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

Abstract

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.