Regulation and drug resistance mechanisms of mammalian ribonucleotide reductase, and the significance to DNA synthesis'

Departments of Biochemistry and Microbiology, and Manitoba Institute of Cell Biology, University of Manitoba, 100 Olivia Street, Winnipeg, Man., Canada R3E OV9

Biochem. Cell Biol. Downloaded from by University of Otago on 01/01/15 For personal use only.

Received April 6, 1990 WRIGHT,J. A., CHAN,A. K., CHOY,B. K., HURTA,R. A. R., MCCLARTY,G. A., and TAGGER,A. Y. 1990. Regulation and drug resistance mechanisms of mammalian ribonucleotide reductase, and the significance to DNA synthesis. Biochem. Cell Biol. 68: 1364-1371. Mammalian ribonucleotide reductase, which occupies a key position in the synthesis of DNA, is a highly controlled enzyme activity, because it is solely responsible for the de novo reduction of ribonucleoside diphosphates to their corresponding deoxyribonucleoside diphosphate forms, required for DNA synthesis. Ribonucleotide reductase consists of two dissimilar protein components often called M1 and M2, which are independently regulated during cell proliferation. The M1 component contains multiple effector binding sites and is responsible for the complex allosteric regulation of the enzyme, whereas the M2 protein contains nonheme iron and a unique tyrosyl-free radical required for ribonucleotide reduction. Since the reaction is rate limiting for DNA synthesis, ribonucleotide reductase plays an important role in regulating cell division, and hence, cell proliferation. There are many inhibitors of ribonucleotide reductase and perhaps the most valuable one from a cell biology, biochemistry, and clinical point of view is the hydroxamic acid, hydroxyurea. This drug has also been very useful as a selective agent for isolating a variety of mammalian mutant cell lines altered in ribonucleotide reductase gene expression. Regulatory, structural, and biological characteristics of ribonucleotide reductase are reviewed, including evidence that ribonucleotide reductase, particularly the M2 protein, has an important early role to play in tumor promotion. In addition, modifications in the expressions of genes altered in hydroxyurearesistant mutants and cultured in the absence or presence of hydroxyurea are discussed, with emphasis on changes in M2 protein, M1 protein, and the iron-storage protein ferritin. Several regulatory models are presented, including a model showing the relationships between M2 protein levels, deoxyribonucleotide pools, and DNA synthesis, and a model demonstrating a linkage between M2 and ferritin proteins in regulating DNA synthesis in normal and hydroxyurearesistant mammalian cells. Key words: DNA synthesis, cell proliferation, ribonucleotide reductase, drug resistance. WRIGHT,J. A., CHAN,A. K., CHOY, B. K., HURTA.R. A. R., MCCLARTY,G. A., et TAGGER,A. Y. 1990. Regulation and drug resistance mechanisms of mammalian ribonucleotide reductase, and the significance to DNA . synthesis. Biochem. Cell Biol. 68 : 1364-1371. La ribonucleotide reductase mammalienne est une enzyme fortement contr81Ce qui occupe une position clC dans la synthkse du DNA car elle est uniquement responsable de la reduction de novo de ribonucleoside diphosphates en leurs desoxyribonucleoside diphosphates correspondants, requis pour la synthkse du DNA. La ribonucleotide reductase est formke de deux proteines differentes, souvent nommees M1 et M2, qui sont contr8lbes de f a ~ o nindependante durant la proliferation cellulaire. La protkine M1 renferme de multiples sites de liaison de l'effecteur et elle est responsable de la regulation allostCrique complexe de l'enzyme alors que la protkine M2 contient le fer non hemique et un seul radical tyrosyl libre nkcessaire pour la reduction des ribonuclCotides. Comme la reaction est limitante de la synthtse du DNA, la ribonucleotide reductase joue un r81e important dans la regulation de la division cellulaire et par le fait mCme, de la proliferation cellulaire. I1 existe plusieurs inhibiteurs de la ribonucleotide reductase et du point de vue de la biologie cellulaire, de la biochimie et de la clinique, le plus utile est peut-Etre l'acide hydroxamique, l'hydroxyurke. Cette substance est Cgalement trks utile comme agent stlecteur pour isoler diverses lignkes cellulaires mutantes mammaliennes dont l'expression gknique de la ribonucleotide reductase est alterke. Nous passons en revue les caracteristiques rkgulatrices, structurales et biologiques de la ribonucleotide reductase y compris le fait que cette enzyme, particulikrement la prottine M2, joue rapidement un r61e important dans le developpement des tumeurs. De plus, nous discutons des modifications de l'expression des gknes alterks chez les mutants resistants a l'hydroxyuree, cultives en absence ou en presence d'hydroxyuree, en mettant I'accent sur les changements que subissent la proteine M2, la proteine M1 et la ferritine, proteine d'emmagasinage du fer. Plusieurs modkles rkgulateurs sont presentis dont un montrant les relations entre les taux de la protkine M2, les pools de dCsoxyribonucleotides et la synthkse du DNA et un autre dkmontrant une liaison entre la proteine M2 et la ferritine dans la regulation de la synthkse du DNA dans les cellules mammaliennes normales et les cellules mammaliennes resistantes a l'hydroxyuree. Mots clPs : synthkse du DNA, proliferation cellulaire, ribonuclCotide reductase, resistance des drogues. [Traduit par la revue]

Introduction to regulatory, structural, and biological features of ribonucleotide reductase DNA synthesis requires a continuous and balanced supply of the four deoxyribonucleoside triphosphates that are derived from the direct reduction of the 2'-carbon atom on the ribose moiety of ribonucleotides. In mammalian cells, this reduction occurs at the diphosphate level in the presence of ribonucleotide reductase, and with one exception, a single enzyme activABBREVIATIONS: TPA, 12-0-tetradecanoylphorbol-13-acetate;ODC, ornithine decarboxylase. h he substance of this paper was presented at the Fourth Biennial Rossiter Research Conference "Molecular cell biology and disease," held September 29 - October 2, 1989, at Geneva Park, Lake Couchiching, Ont. ' ~ u t h o rto whom correspondence should be addressed. Printed in Canada / Imprim6 au Canada









Biochem. Cell Biol. Downloaded from by University of Otago on 01/01/15 For personal use only.




