Molecular Mechanisms of Drug Resistance Involving Ribonucleotide ReducÃ-ase: Hydroxyurea Resistance in a Series of Clonally Related Mouse Cell Lines Selected in the Presence of Increasing Drug Concentrations1

Mammalian ribonucleotide reducÃ-ase is a highly regulated, rate-lim iting activity responsible for converting ribonucleoside diphosphates to the deoxyribonucleotide precursors of DNA. The enzyme consists of two nonidentical proteins often called Ml and M2, both of which are required for activity. Hydroxyurea is an antitumor agent which inhibits ribonucle otide reducÃ-ase by interacting with the M2 component specifically at a unique tyrosyl free radical. To obtain further information about drug resistance mechanisms, we have used Ml 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 reducÃ-ase 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 UNA. 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 amplifi cation, posttranscriptional modification also occurred during drug selec tion. 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 HIM exhibited changes that involved M2, without detectable changes to Ml. These results provide further evidence that Ml and M2 levels are controlled by different mechanisms in mammalian cells. Even tually, however, cells required an elevation in the Ml protein, as well as the M2 protein, to survive in a hydroxyurea concentration of 5 HIM. 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.