ALTERATIONS IN THE ACTIVITY AND REGULATION OF MAMMALIAN RIBONUCLEOTIDE REDUCTASE BY CHLORAMBUCIL, A DNA DAMAGING AGENT

Ribonucleotide reductase provides the four deoxyribonucleotides requir ed for the synthesis of DNA. In this study, we examined the hypothesis that alterations in the regulation of ribonucleotide reductase activi ty may be necessary to provide the deoxyribonucleotides required for D NA repair, following exposure of mammalian cells to DNA damaging agent s such as the antitumor agent chlorambucil. We observed a marked trans ient increase in ribonucleotide reductase activity within 2 h of expos ing BALB/c 3T3 mouse cells to DNA damaging concentrations of chlorambu cil. Northern blot analysis showed that elevations in activity were ac companied 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 cultu res. The chlorambucil effects upon activity and regulation of ribonucl eotide reductase occurred without any detectable changes in the rate o f DNA synthesis, as would be expected if the elevation in enzyme activ ity is required for DNA repair. The chlorambucil-induced elevations in R1 and R2 message levels were blocked by treatment of cells with acti nomycin D or the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate, indicating the importance of the reductase transcriptional process in responding to the action of chlorambucil and providing evidence for t he involvement of a protein kinase C pathway in the regulation of mamm alian ribonucleotide reductase. In addition to the chlorambucil-induce d elevations in enzyme activity, message, and protein levels, the drug was also shown to be an inhibitor of ribonucleotide reductase activit y in cell-free preparations. Separation of ribonucleotide components o n an affinity column followed by selective exposure of the protein com ponents to chlorambucil showed that both R1 and R2 proteins were targe ts for chlorambucil, in keeping with the known alkylating abilities of the drug. These observations provide the first direct demonstration o f a link between the regulation of mammalian ribonucleotide reductase and the process of DNA repair and contribute to our understanding of t he mode of action of a class of drugs represented by chlorambucil, in which chemotherapeutic activity has been attributed to DNA damaging ef fects.