---- + DNA







Allorteric regulation of mammalian ribonucleotide reductase. Inhibitory effects are indicated by the bars and the nucleotides shown in the arrows act as positive effectors. For further information, see Wright (1989).

ity (nucleoside diphosphate kinase) is required to provide the four substrates for DNA polymerase (Wright 1989; Wright et al. 1989). The exception is dTTP, which comes from the reduction of either UDP or CDP, with the introduction of a methyl group at the monophosphate level. Since ribonucleotide reductase is entirely responsible for the conversion of ribonucleotides to deoxyribonucleotides, it occupies a crucial rate-limiting position in the synthesis of DNA. In keeping with its importance in this process, enzyme substrate specificity and activity is strictly regulated in a complex manner by a variety of nucleotides (Fig. 1). For example, reduction of CDP to dCDP and UDP to dUDP occurs in the presence of an ATP-activated enzyme. GDP to dGDP conversion requires dTTP and reduction of ADP to dADP requires dGTP. Interestingly, dATP acts as an overall negative effector through inhibition of all four ribonucleotide reductions. This regulatory scheme suggests that ribonucleotide conversion may begin with a reduction of CDP and UDP by an ATP-activated enzyme, proceed to GDP reduction through a dTTP-regulated activity, and finally involve ADP reduction via a dGTP-activated enzyme activity. A decline in DNA synthesis leads to the accumulation of dATP which turns off ribonucleotide reduction, since this compound is a potent inhibitor of the reduction of all four ribonucleotide substrates (Fig. 1). Furthermore, dTTP is an inhibitor of pyrimidine reduction and dGTP is a negative feedback inhibitor of GDP reduction and inhibits the reduction of pyrimidines. Actually, the allosteric regulation of mammalian ribonucleotide reductase appears to be even more complex than described above (Hards and Wright 1984; Fox 1989), but this general model (Fig. I), although not entirely satisfying, has been very useful for investigations involving many types of mammalian cells (Wright 1989). Structural features of the enzyme are also quite interesting. Like the enzyme from Escherichia coli, the mammalian ribonucleotide reductase can be separated into two dissimilar protein components (Fig. 2) that are often referred to as M1 and M2 (Wright 1989; Wright et al. 1989; Hopper 1972). Substrates and effectors bind to protein MI, which exists as a dimer with a molecular weight of 170 000 (Thelander et al. 1980). Protein M2 is a dimer of molecular weight of

A Substrate binding site

FIG. 2. Mammalian ribonucleotide reductase model showing the MI and M2 components. Note the M1 activity (0) and ) and the M2 nonheme iron and tyrosylsubstrate s~ecificitv(0sites. free radical. For furtkr information, see Wright (1989). recent study has indicated that the iron content approaches four per B2 subunit (M2 equivalent) from E. coli (Lynch et al. 1989), suggesting that the iron content per M2 component may be higher than shown in the figure. ~~


88 000 (Thelander et al. 1985; McClarty et al. 1987b) and contains nonheme iron that stabilizes a unique tyrosyl-free radical (McClarty et al. 1990), essential for ribonucleotide reduction (McClarty et al. 19876). The existence of an organic free radical as part of the functional M2 component makes it possible to measure active M2 protein in cells by electron paramagnetic resonance spectroscopy (McClarty et al. 1987b; Wright et al. 1987). Enzyme activity requires the presence of both M1 and M2, but different mechanisms regulate the levels of these two proteins during cell growth (Choy et al. 1988; Eriksson and Martin 1981). This is in contrast to the situation in E. coli, where the two genes encoding proteins equivalent to M1 and M2 (B1 and B2) are found in a single operon and their syntheses are coordinately controlled (Hanke and Fuchs 1983; Carlson et al. 1984). Furthermore, it has been found that the mammalian genes encoding M1 and M2 are located on different chromosomes (Tonin et al. 1987; Yang-Feng et al. 1987) and there are pseudogenes for M2 (Wright et al. 1987; Yang-Feng et al. 1987). There are many reports describing interesting correlations between alterations in ribonucleotide reductase activity and modifications in important biological properties of cells (reviewed in Wright 1989; Wright et al. 1989). For example, elevations in ribonucleotide reductase activity have been reported to be linked to neoplastic properties of cells (Weber 1983), and attempts are being made to exploit the key role of ribonucleotide reduction in cell proliferation by developing antitumor drugs that selectively inhibit the reductase activity (e.g., Veale et al. 1988; Weckbecker et al. 1988). Ribonucleotide reductase has been described as a mutator gene, since alterations in activity leading to changes in deoxyribonucleotide pools, the substrates for DNA polymerase, can significantly modify spontaneous mutation rates of cells (Weinberg et al. 1981). In addition, it has been reported that a combination of the complex allosteric properties of ribonucleotide reductase (Fig. 1) and abnormal deoxyribonucleotide concentrations brought about by alterations in other enzyme activities are important in certain immunodeficiency diseases in man (Ullman et al. 1979; Williams et al. 1987). Very recent studies have suggested an early key



M1 and M2


Ribonucleotide Reduction

Biochem. Cell Biol. Downloaded from by University of Otago on 01/01/15 For personal use only.




Hours of Treatment

FIG. 4. A model for the regulation of DNA synthesis in proliferating cells by M2 protein levels and deoxyribonucleotides. M21, increased levels of M2 protein; ribonucleotide reduction 1,elevation in ribonucleotide reductase activity.

