acute nonlymphocytic leukemia the intracellular araCTP concentration was not linearly correlated with the incorporation of araCTP (into DNA). This finding may be the result of increasing inhibition of DNA polymerase ~ by araCTP with an increase in the intracellular concentration of araCTP. 2.5.4. Inhibition o f Membrane Precursor Synthesis AraC has been shown to inhibit enzymes involved in the synthesis of glycoproteins and glycolipids in mammalian cells (Hawtrey et al., 1974a,b; MyersRobfogel and Spataro, 1980). Since these are essential components of the cell membrane, araC may alter the composition of the membrane and, subsequently, its function. AraC inhibits the incorporation of glucosamine into glycoproteins and glycolipids (Hawtrey et al., 1974a,b) and the transfer of galactose, Nacetyl-D-glucosamine, and sialic acid from their respective nucleotide carrier to glycoproteins (Hawtrey
Pyrimidine analogues and their nucleosides
NH2
NH2 0
211
N.~
2~r "NH HOC~ OH OH {A)
HOCH2
(B)
{C)
FIG. 11. Reversible spontaneous decomposition of azacytidine (A) to N-(formylamidino)-N'-fl-D-ribofuranosylurea (B) which may further decompose to 1-fl-D-ribofuranosyl-3-guanylurea (C).
et al., 1974a,b; Klohs et al., 1979). AraCMP and
araCTP also competitively inhibit sialytransferases which synthesize sialylglycoproteins (Myers-Robfogel and Spataro, 1980). Inhibition of membrane precursor synthesis appears to occur at high araC concentrations which also inhibit D N A synthesis and, thus, its relationship to and impact on cell survival are difficult to determine. In addition, araCTP serves as a substrate for the synthesis of araCDP-choline (Lauzon et al., 1978a,b) and, therfore, may serve as a substrate for membrane phospholipid synthesis. The rapid lysis of leukemic blasts noted in clinical trials with high-dose araC (Capizzi et al., 1984) suggests that such a mechanism may exist in addition to inhibition of DNA synthesis.
utilized this synthetic azaCyd to demonstrate that azaCyd produced growth inhibition in bacteria. Two years later, azaCyd was also isolated as an antibiotic from fungal cultures (Hanka et al., 1966). Based on the concept that initiated the synthesis of azaCyd several other nucleosides closely related to azaCyd have also been synthesized and investigated for antitumor activity. These include 2'-deoxy-5-azacytidine (Pliml and Sorm, 1964), 5,6-dihydro-5-azacytidine (Beisler et al., 1976), and the arabinosyl nucleoside of azaCyd, arabinosyl-5-azacytidine (Beisler et al., 1977). At present, the metabolism, mechanism of action, or therapeutic efficacy of these compounds relative to azaCyd has not been thoroughly evaluated. 3.2. CHEMISTRY
3. AZACYTIDINE The success of araC as an antileukemic drug stimulated the research for other cytidine analogues that may have antitumor activity. Of particular interest were analogues that did not require activation by deoxycytidine kinase since its activity is decreased in many of the araC-resistant tumors. Therefore, cytidine analogues were examined with substitutions in the pyrimidine ring rather than in the ribose moiety since these analogues would most likely be activated by uridine/cytidine kinase instead of deoxycytidine kinase. This rationale led to the development of azacytidine (azaCyd; 5-azacytidine). Although azaCyd was shown to have antitumor activity in initial tumor screening, clinical trials have been less impressive limiting enthusiasm for this drug as an antitumor agent (Karon et al., 1973; McCredie et al., 1973; Vogler et al., 1976). Occasional responses have been reported in patients with solid tumors (e.g. breast, colon, and malignant melanoma) but the therapeutic efficacy was very low and further clinical trials were not warranted (Von Holt et al., 1976). The only therapeutic role for azaCyd today is in the treatment of acute myleoblastic leukemia. 3.1. HISTORICALPERSPECTIVE AzaCyd was first synthesized in 1964 by Sorm and his colleagues (Sorm et al., 1964). Sorm et al. (1964)
AzaCyd is an analogue of cytidine and differs from the de novo nucleoside by the presence of a nitrogen in the pyrimidine ring (Fig. 9). This substitution destabilizes the heterocyclic ring and leads to spontaneous decomposition of azaCyd in neutral or alkaline solutions (Fig. 11) with a half-life of approximately 4 hr (Beisler, 1978). At acidic pH or in buffered solutions azaCyd is much more stable with a half-life of 65 hr at 25°C and 94 hr at 20°C (Israili et al., 1976). 3.3. UPTAKEINTOCELLS AzaCyd readily enters all mammalian cells by the same facilitated nucleoside transporter as the de novo nucleosides, uridine and cytidine (Notari and DeYoung, 1975). This transport system was discussed in detail previously in Sections 1.3 and 2.3. 3.4. METABOLISM As discussed previously for both the fluoropyrimidines (Section 1.4) and araC (Section 2.4), the metabolism of azaCyd by host and tumor cells is an important aspect of its usefulness as an antitumor agent. As with the previous antimetabolites, the close structural similarity of azaCyd to physiological nucleosides (Fig. 9) results in its metabolism by the same enzymes that metabolize cytidine.
212
G.C. DAHERet al. .HH2
.NH2
(~'~N~1 0 ~ j 3
I 4 I
.NH2
.
¢.J
/\ RNA
DNA
¢.2
FIG. 12. Metabolism of azacytidine (azaCyd) in humans. Anabolism of azaCyd is catalyzed initially by uridine/cytidine kinase (I) to azacytidine monophosphate (azaCMP) followed by nucleotide kinases (2) which ultimately form azacytidine triphosphate (azaCTP). AzaCTP may be incorporated into DNA or RNA leading to inhibition of nucleic acid synthesis. Catabolism of azaCyd primarily occurs via cytidine deaminase (3) to form azauridine. Deamination of azaCMP to azauridine monophosphate by deoxycytidylate deaminase (4) is also suspected. 3.4.1. Catabolism
3.4.2. Anabolism
In addition to spontaneous decomposition in neutral or alkaline solutions, azaCyd may be catabolized to inactive products (Fig. 12) via the enzymes, cytidine deaminase (EC 3.5.4.5) and deoxycytidylate deaminase (EC 3.5.4.12). These enzymes were discussed in the sections on araC catabolism (see Sections 2.4.1.1 and 2.4.1.2). The importance of deamination in azaCyd pharmacokinetics has been demonstrated by studies with THU (Neil et al., 1975). As noted earlier, THU is a potent cytidine deaminase inhibitor. These studies demonstrate that THU increases the plasma concentration of azaCyd as well as the toxicity and therapeutic activity of orally administered azaCyd. The role of cytidine deaminase in the development of resistance to azaCyd has not been determined.
The initial step in the activation of azaCyd is its conversion to a monophosphate (azaCMP) by uridine/cytidine kinase with subsequent conversion to azaCyd triphosphate (azaCTP) by nucleotide kinases (Fig. 12).
NH2
3.4.2.1. Uridine/cytidine kinase. Uridine/cytidine kinase (EC 2.7.1.48) is responsible for the conversion of either uridine or cytidine to their respective monophosphates, uridine monophosphate (UMP) or cytidine monophosphate (CMP). In addition, when cells are exposed to azaCyd, uridine/cytidine kinase catalyzes the conversion of azaCyd to azaCMP (eqn 15). Uridine/cytidine kinase has a lower affinity for azaCyd with a Km between 0.2 and 11 mM than for NH 2
H
OHOH
H
OHOH
(15)
213
Pyrimidine analogues and their nucleosides uridine or cytidine with a Km of 0.05 M (Drake et al., 1977; Lee et al., 1974) and is the rate-limiting step in azaCyd anabolism. Both uridine (Vadlamudi et al., 1970b) and cytidine competitively inhibit the anabolism of azaCyd and have been shown to prevent azaCyd toxicity in whole animals and tissue culture (Vadlamudi et al., 1970a). Deletion of uridine/ cytidine kinase has been observed in mutant Novikoff hepatoma cells that were resistant to azaCyd (Plagemann et al., 1978a). 3.4.2.2. Nucleotide kinases. Further activation of azaCyd involves the conversion of azaCMP to azaCDP and, subsequently, azaCTP. These reactions are most likely catalyzed by the enzymes deoxcytidylate kinase and N D P kinase, respectively. Deoxycytidylate kinase was previously discussed in regard to araC metabolism in Section 2.4.2.2 and NDP kinase was previously discussed in Sections 1.4.2.6 and 2.4.2.3.
