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 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)

Generation of metastatic variants in populations of mutator and amplificator mutants.

Genetic instability has been hypothesized by P. C. Nowell and other investigators to be an important aspect of tumor progression that leads to the generation of metastatic variants. In this study we examined the rate of generation of metastatic variants in mutant cell lines having increased rates of spontaneous mutation and gene amplification. Parallel clonal populations of the spontaneous mutation rate mutant thy-49 and the gene amplification mutants YMP1 and YMP7 and their respective wild types were generated and grown to a critical population size. The number of metastatic variants in each clonal population was then determined following iv injection into nude mice. Lung tumors were scored 3-4 weeks after injection of cells, and the mean number per clonal population was determined. Analysis of the means with the Luria-Delbruck fluctuation test showed no significant differences in the rate of generation of metastatic variants produced in the genetically unstable lines compared to their normal counterparts. This study suggests that increased spontaneous mutation and gene amplification rates in mammalian cells are not sufficient on their own to increase the rate of generation of metastatic variants.

Lack of correlation between deoxyribonucleotide pool sizes, spontaneous mutation rates and malignant potential in Chinese hamster ovary cells.

To examine the relationship between altered spontaneous mutation rates and malignant characteristics of cells, two hydroxyurea-resistant Chinese hamster ovary cell lines, with alterations in ribonucleotide reductase, were examined for their rates of spontaneous mutation to 6-thioguanine and ouabain resistance, tumor growth rates and their ability to form experimental lung metastases. The most resistant cell line, HR-R2T, showed no changes in the rate of spontaneous mutation to 6-thioguanine or ouabain resistance compared to the parental wild-type cell line; however, the mutant line formed lung metastases in experimental metastasis assays with BALB/c nu/nu mice, and exhibited metastatic abilities significantly different from the wild-type population. Furthermore, the HR-R2T population did not show imbalances in any of the deoxyribonucleoside triphosphate pool sizes, which are frequently observed in cells altered in ribonucleotide reductase activity. The second hydroxyurea-resistant line, HNR-AT, had gross alterations in dCTP and dGTP pools and although the rate of spontaneous mutation to 6-thioguanione resistance was unaltered, it showed a moderate decrease in the rate of spontaneous mutation to ouabain resistance when compared to the parental wild-type population. Interestingly, the HNR-AT cell line did not form any lung metastases in the experimental metastasis assay. Both mutant cell lines, HR-R2T, and HNR-AT, had increased tumor growth rates in C57 BALB/c "beige" nude (nu/nu) mice as compared to the parental wild-type population. In total, the results obtained with the two mutant cell lines question the association of altered mutation rates with increased metastatic potential. Although several explanations are possible for the altered malignant properties exhibited by HR-R2T and HNR-AT cells, it is interesting to note that the results are consistent with earlier suggestions that changes in ribonucleotide reductase may accompany modifications in the malignant characteristics of cells.

Molecular and cellular characterization of drug resistant hamster cell lines with alterations in ribonucleotide reductase.

Ribonucleotide reductase consists of 2 protein components frequently called M1 and M2. Hydroxyurea specifically inhibits DNA synthesis by interacting with the M2 protein and destroying a unique tyrosyl-free radical. We have carried out a molecular and cellular characterization of 2 Chinese hamster ovary cell lines exhibiting either low (HN(R)-AT) or relatively high (H(R)-R2T) resistance to the cytotoxic effects of hydroxyurea. Both drug-resistant lines have an increased level of ribonucleotide reductase activity. EPR measurements for tyrosyl-free radical content and studies with M1-specific antibodies indicated that the elevation in enzyme activity was entirely due to an increase in the M2 component. Studies with M1 cDNA showed that both drug-resistant cell lines contained a wild-type level of M1 mRNA and a wild-type M1 gene copy number. Studies with M2 cDNA indicated that the 2 drug-resistant lines possessed elevated levels of M2 message that could explain the observed increase in M2 component. The elevation of M2 mRNA in the most resistant line, H(R)-R2T, was due to an increase in M2 gene copy number. The low resistant cell line, HN(R)-AT, exhibited a wild-type M2 gene copy number, indicating that the increase in M2 gene message occurred through a process other than gene amplification. Enzyme kinetic studies with partially purified preparations from both drug resistant lines showed reduced sensitivity to hydroxyurea and to the negative allosteric effector, dATP. In addition to hydroxyurea, H(R)-R2T cells were also resistant to several other drugs whose site of action is the M2 component. Furthermore, H(R)-R2T cells were not cross-resistant to colchicine or puromycin, suggesting that hydroxyurea-resistant cells do not share the multi-drug resistance phenotype, which is frequently associated with cross-resistance to these drugs.