FIG. 3. Ribonucleotide reductase activity and Northern blot analysis of BALB/c 3T3 fibroblasts treated with 0.1 pM TPA for various periods of time, as shown. (A) CDP reductase activity was measured and expressed relative to enzyme activity in untreated cells. (B, C) Northern blot analysis of (B) M2 mRNA levels and (C) glyceraldehyde-3-phosphate dehydrogenase mRNA levels as a control for loading was performed as described (Choy et al. 1989). The positions of 18s and 28s rRNA are indicated. For further information, see Choy et al. (1989).

role for ribonucleotide reductase, particularly the M2 protein, in mechanisms in tumor promotion (Choy et al. 1989). Figure 3 shows that a rapid transient increase in ribonucleotide reductase activity is observed with mouse fibroblasts, within 0.5 h of treatment of the cells with TPA. Northern blot analysis of the two enzyme components showed a slight transient increase of M1 mRNA and a dramatic transient elevation of M2 mRNA within 0.5 h of TPA treatment (Fig. 3). Similar changes in M2 protein levels were observed in Western blot experiments as well (Choy et al. 1989). It is clear from the above examples that mammalian ribonucleotide reductase is interesting from a regulatory, structural, and cell biology point of view.

Mammalian cell mutants altered in ribonucleotide reductase Many years ago, our laboratory initiated a genetic study of ribonucleotide reductase (Wright and Lewis 1974; Lewis and Wright 1974). The multiplicity and specificity of modifications that can occur from mutational alterations far exceed those that can be induced by physical or chemical agents (Till et al. 1973), and we reasoned that mutants altered in ribonucleotide reductase would be valuable tools for analyzing the complex properties of the mammalian enzyme (Wright et al. 1980, 1981; Lewis and Wright 1978a). In general, cytotoxic drugs whose intracellular target is ribonucleotide reductase have been used as selective agents in cell culture to isolate drug-resistant cell lines with specific alterations in ribonucleotide reductase (Wright 1989; Wright et al. 1989). Although many potentially useful drugs are available and some have been used in mutant selections (Wright 1989), the antitumor agent hydroxyurea has turned out to be one of the most valuable. Hydroxyurea enters mammalian cells by a diffusion process (Tagger et al. 1987), is cytotoxic for proliferating cells (Wright and Lewis 1974), and specifically inhibits ribonucleotide reductase by destabilizing the iron centre of protein M2 and thereby destroying the tyrosyl-free radical needed for enzyme activity (McClarty et al. 1990). Biochemists and cell biologists are familiar with hydroxyurea as a DNA synthesis inhibitor and cell-synchronizing agent (Ashihara and Baserga 1979). The drug has also been useful from a clinical point of view, in the treatment of solid tumors as well as acute and chronic leukemia (Bolin et al. 1982), in the treatment of polycythemia Vera (Donovan et al. 1984), in controlling the proliferation of psoriasis (McDonald 1981), and as a radiation potentiator (Piver et al. 1983). Drug resistance may occur by a number of different mechanisms (Wright et al. 1980), and our biochemical studies have shown that hydroxyurea-resistant cell lines contain elevated levels of ribonucleotide reductase activity exhibiting either a wild-type sensitivity or a decreased sensitivity to hydroxyurea inhibition (e.g., Wright 1989; Wright et al. 1989; Dick and Wright 1984; Lewis and Wright 1979, 1978b; McClarty et al. 1986a; Tagger and Wright 1988). Molecular studies with M1- and M2-specific cDNAs and monoclonal


TABLE1. A general summary of some of the types of changes in protein, mRNA levels, and gene copy number observed as mammalian cells become progressively more resistant t o the cytotoxic effects of hydroxyurea

Biochem. Cell Biol. Downloaded from by University of Otago on 01/01/15 For personal use only.



ODC, p.5-8

h4 1


Gene No.



Gene No.

Gene No.

NOTE: Drug resistance characteristics increase from top to bottom as shown by the arrow on the left-hand side. t , increased concentration or increased gene copy number as compared with wild-type cells. Changes in ferritin expression are not shown.

or polyclonal antibodies have shown that low-level resistance to hydroxyurea can occur by a relatively straightforward mechanism, involving elevations in M2 message and protein levels, without any changes in M1 gene expression (Wright 1989; Wright et al. 1987; Choy et al. 1988; Tagger and Wright 1988). These M2 alterations are usually, but not always, accompanied by M2 gene amplification (Wright et al. 1987; Choy et al. 1988; Tagger and Wright 1988). Furthermore, reversion from M2 gene amplified drug resistance to drug sensitivity is accompanied by a decline in M2 gene copy number and M2 message (McClarty et al. 1987a). In keeping with the findings observed with mammalian cells, studies with the yeast Saccharomyces cerevisiae have also shown that sensitivity to the cytotoxic effects of hydroxyurea is dependent upon the message levels for the small subunit (M2 equivalent) of ribonucleotide reductase (Rittberg and Wright 1989). DNA amplification studies with hydroxyurearesistant and revertant mammalian cell lines and genemapping experiments have shown that the genes encoding M2, ornithine decarboxylase and p5-8 (a gene of unknown function), are closely linked and form part of a single amplicon in human and hamster cells (Tonin et al. 1987; Srinivasan et al. 1987; McClarty et al. 1988b; Tonin et al. 1989). There is also evidence that the myc gene may be amplified and part of a M2 amplicon in some hydroxyurearesistant cell lines (Tonin et al. 1989). The hydroxyurearesistant and revertant cell lines are interesting model systems, potentially useful for studies aimed at gaining information on molecular mechanisms responsible for gene coamplification (McClarty et al. 1988b). The finding that low to intermediate hydroxyurea resistance in a variety of mammalian cell lines (human, hamster, rat, and mouse) involves changes in ribonucleotide reductase through elevations in M2 gene expression, without changes in M1 gene expression (Wright 1989; Wright et al. 1987; Choy et al. 1988; Tagger and Wright 1988), supports the concept that the M2 protein component is limiting for ribonucleotide reduction in proliferating cells and the levels of M2 protein are critically important for the synthesis of DNA and cell proliferation. This point is emphasized in the model presented in Fig. 4. Prior to S phase, an elevation in M2 protein is required and the subsequent association between M1 and M2 components leads to ribonucleotide reduction. This activity occurs in the cytoplasm of the cell (Engstrom et al. 1984) and the deoxyribonucleotides formed have two important roles to play. They can enter the nucleus, eventually becoming substrates for DNA polymerase, and they can act as positive and (or) negative effectors in the