3.5. METABOLICSITESOF ACTION Over the past 25 years there have been several studies on the cytotoxic effects of azaCyd. Both drug concentration and duration of exposure to azaCyd greatly influence the cytotoxicity observed in cultured cells (Chabner, 1982). AzaCyd is primarily cytotoxic to cells in the S phase of the cell cycle with little, if any, effect against nondividing cells (Li et aL, 1970; Lloyd et al., 1972). These findings would suggest that one of the effects of azaCyd is on D N A synthesis (Li et al,, 1970). Dose-response curves in both normal and tumor cell cultures are biphasic indicating more than one mechanism is probably responsible for the cytotoxicity of azaCyd (Chabner, 1982). The incorporation of azaCyd (or more specifically, azaCTP) into R N A and D N A was demonstrated in both cultured rhesus monkey kidney cells (Sorm et al., 1964) and L1210 cells (Li et al., 1970). In both cases, inhibition of D N A synthesis was more pronounced than R N A synthesis. While incorporation of azaCTP into D N A is one possible mechanism for the inhibition of D N A synthesis, another is by the competition between azaCTP and dCTP for D N A polymerase as was shown to occur with araCTP. However, if this were one of the major mechanisms for the inhibition of D N A synthesis deoxycytidine would be expected to alleviate azaCyd inhibition of D N A synthesis which is not the case (Li et al., 1970). Therefore, interference with the synthesis of de novo pyrimidine nucleotides and the inhibition of D N A synthesis due to incorporation of azaCTP appear to be the major mechanisms of cytotoxicity following exposure to azaCyd. In addition to its cytotoxic effects, azaCyd has other biological actions that may be of clinical importance. AzaCyd stimulates the activity of various hepatic enzymes including tyrosine aminotransferase (Cihak et al., 1973) and uridine/cytidine kinase (Cihak and Vesely, 1973). It also stimulates differentiation in certain mouse embryo cells in culture (Constantinides et al., 1977). This 'differentiating' effect of azaCyd has not been thoroughly examined
in tumor cells. AzaCyd also has mutagenic and teratogenic effects. Chromatid breaks have been observed in cultured hamster fibroblasts (Karon and Benedict, 1972) and LI210 cells (Li et al., 1970). However, no secondary malignancies related to azaCyd treatment have been described in animals or humans. REFERENCES ABSIL,J., TUILIE,M. and Roux, J. M. (1980) Electrophoretitally distinct forms of uridine kinase in the rat. Tissue distribution and age-dependence. Biochem. J. 185: 273-276. ArtM~D, N. K. (1982) Multiple forms and inhibitors of uridine-cytidine kinase in neoplastic cells. Int. J. Biochem. 14: 259-262. ANDERSON,E. P. (1973) Nucleoside and nucleotide kinases. In: The Enzymes, pp. 49-96, BOYER,P. D. (ed.) Academic Press, New York. ARDALAN, B. and GLAZER,R. (1981) An update on the biochemistry of 5-fluorouracil. Cancer Treat. Rev. 8: 157-167. ARDALAN,B., COONEY,D. A., JAYARAM,H. N,, CARRICO,C. K., GLAZER, R. I., MACDONALD, J. and SCHEIN, P. S. (1980) Mechanism of sensitivity and resistance of murine tumors to 5-fluorouracil. Cancer Res. 40: 1431-1437. ARDALAN, B., VILLACORTE,O., HECK, D. and CORBETT,T. (1982) Phosphoribosyl pyrophosphate, pool size and tissue levels as a determinant of 5-fluorouracil response in murine colonic adenocarcinomas. Biochem. Pharmac. 31: 1989-1992. ARIMA, T., AKIYOSHI,H. and FUJII, S. (1977) Characterization of pyrimidine monophosphokinase in normal and malignant tissues. Cancer Res. 37:1593-1597. ARMSTRONG,R. D. and DIASIO,R. B. (1980) Metabolism and biological activity of 5'-deoxy-5-fluorouridine, a novel fluoropyrimidine. Cancer Res. 40: 3333-3338. ARMSTRONG,R. D., LEWIS,M., STERN,S. G. and CADMAN, E. C. (1986) Acute effect of 5-fluorouracil on cytoplasmic and nuclear dihydrofolate reductase messenger RNA metabolism. J. biol. Chem. 261: 7366-7371. ASHLEY,G. W. and BARTLETT,P. A. 0984) Purification and properties of cytidine deaminase from Escherichia coil J. biol. Chem. 259: 13615-13620. Au, J. L., Wu, A. T., FRIEDMAN,M. A. and SADEE,W. (1979) Pharmacokinetics and metabolism of ftorafur in man. Cancer Treat. Rep. 63: 343-350. AU, J. L., RUSTUM,Y. M., LEDESMA,E. J., MITTELMAN,A. and CR~.AVEN,P. J. (1982) Clinical pharmacological studies of concurrent infusion of 5-fluorouracil and thymidine in treatment of colorectal carcinoma. Cancer Res. 42: 2930-2937. AVRAMIS,V. I. and POWELL,W. (1987) Pharmacology of combination chemotherapy of cytosine arabinoside (araC) and uracil arabinoside (ara-U) or tetrahydrouridine (THU) against murine leukemia LI210/0 in tumorbearing mice. Cancer Invest. 5: 293-299. BAND, P. R., HOLLAND, J. F., BERNARD, J., WEIL, M., WALKER, M. and RALL, D. (1973) Treatment of central nervous system leukemia with intrathecal cytosine arabinoside. Cancer 32: 744-748. BEISLER,J. A. (1978) Isolation, characterization, and properties of a labile hydrolysis product of the antitumor nucleoside, 5-azacytidine. J. reed. Chem. 21: 204-208. BEISLER, J. A., ABBASI,M. M. and DR1SCOLL,J. S. (1976) Dihydro-5-azacytidine hydrochloride, a biologically active and chemically stable analog of 5-azacytidine. Cancer Treat Rep. 60: 1671-1674. BEISLER,J. A., ABBASI,M. M. and DRISCOLL,J.S. (1977) The synthesis and antitumor activity of arabinosyl-5-azacytosine. Biochem. Pharmac. 26: 2469-2472.
214
G.C. DAI~R et al.
BENVENUTO, J., LU, K., HALL, S. W., BENJAMIN, R. S. and Loo, T. L. (1978) Metabolism of 1-(tetrahydro-2-furanyl)5-fluorouracil (ftorafur). Cancer Res. 38: 3867-3870. BERENS, K. and SHUGAR,D. (1963) Ultraviolet absorption spectra and structure of halogenated uracils and their glycosides. Acta biochimica polonica 10: 25-47. BERGMANN,W. and FEENEY, R. J. (1950) The isolation of a new thymine pentoside from sponges. J. Am. chem. Soc. 72: 2809-2810. BERGMANN, W. and FEENEY, R. J. (1951) Contributions to the study of marine products. XXXII. The nucleosides of sponges. J. org. Chem. 16: 981. BERg, A. J. and CLAYTON,D. A. (1973) A genetically distinct thymidine kinase in mammalian mitochondria. Exclusive labelling of mitochondrial deoxyribonucleic acid. J. biol. Chem. 248: 2722-2729. BIANCHI, P. A., FARINA, M. V. and POLLI, E. (1964) Phosphorylation of thymidine diphosphate in resting and proliferating mammalian cells. Biochim. biophys. Acta 91: 323-325. BLEYER, W. A. (1989) New vistas for leukovorin in cancer chemotherapy. Cancer 63: 995-1007. BOLLAG, W. and HARTMANN,H. R. (1980) Tumour inhibitory effects of a new fluorouracil derivative: 5'-deoxy-5fluorouridine. Eur. J. Cancer 16: 427-432. BOURTOURAULT,M., MAHOUDO, H., HARAS, D., SAMPEREZ, S. and JOUAN, P. (1984) Stimulation by estradiol-17fl of thymidine kinase activity in the rat uterus. J. Steroid Biochem. 21: 613~20. BOWEN, D., WHITE, J. C. and GOLDMAN,I. D. (1978) A basis for fluoropyrimidine-induced antagonism to methotrexate in Ehrlich ascites tumor cells in vitro. Cancer Res. 38: 219-222. BREITMAN, T. R. (1963) The feedback inhibition of thymidine kinase. Biochim. biophys. Acta 67: 153-155. BREZNICK, E, and KARJALA, R. J. (1964) End-product inhibition of thymidine kinase activity in normal and leukemic human leukocytes. Cancer Res. 24: 841-846. BROOKS, K. P., KIM, B. D. and SANDER, E. G. (1979) Dihydropyrimidine amidohydrolase is a zinc metalloenzyme. Biochim. biophys. Acta 570: 213-214. BROOKS, K. P., JONES, E. A., KIM, B. D. and SANDER,E. G. (1983) Bovine liver dihydropyrimidine amidohydrolase: Purification, properties, and characterization as a zinc metalloenzyme. Archs Biochem, Biophys. 226: 469-483. BRYAN,J. H., HENDERSON,E. S. and LEVENTHAL,B. G. (1974) Cytosine arabinoside and 6-thioguanine in refractory acute lymphocytic leukemia. Cancer 33: 539-544. CAMIENER, G. W. (1967) Studies on the enzymatic deamination of cytosine arabinoside. II. Properties of the deaminase of human liver. Biochem. Pharmac. 16: 168 I-1689.
CAMIENER,G. W. and SMITH, C. G. (1965) Studies of the enzymatic deamination of cytosine arabinoside. I. Enzyme distribution and species specificity. Biochem. Pharmac. 14: 1405-1416. CANELLAKIS, E. S. (1956) Pyrimidine metabolism. I. Enzymatic pathways of uracil and thymine degradation. J. biol. Chem. 221: 315-321. CAPIZZI, R. L., YANG, J. L., CHENG, E., BJORNSSON, T., SAHASRABUDHE,D., TAN, R.-S. and CHENG, Y.-C. (1983) Alteration in the pharmacokinetics of high dose ara-C by its metabolite, ara-U, in patients with acute leukemia. J. c/in. Oncol. l: 763-771. CAPIZZI,R. L., POOLE, M., COOPER,M. R., R1CHARDS,F., II, STUART, J. J., JACKSON, D. V., JR, WHITE, D. R., SPURR, C. L., HOPKINS, J. O., Muss, M. B., RUDNICK, S. A., WELLS,R., GABRIEL,D. and Ross, D. (1984) Treatment of poor risk acute leukemia with sequential high dose araC and asparaginase. Blood 63: 694-700. CARADONNA,S. J. and CnENG, Y.-C. (1980a) The role of deoxyuridine triphosphate nucleotidohydrolase, uracilDNA glycosylase, and DNA glycosylase, and DNA
polymerase ct in the metabolism of FUdR in human tumor cells. Molee. Pharmac. 18: 513-520. CARADONNA, S. J. and CnENG, Y.-C. (1980b) Uracil DNAglycosylase. Purification and properties of this enzyme isolated from blast cells of acute myelocytic leukemia patients. J. biol. Chem. 255: 2293-2300. CARTER, G. L. and CoRv, J. G. (1988) Cross-resistance patterns in hydroxyurea-resistant leukemia LI210 cells. Cancer Res. 48: 5796-5799. CASS, C. E., KOLASSA,N., UEHARA,Y., DAHLING-HARLEY,E., HARLEY, E. and PATERSON, A. R. P. (1981) Absence of binding sites for the transport inhibitor nitrobenzylthioinosine on nucleoside transport deficient mouse lymphoma cells. Biochem. biophys. Acta 649: 769-777. CHABNER, B. A. (1982) Pyrimidine antagonists. In: Pharmacologic Principles of Cancer Treatment, pp. 183-212, CHABNER, B. A. (ed.) W. B. Saunders Co., Philadelphia. CHAaNER, B. A. and MYERS, C. E. (1985) Clinical pharmacology of cancer chemotherapy. In: Cancer--Principles and Practice of Oncology (2nd edn), pp. 287-328, DEVITA, V. T., JR, HELLMAN,S. and ROSENaERG,S. A. (eds) Lippincott, Philadelphia. CHABNER, B. A., DRAKE, J. C. and JOHNS, D. G. (1973) Deamination of 5-azacytidine by a human leukemia cell cytidine deaminase. Biochem. Pharmac. 22: 27632765. CHABNER, I . A., JOHNS, D. G., COLEMAN,C. N., DRAKE,J. C. and EVANS, W. H. (1974) Purification and properties of cytidine deaminase from normal and leukemic granulocytes. J. clin. Invest. 53:922-93 I. CHABOT, G. G., BOUCHARD,J. and MOMPARLER,R. L. (1983) Kinetics of deamination of 5-aza-2'-deoxycytidine and cytosine arabinoside by human liver cytidine deaminase and its inhibition by 3-deazauridine, thymidine, or uracil arabinoside. Biochem. Pharmac. 32:1327-1328. CHAUDHURI, N. K., MONTAG, B. J. and HEIDELBERGER, C. (1958) Studies on fuorinated pyrimidines III. The metabolism of 5-fuorouracil-2-C14 acid in vivo. Cancer Res. 18: 318-328. CI-IENG, N., PAYNE, R. C. and TRAUT, T. W. (1986) Regulation of uridine kinase: Evidence for a regulatory site. J. biol. Chem. 261: 13006-13012. CHENG, Y.-C. and CAPIZZI, R. L. (1982) Enzymology of cytosine arabinoside. Med. Pediat. Oncol. 10 (Suppl. I): 27-31. CHIGA, M., ODA, A. and HOLTZER,R. L. (1963) The activities of certain nucleoside diphosphokinases of normal and regenerating rat liver. Archs Biochem. Biophys. 103: 366-370. CHOU, T.-C., HUTCHISON, D. J., SCHMID, F. A. and PHILIPS, F. S. (1975) Metabolism and selective effects of 1-fl-D-arabinofuranosylcytosine in LI210 and host tissues in vivo. Cancer Res. 35: 225-236. CHU, M. Y. and FISCHER, G. A. (1962) A proposed mechanism of action of l-fl-D-arabinofuranosylcytosine as an inhibitor of the growth of leukemic cells. Biochem. Pharmac. 11: 423-430. CHU, M. Y. and FISCH/~R,G. A. (1965) Comparative studies of leukemic cells sensitive and resistant to cytosine arabinoside. Biochem. Pharmac. 14: 333-341. CHU, M. Y., NAGUIB, N. M., ILTZSCH, L. H., EL KOUNI, M. H., CHU, S. H. and CALABRESI,P. (1984) Potentiation of 5-fluoro-2'-deoxyuridine antineoplastic activity by the uridine phosphorylase inhibitors benzylacyclouridine and benzyloxybenzylacyclouridine. Cancer Res. 44:1852-1856. CIHAK,A. and VESELY,J. (1973) Enhanced uridine kinase in rat liver following 5-azacytidine administration. J. biol. Chem. 248: 1307-1313. CIHAK, A., LAMAR,C. and PITOT, H. C. (1973) Studies on the mechanism of the stimulation of tyrosine aminotransferase activity in vivo by pyrimidine analogs: The role of enzyme synthesis and degradation. Archs Biochem. BiDphys. 156: 176-187.
