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

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-tetradecanoyl-phorbol-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.PT: J; CR: BANK BB, 1989, CANCER RES, V49, P554 BLAKE MS, 1984, ANAL BIOCHEM, V136, P175 CARLSON J, 1984, P NATL ACAD SCI USA, V81, P4294 CHOY BK, 1988, CANCER RES, V48, P2029 CHOY BK, 1989, BIOCHEM BIOPH RES CO, V162, P1417 EDWARDS DR, 1985, MOL CELL BIOL, V5, P3280 ELLEDGE SJ, 1989, MOL CELL BIOL, V9, P4932 ENDRESEN L, 1983, CANCER RES, V43, P2918 GOUGH NM, 1988, ANAL BIOCHEM, V173, P93 HURTA RAR, 1990, BIOCHIM BIOPHYS ACTA, V1087, P165 HURTA RAR, 1991, J BIOL CHEM, V266, P24097 JIANG BZ, 1989, CANCER RES, V49, P5514 JOHNSTON JB, 1990, J NATL CANCER I, V82, P776 LEWIS WH, 1978, J CELL PHYSL, V94, P287 MCCLARTY GA, 1987, BIOCHEMISTRY-US, V26, P8004 MCCLARTY GA, 1988, BIOCHEMISTRY-US, V27, P7524 MCCLARTY GA, 1990, J BIOL CHEM, V265, P7539 NISHIZUKA Y, 1986, SCIENCE, V233, P305 PHILLIPS DR, 1986, BIOCHEMISTRY-US, V25, P7355 ROBSON CN, 1986, CANCER RES 1, V46, P6290 RUBIN EH, 1986, CANCER RES 1, V46, P6165 STEEPER JR, 1970, ANAL BIOCHEM, V34, P123 THELANDER L, 1980, J BIOL CHEM, V255, P7426 TOWBIN H, 1979, P NATL ACAD SCI USA, V76, P4350 ULLMAN B, 1980, J BIOL CHEM, V255, P8308 ULLMAN B, 1989, DRUG RESISTANCE MAMM, V1, P69 WEBER G, 1983, CANCER RES, V43, P3466 WEINBERG G, 1981, P NATL ACAD SCI USA, V78, P2447 WRIGHT JA, 1987, SOMAT CELL MOLEC GEN, V13, P155 WRIGHT JA, 1989, DRUG RESISTANCE MAMM, V1, P15 WRIGHT JA, 1989, INT ENCY PHARM THERA, V128, P89 WRIGHT JA, 1990, BIOCH CELL BIOL, V68, P1364 YOUNG S, 1987, BIOCHEM J, V244, P775; NR: 33; TC: 65; J9: J BIOL CHEM; PG: 6; GA: HM053Source type: Electronic(1