Alterations in the activity and regulation of mammalian ribonucleotide reductase by chlorambucil, a DNA damaging agent

Abstract

Ribonucleotide reductase provides the four deoxyribonucleotides required for the synthesis of DNA. In this study, we examined the hypothesis that alterations in the regulation of ribonucleotide reductase activity may be necessary to provide the deoxyribonucleotides required for DNA repair, following exposure of mammalian cells to DNA damaging agents such as the antitumor agent chlorambucil. We observed a marked transient increase in ribonucleotide reductase activity within 2 h of exposing BALB/c 3T3 mouse cells to DNA damaging concentrations of chlorambucil. Northern blot analysis showed that elevations in activity were accompanied by transient increases in the mRNA levels of both genes (R1 and R2) that code for ribonucleotide reductase. Western blot analysis indicated that only the protein for the limiting component for enzyme activity, R2, was significantly elevated in chlorambucil treated cultures. The chlorambucil effects upon activity and regulation of ribonucleotide reductase occurred without any detectable changes in the rate of DNA synthesis, as would be expected if the elevation in enzyme activity is required for DNA repair. The chlorambucil-induced elevations in R1 and R2 message levels were blocked by treatment of cells with actinomycin D or the tumor promoter 12-O-tetradecanoylphorbol-13-acetate, indicating the importance of the reductase transcriptional process in responding to the action of chlorambucil and providing evidence for the involvement of a protein kinase C pathway in the regulation of mammalian ribonucleotide reductase. In addition to the chlorambucil-induced elevations in enzyme activity, message, and protein levels, the drug was also shown to be an inhibitor of ribonucleotide reductase activity in cell-free preparations. Separation of ribonucleotide components on an affinity column followed by selective exposure of the protein components to chlorambucil showed that both R1 and R2 proteins were targets for chlorambucil, in keeping with the known alkylating abilities of the drug. These observations provide the first direct demonstration of a link between the regulation of mammalian ribonucleotide reductase and the process of DNA repair and contribute to our understanding of the mode of action of a class of drugs represented by chlorambucil, in which chemotherapeutic activity has been attributed to DNA damaging effects.