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.

The in Vivo Toxicity of Hydroxyurea Depends on Its Direct Target Catalase

“…To determine whether HU resistance was caused by an increase in RNR activity or by RNR activity resistant to HU inhibition, as previously described (9, 17–19), we compared RNR activity between wild-type plants and HU-resistant mutants. RNR enzyme activity in resistant mutants could be inhibited by HU and was not higher than in wild-type plants (Fig.…”

Characterization of the human ribonucleotide reductase M2 subunit gene; genomic structure and promoter analyses

“…The mechanism of HU resistance is known to result from an overproduction of the M2 mRNA leading to an increase in RR protein (Tagger and Wright, 1988;Yen et al, 1994).…”

A phase I pharmacodynamic study of GTI-2040, an antisense oligonucleotide against ribonuclotide reductase, in acute leukemias: a California Cancer Consortium study

“…This low potency allows human tumors to rapidly develop resistance (9-13). Overexpression of M2 mRNA and RR protein has been demonstrated to be a mechanism of HU resistance (5, 14). It is well established that RR activity is closely correlated with DNA synthesis and cell proliferation (15, 16).…”