Inhibition of mammalian ribonucleotide reductase by cis-diamminedichloroplatinum(11) CHRIST~NE S. M. CHIU,ARTHUR K. CHAN,AND JIM A.

WRIGHT'

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Department of Biochemistry and Molecular Biology, University of Manitoba and the Manitoba Institute of Cell Biology, 100 Olivia Street, Winnipeg, Man., Canada R3E OV9 Received June 12, 1992 CHIU, C. S. M., CHAN,A. K., and WRIGHT,J. A. 1992. Inhibition of mammalian ribonucleotide reductase by cis-diamminedichloroplatinum(II). Biochem. Cell Biol. 70: 1332-1338. Ribonucleotide reductase is a highly regulated, rate-limiting activity in the synthesis of DNA. A previous study has shown that the Escherichia coli enzyme is inhibited by the clinically important antitumor agent cis-diamminedichloroplatinum(I1) (DDP), and this has led to the hypothesis that ribonucleotide reductase is an important site of action for this chemotherapeutic agent. This hypothesis has been directly tested in this investigation. We observed that DDP inhibits the mammalian ribonucleotide reductase, with 50% inhibition occurring at 0.3 mM. Unlike the E. coli enzyme where only one of the two protein components is targeted by DDP, we observed that both of the mammalian proteins (R1 and R2) were sites for the inhibitory activity of the drug. Colony-forming experiments, enzyme activity studies, and analyses of R1 and R2 message levels in mutant cell lines containing either high levels of ribonucleotide reductase activity or exhibiting resistance to the cytotoxic effects of DDP were used to further investigate the potential role of ribonucleotide reductase in DDP cytotoxic action and drug resistance. These studies did not support a hypothesis formulated in the earlier investigation that inhibition of ribonucleotide reductase is an important component of DDP cytotoxic activity or that it is a major participant in DDP resistance mechanisms. From a biological point of view, DDP is a very active drug, and in addition to its cytotoxic effects it is capable of inducing a variety of cellular changes. Whether or not the inhibition of mammalian ribonucleotide reductase activity that we have described in this study plays a role in mediating any of these other effects remains to be determined. Key words: cisplatin, ribonucleotide reductase, drug resistance, hydroxyurea. CHIU,C. S. M., CHAN,A. K., et WRIGHT,J. A. 1992. Inhibition of mammalian ribonucleotide reductase by cis-diamminedichloroplatinum(I1). Biochem. Cell Biol. 70 : 1332-1338. La ribonucleotide reductase exerce une activitk fortement contr61Ce et limitant la vitesse dans la synthkse du DNA. Une etude anterieure a montre que l'enzyme de Escherichia coli est inhibee par un agent antitumoral cliniquement important, le cis-diarnrninedichloroplatine(1I)(DDP), d'ou l'hypothese que la ribonucleotide rtductase est un site d'action essentiel pour cet agent chimiotherapeutique. Ce travail a pour but de verifier directement cette hypothese. Nous avons observe que le DDP inhibe la ribonucleotide reductase mammalienne; une concentration de 0,3 mM produit 50% d'inhibition. Au contraire de I'enzyme de E. coli ou un seul des deux constituants prottiques est touche par le DDP, nous avons observe que les deux proteines mammaliennes (R1 et R2) sont des sites de l'activite inhibitrice de cette drogue. Pour etudier davantage le r6le potentiel de la ribonucleotide reductase dans l'action cytotoxique du DDP et la resistance a cette drogue, nous avons effectue des experiences de formation de colonies, des etudes d'activitk enzymatique et des analyses des taux du message des prottines R1 et R2 dans des lignkes cellulaires mutantes renfermant des valeurs ClevCes de I'activiti de la ribonucleotide reductase ou montrant une resistance aux effets cytotoxiques du DDP. Ces etudes ne corroborent pas l'hypothbe anterieure voulant que I'inhibition de la ribonucleotide reductase soit une composante importante de I'activitk cytotoxique du DDP ou qu'elle soit une reaction majeure dans les mecanismes de resistance au DDP. Du point de vue biologique, le DDP est une drogue t r b active et, en plus de ses effets cytotoxiques, elle est capable d'induire divers changements cellulaires. I1 reste a determiner si oui ou non l'inhibition de l'activite de la ribonuclCotide reductase mammalienne decrite ici joue un r61e dans la production d'un de ces autres effets. Mots eels : cisplatine, ribonucleotide rkductase, resistance aux drogues, hydroxyurte. [Traduit par la redaction]

