The regulation of ribonucleoside diphosphate reductase by the tumor promoter orotic acid in normal rat liver in vivo

Our earlier studies have shown that in normal hepatocytes, erotic acid (OA) inhibits DNA synthesis induced by several growth factors in vitro and afte r two-thirds partial hepatectomy (PH) in vivo. As in the normal liver OA in duces an imbalance in nucleotide pools (specifically, an increase in uridin e nucleotides, including deoxyuridine nucleotides, and a decrease in adenos ine nucleotides, including ATP) and creation of this imbalance is crucial f or the mitoinhibitory effects of OA, we hypothesized that ribonucleoside di phosphate reductase (RNR), a key enzyme in DNA synthesis that is regulated by nucleotide/deoxynucleotide levels, might be one of the targets for the i nhibition of DNA synthesis by OA. To test this hypothesis, we subjected mal e Fischer 344 rats (130-150 g) to two-thirds PH in the absence or in the pr esence of OA (a 300-mg tablet of OA methyl ester implanted intraperitoneall y at the time of two-thirds PH). The rats were killed at different times la ter, and their livers were processed for analysis of levels of RNR enzyme a ctivity, protein, and mRNA transcripts. The results obtained indicated that treatment with OA resulted in a near-100% inhibition of RNR induced by two -thirds PH in rat liver, as monitored by enzyme activity and protein level. Furthermore, this inhibition was paralleled by a decrease in the mRNA tran scripts for both the M1 and M2 subunits of RNR. Nuclear run-off assays indi cated that this decrease in the levels of mRNA transcripts could not be att ributed to an effect on transcription. However, administration of OA 20 h a fter two-thirds PH, when RNR mRNA transcripts were maximally induced, resul ted in increased degradation of the RNR M1 and M2 subunits. Taken together, these results indicate that OA treatment decreases RNR levels induced by t wo-thirds PH, at the levels of enzyme activity, protein, and mRNA transcrip ts, and the decreased levels of mRNA transcripts appeared to be due to incr eased degradation of the transcripts. Mol. Carcinog. 24:188-196, 1999. (C) 1999 Wiley-Liss, Inc.