cytoplasm by regulating substrate specificity and overall activity of ribonucleotide reductase. This fine-tuned mechanism ensures that there will be a continuous supply and a proper balance of the critical deoxyribonucleotides required for DNA synthesis. One of the aims in isolating hydroxyurea-resistant mutant lines was to obtain cell lines altered in a variety of regulatory mechanisms affecting ribonucleotide reduction. Studies carried out with lines exhibiting low to very high drug resistance characteristics have indeed revealed many different and interesting mechanisms for modifying the expression of the two components of ribonucleotide reductase. As noted earlier, low to intermediate drug resistance mechanisms involved alterations in M2 gene expression without changes in M1. However, high to very high drug resistance mechanisms were accompanied by interesting alterations in M1 gene expression as well (McClarty et al. 1987b; Choy et al. 1988; Hurta and Wright 1990a, 1990b; Cocking et al. 1987). As a general rule, as cells become more resistant to the cytotoxic effects of hydroxyurea, the alterations in M2 gene expression and eventually M1 gene expression become progressively more complex; the mechanisms affected included M2 transcription, M2 gene amplification, M2 translation, M1 transcription, and eventually M1 gene amplification (McClarty et al. 19876; Wright et al. 1987; Choy et al. 1988; Tagger and Wright 1988; Hurta and Wright 1990a, 1990b). A general summary of this concept is shown in Table 1. Clearly, mutants overproducing M1 and (or) M2 proteins provide a novel opportunity to investigate ribonucleotide reduction and its relationship to DNA synthesis, at many different levels of regulation. Such studies are now underway in several laboratories (e.g., Wright 1989; Wright et al. 1989; Cocking et al. 1987; Albert et al. 1987; Carter et al. 1989; Thelander and Berg 1986). Modulation of ribonucleotide reductase expression by hydroxyurea We have noticed that ribonucleotide reductase activity is elevated in cells that have been cultured in the presence of hydroxyurea (Wright et al. 1980, 1981; McClarty et al. 1986~)and we have been interested in determining the mechanism responsible for these observations. Recent studies have been carried out with a hydroxyurea-resistant mouse cell line that contains increased levels of M1 and M2 proteins owing to increased concentrations of mRNAs for both components (McClarty et al. 1987b). Interestingly, we observed (McClarty et al. 1988a) that the levels of both protein components were further elevated in the presence of




Biochem. Cell Biol. Downloaded from by University of Otago on 01/01/15 For personal use only.


b -


H- Ferritin



FIG. 5. Hydroxyurea effects on M1 and M2 protein levels. Western blot analysis was carried out with cell extracts prepared from a hydroxyurea-resistant mouse L cell line called HR-5.OSC2, which contains elevated levels of M1 (2- to 3-fold) and M2 (about 50-fold) proteins, even when cells are cultured in the absence of hydroxyurea. (A) Western blot analysis for protein MI with 25 pg cell extract protein loaded in each lane. (B) Western blot analysis for protein M2 with 10 yg cell extract protein loaded in each lane. Individual lanes represent protein extract prepared from cells grown in the absence (a), and presence of 1.0 (b), 5.0 (c), and 10 ( d ) mM hydroxyurea for 3 days. For further information about H ~ - 5 . 0 cells ~ ~ 2and the procedures see McClarty et al. (1986b, 19876, 1988a, 1990). hydroxyurea, in a time-dependent and drug concentration manner (Fig. 5 ) , and this elevation was not accompanied by increases in their corresponding mRNAs. Therefore, hydroxyurea is capable of modifying ribonucleotide reductase expression posttranscriptionally (McClarty et al. 1988a). This mechanism(s) would be important in allowing cells to survive in normally cytotoxic concentrations of hydroxyurea and could point to normal mechanisms involved in controlling the levels of the two enzyme components. Further work has shown that two processes are important. Drug-induced protein elevations were brought about by an increase in the half-lives of the M1 and M2 components and by increasing their rates of biosynthesis (McClarty et al. 1988a). Total cellular protein synthesis rates were essentially unaffected by hydroxyurea treatment, indicating some specificity for the M1 and M2 proteins. Increased protein stabilization may be due to decreased susceptibility of M1 and M2 to proteolytic degradation in the presence of hydroxyurea, similar to the way in which metabolic inhibitors affect the rate of degradation of dihydrofolate reductase (Cowan et al. 1986), and it is possible that alterations in deoxyribonucleotide pools in hydroxyurea-treated cells (Richard 1988) may mediate the drug effects observed on M1 and M2 protein biosynthesis, much the way in which polyamine pools may regulate ornithine decarboxylase biosynthesis (McConlogue et al. 1986). The precise mechanisms responsible for these drug-induced alterations are certain to be interesting, but are not yet fully understood.

Iron, ferritin, and ribonucleotide reductase Cell proliferation is dependent upon the availability of iron in a soluble form (Theil 1987; Drysdale 1988), due to the iron requirements of proteins participating in oxygen activation, electron transfer, and ribonucleotide reduction.

