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 hydroxyurea-resistant 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.(ABSTRACT TRUNCATED AT 250 WORDS)

In the present study, we show that hydroxyurea-inactivated ribonucleotide reductase protein M2 has a destabilized iron center, which readily releases iron. In addition, evidence is presented which indicates that single or multistep selection for hydroxyurea resistance, in a variety of mammalian cell lines, leads to alterations in the expression of the gene for the iron storage protein, ferritin. In all hydroxyurea-resistant cell lines examined, including human, hamster, rat, and mouse, there was an elevation in ferritin heavy (H)- and/or light (L)-mRNA levels, but no change in the corresponding gene copy number. A detailed analysis of ferritin expression in a hydroxyurea-resistant mouse L cell line showed that when compared to its wild type counterpart, there was an increase in H subunit concentration but no significant change in L subunit levels. The increased H/L subunit ratio was not brought about by specific changes in the rates of ferritin subunit biosynthesis, but rather resulted from changes in the post-translational stability of H subunits relative to L subunits in the resistant cell line compared to its parental wild type. Also, we show that treatment of cells with hydroxyurea results in an increased rate of ferritin biosynthesis in the absence of changes in H- or L-mRNA levels. These results indicate that the development of even low level hydroxyurea resistance in mammalian cells may require alterations in ferritin gene expression, and they show an interesting relationship between the expressions of two highly regulated activities, ribonucleotide reductase and ferritin.

A rapid elevation of ribonucleotide reductase activity was observed with BALB/c 3T3 fibroblasts within 12 to 1 hour treatment with 0.1 μM 12-O-tetradecanoylphorbol-13-acetate (TPA). This increase in activity was transient, and returned to about normal levels within 24 to 48 hours. Northern analysis of the two components of ribonucleotide reductase showed a slight transient elevation of M1 mRNA and a marked transient elevation of M2 mRNA after 12 hour TPA treatment. As a positive control, ornithine decarboxylase message levels were also observed to be transiently elevated following identical treatment with TPA. Western blot analysis with M1 and M2 specific monoclonal antibodies indicated that the increase in ribonucleotide reductase activity was primarily due to the transient elevation of the M2 but not the M1 protein during treatment with 0.1 μM TPA. This first demonstration that the tumor promotor, TPA, can cause rapid and transient alterations in ribonucleotide reductase suggests that the enzyme, particularly the M2 component, may play an important role in the critical events involved in the process of tumor promotion.

Mammalian ribonucleotide reductase is a highly regulated, rate-limiting activity responsible for converting ribonucleoside diphosphates to the deoxyribonucleotide precursors of DNA. The enzyme consists of two nonidentical proteins often called M1 and M2, both of which are required for activity. Hydroxyurea is an antitumor agent which inhibits ribonucleotide reductase by interacting with the M2 component specifically at a unique tyrosyl free radical. To obtain further information about drug resistance mechanisms, we have used M1 and M2 complementary DNAs and monoclonal antibodies to investigate the properties of a series of clonally related drug-resistant mouse cell lines, selected by a step-wise procedure for increasing levels of resistance to the cytotoxic effects of hydroxyurea. Several interesting mechanisms have been identified. Each successive drug selection step leading to the isolation of highly resistant cells was accompanied by stable elevations in cellular resistance and ribonucleotide reductase activities. The changes that occurred at each step involved the M2 component. A very early event, occurring at the first step in the selection process, was the amplification of the M2 gene accompanied by an increase in M2 messenger RNA. Although cellular resistance and M2 protein levels increased significantly during drug selection, only a modest change in M2 gene copy number was observed after the initial selection step. Analysis of wild type, moderately resistant, and highly resistant cells indicated that, in addition to M2 gene amplification, posttranscriptional modification also occurred during drug selection. This second mechanism was not due to alterations in protein M2 half-life, but involved an increase in translational efficiency. By increasing the rate of M2 synthesis, without altering degradation rates, resistant cells were able to accumulate high levels of this key regulatory protein. Cells selected for the ability to proliferate in concentrations of drug as high as 4 mM exhibited changes that involved M2, without detectable changes to M1. These results provide further evidence that M1 and M2 levels are controlled by different mechanisms in mammalian cells. Eventually, however, cells required an elevation in the M1 protein, as well as the M2 protein, to survive in a hydroxyurea concentration of 5 mM. These results illustrate the complexity of the drug-resistant phenotype and provide further information about the molecular processes that lead to the development of cells resistant to low, intermediate, and high concentrations of hydroxyurea.

Ribonucleotide reductase, which catalyzes the formation of deoxyribonucleotides from ribonucleoside diphosphate precursors, is the rate-limiting enzyme in DNA synthesis. The enzyme consists of two nonidentical subunits called M1 and M2, both of which are required for activity. Hydroxyurea, a specific inhibitor of DNA synthesis, acts by destroying the unique tyrosyl free radical of protein M2. Previously, we have described a mouse L cell line which exhibited a stable resistance to high concentrations of hydroxyurea. This mutant cell line contains elevated quantities of both proteins M1 and M2 as a result of corresponding increases in the levels of mRNAs for both subunits. Interestingly, both M1 and M2 protein levels were further elevated when mutant cells were cultured in the presence of hydroxyurea, and this elevation was not accompanied by increases in their corresponding mRNAs. These results indicated that hydroxyurea can modulate ribonucleotide reductase expression posttranscriptionally. In this report, we show that the level of both subunits of ribonucleotide reductase responds to hydroxyurea in a drug concentration dependent manner. Furthermore, results from kinetic studies indicate that protein M2 levels rise much more rapidly than protein M1. Pulse-chase experiments indicated that the half-lives of both the M1 and M2 polypeptides are increased by approximately 2-fold when the mutant cells are cultured in the presence of hydroxyurea. We also present evidence indicating that exposure of these cells to hydroxyurea leads to a relatively slow but specific increase in the rate of biosynthesis of both proteins M1 and M2, as assayed by pulse labeling.(ABSTRACT TRUNCATED AT 250 WORDS)

A key rate-limiting reaction in the synthesis of DNA is catalyzed by ribonucleotide reductase, the enzyme which reduces ribonucleotides to provide the deoxyribonucleotide precursors of DNA. The antitumor agent, hydroxyurea, is a specific inhibitor of this enzyme and has been used in the selection of drug resistant mammalian cell lines altered in ribonucleotide reductase activity. An unstable hydroxyurea resistant population of mammalian cells with elevated ribonucleotide reductase activity has been used to isolate three stable subclones with varying sensitivities to hydroxyurea cytotoxicity and levels of ribonucleotide reductase activities. These subclones have been analyzed at the molecular level with cDNA probes encoding the two nonidentical subunits of ribonucleotide reductase (M1 and M2). Although no significant differences in M1 mRNA levels or gene copy numbers were detected between the three cell lines, a strong correlation between cellular resistance, enzyme activity, M2 mRNA and M2 gene copies was observed. This is the first demonstration that reversion of hydroxyurea resistance is directly linked to a decrease in M2 mRNA levels and M2 gene copy number, and strongly supports the concept that M2 gene amplification is an important mechanism for achieving resistance to this antitumor agent through elevations in ribonucleotide reductase.