Pyrimidine analogues and their nucleosides
215
CISNEROS, R. J., SILKS, L. A. and DUNLAP, R. B. (1988) DESGRANGES, C., RAZAKA, G., RABAUD, i . , PICARD, P., DUPUCH, F. and BRICAUD,H. (1982) The human blood Mechanistic aspects of thymidylate synthase: Molecular platelet: A cellular model to study the degradation of basis for drug design. Drugs of the Future 13: 859-881. thymidine and its inhibition. Biochem. Pharmac. 31: COl-raN, A., ULLMAN,B. and MARTIN,D. W. (1979) Charac2755-2759. terization of a mutant mouse lymphoma cell line with DESGRANGES,C., RAZAKA,G., DE CLERCQ,E., HERDEWIJN, deficient transport of purine and pyrimidine nucleosides. P., BALZARINI,J., DROUILLET,F. and BRICAUD,H. (1986) J. biol. Chem. 254: 110-116. Effect of (E)-5-(2-bromovinyl)uracil on the catabolism COHEN, S. S. (1966) Introduction to the biochemistry of and antitumor activity of 5-fluorouracil in rats and D-arabinosyl nucleosides. In: Progress in Nucleic Acid leukemic mice. Cancer Res. 46:1094-1101. Research and Molecular Biology, pp. 1-88, DAV1DSON, DIASIO, R. B., BEAVERS,T. L. and CARPENTER,J. T. (1988) J. N. and COHN,W. E. (eds) Academic Press, New York. Familial deficiency of dihydropyrimidine dehydroCOLEMAN,C. N., STOLLER,R. G., DRAKE,J. C. and CHABNER, genase. Biochemical basis for familial pyrimidinemia B. A. (1975) Deoxycytidine kinase: properties of the and severe 5-fluorouracil-induced toxicity. J. clin. Invest. enzyme from human leukemic granulocytes. Blood 46: 81: 47-51. 791-803. DIXON, R. L. and ADAMSON,R. H. (1965) Antitumor activity CONSTANTINIDES,P. G., JONES,P. A. and GEVERS,W. (1977) and pharmacologic disposition of cytosine arabinoside Functional striated muscle cells from non-myoblast pre(NSC 63878). Cancer Chemother. Rep. 48: 11-16. cursors following 5-azacytidine treatment. Nature 267: DRAHOVSKY, D. and KKEIS, W. (1970) Studies on drug 364-366. resistance. II. Kinase patterns in P815 neoplasms sensitive COOPER, G. M., DUNNING,W. F. and GREER,S. (1972) Role and resistant to l-fl-o-arabinofuranosylcytosine. BiDof catabolism in pyrimidine utilization for nucleic acid chem. Pharmac. 19: 940-944. synthesis in vivo. Cancer Res. 32: 390-397. CORY, J. G. and CARTER,G. L. (1988) Leukemia L1210 cell DRAKE, J. C., STOLLER,R. G. and CHAnNER, B. A. (1977) Characteristics of the enzyme ufidine-cytidine kinase lines resistant to ribonucleotide reductase inhibitors. isolated from a cultured human cell line. Biochem. PharCancer Res. 48: 839-843. mac. 26: 64-66. CORY, J. G. and FLEISHER,A. E. (1979) Specific inhibitors directed at the individual components of ribonucleotide DRAKE, J. C., HANDE,K. R., FULLER,R. W. and CHABNER, B. A. (1980) Cytidine and deoxycytidylate deaminase reductase as an approach to combination chemotherapy. inhibition by uridine analogs. Biochem. Pharmac. 29: Cancer Res. 39: 4600-5604. 807-811. CREAS~Y,W. A. (1963) Studies on the metabolism of 5-iodoDUDLEY, K. H., BUTLER,T. C. and BIDS,D. L. (1974) The 2'-deoxycytidine in vitro. Purification of nucleoside deamrole of dihydropyrimidinase in the metabolism of some inase from mouse kidney. J. biol. Chem. 238: 1772-1776. hydantoin and succinimide drugs. Drug Metab. Dispos. 6: DANENBERG,P. V. and DANENBERG,K. D. (1978) Effects of 601~505. 5,10-methylenetetrahydrofolate on the dissociation of 5fluoro-2'-deoxyuridylate from thymidylate synthase: Evi- DUNN, W. C. and REGAN,J. D. (1979) Inhibition of DNA excision repair in human cells by arabinofuranosyl cydence for an ordered mechanism. Biochemistry 17: tosine: Effect on normal and xeroderma pigmentosa cells. 4018-4024. Molec. Pharmac. 15: 367-374. DANENBERG,P. V. and LOCKSH1N,A. (1981) Fluorinated pyrimidines as tight-binding inhibitors of thymidylate DUSCHINSKY,R., PLEVEN,E. and HEIDELBERGER,C. (1957) synthase. Pharmac. Ther. 13: 69-90. The synthesis of 5-fluoropyrimidines. J. Am. chem. Soc. 79: 4559-4560. DANENBERG,P. V., HEIDELBERGER,C., MULKINS,M. k. and PETERSON,A. R. (1981) The incorporation of 5-fluoro-2'- EL HIYANI,L., SAMPEREZ,S. and JOUAN,P. (1987) Inhibition deoxyuridine into DNA of mammalian tumor cells. by Celeptium~ of the fetal thymidine kinase synthesis induced by estrogens in the rat uterus. Chem.-Biol. Biomed. biophys. Res. Commun. 102: 654q558. DANENBERG,P. V,, SHEA,L. C. C. and DANENBERG,K. (1990) Interact. 62: 167-178. ELLIMS, P. H., KAO, A. Y. and CHABNER,B. A. (1981) Effect of 5-fluorouracil substitution on the self-splicing activity of tetrahymena ribosomal RNA. Cancer Res. 50: Deoxycytidylate deaminase: Purification and some properties of the enzyme isolated from human spleen. J. biol. 1757-1763. DANHAUSER,L. L. and RUSTUM,Y. M. (1979) A method for Chem. 256: 6335~5340. continuous drug infusion in unrestrained rats: Its appli- ELLIMS,P. H., KAO,A. Y. and CHABNER,B. A. (1983) Kinetic cation in evaluating the toxicity of 5-fluorouracil/ behaviour and allosteric regulation of human deoxythymidine combination. J. Lab. clin. Med. 93: 1047-1053. cytidylate deaminase derived from leukemic cells. Molec. DANHAUSER, L. L. and RUSTUM,Y. M. (1980) Effect of cell. Biochem. 57: 185-190. thymidine on the toxicity, antitumor activity and metabELLISON, R. R., HOLLAND,J. F., WELL,M., JACQUILLAT,C., olism of l-fl-o-arabinofuranosylcytosine in rats bearing a BOIRON,M., BERNARD,J., SAWITSKY,A., ROSNER,F., GUSchemically induced colon carcinoma. Cancer Res. 40: SOFF,B., SILVER,R. m., KARANAS,k., CUTTNER,J., SPURR, 1274-1280. C., HAYES, D. M., BLOM, J., LEONE, L. A., HAUVANI,F., DANHAUSER,L. L. and RUSTUM,Y. M. (1984) ChemotheraKYLE, R., HUTCmSON, J. U, FORClER, R. J. and MOON, peutic efficacy of 5-fluorouracil with concurrent J. H. (1968) Arabinosyl cytosine: a useful agent in the thymidine infusion against transplantable colon tumor in treatment of acute leukemia in adults. Blood 32: 507-523. rodents. Cancer Drug Delivery 1: 269-282. ENGSTRrM, Y. and ROZELL,B. (1988) Immunocytochemical DARNOWSKI, J. W. and HANDSCHUMACHER,R. E. (1985) evidence for cytoplasmic localization and differential exTissue-specific enhancement of uridine utilisation and pression during the cell cycle of M 1 and M2 subunits of 5-fluorouracil therapy in mice by benzylacyclouridine. mammalian ribonucleotide reductase. EMBO J. 7: Cancer Res. 44: 5364-5368. 1615-1620. DATTA, N. S., SHEWACH,D. S., HURLEY,M. C., MITCHELL, ENGSTROM, Y., ROZELL, B., HANSSON,H., STEMME,S. and B. S. and Fox, I. H. (1989) Human T-lymphoblast deoxyTHELANDER,L. (1984) Localization of ribonucleotide recytidine kinase: Purification and properties. Biochemistry ductase in mammalian cells. EMBO J. 3: 863-867. 28:114-123. ERIKSSON,S., GR.~SLUND,A., SKOG, S., THELANDER,L. and DESGRANGES,C., RAZAKA,G., RABAUD,M. and BRICAUD,H. TRIBUKAIT,B. (1984) Cell cycle-dependent regulation of (1981) Catabolism of thymidine in human blood platelets: mammalian ribonucleotide reductase. The S phase-correPurification and properties of thymidine phosphorylase. lated increase in subunit M2 is regulated by de novo Biochem. biophys. A cta 654:211-218. protein synthesis. J. biol. Chem. 259:11695-11700.
216
G.C. DAHER et al.