FIG. 6. Northern blot analysis of H-ferritin mRNA levels in (A) wild-type Chinese hamster ovary cells and (B) Chinese hamster ovary hydroxyurea-resistant cells called H ~ - 1 2 ~Northern ~8. blot analysis of L-ferritin mRNA levels in (C) wild-type rat L6 cells and (D) rat L6 hydroxyurea resistant cells called H ~ - 1 As . controls, the filters were probed with P-actin 32~-labelled cDNA. The position of the 18s rRNA is indicated. For further information, see McClarty et al. (1988~). The protein that maintains iron in a form available for these processes is ferritin. This protein is a highly conserved heteropolymer of 24 subunits, which are products of the ferritin H-chain and the ferritin L-chain genes (Theil 1987; Drysdale 1988). Among the functions of ferritin, it is worth noting that it stores, detoxifies, and transports iron, and in the presence of a reductant it can release Fe2+ and facilitate free radical generation (Theil 1987; Thomas and Aust 1986). Hydroxyurea treatment of cells or extracts leads to a loss of ribonucleotide reductase activity due to destruction of the tyrosyl-free radical of the iron-containing M2 component (McClarty et al. 1987b; Wright et al. 1987). In a recent report, we have demonstrated that hydroxyurea-inactivated protein M2 contains a destabilized iron centre, which can readily lose its iron (McClarty et al. 1990). The radical of the mammalian protein can be regenerated by the addition of dithiothreitol and iron in the presence of oxygen (McClarty et al. 1987b). These observations suggest that drug-induced inactivation of protein M2 involves a release of ~ e and ~ regeneration + of a functional M2 protein requires acquisition of Fez+ from a suitable donor (McClarty et al. 19876, 1990). In mammalian cells, it is tempting to suggest that ferritin may be a donor of ferrous iron for regeneration of functional M2 protein. If this relationship between M2 and ferritin is correct, there are at least two reasons to expect ferritin gene expression to be altered in cell lines selected for resistance to hydroxyurea. The increased levels of M2 protein observed in all hydroxyurearesistant cell lines (Wright 1989; Wright el al. 1989) may require increases in ferritin, to detoxify iron released during growth in drug supplemented medium and to provide iron



+ Ferritin

Mp- Iron (active)



MI ==*





Biochem. Cell Biol. Downloaded from by University of Otago on 01/01/15 For personal use only.

(active) (inactive) FIG. 7. Relationships between hydroxyurea effects, MI,M2, ferritin, and DNA synthesis. M21, increased level of M2 protein; ferritin t , increased level of ferritin; HU, hydroxyurea. The broken arrow between iron and ferritin indicates that iron induces ferritin biosynthesis and increases cellular ferritin levels.

required for functional M2 free radical generation. We have examined H- and L-ferritin gene expression in 14 independent hydroxyurea-resistant human, hamster, rat, and mouse cell lines cultured in drug-free medium (McClarty et al. 1990; Hurta and Wright 1990b), and in every case we observed an elevation in H- and (or) L-ferritin mRNA levels (Fig. 6). This increase in ferritin message level usually occurred in the absence of ferritin gene amplification (McClarty et al. 1990), but two very highly hydroxyurea-resistant lines contained amplifications of both the H- and the L-ferritin genes (Hurta and Wright 1990b). Elevation of ferritin protein levels in resistant cells was also observed (McClarty et al. 1990; Hurta and Wright 1990b). In addition, we have recently found that reversion from resistance to hydroxyurea sensitivity correlated with a decline in ferritin message and protein levels (R.A.R. Hurta and J.A. Wright, unpublished data). Interestingly, selection for hydroxyurea resistance leads to the isolation of mutants altered in ferritin gene expression, and to the best of our knowledge, these are the only somatic cell mutants that have been isolated with modifications in expression of this highly regulated protein. One other interesting relationship between hydroxyurea, ribonucleotide reductase, and ferritin is our observation that hydroxyurea treatment of cells leads to an increased rate of ferritin biosynthesis in the absence of changes in H- or L-ferritin mRNA levels (McClarty et al. 1990). This bears a striking resemblance to our previous finding, discussed earlier, that hydroxyurea treatment also leads to elevation in the rates of M1 and M2 protein biosynthesis (McClarty et al. 1988a). The various relationships described above give the strong impression that the regulation of mammalian ribonucleotide reductase is very much dependent upon the presence and regulation of ferritin, the iron-storage protein. Hydroxyurea effects and relationships between MI, M2, ferritin and DNA synthesis: a possible model Using the observations outlined in this review, a possible model has been developed that may help explain the interactions between ribonucleotide reductase, ferritin, and their relationships to the synthesis of DNA, in normal and hydroxyurea-resistant cell lines (Fig. 7). The synthesis of a functional M2 component requires iron to generate the essential tyrosyl-free radical. We suggest that this is provided either directly or indirectly by ferritin. This active iron-containing form of M2 associates with M1 protein during S phase to provide the deoxyribonucleotides required for DNA synthesis. The levels of functional M2