Hydroxy[14C]urea uptake by normal and transformed human cells: evidence for a mechanism of passive diffusion.

The antitumor agent hydroxyurea is a potent inhibitor of cell division and selectivity toxic for rapidly proliferating cells. This drug has been used in the treatment of human cancer and, since drug transport is an important aspect of drug action, we investigated the mechanism of hydroxy[14C]urea uptake by human diploid fibroblasts and their SV40-virus-transformed counterparts. Kinetic analysis of drug uptake, studies with metabolic inhibitors, and estimates of cell/medium distribution ratios and temperature coefficient (Q10) values indicated that hydroxyurea enters normal and SV40-virus-transformed human cells by a mechanism of diffusion.

Altered expression of ribonucleotide reductase and role of M2 gene amplification in hydroxyurea-resistant hamster, mouse, rat, and human cell lines.

Five hamster, mouse, and rat cell lines resistant to the cytotoxic effects of hydroxyurea have been characterized. All cell lines contained increased ribonucleotide reductase activity, elevated levels of the M2 component of ribonucleotide reductase as judged by electron paramagnetic resonance spectroscopy, and increased copies of M2 mRNA as determined by Northern blot analysis. Two species of M2 mRNA were detected in rodent cell lines, a high-molecular-weight species of approximately 3.4 kb in hamster and rat cells and about 2.1 kb in mouse cells. The low molecular-weight M2 mRNA was about 1.6 kb in all rodent lines. Northern blot analysis showed that the mRNA for the other component of ribonucleotide reductase, M1, was not markedly elevated in the drug-resistant cells and existed as a single 3.1-kb species. Four of the five resistant lines contained an M2 gene amplification as determined by Southern blot analysis, providing direct evidence to support earlier suggestions that hydroxyurea resistance is often accompanied by amplification of a ribonucleotide reductase gene. An increase in gene dosage was detected even in cells exhibiting only modest drug-resistance properties. No evidence for amplification of the M1 gene of ribonucleotide reductase was found. In keeping with these observations with drug-resistant rodent lines, a human (HeLa) cell line resistant to hydroxyurea was also found to contain increased levels of two M2 mRNA species (about 3.4 and 1.6 kb) and exhibited M2 gene amplification. One hamster cell line resembled the other resistant rodent lines in cellular characteristics but did not show amplification of either the M1 or M2 gene, providing an example of a drug-resistant mechanism in which an elevation of M2 mRNA has occurred without a concomitant increase in M2 gene copy number.

A comparison of ribonucleotide reductase activities in normal human fibroblast strains with their transformed counterparts.

The objective of this investigation was to examine the relationship between levels of ribonucleotide reductase activity and transformation of two human cell strains. Enzyme activity levels were elevated by 3.2- to 3.5-fold in transformed cells compared directly to the normal human fibroblast strains from which they were derived. There did not appear to be a general correlation between elevated ribonucleotide reductase and increased proliferation abilities as has been previously observed with some rodent tumor cell lines. In keeping with the rise in reductase activity, human transformed cells were relatively more resistant to the cytotoxic effects of hydroxyurea, an antitumor agent whose site of action is ribonucleotide reductase. This indicates that an important point to be considered during drug therapy aimed at the reductase, is the greater sensitivity of normal compared to transformed cells due to differences in enzyme activity. The results of this investigation support the view that an increased ability to reduce ribonucleotides is an important step towards the development of a neoplastic program in human cells.