ERLICHMAN, C., FINE, S. and ELHAKIM,T. (1986) Plasma pharmacokinetics of 5-fluorouracil by continuous infusion with allopurinol. Cancer Treat. Rep. 70: 903-904. EVANS,J. S., MUSSER,E. A., MENGEL,G. D., FORSBLAD,K. R. and HUNTER,J. H. (1961) Antitumor activity of 1-fl-Darabinofuranosylcytosine hydrochloride. Proc. Soc. exp. Biol. Med. 106: 350-353. EVANS, J. S., MUSSER, E. A., BOSTWICK, L. and MENGEL, G. D. (1964) The effect of l-fl-D-arabinofuranosylcytosine hydrochloride on murine neoplasms. Cancer Res. 24: 1285-1293. FANUCCHI,M. P., WATANABE,K. A., Fox, J. J. and CHOU, T.-C. (1986) Kinetics and substrate specificity of human and canine cytidine deaminase. Biochem. Pharmac. 35: 1199-1201. FIALA, S., F1ALA,A., TOBAR,G. and McQUILLA, H. (1962) Deoxynucleotidase activity in rat liver and certain tumors. J. natn. Cancer Inst. 28: 1269-1289. FISHER, P. H. and BAXTER,D. (1981) Enzyme regulatory site-directed drugs. Modulation of thymidine triphosphate inhibition of thymidine kinase by 5'-amino-5'deoxythymidine. Molec. Pharmac. 22: 231-234. FISHER, P. H. and PHILLIPS,A. W. (1984) Antagonism of feedback inhibition. Stimulation of the phosphorylation of thymidine and 5-iodo-2'-deoxyuridine by 5-iodo-5'amino-2',5'-dideoxyuridine. Molec. Pharmac. 25: 446-451. FISHER, P. H., WEDDLE, M. A. and MOSSIE, R. D, (1983) Modulation of the metabolism and cytotoxicity of iododeoxyuridine by 5'-amino-5'-deoxythymidine. Molec. Pharmac. 23: 709-716. FISHER, P. H., FANG, T.-T., LIN, T.-S., HAMPTON, A. and BRUGGINK,J. (1988) Structure-activity analysis of antagonism of the feedback inhibition of thymidine kinase. Biochem. Pharmac. 37: 1293-1298. Fox, R. M., PIDOINGTON,S. K., TRIPP, E. H., DUNMAN,N. P. and TATTERSALL,M. H. N. (1979) Thymidine sensitivity of cultured leukaemic lymphocytes. Lancet 2: 391-393. FRAM, R. J. and KUFE, O. W. (1982) DNA strand breaks caused by inhibitors of DNA synthesis: l-fl-a-arabinofuranosylcytosine and aphidicolin. Cancer Res. 42: 4050-4053. FRIDLAND,A. and VERHOEF,V. (1987) Mechanism for araCTP catabolism in human leukemic cells and effect of deaminase inhibitors on this process. Sem. Oncol. 14 (Suppl. 1): 262-268. FRITZSON, P. (1960) Properties and assay of dihydrouracil dehydrogenase of rat liver. J. biol. Chem. 235: 719-725. FRITZSON,P. (1962) The relation between uracil-catabolizing enzymes and rate of rat liver regeneration. J. biol. Chem. 237: 150-156, FULCH1GNONI-LATAUD,M.-C., TUILIE,M. and Roux, J.-M. (1976) Uridine-cytidine kinase from foetal and adult rat liver: Purification and study of some properties. Fur. J. Biochem. 69: 217-222. FURTH, J. J. and COHEN, S. S, (1968) Inhibition of mammalian DNA polymerase by the 5'-triphosphate of 1-fl-n-arabinofuranosylcytosine and the 5'-triphosphate of 9-/~-o-arabinofuranosyladenine. Cancer Res. 28: 2061-2067. GALLO, R. C. and SEYMOUR,P. (1969a) The enzymatic mechanisms for deoxythymidine synthesis in human leukocytes. IV. Comparisons between normal and leukemic leukocytes. J. clin. Invest. 48: 105-116. GALLO, R. C. and SEYMOUR,P. (1969b) Enzyme abnormality in human leukaemia. Nature 218: 465-466. GARMONT, A. D., KRULIK, M., CADY, J., LAGADEC, B., MAISANI, J.-E., LOISEAU,J.-P., GRANGE,J.-D., GUSTAVO, G.-C., BENEDICTE,D., LOUVET,C., SEROKA,J., DRAY, C. and DEBRAY,J. (1988) High-dose folonic acid and 5fluorouracil bolus and continuous infusion in advanced colorectal cancer. Eur. J. din. Onco/. 9: 1499-1503.
GEORGE,C. B. and CORY,J. G. (1979) Activation of deoxycytidylate deaminase by 1-fl-o-arabinofuranosylcytosine5'-triphosphate. Biochem. Pharmac. 28: 1699-1701. GERACI,G., ROSSI,M. and SCARANO,E. (1967) Deoxycytidylate aminohydrolase. I. Preparation and properties of the homogeneous enzyme. Biochemistry 6: 183-191. GRAHAM, F. L. and WHITMORE,G. F. (1970a) The effect of 1-//-arabinofuranosylcytosine on growth, viability, and DNA synthesis of mouse L-cells. Cancer Res. 30: 2627-2635. GRAHAM,F. L. and WHITMORE,G. F. (1970b) Studies in the mouse L-cells on the incorporation of 1-fl-D-arabinofuranosylcytosine into DNA and on inhibition of DNA polymerase by 1-//-o-arabinofuranosylcytosine-5'-triphosphate. Cancer Res. 30: 2636-2644. GRAY, H. J. and SMELLIE,R. M. S. (1963) The mechanism of formation of thymidine 5'-triphosphate by enzymes from Landschutz ascites-tumor cells. Biochem. J. 89: 486-491, GREENBERG,N., SCHUMM,D. E. and WEBa, T. (1977) Uridine kinase activities and pyrimidine nucleoside phosphorylation in fluoropyrimidine-sensitive and resistant cell lines of the Novikoff hepatoma. Biochem. J. 164: 379-387. HALL, S. W., VALDIVIESO,M. and BENJAMIN,R. S. (1977) Intermittent high single dose ftorafur. A phase I clinical trial with pharmacologic-toxicity correlations. Cancer Treat. Rep. 61: 1495-1498. HANDE, K. R. and CHABNER,B. A. 0978) Pyrimidine nucleoside monophosphate kinase from human leukemic blast cells. Cancer Res. 38: 579-585. HANKA, L. J., EVANS,J. S., MASON, D. J. and DIETZ, A. (1966) Microbiological production of 5-azacytidine. 1. Production and biological activity. Antimicrob. Ag. Chemother. 619-624. HARDY, L. W., FINER-MOORE, J. S., MONTFORT, W. R., JONES, M. O., SANTI, D. V. and STROUD,R. M. (1987) Atomic structure of thymidylate synthase: Target for rational drug design. Science 235: 448-455. HARRIS, A. L., GRAHAME-SMITH,D. G., POTTER, C. G. and BUNCH, C. (1981) Cytosine arabinoside deamination in human leukaemic myeloblasts and resistance to cytosine arabinoside therapy. Clin. Sci. 60: 191-198. HARRIS, B. E., SONG, R., HE, Y.-J., SOONG,S.-J. and DIASIO, R. B. (1988) Circadian rhythm of rat liver dihydropyrimidine dehydrogenase. Possible relevance to fluoropyrimidine chemotherapy. Biochem. Pharmac. 37: 4759-4762. HARRIS, B. E., SONG, R., SOONG, S.-J. and DIAS10, R. B. (1990) Relationship between dihydropyrimidine dehydrogenase activity and plasma 5-fluorouracil levels: Evidence for circadian variation of plasma drug levels in cancer patients receiving 5-fluorouracil by protracted continuous infusion. Cancer Res. 50: 197-201. HART, J. S., HO, D. H., GEORGE,S. L., SALEM,P., GOTTLIEB, J. A. and FREI, E. I. (1972) Cytokinetic and molecular pharmacology studies of arabinosylcytosine in metastatic melanoma. Cancer Res. 22: 2763-2765. HARTMANN,K. V. and HEIDELBERGER,C. (1961) Studies on fluorinated pyrimidines XIII. Inhibition of thymidylate synthetase. J. biol. Chem. 236: 3006-3013. HAWTREY,A. O., SCOTT-BURDEN,T., JONES,P. and ROBERTSON, G. (1974a) Inhibition by l-fl-D-arabinofuranosylcytosine of the incorporation of N-acetylneuraminic acid into glycolipids and glycoproteins of hamster embryo cells. Biochem. biophys. Res. Commun. 54: 1282-1287. HAWTP~Y, A. O., SCOTT-BUROEN,T. and ROBERTSON,G. (1974b) Inhibition of glycoprotein and glycolipid synthesis in hamster embryo cells by cytosine arabinoside and hydroxyurea. Nature 252: 58-60. HEGGIE, G. D., SAMMADOSSI,J. P., CROSS, D. S., HUSTER, W. J. and DIASlO,R. B. (1987) Clinical pharmacokinetics of 5-fluorouracil and its metabolites in plasma, urine, and bile. Cancer Res. 47: 2203-2206. HEICHAL,O., IsH-SHALOM,D., KOREN, R. and STEIN,W. D. (1979) The kinetic dissection of transport from metabolic
Pyrimidine analogues and their nucleosides trapping during substrate uptake by intact cells. Uridine uptake by quiescent and serum-activated Nil 8 hamster cells and their murine sarcoma virus-transformed counterparts. Biochim. biophys. Acta 551: 169-186. HEIDELBERGER, C. (1975) Fluorinated pyrimidines and their nucleosides. In: Antineoplastic and Immunosuppressive Agents Part 11, pp. 193-231, SARTORELLi,A. C. and JOHNS, D. G. (eds) Springer-Verlag, New York. HEIDELBERGER, C., DANENBERG, P. V. and MORAN, R. G. (1983) Fluorinated pyrimidines and their nucleosides. Adv. Enzymol. 54:57-119. HERRICK, n . J., MAJOR, P. P. and KUFE, D. W. (1982) Effect of methotrexate on incorporation and excision of 5fluorouracil residues in human breast carcinoma DNA. Cancer Res. 42: 5015-5017. Hiss, E. A. and PRESTON, R. J. (1978) The effect of cytosine arabinoside on the presence of single strand breaks in DNA of mammalian cells following irradiation or chemical treatment. Biochem. biophys. Acta 478: 1-8. Ho, D. H. and FREI, E. I. (1971) Clinical pharmacology of l-fl-D-arabinofuranosyl cytosine. Clin. Pharmac. Ther. 12: 944-954. Ho, D. H. W. and FREIREICH, E. J. (1975) Clinical pharmacology of arabinosylcytosine. In: Handbook of Experimental Pharmacology: Antineoplastic and lmmunosuppressive Agents (Part H), pp. 232-256, SARTORELLI,A. C. and JOHNS, D. G. (eds) Springer-Verlag, New York. Ho, D. H. W., CARTER, C. J. and Loo, T. L. (1975) Effects of tetrahydrouridine (THU) on the uptake and metabolism of arabinosylcytosine by human acute myelogenous leukemia (AML) cells. Proc. Am. Ass. Cancer Res. 16: 57. HOUGHTON, J. A. and HOUGHTON,P. J. (1983) Elucidation of pathways of 5-fluorouracil metabolism in xenografts of human coiorectal adenocarcinoma. Fur. J. Cancer. clin. Oncol. 19:807-815. HOUGHTON, J. A., MARODA, S. J., PHILIPS, J. D. and HOUGHTON, P. J. (1981) Biochemical determinants of responsiveness to 5-fluorouracil and its derivatives in xenografts of human colorectal adenocarcinomas in mice. Cancer Res. 41: 144-149. IIGO, M., ARAKI, E., NAKAJ1MA, Y., HOSHI, A. and DE CLERCQ, E. (1988) Enhancing effect of bromovinyldeoxyuridine on antitumor activity of 5-fluorouracil against adenocarcinoma 755 in mice: Increased therapeutic index and correlation with increased plasma 5fluorouracil level. Biochem. Pharmac. 37: 1609-1613. ILTZSCH, M. H., EL KOUNL M. H. and CHA, S. (1985) Kinetic studies of thymidine phosphorylase from mouse liver. Biochemistry 24: 6799-6807. IMAZAWA,M. and ECKSTEIN, F. (1979) Synthesis of sugarmodified nucleoside 5'-triphosphates with partially purified nucleotide kinases from calf thymus. Biochim. biophys. Acta 570: 284-290. IMONDL A. R., BALLS,M. E. and LIPKIN, M. (1969) Changes in enzyme levels accompanying differentiation of intestinal epithelial cells. Exp Cell Res. 58: 323-330. INAGATI, A., NAKAMERA, T. and WAKINSOKA,G. (1970) Studies on the mechanism of action of 1-beta-o-arabinofuranosylcytosine as an inhibitor of DNA synthesis in human leukemic leukocytes. Cancer Res. 29: 2169-2176. INGRAHAM, H. A., TSENG, B. Y. and GOULIAN, M. (1980) Mechanism for exclusion of 5-fluorouracil from DNA. Cancer Res. 40: 998-1001. INGRAHAM, H. A., TSENG, B. Y. and GOULIAN, M. (1982) Nucleotide levels and incorporation of 5-fluorouracil and uracil into DNA of cells treated with 5-fluorodeoxyuridine. Molec. Pharmac. 21: 211-216.