protein are rate limiting for DNA synthesis, and therefore, cell proliferation. In the presence of hydroxyurea and related drugs (Wright 1989; Wright et al. 1989), the iron centre of M2 is destabilized, iron is lost, and the tyrosyl-free radical is destroyed, leading to a loss of ribonucleotide reductase activity and cell death. Hydroxyurea-resistant cell lines are able to proliferate in the presence of drug because enough functional iron-containing M2 protein is available to combine with M1 to carry out ribonucleotide reduction. This occurs for several reasons. Clearly, the abnormally high levels of M2 protein in drug-resistant lines and the accompanying increases in M1 protein observed in very highly resistant lines are essential for cell survival. Furthermore, hydroxyurea-resistant cells cultured in the presence of drug exhibit another increase in M2 protein and elevate M1 protein as well, through increased protein stabilization and increased protein biosynthetic rates. The additional increase in M1 protein may be advantageous because M1 components are probably removed from participating in ribonucleotide reduction, due to M1 interactions with elevated levels of inactive iron-lacking M2 proteins. An increase in M1 biosynthesis in the presence of hydroxyurea would, therefore, provide a larger pool of free M1 components. Although the elevations in M2 protein are required for cell survival in the presence of drug, we also know that much of the M2 protein in drug-resistant cells is in an inactive iron-decreased state, when cells are cultured in the presence of hydroxyurea (McClarty et al. 1987b). Therefore, at least one other alteration is required and responsible for converting inactive M2 to an active functional protein. Ferritin levels are elevated in hydroxyurea-resistant cell lines and ferritin biosynthetic rates are also increased in cells grown in the presence of hydroxyurea. This favours conversion of inactive (iron deficient) M2 protein to a functional form with the iron centre stabilizing a tyrosyl-free radical, capable of associating with unbound M1 and resulting in ribonucleotide reduction, DNA synthesis, and eventually cell proliferation. The balance between active and inactive M2 in any cell line would be regulatory for DNA synthesis and would determine the concentrations of hydroxyurea allowable for proliferation in the presence of hydroxyurea. The model also leads to an intriguing and very speculative question. Is hydroxyurea actually mimicking the effects of a natural cellular compound that may be involved in the regulation of DNA synthesis, through hydroxyurea-like effects upon ribonucleotide reduction? Acknowledgements This work was supported by funds from the National Cancer Institute of Canada and the Natural Sciences and Engineering Research Council of Canada (NSERC) to J.A.W. B.K.C. thanks the Manitoba Health Research Council (M.H.R.C.) for a graduate student fellowship; R.A.R.H. thanks the Sellers Foundation Department of Internal Medicine, University of Manitoba, for a postdoctoral fellowship; G.A.M. thanks M.H.R.C. and the Medical Research Council (Canada) for previous postdoctoral fellowship support; and A.Y.T. thanks the Manitoba Cancer Treatment and Research Foundation and NSERC for previous graduate student fellowships. J.A.W. is a Terry Fox Senior Research Scientist of the National Cancer Institute of Canada.

Biochem. Cell Biol. Downloaded from by University of Otago on 01/01/15 For personal use only.



ALBERT,D.A., GUDAS,L.J., and NODZENSKI, E. 1987. Deoxyribonucleotide metabolism and cyclic AMP resistance in hydroxyurea resistant S49 T-lymphoma cells. J. Cell. Physiol. 130: 262-269. ASHIHARA,T., and BASERGA,R. 1979. Cell synchronization. Methods Enzymol. 58: 248-262. BOLIN,R.W., ROBINSON, W.A., SUTHERLAND, J., and HAMMAN, R.E. 1982. Busulfin versus hydroxyurea in long-term therapy of chronic myelogenous leukemia. Cancer (Philadelphia), 50: 1683-1686. CARLSON, J., FUCHS,J.A., and MESSING, J. 1984. Primary structure of the Escherichia coli ribonucleoside diphosphate reductase operon. Proc. Natl. Acad. Sci. U.S.A. 81: 4294-4297. CARTER,G.L., THOMPSON, D.P., and CORY,J.G. 1989. Mechanisms of drug resistance to inhibitors directed to the individual subunits of ribonucleotide reductase. Cancer Commun. 1: 13-20. CHOY,B.K., MCCLARTY, G.A., CHAN,A.K., THELANDER, L., and WRIGHT,J.A. 1988. Molecular mechanisms of drug resistance involving ribonucleotide reductase: hydroxyurea resistance in a series of clonally related mouse cell lines selected in the presence of increasing drug concentrations. Cancer Res. 48: 2029-2035. CHOY,B.K., MCCLARTY, G.A., and WRIGHT,J.A. 1989. Transient elevation of ribonucleotide reductase activity, M2 mRNA and M2 protein in BALB/c 3T3 fibroblasts in the presence of 12-0-tetradecanoylphorbol-13-acetate.Biochem. Biophys. Res. Commun. 162: 1417-1424. COCKING, J.M., TONIN,P.N., STOKOE,N.M., WENSING,E. J., LEWIS,W.H., and SRINIVASAN, P.R. 1987. The gene for the M1 subunit of ribonucleotide reductase is amplified in hydroxyurearesistant hamster cells. Somatic Cell. Mol. Genet. 13: 221-233. COWAN,K., GOLDSMITH, M., RICCIARDONE, M., LEVINE,R., E., and JOLIVET,J. 1986. Regulation of RUBALCABA, dihydrofolate reductase in human breast cancer cells and in mutant hamster cells transfected with a human dihydrofolate reductase minigene. Mol. Pharmacol. 30: 69-76. DICK,J.E., and WRIGHT,J.A. 1984. Human diploid fibroblasts with alterations in ribonucleotide reductase activity, deoxyribonucleotide pools and in vitro lifespan. Mech. Ageing Dev. 26: 37-49. DONOVAN, P.B., KAPLAN,M.E., GOLDBERG, J.D., TATARSKY, I., E.B., KNOSPE,W.H., LASZLO,J., NAJEAN,Y., STILBERSTEIN, MACK,K., BERK,P.D., and WASSERMAN, L.R. 1984. Treatment of polycythemia Vera with hydroxyurea. Am. J. Hematol. 17: 329-334. DRYSDALE, J.W. 1988. Human ferritin gene expression. Prog. Nucleic Acid Res. Mol. Biol. 35: 127-155. Y., ROZELL,B., HANSSON, H.-A., STEMME, S., and ENGSTROM, THELANDER, L. 1984. Localization of ribonucleotide reductase in mammalian cells. EMBO J. 3: 863-867. ERIKSSON,S., and MARTIN,D.W., JR. 1981. Ribonucleotide reductase in cultured mouse lymphoma cells. Cell cycle-dependent variation in the activity of subunit protein M2. J. Biol. Chem. 256: 9436-9440. FOX,R.M. 1989. Changes in deoxynucleoside triphosphate pools induced by inhibitors and modulators of ribonucleotide reductase. Int. Encycl. Pharmacol. Ther. 128: 113-125. HANKE,P.D., and F u c ~ s ,J.A. 1983. Characterization of the mRNA coding for ribonucleoside diphosphate reductase in Escherichia coli. J. Bacteriol. 156: 1192-1 197. HARDS,R.G., and WRIGHT,J.A. 1984. Regulation of ribonucleotide reductase activity in intact mammalian cells. Arch. Biochem. Biophys. 231: 9-16. HOPPER,S. 1972. Ribonucleotide reductase of rabbit bone marrow. I. Purification, properties, and separation into two protein fractions. J. Biol. Chem. 247: 3336-3340. HURTA,R.A.R., and WRIGHT,J.A. 1990a. Amplification of the genes for both components of ribonucleotide reductase in hydroxyurea resistant mammalian cells. Biochem. Biophys. Res. Commun. 167: 258-264.