Introduction cis-Diamminedichloroplatinum(11) is an important antineoplastic agent used in the treatment of a variety of tumors, alone or in combination with other chemotherapeutic compounds (Loehrer and Einhorn 1984; Beppu et al. 1991; Hainsworth et al. 1988). A major problem in chemotherapy, and this includes the use of DDP, is that in many cases an initial positive response is followed by relapse and failure of tumors to respond to further drug treatment (Hainsworth et al. 1988; Scanlon et al. 1989a). These observations have led to considerable interest in determining the mode of action of antineoplastic agents like DDP and the mechanisms responsible for drug resistance (Scanlon et al. 1989a). From a biological point of view, DDP is a very active ABBREVIATIONS: DDP, cis-diamminedich1oroplatinum(II); a-MEM, alpha minimal essential medium. l ~ u t h o rto whom all correspondence should be addressed. Printed in Canada / Im~rimeau Canada

drug and has been shown to bind and damage DNA (Weiss and Poster 1982; Plooy et al. 1985) and to cause chromosomal abberations in patients receiving the drug (Gundy et al. 1990). In addition, DDP is mutagenic (Beck and Fisch 1980; Taylor et al. 1979), is a potent inducer of cell transformation (Hall and Hei 1985), appears to act like an initiator in in vivo studies of carcinogenesis (Hennings et al. 1990), and induces malignancies in laboratory animals (Hennings et al. 1990; Kempf and Ivankovic 1986). Indeed, there is some concern about possible DDP carcinogenicity in patients that have previously undergone antineoplastic therapy (Greene 1992). Clearly, it is important to determine the cellular targets that interact with the drug, if we are to understand the mechanisms that mediate the many biological changes that occur to mammalian cells in response to DDP exposure. From this point of view, an interesting report by Smith and Douglas (1989) has appeared showing that the ribonucleotide reductase activity prepared from Escherichia

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CHIU ET AL.

coli is inhibited by DDP. The authors suggested that inhibition of this enzyme could be important in the cytotoxic action of this chemotherapeutic compound, but no studies were performed with the mammalian enzyme to test this hypothesis. Since this question is of some importance to the mode of action of DDP and to the regulation of ribonucleotide reductase, a rate-limiting activity required for the synthesis of DNA (Wright 1989; Wright et al. 1990), we have investigated the effects of DDP on the activity of mammalian ribonucleotide reductase and its two protein compounds (R1 and R2). In addition, we have used mutant cell lines either altered in ribonucleotide reductase gene expression or modified in sensitivity to the cytotoxic effects of DDP, to further test the hypothesis that ribonucleotide reductase is an important site of action for the cytotoxic activity of platinum based drugs like DDP (Smith and Douglas 1989).