ISRAILI,Z. H., VOGLER,W. R., MINGIOLI,E. S., PIRKLE,J. L., SMITHWICK, R. W. and GOLDSTEIN, J. H. (1976) The disposition of pharmacokinetics in humans of 5-azacytidine administered intravenously as a bolus or by continuous infusion. Cancer Res. 36: 1453-1461.
217
IvEs, D. H. (1965) Evidence for thymidine diphosphate as the precursor of thymidine triphosphate in tumor. J. biol. Chem. 240: 819-824. IRES, D. H., MORSE, P. A. J. and PORTER, V. R. (1963) Feedback inhibition of thymidine kinase by thymidine triphosphate. J. biol. Chem. 238: 1467-1474. JAcQtmZ, J. A. (1962) Permeability of Ehrlich cells to uracil, thymine, and fluorouracil. Proc. Soc. exp. Biol. Med. 109: 132-135. JOHNSON, R. K., SWYRYD, E. A. and STARK, G. R. (1978) Effects of N-(phosphonacetlyl)-L-aspartate on murine tumors and normal tissues in vivo and in vitro and the relationship of sensitivity to rate of proliferation and level of aspartate transcarbamylase. Cancer Res. 38: 371-378. KANAMARU,R. and WAKUI,A. (1988) Mechanism of action of anti-cancer drugs from the viewpoint of RNA metabolism. Jap. J. Cancer Chemother. 15:1011-1018. KANAMARO, R., KAKUTA,H., SATO, T., ISmOKA, C. and WAKUI,A. (1986) The inhibitory effects of 5-fluorouracil on the metabolism of preribosomal and ribosomal RNA in L-1210 cells in vitro. Cancer Chemother. Pharmac. 17: 43-46. KARON, M. and BENEDICT,W. (1972) Chromatid breakage: differential effect of inhibitors of DNA synthesis during G 2 phase. Science 178: 62.
KARON, i . , SIEGER,L., LEIMBROCK,S., FINKLESTEIN,J. Z., NESmT, M. E. and SWANEY,J. J. (1973) 5-Azacytidine: a new active agent for the treatment of acute leukemia. Blood 42: 359-365. KAWAI,i . , ROSENFELD,J., MCCULLOCH,P. and HILLCOAT, B. L. (1976) Blood levels of 5-fluorouracil during intravenous infusion. Br. J. Cancer 36: 346-347. KAYSEN, J., SPRIGGS,D. and KUFE, D. (1986) Incorporation of 5-fluorodeoxycytidine and metabolites into nucleic acids of human MCF-7 breast carcinoma cells. Cancer Res. 46: 4534-4538. KEE~R, R. C., MCNAMARA, D. J. and WEaa, T. E. (1975) The separation of two forms of uridine kinase from the Novikoff hepatoma. Cancer Biochem. Biophys. 1: 107-110. KESSEL, D. (1980) Cell surface alterations associated with exposure of leukemia LI 210 cells to fluorouracil. Cancer Res. 40: 322-324. KESSEL, D. and HALL, T. C. (1967) Studies on drug transport by normal human leukocytes. Biochem. Pharmac. 16: 2395-2403. KESSEL, D. and SHUmN, S. B. (1968) Transport of two non-metabolized nucleosides, deoxycytidine and cytosine arabinoside, in a sub-line of the L1210 murine leukemia. Biochim. biophys. Acta 163: 179-187. KESSEL, D., DEACON, J., COFFEY, B. and BAKAMAJIAN,A. (1972) Some properties of a pyrimidine phosphoribosyltransferase from murine leukemia cells. Molec. Pharmac. g: 731-739. KEYOMARSI, K. and MORAN, R. G. (1988) Mechanism of cytotoxic synergism of fluoropyrimidines and folinic acid in mouse leukemia cells. J. biol. Chem. 263: 14402-14409.
KIKUGAWA,M., FUJIMOTO,S., MIZOTA,C. and TAMAKI,N. (1988) Bisfunction of proprionic acid on purified rat liver fl-ureidopropionase. FEBS Lett. 229: 345-348. KIM, B. D., KEENAN, S., BODNAR, J. K. and SANDER,E. G. (1976) Role of enzymatically catalyzed 5-iodo-5,6-dihydrouracil ring hydrolysis on the dehalogenation of 5iodouracil. J. biol. Chem. 251: 6909-6914. KIMBALL,A. P. and WILSON, M. J. (1968) Inhibition of DNA polymerase by fl-D-arabinosylcytosine and reversal of inhibition of deoxycytidine-5'-triphosphate. Proc. Soc. exp. Biol. Med. 127: 429432. KINAHAN, J. J., OTTEN, M. and GRINDEY, G. B. (1979) Evaluation of ribonucleoside and deoxyribonucleoside triphosphate pools in cultured leukemia cells during exposure to methotrexate or methotrexate plus thymidine. Cancer Res. 39: 3531-3539.
218
G.C. DAI-'IERet al.
KIT, S. (1976) Thymidine kinase, DNA synthesis and cancer. Molec. cell. Biochem. 11: 161-182. KIT, S. and LEUNO,W.-C. (1974) Submitochondrial localization and characteristics of thymidine kinase molecular forms in parental and kinase-deficient HeLa cells. BiDchem. Genetics 11: 231-347. KLOHS, W. D., BERNACKI,R. J. and KORYTNYK,W. (1979) Effects of nucleotides and nucleotide-analogs on human serum sialytransferase. Cancer Res. 39: 1231-1238. KONO, A., HARA, Y., SUGATA,S., KAUBE,Y., MATSUSHIMA, Y. and ISH1TSUKA,H. (1983) Activation of 5'-deoxy-5fluorouridine by thymidine phosphorylase in human tumors. Chem. pharm. Bull. 31: 175-178. KRENITSKY,T. A. (1968) Pentosyl transfer mechanisms of the mammalian nucleoside phosphorylases. J. biol. Chem. 243: 2871-2875. KRYSTAL, G. and SCHOLEEmLD,P. G. (1973) The partial purification and properties of uridine kinases from Ehrlich ascites tumor cells. Can. J. Biochem. 51: 379-389. KUmLUS,J., LEE, L. D. and BADEN,H. P. (1978) Purification of thymidine phosphorylase from human amniochorion. Biochem. biophys. Acta 527: 221-228. KUEE, D. W. and MAJOR, P. P. (1982) Studies on the mechanism of action of cytosine arabinoside. Med. Pedlar. Oncol. 1: 49-67. KUFE, D. W., BEARDSLEY, P., KARP, D., PARKER, L., ROSOWSKY, A., CANELLOS,G. and FP,EI, E., III (1980a) High-dose thymidine infusions in patients with leukemia and lymphoma. Blood 55: 580°589. KUFE, D. W., MAJOR, P. P., EGAN, E. M. and BEARDSLEY, G. P. (1980b) Correlation of cytotoxicity with incorporation of araC into DNA. J. biol. Chem. 255: 8897-9000. KUFE, D. W., MAJOR,P. P., EGAN,E. M. and Lo,, E. (1981) 5-Fluoro-2'-deoxyuridine. Incorporation in L1012 DNA. J. biol. Chem. 256: 8885-8888. KUFE, D. W., SCOTT, P., FRAM, R. and MAJOR, P. (1983) Biological effect of 5-fluoro-2'-deoxyuridine incorporation in L1210 deoxyribonucleic acid. Biochem. Pharmac. 32: 1337-1340. LAUFMAN,L. R., KRZECZOWSKI,R. R. and SEGAL,M. (1987) Leucovorin plus 5-fluorouracil: An effective treatment for metastatic colon cancer. J. clin. Oncol. 5: 1394-1400. LAUZON,(3. J., PARAN,J. H. and PATERSON,A. R. P. (1978a) Formation of l-fl-n-arabinofuranosylcytosine diphosphate choline in cultured human leukemic RPMI 6410 cells. Cancer Res. 38: 1723-1729. LAUZON,(3. J., PATERSON,A. R. P. and BELCH,A. W. (1978b) Formation of 1-fl-n-arabinofuranosylcytosine diphosphate choline in neoplastic and normal cells. Cancer Res. 38:1730-1733. LEE, M. H., COWLING,R. A., SANDER,E. G. and PETTIGREW, D. W. (1986) Bovine liver dihydropyrimidine amidohydrolase: pH dependencies of inactivation by chelators and steady state kinetic properties. Archs Biochem. Biophys. 248: 368-378. LEE, M. H., PETTIGREW,D. W., SANDER,E. G. and NOWAK, T. (1987) Bovine liver dihydropyrimidine amidohydrolase: pH dependencies of the steady-state kinetic and proton relaxation rate properties of the Mn(lI)-containing enzyme. Archs Biochem. Biophys. 259: 597-604. LEE, T., KARON,M. and MOMPARLER,R. L. (1974) Kinetic studies on phosphorylation of 5-azacytidine with the purified uridine-cytidine kinase from calf thymus. Cancer Res. 34: 2481-2488. LEVINSON, B. S., ULLMAN, B. and MARTIN, D. W. (1979) Pyrimidine pathway variants of cultured mouse lymphoma cells with altered levels of both orotate phosphoribosyltransferase and orotidylate decarboxylase. J. biol. Chem. 254: 4396-4401. LI, L. H., OLIN, E. J., FRASER,T. J. and BHUYAN,B. K. (1970) Phase specificity of 5-azacytidine against mammalian cells in tissue culture. Cancer Res. 30: 277002775.
