Involvement of ribonucleotide reductase in cellular differentiation

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.

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

Tagger

Damen

Greenberg

et al. 1989

J Cancer Res Clin Oncol

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Altered expression of ribonucleotide reductase and role ofM2 gene amplification in hydroxyurea-resistant hamster, mouse, rat, and human cell lines

Wright

Alam

McClarty

et al. 1987

Somat Cell Mol Genet

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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.

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Characterization of a mouse cell line selected for hydroxyurea resistance by a stepwise procedure: Drug-dependent overproduction of ribonucleotide reductase activity

McClarty

Chan

Wright

1986

Somat Cell Mol Genet

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Hydroxyurea was used as a selective agent in culture, to isolate by a stepwise procedure, a unique mouse L cell line called LHF which exhibited a stable resistance to high concentrations of drug (5 mM). LHF cells contained an elevation in ribonucleotide reductase activity which depended upon whether cells were previously cultured in the presence or absence of hydroxyurea. M1 immunoprecipitation and M2 titration experiments indicated that both ribonucleotide reductase subunits were elevated in drug-resistant cells. Interestingly, a very large drug-dependent change in the M2 activity (about a 100-fold) was observed. Studies on enzyme activity with cycloheximide and actinomycin D indicated that the hydroxyurea-dependent increase in activity required de novo protein synthesis and transcriptional activity. These results are different from other ribonucleotide reductase overproducing cell lines previously described, and indicate that hydroxyurea modulates enzyme activity by an interesting mechanism.

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Involvement of ribonucleotide reductase in cellular differentiation

Cited by 10 publications

References 38 publications

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

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

Altered expression of ribonucleotide reductase and role ofM2 gene amplification in hydroxyurea-resistant hamster, mouse, rat, and human cell lines

Characterization of a mouse cell line selected for hydroxyurea resistance by a stepwise procedure: Drug-dependent overproduction of ribonucleotide reductase activity

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