VOL. 68, 1990

-1990b. Mammalian drug resistant mutants with multivle

gene amplifications: Genes coding the M1 component -of ribonucleotide reductase. the M2 comDonent of ribonucleotide reductase, ornithine decarboxylase, p5-8, the H-subunit of ferritin, and the L-subunit of ferritin. Biochim. Biophys. Acta. In press. LEWIS,W.H., and WRIGHT,J.A. 1974. Altered ribonucleotide reductase activity in mammalian tissue culture cells resistant to hydroxyurea. Biochem. Biophys. Res. Commun. 60: 926-933. -1978a. Genetic characterization of hydroxyurea resistance in Chinese hamster ovary cells. J. Cell. Physiol. 97: 73-86. -197%. Ribonucleotide reductase from wild type and hydroxyurea-resistant Chinese hamster ovary cells. J. Cell. Physiol. 97: 87-98. -1979. Isolation of hydroxyurea-resistant CHO cells with altered levels of ribonucleotide reductase. Somatic Cell Genet. 5: 83-96. LYNCH,J.B., JUAREZ-GARCIA, C., MUNCH,E., and QUE,L., JR. 1989. Mossbauer and EPR studies of the binuclear iron center in ribonucleotide reductase from Escherichia coli. J. Biol. Chem. 264: 8091-8096. MCCLARTY,G.A., CHAN,A.K.M., and WRIGHT,J.A. 1986a. Characterization of a mouse cell line selected for hydroxyurea resistance by a stepwise procedure: drug dependent overproduction of ribonucleotide reductase activity. Somatic Cell Mol. Genet. 12: 121-131. -1986b. Hydroxyurea-induced conversion of mammalian ribonucleotide reductase to a form hypersensitive to bleomycin. Cancer Res. 46: 45 16-4521. MCCLARTY, G.A., CHAN,A.K., CHOY,B.K., and WRIGHT,J.A. 1987a. Reversion of hydroxyurea resistance, decline in ribonucleotide reductase activity and loss of M2 gene amplification. Biochem. Biophys. Res. Commun. 145: 1276-1282. Y ., WRIGHT,J.A., MCCLARTY, G.A., CHAN,A.K., ENCSTROM, and THELANDER, L. 19876. Elevated expression of M1 and M2 components and drug-induced posttranscriptional modulation of ribonucleotide reductase in a hydroxyurea-resistant mouse cell line. Biochemistry, 26: 8004-801 1. L., MCCLARTY, G.A., CHAN,A.K., CHOY,B.K., THELANDER, and WRIGHT,J.A. 1988a. Molecular mechanisms responsible for the drug-induced posttranscriptional modulation of ribonucleotide reductase levels in a hydroxyurea-resistant mouse L cell line. Biochemistry, 27: 7524-7531. MCCLARTY,G.A., TONIN,P.N., SRINIVASAN, P.R., and WRIGHT, J.A. 19886. Relationships between reversion of hydroxyurea resistance in hamster cells and the co-amplification of ribonucleotide reductase M2 component, ornithine decarboxylase and P5-8 genes. Biochem. Biophys. Res. Commun. 154: 975-981. MCCLARTY, G.A., CHAN,A.K., CHOY,B.K., and WRIGHT,J.A. 1990. Increased ferritin gene expression is associated with increased ribonucleotide reductase gene expression and the establishment of hydroxyurea resistance in mammalian cells. J. Biol. Chem. 265: 7539-7547. MCCONLOGUE, L., DANA,S., and COFFINO,P. 1986. Multiple mechanisms are responsible for altered expression of ornithine decarboxylase in overproducing variant cells. Mol. Cell. Biol. 6: 2865-2871. MCDONALD, C.J. 1981. The uses of systemic chemotherapeutic agents in psoriasis. Pharmacol. Ther. 14: 1-24. PIVER,M S . , BARLOW, J.J., VONGTAMA, V., and BLUMENSON, L. 1983. Hydroxyurea: a radiation potentiator in carcinoma of the uterine cervix. Am. J. Obstet. Gynecol. 147: 803-808. RICHARD,P. 1988. Interactions between deoxyribonucleotide and DNA synthesis. Annu. Rev. Biochem. 57: 349-374. RITTBERG, D.A., and WRIGHT,J.A. 1989. Relationships between sensitivity to hydroxyurea and 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone (MAIQ) and ribonucleotide reductase RNR2 mRNA levels in strains of Saccharomyces cerevisiae. Biochem. Cell Biol. 67: 352-357.


Biochem. Cell Biol. Downloaded from by University of Otago on 01/01/15 For personal use only.