LLOYD, H. H., DALMADGE,E. A. and WIKOFF,L. J. (1972) Kinetics of the reduction in viability of cultured L1210 leukemic cells exposed to 5-azacytidine (NSC-102816). Cancer Chemother. Rep. 56: 585-591. LOCKSmN, A. and DANENBERG,P. V. (1981) Biochemical factors affecting the tightness of 5-fluorodeoxyuridylatc binding to human thymidylate synthetase. Biochem. Pharmac. 30: 247-257. LOZERON, H. A., GORDON, M. P., GABRIEL,T., TAUTZ, W. and DUSCHINSKY,R. (1964) The photochemistry of 5fluorouracil. Biochemistry 3: 1844-1850. MAEHERA,Y., NAGAYAMA,S., OKAZAKI,H., NAKAMURA,H., SHIRAZAKA, T. and FUJI, S. (1981) Metabolism of 5fluorouracil in various human normal and tumor tissues, Gann 72: 824-827. MAGUIRE, J. H. and DUDLEY,K. H. (1978) Partial purification and characterization of dihydropyrimidinases from calf and rat liver. Drug Metab. Dispos. 6: 601--605. MAJOR, P., EGAN,E. M., BEARDSLEY,G. P., MINDEN,M. D. and KUFE, D. W. (1981) Lethality of human myeloblasts correlates with the incorporation of araC into DNA. Proc. natn. Acad. Sci. U.S.A. 78: 3235-3239. MALATHI, V. G. and SILBER, R. (1971) Effects of viral leukemia on spleen nucleoside deaminase: Purification and properties of the enzyme from leukemic spleen. Biochem. biophys. Acta 238: 377-387. MALEY, G. F. and MALEY, F. (1964) The purification and properties of deoxycytidylate deaminase from chick embryo extracts. J. biol. Chem. 239: I168-I 176. MALEY,G. F. and MALEY,F. (1968) Regulatory properties and subunit structure of chick embryo deoxycytidylate deaminase. J. biol. Chem. 243: 4506-4512. MANCINI, W. R. and CrtENG, Y.-C. (1983) Human deoxycytidylate deaminase: Substrate and regulator specificities and their chemotherapeutic implications. Molec. Pharmac. 23: 159-164. MANE.KS,P. and ORENGO,A. (1975) A pyrimidine nucleoside monophosphate kinase from rat liver. Biochemistry 14: 1484-1489. MANESS, P. and ORENGO, A. (1976) Differences between pyrimidine nucleoside monophosphate kinase from rat Novikoff ascites hepatoma and rat liver. Cancer Res. 36: 2312-2316. MANN, G. J., DYNE, M. and MUSGROVE,E. A. (1987) Immunofluorescent quantification of ribonucleotide reductase Ml subunit and correlation with DNA content by flow cytometry. Cytometry 8: 509-517. MANN, G. J., MUSGROVE,E. A., FOX, R. M. and THELANDER, L. (1988) Ribonucleotide reductase M1 subunit in cellular proliferation, quiescence, and differentiation. Cancer Res. 48: 5151-5156. MARSH,J. C. and PERRY,S. (1964) Thymidine catabolism by normal and leukemic human leukocytes. J. clin. Invest. 43: 267-278. MARZ, R., WOHLHUETER,R. M. and PLAGEMANN,P. G. W. (1977) Relationship between thymidine transport and phosphorylation in Novikoff rat hepatoma cells as analyzed by a rapid sampling method. J. supramolec. Struct. 6: 433-440. MASAAK1,I., ARAKI, E., NAKAJIMA,Y. and DE CLERCQ, E. (1988) Enhancing effects of bromovinyldeoxyuridine on antitumor activity of 5-fluorouracil against adenocarcinoma 755 in mice. Increased therapeutic index and correlation with increased plasma 5-fluorouracil levels. Biochem. Pharmac. 37: 1609-1613. MASUI, H. and GARREN,L. D. (1971) On the mechanism of action of adrenocorticotropic hormone. The stimulation of thymidine kinase activity with altered properties and changed subcellular distribution. J. biol. Chem. 246: 5407-5413. MATTHES, E., BARWOLFF,D. and LANGEN,P. (1974) Inhibition by 6-aminothymine of the degradation of nucleosides (5-iododeoxyuridine, thymidine) and pyrimidine
Pyrimidine analogues and their nucleosides bases (5-iodouracil, uracil and 5-fluorouracil) in vivo. Acta biol. med. germ. 32: 483-502. MATTHEWS, M. M. and TRAUT, T. W. (1987) Regulation of N-carbamoyl-fl-alanine amidohydrolase, the terminal enzyme in pyrimidine catabolism, by ligand-induced change in polymerization. J. biol. Chem. 262: 72327237. MCCLARTY, G. A., CHAN, A. K., ENGSTROM,Y., WRIGHT, J. A. and THELANDER,L. (1987) Elevated expression and M1 and M2 components and drug-induced posttranscriptional modulation of ribonucleotide reductase in a hydroxyurea-resistant mouse cell line. Biochemistry 26: 8004-8011. McCREDIE, K. B., BODEY, G. P., BURGESS, M. A., GUTTERMAN,J. U., RODRIGUEZ,V., SULLIVAN,M. P. and FREIREICH,E. J. (1973) Treatment of acute leukemia with 5-azacytidine (NSC-102816). Cancer Chemother. Rep. 57: 319-323. MENTRE, F., STEIMER,J.-L., SOMMADOSSI,J.-P., DIASIO,R. B. and CANO, J. P. (1984) A mathematical model of the kinetics of 5-fluorouracil and its catabolites in freshly isolated rat hepatocytes. Biochem. Pharrnac. 33: 2727-2732. MILLS-YAMAMOTO,C., LAUZON,G. J. and PATERSON,A. R. P. (1978) Toxicity of combinations of arabinosylcytosine and 3-deazuridine toward neoplastic cells in culture. Biochem. Pharmac. 27: 181-186. MOLLGAARD,H. and NEUHARD,J. (1978) Deoxycytidylate deaminase from Bacillus subtilis. J. biol. Chem. 253: 3536-3542. MOMPARLER, R. (1972) Kinetic and template studies with l-fl-o-arabinofuranosylcytosine 5'-triphosphate and mammalian deoxyribonucleic acid polymerase. Molec. Pharmac. 8: 362-370. MOMPARLER,R. L., CHU, M. Y. and FISCHER,G. A. (1968) Studies on a new mechanism of resistance of L5178Y murine leukemia cells to cytosine arabinoside. Biochem. biophys. Acta 161: 481-493. MONOD, J., CHANGEAUX,J. and JACOB,J. (1963) Allosteric proteins and cellular control systems. J. molec. Biol. 6" 30(~329. MORAN, R. G. (1988) The biochemistry of leucovorin potentiation of 5-FU. Advances in Chemotherapy: Status of 5-fluorouracil / leucovorin in advanced malignancies, pp. 8-9, American Cancer Society, Cleveland, OH. MOURAD, N. and PARKS, R. E. J. (1966) Erythrocytic nucleoside diphosphokinase II. Isolation and kinetics. J. biol. Chem. 241: 271-278. MOVER,J. D. and HANDSCHUMACHER,R. E. (1979) Selective inhibition of pyrimidine synthesis and depletion of nucleotide pools by N-(phosphonacetyl)-L-aspartate. Cancer Res. 39: 3089-3094. MULDER, J. H. and HARRAP, K. R. (1975) Cytosine arabinoside uptake by tumor cells in vitro. Fur. J. Cancer ll: 373-379. MULKINS,M. A. and HEIDELBERGER,C. (1982) Biochemical characterization of fluoropyrimidine-resistant murine leukemic cell lines. Cancer Res. 42: 965-973. MULLER, W. E. G. (1977) Rational design of arabinosyl nucleosides as antitumor and antiviral agents. J. Antibiot., Tokyo 30 (Suppl.): 104-120. MYERS-ROBFOGEL,M. W. and SPATARO,A. C. (1980) 1-fl-Darabinofuranosylcytosine nucleotide inhibition of sialic acid metabolism in WI-38 cells. Cancer Res. 40: 1940-1943. NAGUIB, F. N. M., EL KOUNI, M. H. and CHA, S. (1985) Enzymes of uracil catabolism in normal and neoplastic human tissues. Cancer Res. 45: 5405-5412. NAGUIB, F. N. M., NIEDZWICKI, J. G., ILTZSCH, M. H., WmMANN,M. C., EL KOUNI,M. and SUNGMAN,C. (1987a) Effects of N,N-dimethylformamide and sodium butyrate on enzymes of pyrimidine metabolism in cultured human tumor cells. Leukemia Res. 11: 855-861.