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Materials and methods Cell lines and growth conditions Mouse L cell lines were routinely cultured at 37OC on plastic tissue culture plates (Lux Scientific, Ltd.) in a-MEM (Flow Laboratories Ltd.) supplemented with antibiotics and 10% (v/v) fetal bovine serum (Gibco Ltd.), as previously described (Hurta and Wright 1990; McClarty et al. 1987). The hydroxyurea-resistant mouse cell lines called SC2 and H ~ - 3 0contain elevated levels of ribonucleotide reductase activity and have been described in detail elsewhere (Hurta and Wright 1990; McClarty et al. 1987). A2780S and A2780DDP are human ovarian carcinoma cell lines sensitive and resistant to the cytotoxic effects of DDP, respectively (Scanlon and Kashani-Sabet 1988; Lu et al. 1988). HCT8S and HCT8DDP are human colon carcinoma cell lines sensitive and resistant to DDP, respectively (Scanlon et al. 1989b, 1989~).The human cell lines were grown in RPMI 1640 medium (Flow Laboratories Ltd.) supplemented with antibiotics and 10% (v/v) fetal bovine serum as described (Lu et al. 1988; Scanlon et al. 1989b). The relative colony-forming efficiency was defined as the ability to form colonies in the presence of drug divided by the ability to form colonies in the absence of drug (Hards and Wright 1981). The Dlo value is the drug concentration that reduces the relative colony forming efficiency to 10% (Hards and Wright 1981). Ribonucleotide reductase assay Enzyme preparations containing 1-3 mg protein/mL (Hurta and Wright 1992) were used to determine ribonucleotide reductase activity by the method of Steeper and Stuart (1970), using [ I 4 c ] c ~ p (Moravek Biochemicals Inc.) as substrate and Crotalus atrox snake venom dissolved in 0.1 M Hepes (pH 8.0), containing 10 mM MgCl, to hydrolyze the nucleotides to nucleosides, as we have previously described (Hards and Wright 1981; Hurta and Wright 1992; Lewis et al. 1978). The reaction mixture contained 50 pM [ I 4 c ] c ~ p(0.05 ~ c i ;1 Ci = 37 GBq); dithiothreitol (6 mM); magnesium acetate (4 mM), ATP (2 h ~ ) and , an app;opriate quantity of enzyme preparation in a final volume of 150 pL. Reactions were initiated by the addition of enzyme and then carried out for 20 min at 37°C for CDP reduction. The reaction was terminated by boiling for 5 min. The nucleotides were converted to nucleosides by treatment for 1 h at 37°C with 2 mg/assay of Crotalus atrox venom. The reaction was again terminated by boiling for 5 min and then 0.5 mL distilled water was added to each assay tube. The heat-precipitablematerial was removed by centrifugation. The supernatant was retained and the deoxycytidine was separated from the cytidine compounds on 5 x 80 mM Dowex-1-borate columns (Bio-Rad). Briefly, the separation depends on the formation of a complex between ribonucleosides and borate ions in the column resin. Deoxyribonucleosides do not possess a cis-diol and hence cannot form a complex. Deoxycytidine was eluted from the column with 5 mL of distilled water. Scintiverse (Fisher Scientific)

DDP (mM)

FIG. 1. Inhibition of ribonucleotide reductase activity by DDP under normal conditions ( @ ) and under highly reduced conditions achieved with nitrogen gas (A). Each data point was the average of at least two independent determinations. No statistically significant differences in slope or intercept were observed when inhibition under the two conditions was analyzed. was then added and radioactivity was determined in a Beckman model LS7800 liquid scintillation spectrophotometer. To determine ribonucleotide reductase activity under highly reduced anaerobically enriched conditions, enzyme preparations on ice were flushed with nitrogen gas. Various concentrations of DDP were added to microfuge tubes containing the enzyme. DDP treatment under oxygen-reduced conditions was continued by sparging continuously each microfuge tube with nitrogen gas in a nitrogen-saturated container. Incubation of enzyme with DDP was performed at room temperature for 10 rnin and enzyme activity was determined as described above. In some experiments, partially purified preparations containing either the R1 or R2 protein component of ribonucleotide reductase were prepared, mixed, and assayed for e w e activity as we have previously done (Hurta and Wright 1992). The R I and R2 proteins were separated from enzyme preparations obtained from logarithmically growing cuttures of mouse cells, SC2 and H~-30. shown previously to have elevated levels of R1 and R2 proteins (Hurta and Wright 1990; McCIarty et al. 1987). The protein cornponents were separated by affinity chromatography on blue dextran-Sepharose as described (Rubin and Cory 1986), Protein concentrations were estimated with the Bio-Rad determination kit (Technical Bulletin 1051)using bovine serum albumin as a standard. Northern blot analysis Total cellular RNA was extracted from logarithmically growing cells using the guanidinium isothiocyanate - cesium chloride method (Chirgwin er 01. 1979), as we have done in other studies (McCfarty et 01. 1987). RNA was subjected to electrophoresis through 1% formaldehyde-agarose gels followed by transfer to nylon membranes (Nytran) (Schleicher and Schuell). AIl blots were prehybridized at 4Z°C for 24 h in a mixture of 50% formamide, 5 x SSC ( J x SSC is 0.15 M NaCl - I5 rnM sodium citrate, pH 7.0), 7.5 x Denhardt's solution (1 x Denhardt's solution contains 20 mg each of Ficoll, polyvinylpyrrolidone, and bovine serum albumin in 100 mL of water), 50 mM sodium phosphate (pH 7.0), 0.1% SDS, 50% dextran sulfate solution, and 100 pg/mL of denatured salmon sperm DNA. Hybridization was performed in the same solution for 24 h with 1 x lo6 - 2 lo6 cpm/mL of a 32~-labelled NcoI-generated fragment containing the cDNA

