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.