219
NAGUIB,F. N. M., EL KOUNI,M. H., CHU, S. H. and CHA, S. (1987b) New analogues of benzylacylcycloufidines, specific and potent inhibitors of uridine phosphorylase from human and mouse livers. Biochem. Pharmac. 36: 2195-2201. NAGUIB, F. N. M., EL KOUNI, M. H. and CHA, S. (1989) Structure-activity relationship of ligands of dihydrouracil dehydrogenase from mouse liver. Biochem. Pharmac. 38: 1471-1480. NAKAMURA,H. and SUGINO,Y. (1966) Metabolism of deoxyribonucleotides. II. Enzymatic phosphorylation of deoxycytidylic acid in normal rat liver and rat ascites hepatoma cells. Cancer Res. 26 (Part 1): 1425-1429. NEIL, G. L., MOXLEY,T. E., KUENTZEL,S. L., MANAK,R. C. and HANKA, L. J. (1975) Enhancement by tetrahydrouridine (NSC-I 12907) of the oral activity of 5-azacytidine (NSC-102816) in L1210 leukemic mice. Cancer Chemother. Rep. 59: 459-465. NmDZWICKI,J. G., EL KOUNI,M. H., CHU, S. H. and C,A, S. (1983) Structure-activity relationship of ligands of the pyrimidines nucleoside phosphorylases. Biochem. Pharmac. 32: 399-415. NIEDZWICKi, J. G., ILTZSCH, M. H., EL KOUNI, M. H. and CHA, S. (1984) Structure-activity relationship of pyrimidine base analogs as ligands of orotate phosphoribosyltransferase. Biochem. Pharmac. 33: 2383-2395. NOTARI, R. E. and DEYOUNG, J. L. (1975) Kinetics and mechanisms of degradation of the antileukemic agent 5-azacytidine in aqueous solutions. J. pharm. Sci. 64: 1148-I157. O'DONOVAN,G. A. and NEUaARD,J. (1970) Pyrimidine metabolism in microorganisms. Bacteriol. Rev. 34: 278-343. PALU, G. (1980) Thymidine phosphorylase: A possible marker of cell maturation in acute myeloid leukaemia. Tumori 66: 35-42. PAYNE,R. C. and TRAUT,T. W. (1982) Regulation of uridine kinase quaternary structure. J. biol. Chem. 257: 12485-12488. PAYNE, R. C., CHENG,N. and TRAUT,T. W. (1985) Uridine kinase from Ehrlich ascites carcinoma: Purification and properties of homogeneous enzyme. J. biol. Chem. 260: 10242-10247. PETERS,G. J., LAURENSSE,E., LANKELMA,J., LEYVA,A. and PINEOO,H. M. (1984) Separation of several 5-fluorouracil metabolites in various melanoma cell lines. Evidence for the synthesis of 5-fluorouracil-nucleotide sugars. Eur. J. Cancer clin. Oncol. 20: 1425-1431. PETIT, E., MILANO,G., LEVI,F., THYSS,A., BA1LLEUL,F. and SCHNEIDER,M. (1988) Circadian rhythm-varying plasma concentration of 5-fluorouracil during a five day continuous venous infusion at a constant rate in cancer patients. Cancer Res. 48: 1676-1679. PETRELLI, N., HERR.ERA, L., RUSTUM, Y., BURKE, P., CREAVEN,P., STULC,J., EMRICH,L, J. and MITTELMAN,A. (1987) A prospective randomized trial of 5-fluorouracil versus 5-fluorouracil and high-dose leucovorin versus 5-fluorouracil and methotrexate in previously untreated patients with advanced colorectal carcinoma. J. clin. Oncol. 5: 1559-1565. PLAGEMANN, P. G. W., BEHRENS, M. and ABRAHAM,D. (1978a) Metabolism and cytotoxicity of 5-azacytidine in cultured Novikoff rat hepatoma and P388 mouse leukemia cells and their enhancement by preincubation with pyrazofurin. Cancer Res. 38: 2458-2466. PLAGEMANN,P. G. W., MARZ, R. and WOHLHUETER,R. M. (1978b) Transport and metabolism of deoxycytidine and I-~-D-arabinofuranosylcytosine into cultured Novikoff rat hepatoma cells, relationship to phosphorylation, and regulation of triphosphate synthesis. Cancer Res. 38: 978-989. PLIML, J. and SORM,F. (1964) Synthesis of 2'-deoxy-D-furanosyl-5-azacytosine. Colin Czech. chem. Commun. 29: 2576-2578.
220
G.C. DArtER et al.
PRE1SLER, H. D., RUSTUM, Y. and PRIORE, R. L. (1985) Relationship between leukemic cell retention of cytosine arabinoside triphosphate and the duration of remission in patients with acute non-lymphocytic leukemia. Eur. J. Cancer din. Oncol. 21: 23-30. QUEENER, S. F., MORIS, H. P. and WEBER, G. (1971) Dihydrouracil dehydrogenase activity in normal, differentiating, and regenerating liver and in hepatomas. Cancer Res. 31: 1004-1009. RABINOW1TZ,Y. and WILHITE,B. A. (1969) Thymidine salvage pathway in normal and leukemic leukocytes with effects of ATP on enzyme control. Blood 33: 759-771. REYES, P. (1969) The synthesis of 5-fluorouridine 5'-phosphate by a pyrimidine phosphoribosyltransferase of mammalian origin. I. Some properties of the enzyme from P1534J mouse leukemic cells. Biochemistry g: 2057-2062. REYES, P. and GU~ANIG, M. E. (1975) Studies on a pyrimidine phosphoribosyltransferase from murine leukemia PI534J. J. biol. Chem. 250: 5097-5108. RICHARD, P., SKOLD, O., KLIEN, G., R&v~sz, U and MAGNUSSON, P.-H. (1962) Studies on resistance against 5fluorouracil I. Enzymes of the uracil pathway during development of resistance. Cancer Res. 22: 235-243. Ross, D. D., THOMPSON, B. W., JONECKIS, C. C., AKMAN, S. A. and SCmFFER, C. A. (1987) Studies of araC metabolism in acute leukemia. Sem. Oncol. 14 (Suppl. 1): 182-191. RUSTUM, Y. M. and PREISLER, H. D. (1979) Correlation between leukemic cell retention of 1-fl-D-arabinofuranosylcytosine 5'-triphosphate and response to therapy. Cancer Res. 39: 42~,9. SANNO, Y., HOLZER, M. and SCHIMKE, R. T. (1970) Studies of a mutation affecting pyrimidine degradation in inbred mice. J. biol. Chem. 245: 5668-5676. SANTI, D. V. and DANENBERG,P. V. (1984) Folates in pyrimidine nucleotide biosynthesis. In: Folates and Pterins, pp. 345-398, BLAKLEY, R. L. and BENKOVIC, S. J. (eds) John Wiley & Sons, New York. SANTI, D. V. and HARDY, L. W. (1987) Catalytic mechanism and inhibition of tRNA (uracil-5-)methyltransferase: Evidence for covalent catalysis. Biochemistry 26: 85998606. SAWYER,R. C., STOLFI,R. L., MARTIN, D. S. and SPIEGELMAN, S. (1984) Incorporation of 5-fluorouracil into murine bone marrow DNA in vivo. Cancer Res. 44: 1847 1851. SCHOLAR,E. and CALABRESI,P. (1973) Identification of the enzymatic pathways of nucleoside metabolism in human lymphocytes and leukemic ceils. Cancer Res. 33: 94-103. SCHRECKER, A. W. and URSHEL, M. J. (1968) Metabolism of 1-fl-D-arabinofuranosylcytosine nucleoside inhibition of sialic acid metabolism of WI-38 cells. Cancer Res. 28: 793-810. SCHUETZ, J. D. and DIASIO, R. B. (1985) The effect of 5-fluorouracil on DNA chain elongation in intact bone marrow ceils. Biochem. biophys. Res. Commun. 133: 361-367. SCHUETZ, J. D., COLLINS, J. M., WALLACE, H..l. and DIASlO, R. B. (1986) Alteration of the secondary structure of newly synthesized DNA from murine bone marrow cells by 5-fluorouracil. Cancer Res. 46:119 123. SCHUETZ, J. D., WALLACE,H. J. and D1ASlO, R. B. (1988) DNA repair following incorporation of 5-fluorouracil into DNA of mouse bone marrow ceils. Cancer Chemother. Pharmac. 21: 208-210. SCHWARTZ, P. M., MOIR, R. D., HYDE, C. M., TUREK, P. J. and HANDSCHUMACHER, R. E. (1985) Role of uridine phosphorylase in the anabolism of 5-fluorouracil. Biochem. Pharmac. 34: 3585-3589. SERGOTT, R. C., DEBEER, L. J. and BESSMAN,M. J. (1971) On the regulation of bacterial deoxycytidylate deaminase. J. biol. Chem. 246: 7755-7758.
SHIOTANI,T. and WEBER,G. (1981) Purification and properties of dihydrothymine dehydrogenase from rat liver. J. biol. Chem. 256: 219-224.
SHOUVAL, D., ADLER, R., WANDS, J. R. and HURWITZ, E. (1986) Conjugates between monoclonal antibodies to HBsAg and cytosine arabinoside. J. Hepatol. 3 (Suppl. 2): $87-95. Snow, T. B., ALPER, C. A., BOOTSMA, D., DORF, M., DOUGLAS,T., HUISMAN, T., KIT, S., KLINGER, H. P., KOZAK, P. A., LALLEY, P. A., LINDLEY, D., McALPINE, P. J., McDOUGALL, J. K., MEERAKHAN, P., MEISLER, M., MORTON, N. E., OPlTZ, C. W., PARTRIDGE,C. W., PAYNE, R., RODERICK, T. H., RUBINSTEIN, P., RUDDLE, F. H., SHAW, M., SPRANGER, J. W. and WEISS, K. (1979) International system for human gene nomenclature. Cytogenet. Cell Genet. 25:96-116. SKrLD, O. (1958) Enzymic ribosidation and ribotidation of 5-fluorouracil by extracts of the Ehrlich-ascites tumor. Biochim. biophys. Acta 29: 651. SKOLD, O. (1963) Studies on resistance against 5-fluorouracil IV. Evidence for an altered uridine kinase in resistant cells. Biochim. biophys. Acta 76: 160-162. SLECHTA, L. (1961) Effect of arabinonucleotides on growth and metabolism of E. coll. Fedn Proc. 20: 357. SMITH, C. G., LUMMIS,W. L. and GRADY, J. E. (1959) An improved tissue culture assay. II. Cytotoxicity studies with antibiotics, chemicals and solvents. Cancer Res. 19: 847-852. SMYTH, J. F., ROmNS, A. B. and LEESE, L. L. (1976) The metabolism of cytosine arabinoside as a predictive test for clinical response to the drug in acute myeloid leukaemia. Eur. J. Cancer 12: 567-575. SOCCA, J. J., PANNY, S. A. and BESSMAN,M. J. (1969) Studies of deoxycytidylate deaminase from T4-infected Escherichia coli. J. biol. Chem. 244: 3698-3706. SOMMADOSSI,J.-P., GEWIRTZ, D. A., DIASIO, R. B., AUaERT, C., CANO, J. P. and GOLDMAN, I. D. (1982) Rapid catabolism of 5-fluorouracil in freshly isolated hepatocytes as analyzed by high performance liquid chromatography. J. biol. Chem. 257: 8171-8176. SORM, F., PISKALA, A., CIHAK, A. and VESELY, J. (1964) 5-Azacytidine, a new highly effective cancerostatic. Experientia 20: 202-203. SPIEGELMAN, S., NAYAK, R., SAWYER, R., STOLFI, R. and MARTIN, D. (1980a) Potentiation of the anti-tumor activity of 5-FU by thymidine and its correlation with the formation of (5FU) RNA. Cancer 45:1129-1134. SPIEGELMAN, S., SAWYER, R., NAYAK, R., RITZ1, E., STOLFI, R. and MARTIN, D. (1980b) Improving the anti-tumor activity of 5-fluorouracil by increasing its incorporation into RNA via metabolic modulation. Proc. natn. Acad. Sci. U.S.A. 77: 4966-4970. SPlERS, A. S. D. and FIRTH, J. L. (1972) Treating the nervous system in acute leukemia. Lancet 2: 1433. STENSTROM, M. L., EDELSTEIN, M. and GRISHAM, J. W. (1974) Effect of ara-CTP on DNA replication and repair in isolated hepatocyte nuclei. Expl Cell Res. 89: 439-442. STERNBERG, A., PETRELLL N., AU, J. L.-S., RUSTUM, Y. M., MITTELMAN, A. and CREAVEN,P. (1984) A combination of 5-fluorouracil and thymidine in advanced colorectal carcinoma. Cancer Chemother. Pharmac. 13: 218-222. STEUART,C. D. and BURKE,P. J. (1971) Cytidine deaminase and the development of resistance to arabinosyl cytosine. Nature New Biol. 233: 109-110. STOLLER, R. G., MYERS, C. E. and CHABNER, B. A. (1978) Analysis of cytidine deaminase and tetrahydrouridine interaction by use of ligand techniques. Biochem. Pharmac. 27:53 59. SUGINO, Y., TERAOKA, H. and SHIMONO, H. (1966) Metabolism of deoxyribonucleotides. I. Purification and properties of deoxycytidine monophosphokinase of calf thymus. J. biol. Chem. 241: 961-969.