BIOCHEM. CELL BIOL. VOL. 70, 1992

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R1

R2 Fraction (pg)

Fraction (pg)

s 0.0

1

1

I

I

0.5

1 .O

1.5

2.0

DDP (mM)

0.0

0.5

1.O

1.5

2.0

DDP (mM)

FIG.2. Reconstitution of ribonucleotide reductase activity by adding increasing amounts of R1 protein to a constant amount of R2 protein (84 pg) (A) or by adding increasing amounts of R2 protein to a constant amount of R1 protein (34 pg) (B). Inhibition of ribonucleotide reduclase activity by DDP by adding various drug concentrations to R1 protein (50 pg), removing unbound DDP, and incubating the drug-treated RI with untreated R2 protein (64pg) (C); or by adding various drug concentrations to R2 protein (64 pg), removing unbound DDP,and incubating drug treated R2 with untreated R1 protein (48 pg) (D). The two protein fractions were separated by affinitychromatography as described in Materials and methods. Unbound DDP was removed by gel filtration through a G-25 Sephadex column. Each data point in C and D was the average of two and three independent determinations, respectively. of clone 65 (RI protein) or the PstI fragment of clone 10 (R2 protein) (Hurta and Wright 1990; McClarty et al. 1987; Hurta and Wright 1992). cDNA probes were labelled by the hexadeoxyribonucleotide method of Feinberg and Vogelstein (1983) using I ~ - ' ~ P ] ~ c(specific TP activity, 3000 Ci/mmol; Amersham) to a specificactivity o f 2 x 10' - 1 x 10' cpm/pg. Blots were washed twice Tor 30 mln each in 2 x SSC - 0.1 % 5DS at room temperature and then twice for 30 min each in 0.2 x SSC - 0.1 To SDS at 60°C. Autoradiographyw a carried out at - 70°C using Kodak X-Omat AR film and Cronex Lighting Plus intensifying screens. A piasmid containing the glyceraldehyde-3-phosphatedehydrogenase gene was IabelIed by nick transration and used in loading control hybridizations (Hurta and Wright 1992). Dmsitometric analysis on autoradiograms was done with a Bio-Rad model 620 video densitometer and Amdek Video-300A screen using the one-dimensional Bio-Rad program (Bio-Rad Co.). Statistical analysis

Results were analyzed by covariates analysis of variants or by two-way ANOVA analysis of variants. Results DDP inhibits ribonucleotide reductase activity Ribonucleotide reductase prepared from bacterial and mammalian sources has an active site redox-active dithiol (Wright 1989; Wright et al. 1990; Lammers and Follman 1983). Since DDP contains a platinum(I1) moiety known to have an affinity for -SH groups, Smith and Douglas (1989) examined the possibility that the E. coli ribonucleotide

reductase could be a target for inhibition by DDP. They observed inhibition of the bacterial enzyme, with about 0.4 mM DDP giving 50% inhibition. In addition, they also observed that pretreatment of the E. coli enzyme with nitrogen gas to obtain a fully reduced condition resulted in a further increase in ribonucleotide reductase sensitivity to DDP inhibition. Preparation of mammalian ribonucleotide reductase and assays of its activity are routinely carried out under reducing conditions (in the presence of dithiothreitol) (Hards and Wright 1981; Hurta and Wright 1992; Lewis et al. 1978). Figure 1 shows that the mammalian enzyme was also sensitive to DDP inhibition, with 50% inhibition occurring at 0.3 mM drug. However, since the E. coli enzyme exhibited a greater sensitivity to DDP when pretreated with nitrogen gas, we also performed inhibition studies with mammalian enzyme exposed to nitrogen gas, to be certain that the enzyme was fully reduced and to parallel the conditions used in the previous study (Smith and Douglas 1989). In the mammalian situation, pretreatment with nitrogen did not significantly alter the drug sensitivity of the enzyme, since 50% inhibition was still observed at approximately 0.3 mM DDP (Fig. 1). Ribonucleotide reduction requires the presence of two dissimilar protein components called R1 and R2 (Wright 1989; Wright et al. 1990). The R1 is a dimer with a molecular weight of 170 000 and contains substrate and effector bind-

CHIU

ET AL.