SRINIVASAN, P.R., TONIN,P.N., WENSING,E.J., and LEWIS, W.H. 1987. The gene for ornithine decarboxylase is co-amplified in hydroxyurea-resistant hamster cells. J. Biol. Chem. 262: 12 871 - 12 878. TAGGER,A.Y., and WRIGHT,J.A. 1988. Molecular and cellular characterization of drug resistant hamster cell Iines with alterations in ribonucleotide reductase. Int. J. Cancer, 42: 760-766. TAGGER, A.Y., Boux, J., and WRIGHT,J.A. 1987. Hydroxyurea [I4c]urea uptake by normal and transformed human cells: evidence for a mechanism of passive diffusion. Biochem. Cell Biol. 65: 925-929. THEIL,E.C. 1987. Ferritin: structure, gene regulation, and cellular function in animals, plants and microorganisms. Annu. Rev. Biochem. 56: 289-315. THELANDER, L., and BERG,P. 1986. Isolation and characterization of expressible cDNA clones encoding the M1 and M2 subunits of mouse ribonucleotide reductase. Mol. Cell. Biol. 6: 3433-3442. THELANDER, L., ERIKSSON,S., and AKERMAN,M. 1980. Ribonucleotide reductase from calf thymus. Separation of the enzyme into two identical subunits, proteins M1 and M2. J. Biol. Chem. 255: 7426-7432. THELANDER, M., GRASLUND, A., and THELANDER, L. 1985. Subunit M2 of mammalian ribonucleotide reductase. Characterization of a homogeneous protein isolated from M2 overproducing mouse cells. J. Biol. Chem. 260: 2737-2741. THOMAS,C.E., and AUST,S. 1986. Reductive release of iron from ferritin by cation free radicals of paraquat and other bipyridyls. J. Biol. Chem. 261: 13 064 - 13 070. TILL, J.E., BAKER, R.M., BRUNETTE,D.M., LING, V., THOMPSON, L.H., and WRIGHT,J.A. 1973. Genetic regulation of membrane function in mammalian cells in culture. Fed. Proc. 32: 29-33. TONIN,P.N., STALLING, R.L., CARMAN,M.D., BERTINO,J.R., WRIGHT,J.A., SRINIVASAN, P.R., and LEWIS, W.H. 1987. Chromosomal assignment of amplified genes in hydroxyurearesistant hamster cells. Cytogenet. Cell Genet. 45: 102-108. TONIN,P.N., YEGER,H., STALLINGS, R.L., SRINIVASAN, P.R., and LEWIS, W.H. 1989. Amplification of the N-myc and ornithine decarboxylase genes in human neuroblastoma and hydroxyurea-resistanthamster cell lines. Oncogene, 4: 11 17-1 121. ULLMAN, B., GUDAS,L.J., CLIFT,S.M., and MARTIN,D.W., JR. 1979. Isolation and characterization of purine nucleoside phosphorylation-deficient T lymphoma cells and secondary mutants with altered ribonucleotide reductase: a genetic model for immunodeficiency disease. Proc. Natl. Acad. Sci. U.S.A. 76: 1074-1078. VEALE,D., CARMICHAEL, J., CANTWELL, B.M.J., ELFORD,H.L., BLACKIE, R., KERR,D. J., KAYE,S.B., and HARRIS,A.L. 1988.



Phase 1 and pharmacokinetic study of didox: a ribonucleotide reductase inhibitor. Br. J. Cancer, 58: 70-72. WEBER,G. 1983. Biochemical strategy of cancer cells and the design of chemotherapy: G.H.A. Clowes Memorial Lecture. Cancer Res. 43: 3466-3492. WECKBECKER, G., WECKBECKER, A., LIEN,E.J., and CORY,J.G. 1988. Effect of N-hydroxy-N' -amino-guanidine isoquinoline in combination with other inhibitors of ribonucleotide reductase on L1210 cells. J. Natl. Cancer Inst. 80: 491-496. WEINBERG,G., ULLMAN,B., and MARTIN,D.W., JR. 1981. Mutator phenotypes in mammalian cell mutants with distinct biochemical defects and abnormal deoxyribonucleoside triphosphate pools. Proc. Natl. Acad. Sci. U.S.A. 78: 2447-2451. WLLIAMS,S.R., GEKELER,V., MCIVOR,R.S., and MARTIN, D.W., JR. 1987. A human purine nucleoside phosphorylase deficiency caused by a single base change. J. Biol. Chem. 262: 2332-2338. WRIGHT,J.A. 1989. Altered mammalian ribonucleotide reductase from mutant cell lines. Int. Encycl. Pharmacol. Ther. 128: 89-111. WRIGHT,J.A., and LEWIS, W.H. 1974. Evidence of a common site of action for the antitumor drugs, hydroxyurea and guanazole. J. Cell. Physiol. 83: 437-440. WRIGHT, J.A., LEWIS, W.H., and PARFETT, C.L.J. 1980. Somatic cell genetics: a review of drug resistance, lectin resistance and gene transfer in mammalian cells in culture. Can. J. Genet. Cytol. 22: 443-496. WRIGHT,J.A., HARDS,R.G., and DICK, J.E. 1981. Studies of mammalian ribonucleotide reductase activity in intact permeabilized cells: a genetic approach. Adv. Enzyme Regul. 19: 105-127. WRIGHT,J.A., ALAM.T.G., MCCLARTY,G.A., TAGGER,A.Y ., and THELANDER, L. 1987. Altered expression of ribonucleotide reductase and role of M2 gene amplification in hydroxyurearesistant hamster, mouse, rat and human cell lines. Somatic Cell Mol. Genet. 13: 155-165. WRIGHT, J.A., MCCLARTY,G.A., LEWIS, W.H.. and SRINIVASAN, P.R. 1989. Cellular resistance to hydroxyurea and related compounds. In Drug resistance in mammalian cells. Vol. 1 . Edited by R. Gupta. CRC Press, Inc., Boca Raton, FL. pp. 15-27. YANG-FENG,T.L., BARTON,D.E., THELANDER, L., LEWIS, P.R., and FRANCKE,U. 1987. W.H., - SRINIVASAN, Ribonucleotide reductase M2 subunit mapped to four different chromosomal sites in human and mice: functional locus identified by its amplification in hydroxyurea-resistant cell lines. Genomics, 1: 77-86.

Regulation and drug resistance mechanisms of mammalian ribonucleotide reductase, and the significance to DNA synthesis.

Mammalian ribonucleotide reductase, which occupies a key position in the synthesis of DNA, is a highly controlled enzyme activity, because it is solel...
1MB Sizes 0 Downloads 0 Views