Pyrimidine analogues and their nucleosides SWEENY, D. J., BARNES,S., HEGGIE, G. H. and DIASIO, R. B. (1987) Metabolism of 5-fluorouracil to an N-cholyl-2fluoro-b-alanine conjugate: Previously unrecognized role for bile acids in drug conjugation. Proe. natn. Acad. Sci., U.S.A. 84: 5439-5443. SWEENY, n . J., MARTIN, M. and D1ASIO, R. B. (1988) Nchenodeoxycholyl-2-fluoro-b-alanine: A biliary metabolite of 5-fluorouracil in humans. Drug Metab. Dispos. 16: 892-893. TAGGER, A. Y. and WRIGHT, J. A. (1988) Molecular and cellular characterization of drug resistant hamster cell lines with alterations in ribonucleoside reductase. Int. J. Cancer 42: 760-766. TAGGER, A. Y., Soux, J. and WRIGHT, J. A. (1987) Hydroxy[t4C]urea uptake by normal and transformed human cells: Evidence for a mechanism of passive diffusion. Biochem. Cell Biol. 65: 925-929. TAKIMOTO, C. H., TAN, Y. Y., CADMAN, E. C. and ARMSTRONG, R. D. (1987) Correlation between ribosomal RNA production and RNA-directed fluoropyrimidinecytotoxicity. Biochem. Pharmac. 36: 3243-3248. TAMAKI, N., MIZUTANI, N., KIKUGAWA, M., FUJIMOTO, S. and MIZOTA, C. (1987) Purification and properties of b-ureidopropionase from the rat liver. Eur. J. Biochem. 169: 21-26. TANAKA, M. and YOSH1DA,S. (1987) Formation of cytosine arabinoside-5'-triphosphate in cultured human leukemic cell lines correlates with nucleoside transport capacity. Jap. J. Cancer Res. 78: 851-857. TATTERSALL,M. N. H., GANESHAGURU,K. and HOFFBRAND, A. V. (1974) Mechanisms of resistance of human acute leukaemia cells to cytosine arabinoside. Br. J. Haemat. 27: 39-46. TAYLOR, A. T., STAFFORD, M. A. and JONES, O. W. (1972) Properties of thymidine kinase partially purified from human fetal and adult tissue. J. biol. Chem. 247: 1930-1935. THELANDER, L. and REICHARD, P. (1979) Reduction of ribonucleotides. A. Rev. Biochem. 48: 133-158. THELANDER, L., ERIKSSON,S. and ,~KERMAN,M. (1980) Ribonucleoside reductase from calf thymus. Separation of the enzyme into two nonidentical subunits, proteins MI and M2. J. biol. Chem. 255: 7426-7432. THELANDER, M., GR.~SLUND, A. and THELANDER, L. (1985) Subunits M2 of mammalian ribonucleoside reductase. Characterization of a homogeneous protein isolated from M2-overproducing mouse cells. J. biol. Chem. 260: 2737-2741. TOMCHICK, R., SASLAW, L. D. and WARAVDEKAR, V. S. (1968) Mouse kidney cytidine deaminase. Purification and properties. J. biol. Chem. 243: 2534-2537. TRAUT, T. W. and LOECnEL, S. (1984) Pyrimidine catabolism: Individual characterization of three sequential enzymes with a new assay. Biochemistry 23: 2533-2539. TUCHMAN, M., STOECKELER, J. S., KIANG, D. T., O'DEA, R. F., RAMNARAINE, M. L. and MIRKIN, B. L. (1985) Familial pyrimidinemia and pyrimidinuria associated with severe ttuorouracil toxicity. New Engl. J. Med. 313: 245 249. ULLMAN, B., LEE, M., MARTIN, D. W. and SANTI,D. V. (1978) Cytotoxicity of 5-fluoro-2'-deoxyuridine: Requirement for reduced folate cofactors and antagonism by methotrexate. Proc. natn. Acad. Sci. U.S.A. 75: 980-983. ULLMAN, B., LEVINSON, B. B., ULLMAN, D. H. and MARTIN, D. W. J. (1979) Isolation and characterisation of cultured mouse T-lymphoma deficient in uridine-cytidine kinase. J. biol. Chem. 254: 8736-8739. VADLAMUD1,S., CHOUDRY,J. N., WARAVDEKAR,V. S., KLINE, I. and GOLDIN, A. (1970a) Effect of combination treatment with 5-azacytidine and cytidine on the life span and spleen and bone marrow cells of leukemic (L1210) and non-leukemic mice. Cancer Res. 30: 362-369.
221
VADLAMUDI, S., PADARATHSINGH, M., BONMASSAR,E. and GOLDIN, A. (1970b) Reduction of antileukemic and immunosuppressive activities of 5-azacytidine in mice by concurrent treatment with uridine. Proc. Soe. exp. Biol. Med. 133: 1232-1238. VEREZ,Z., SZINAI,I., SZABOLCS,A., UJSZASZY,K. and DENES, G. (1987) Biological activity of the potent uridine phosphorylase inhibitor 5-ethyl-2,2'-anhydrouridine. Drugs under exp. clin. Res. 13: 615-621. VESELY,J. and SMRT, J. (1977) Enzymes relevant to cancer chemotherapy. II. Heterogeneity of rat kidney uridine kinase following chromatography on sepharose 6B coupled to N-(5-amino-pentyl)-cytidine. Neoplasma (Bristol) 24: 461-467. VESSEY,D. A., CRISSEY, M. H. and ZAKIM,D. (1977) Kinetic studies on the enzymes conjugating bile acids with taurine and glycine in bovine liver. Biochem. J. 163: 181-183. VITA, A., AM1CI, A., CACCIAMANI, T., LANCIOTTI, M. and MAGNI, G. (1985) Cytidine deaminase from Escherichia coil B. Purification and enzymatic and molecular properties. Biochemistry 24: 6020-6024. VITA, A., CACCIAMANI, T., NATALINI, P., RUGGIERI, S., SANTARELLI, I. and MAGNI, G. (1987) Purification of cytidine deaminase from chicken liver. Ital. J. Biochem. 36: 275-282. VOGLER, W. R., MILLER, D. S. and KELLER, J. W. (1976) 5-Azacytidine (NSC-I02816): a new drug for the treatment of myeloblastic leukemia. Blood 48: 331-337. VON HOFF, D. D., SLAVIK,M. and MUGGIA,F. M. (1976) A new anticancer drug with effectiveness in acute myelogenous leukemia. Ann. intern. Med. 85: 237-245. WALLACH, O. P. and SANTIAGO,G. (1957) The purification and properties of hydropyrimidine hydrase. J. biol. Chem. 251: 6909-6914. WALLISER,S. and REDMANN,K. (1978) Effect of 5-fluorouracil and thymidine on the transmembrane potential and 6 potential of HeLa cells. Cancer Res. 38: 3555-3559. WALSH, C. Z., CRAIG, R. W. and AGARWAL, R. P. (1980) Increased activation of 1-fl-arabinofuranosylcytosine by hydroxyurea in L1210 cells. Cancer Res. 40: 3286-3292. WEBER, G. (1980) Recent advances in the design of anticancer chemotherapy. Oneology (Basel)(Suppl. l): 19-24. WECKBECKER,G., LIEN, E. J. and CORY, J. G. (1988) Properties of N-hydroxy-N'-aminoguanidine derivatives as inhibitors of mammalian ribonucleotide reductase. Biochem. Pharmac. 37: 529-534. WILEY, J. S., JONES, P. E., SAWYER, W. H. and PATERSON, A. R. P. (1982) Cytosine arabinoside influx and nucleoside transport sites in acute leukemia. J. clin. Invest. 69: 479-489. WILEY, J. S., JONES,P. E. and SAWYER,W. H. (1983) Cytosine transport by human leukemic cells. Fur. J. Cancer clin. Oncol. 19: 1067-I074. WILEY, J. S., TAUPIN, J., JAMIESON, G. P., SNOOK, M., SAWYER,W. H. and FINCH, L. R. (1985) Cytosine arabinoside transport and metabolism in acute leukemias and T cell lymphoblastic lymphoma. J. clin. Invest. 75: 632-642. WILKINSON, D. S. and CRUMLEY, J. (1977) Metabolism of 5-fluorouracil in sensitive and resistant Novikoff hepatoma cells. J. biol. Chem. 252: 1051-1056. WILKINSON, L. S. and PITOT, H. C. (1973) Inhibition of ribosomal ribonucleic acid maturation on Novikoff hepatoma cells by 5-fluorouracil and 5-fluorouridine. J. biol. Chem. 248: 63-68. WILKINSON, D. S., TlSTY, T. D. and HANAS, R. J. (1975) The inhibition of ribosomal RNA synthesis and maturation in Novikoff hepatoma cells by 5-fluorouridine. Cancer Res. 35: 3014-3020. WISDOM, G. B. and ORSl, B. A. (1969) The purification and properties of cytidine aminohydrolase from sheep liver. Fur. J. Biochem. 7: 223-230.
222
G. C. DAHER et al.
WOHLHUETER, R. M. and PLAGEMANN, P. G. W. (1980) Role of transport and phosphorylation in nutrient uptake in cultured animal cells. Int. Rev. Cytol. 64: 171-240. WOHLHUETER, R. M., MCIVAR, R. S. and PLAGEMAN, P. G. W. (1980) Facilitated transport of uracil and 5fluorouracil, and permeation of orotic acid into cultured mammalian cells. J. cell. Physiol. 194: 309-319. WOODCOCK, T. M., MARTIN, D. S., DAMIN, A. E. M., KEMENY, N. E. and YOUNG, C. W. (1980) Combination clinical trials with thymidine and fluorouracil: A phase 1 and clinical pharmacologic evaluation. Cancer 45: 1135-1143.
WRIGHT, J. A. (1983) Altered forms of mammalian nu-
cleoside diphosphate reductase from mutant cell lines. Pharmac. Ther. 22: 81-102. YANG, J. L., CHENG, E. H., CAPIZZl, R. L., CHENG, Y.-C. and KUTE, T. (1985) Effect of uracil arabinoside on metabolism and cytotoxicity of cytosine arabinoside in L5178Y murine leukemia. J. clin. Invest. 75: 141-146. YOUNG, I., YOUNG, G. J., WILEY, J. S. and VANDER WEYDEN, M. B. (1985) Nucleoside transport and cytosine arabinoside (araC) metabolism in human T lymphoblasts resistant to araC, thymidine and 6-methylmercaptopurine riboside. Eur. d. Cancer clin. OncoL 21: 1077-1082. ZIMMERMAN,M. (1964) II. Nucleoside:pyrimidine deoxyribosyltransferase activity of three partially purified thymidine phosphorylases. J. biol. Chem. 239: 2622-2627.