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TABLE1. Biochemical characteristics of the SC2 and HR-30 cell lines

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Cell lines

Relative increase in enzyme activity"

Relative increase in R1 protein levels

Relative increase in R2 protein levelb

'Enzyme activity was measured as nmol CDP reduced/(hmg protein) and expressed relative to the wild-type condition. b~eterminedfrom densitometric scanning o f data from Western blots, using R1 or R2 specific monoclonal antibodies, and expressed relative to the wild-type condition. 'Results obtained from McClarty et al. (1987). d ~ e s u l t sobtained from Hurta and Wright (1990).

ing sites (Wright 1989; Wright et al. 1990). R2 is also a dimer, has a molecular weight of 88 000, and has nonheme iron and a unique tyrosyl-free radical required for enzyme activity (Wright 1989; Wright et al. 1990). Both proteins contain -SH groups that could possibly be targets for platinum(I1)-based drugs (Wright 1989; Wright et al. 1990). Therefore, to determine the protein component(s) involved in the inhibition of the mammalian reductase, the R1 and R2 proteins from mouse cells that overproduce these components were separated by affinity chromatography, each component was exposed separately to several concentrations of DDP or left untreated, and combinations of the proteins were mixed to analyze enzyme activity. Figures 2A and 2B show that ribonucleotide reductase activity depends upon the presence of both components and that neither preparation of R1 or R2 alone exhibited enzyme activity. Interestingly, the mixing experiments demonstrated that both ribonucleotide reductase proteins are targets for the inhibitory action of DDP (Figs. 2C and 2D). DDP inhibition of colony-forming ability The finding that DDP is an inhibitor of mammalian ribonucleotide reductase, a key rate-limiting enzyme activity in the synthesis of DNA (Wright 1989; Wright et al. 1990), is consistent with the idea that the enzyme may be an interesting in vivo site of action for the cytotoxic effects of this drug. DDP is one of the most important antineoplastic agents used to treat solid tumors, especially ovarian, testicular, and bladder carcinomas (Loehrer and Einhorn 1984; Beppu et al. 1991; Hainsworth et al. 1988). However, in spite of the success of chemotherapy, the remission that may accompany treatment is frequently short and after a relapse the tumor is usually resistant to further DDP treatment. We reasoned that if ribonucleotide reductase is an important intracellular site of action for DDP, then mammalian cell lines containing elevated levels of ribonucleotide reductase activity and gene expression should exhibit significantly decreased sensitivities to the cytotoxic effects of the antineoplastic agent. Cell lines with the properties noted above can be obtained by selection for resistance to hydroxyurea (Wright 1989; Wright et al. 1990). Two mouse cell lines, SC2 and H ~ - 3 0have , been selected for hydroxyurea resistance and their properties have been described in detail (Hurta and Wright 1990; McClarty et al. 1987). Most importantly, both lines contain substantial elevations in the levels of R1 and R2 proteins and exhibit increased ribonucleotide reductase activity (Table 1). Figure 3 shows that the mouse

FIG. 3. Relative colony-forming abilities in the presence of various concentrations of DDP, by parental wild-type mouse L cells ( 0 )and the two hydroxyurea-resistant ribonucleotide reducEach data tase overproducing cell lines SC2 ( v ) and H ~ - 3 0(0). point for the wild-type line was from two to four independent determinations; for the SC2 line each data point was from two to five independent determinations; and for the HR-30 line each data point was from three or four independent determinations. A single line represents the results obtained for the three cell lines, since no statistically significant differences in slope or intercepts were observed when the results obtained with these cell lines were analyzed.

cells were very sensitive to the presence of DDP and exhibited a Dlo value of about 1.4 pM drug in colony-forming experiments. Furthermore, no statistically significant differences in the sensitivity to DDP were observed when parental wild-type cells were compared with the two hydroxyurearesistant ribonucleotide reductase overproducing cell lines. Hydroxyurea sensitivity and ribonucleotide reductase in human DDP-sensitive and -resistant cell lines Mammalian cell lines selected for resistance to DDP in cell culture have been widely used in studying DDP-resistant mechanisms (Scanlon et al. 1989a). Many of these investigations showed a correlation between resistance and an elevation in DDP-targeted molecules like metallothionein or glutathione (Kelley and Rozencweig 1989). Since mammalian ribonucleotide reductase was inhibited by DDP (Fig. l), we examined the possibility that human cell lines selected for DDP resistance would contain alterations in ribonucleotide reductase. This was done in two ways. First we examined the colony-forming abilities of DDP-sensitive and -resistant lines in the presence of hydroxyurea, whose site of action is at mammalian ribonucleotide reductase (Wright 1989; Wright et al. 1990). Cross-resistance to hydroxyurea would occur if the DDP-resistant lines contained modifications in ribonucleotide reductase such as elevated levels of enzyme activity. Second, we directly determined the levels of the enzyme activity and messages of the two components of the enzyme in parental sensitive and DDP-resistant lines. Two human cell lines A2780S and HCTSS, which were used in the selection of the DDP-resistant lines called A2780DDP and HCT8DDP (Scanlon and Kashani-Sabet 1988; Lu and Scanlon 1988; Scanlon et al. 1989b, 1989c), were used in these experiments. The results of a colony-forming study

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TABLE2. Relative colony-forming efficiency of human cell lines in the presence of hydroxyurea Relative plating efficiency + SEa

Hydroxyurea (mM)

A2780S

0.1 0.2 0.3

0.83 0.05 0.28 k 0.02 O.lOkO.01

*

A2780DDP

HCT8S

HCT8DDP

0.97 k 0.08 0.86 k 0.08 0.53 k0.04

0.84k0.13 0.60 k 0.09 0.51 k0.05

0.75 0.02 0.58 k 0.03 0.41 + 0.01

*

'The values presented are the averages of at least three independent determinations for 0.1 and 0.2 mM hydroxyurea, and at least two independent determinations for 0.3 mM hydroxyurea. Statistically significant differences between the A2780S and A2780DDP cell lines were observed at 0.1 mM hydroxyurea ( p = 0.048), 0 . 2 mM hydroxyurea ( p = 0.0001), and 0.3 mM hydroxyurea ( p = 0.0001). N o statistically significant differences between HCT8S and HCT8DDP cell lines were detected at any of the three concentrations of hydroxyurea.

FIG. 4. Ribonucleotide reductase activity in the human wildtype and DDP-resistant cell lines. (A) Specific enzyme activity was determined for A2780S (Loehrer and Einhorn 1984) (1) and A2780DDP (Beppu et al. 1991) (2) cell lines. (B) Specific enzyme activity was determined for HCT8S (Loehrer and Einhorn 1984) (1) and HCT8DDP (Beppu et al. 1991)(2) cell lines. Statistical analysis indicated a trend toward significantly higher levels of enzyme activity in A2780DDP cells when compared with parental A2780S cells ( p = 0.067). No statistically significant differences were found when enzyme levels in HCT8S cells were compared with levels in HCTSDDP cells.

FIG. 5. Northern blot analysis of R2 mRNA levels in human wild-type A2780S (a) and DDP-resistant A2780DDP (6) cell lines. Loading of RNA was estimated by reprobing with glyceraldehyde-3-phosphate dehydrogenase cDNA (Hurta and Wright 1992) and the results are shown below each blot. When corrections for loading obtained by densitometric analysis were taken into account, a 2.1-fold elevation of the R2 mRNA level was observed in the A2780DDP cell line when compared with the A2780S line. Arrows point to the positions of 28s and 18s rRNA.

performed at three concentrations of hydroxyurea are presented in Table 2. Interesting differences in drug sensitivity were observed when A2780DDP cells were compared with parental A2780S cells. The A2780S line was more sensitive t o hydroxyurea cytotoxicity than A2780DDP cells ( p < 0.05 at all hydroxyurea concentrations tested). However, no significant differences in drug sensitivity were observed when HCT8S cells were compared with the DDPresistant HCTSDDP cell line. Studies of hydroxyurea-resistant cell lines have shown that changes in resistance to this drug are accompanied by changes in ribonucleotide reductase activity and gene expression (Wright 1989; Wright et al. 1990; McClarty et al. 1990). All hydroxyurea-resistant cell lines described to date exhibit elevations in the expression of the R2 gene, with or without changes in R1 gene expression (Wright 1989; Wright et al. 1989, 1990). The R2 component is limiting for enzyme activity, so an increase in this component alone can lead to an elevation in ribonucleotide reductase activity (Wright 1989; Wright et al. 1987, 1990). Results of enzyme activity studies carried out with each pair of DDP-sensitive and -resistant human cell lines are provided in Fig. 4. In keeping with the

relationship between hydroxyurea sensitivity and enzyme activity (Wright 1989; Wright et al. 1987, 1990), the A2780DDP line exhibited higher levels of ribonucleotide reductase activity than the parental wild-type A2780S line. As expected from hydroxyurea sensitivity studies, the HCT8S and HCT8DDP cell lines contained approximately the same levels of ribonucleotide reductase activity. Furthermore, consistent with the hydroxyurea sensitivity studies (Table 2) and the enzyme activity results (Fig. 4), and previous studies with hydroxyurea-resistant cell lines (Wright 1989; Hurta and Wright 1990; McClarty et al. 1987; Wright et al. 1987), we observed that the A2780DDP line contained increased levels of R2 mRNA (Fig. 5). Densitometric analysis indicated approximately a 2.1-fold elevation in R2 message when A2780DDP cells were compared with the parental A2780S line. No significant changes in the R1 mRNA levels were detected in A2780DDP cells, and as expected from the hydroxyurea sensitivity and enzyme activity results obtained with the HCT8S and HCT8DDP cell lines, no differences were found in R1 or R2 mRNA levels when these two cell lines were compared (data not shown).

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Discussion DDP inhibits E. coli ribonucleotide reductase through an interaction of the drug with the B1 subunit of the enzyme, without affecting the B2 subunit (Smith and Douglas 1989). In this report we show for the first time that DDP can also inhibit the activity of the mammalian enzyme. Interestingly, in the latter case DDP targets both ribonucleotide reductase proteins, R1 and R2 (which are equivalent to the E. coli B1 and B2 proteins). These observations demonstrate differences between the bacterial and the mammalian enzyme, even though they exhibit many similarities in activity and allosteric regulation (Wright 1989; Wright et al. 1990; Lammers and Follman 1983). However, the results are in keeping with other evidence for differences in structure, including electron paramagnetic resonance spectrums (McClarty et al. 1987; Sahlin et al. 1987), and also the observation that only about 37% homology at the amino acid level exists when E. coli B1 is compared with mouse R1 and even less homology is found between the B2 and R2 proteins (Caras et al, 1985; Thelander and Berg 1986). Inhibition of the R1 protein by DDP is consistent with the finding of active site dithiol groups (Wright 1989; Wright et al. 1990; Stubbe 1990), which should be potential sites of action for DDP. Amino acid analysis of the R2 protein indicates that there are five cysteine residues, and interestingly, one of these (Cys-161) is reasonably close to the active site tyrosylfree radical residue (Tyr-177). It seems possible that DDP may be interacting with the Cys-161 residue and affecting free radical regeneration necessary for enzyme activity (Stubbe 1990; McClarty et al. 1987). It is worth noting that the E. coli B2 subunit does not have an equivalent cysteine residue close to the tyrosyl-free radical, which could perhaps explain why the B2 protein is not a target for DDP inhibition. More information about the relative positions of R2 Cys-161 and Tyr-177 residues in three-dimensional space would be helpful in evaluating these possibilities. Colony-forming experiments performed with mouse cell lines containing increased levels of ribonucleotide reductase activity and protein did not support the idea that ribonucleotide reductase is an important intracellular target for the cytotoxic effects of DDP, since there were no significant differences in the sensitivities of these lines to the drug when compared with parental cells. This was in agreement with the observation that the concentration of DDP which inhibited colony forming ability (Fig. 3) was approximately 100-fold lower than the concentration required to inhibit enzyme activity (Fig. I), and the observation that the human DDP-resistant line HCT8DDP did not show cross-resistance to hydroxyurea and contained similar levels of ribonucleotide reductase activity and message as the wild-type parental line. Results obtained with the A2780S and A2780DDP cell lines were not consistent with the above observations. In this case the DDP-resistant line contained elevated enzyme activity and R2 message levels and was more resistant than the parental wild-type line to the cytotoxic effects of hydroxyurea. Whether these differences are related to the DDP-resistant phenotype exhibited by A2780DDP cells when compared with the parental drug sensitive line is not known, but in view of the results obtained with the HCT8 cell lines noted above, it seems likely that the differences observed with the A2780 lines may be coincidental, perhaps owing to clonal variation and not directly related to DDP resistance. Previous studies have shown a rather

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large number of biochemical and molecular properties have been altered in the two DDP-resistant lines used in this study. These include enhanced DNA repair (Mususda et al. 1990), elevation in DNA polymerase (Scanlon et al. 1989a), increased glutathione levels (Hamilton et al. 1985), enhanced folate metabolism (Lu et al. 1988), and elevated expression of the c-fos and H-ras oncogenes (Scanlon et al. 1989a; Lu and Scanlon 1988). In addition, c-myc mRNA was found to be increased three- to five-fold in A2780DDP cells, but not in HCTSDDP cells (Scanlon et al. 1989b). Many of the alterations shared by the two DDP-resistant cell lines are likely to be important in development of DDP resistance, and the differences between the two lines (e.g., c-myc or ribonucleotide reductase gene expression) would determine unique characteristics that may not play a generally important role in DDP-resistance mechanisms. Our enzyme studies allow us to conclude that the mammalian ribonucleotide reductase like the E. coli enzyme is inhibited by DDP, and this is consistent with the hypothesis of Smith and Douglas (1989). However, studies with mutant cell lines did not generally support their hypothesis that ribonucleotide reductase is an important cellular site of action for the cytotoxic effects of DDP. Whether or not inhibition of mammalian ribonucleotide reductase, as described in this report, has a role to play in mediating any of the other many biological activities of platinum-based drugs like DDP (Greene 1992) is not known, but merits further investigation. Acknowledgements This investigation was supported by funds provided by the National Cancer Institute (Canada) and Natural Sciences and Engineering Research Council to J.A.W. We thank R.A.R. Hurta and B.K. Choy for their helpful comments during the course of this work and M. Cheang for her aid with statistical analysis. We also thank J.G. Cory for generously providing a sample of blue dextran - Sepharose and K.J. Scanlon for sending us the DDP-sensitive and -resistant cell lines (A2780S, A2780DDP, HCT8S, and HCT8DDP). J.A.W. is a Terry Fox Senior Scientist of the National Cancer Institute (Canada). Beck, D.J., and Fisch, J.E. 1980. Mutagenicity of platinum coordination complexes in Salmonella typhimurium. Mutat. Res. 77: 45-54. Beppu, T., Ohara, C., Yamaguchi, Y., Ichihara, T., Yamanaka, T., Katafuchi, S., Ikai, S., Mori, K., Fukushima, S., Nakano, M., and Ogawa, M. 1991. A new approach to chemoembolization for unresectable microspheres in combination with cisplatin suspended in iodized oil. Cancer (Philadelphia), 68: 2555-2560. Caras, I.W., Levinson, B.B., Fabry, M., Williams, S.R., and Martin, D. W., Jr. 1985. Cloned mouse ribonucleotide reductase subunit M1 cDNA reveals amino acid homology with Escherichia coli and herpes virus ribonucleotide reductase. J. Biol. Chem. 260: 7015-7022. Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J., and Rutter, W.J. 1979. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry, 18: 5294-5299. Feinberg, A.P., and Vogelstein, B. 1983. A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132: 6-13. Greene, M.H. 1992. Is cisplatin a human carcinogen? JNCI, J . Natl. Cancer Inst. 84: 306-312. Gundy, S., Baki, M., Bodrogi, I., and Czeibel, A. 1990. Persistence of chromosomal aberrations in blood lymphocytes of testicular

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