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

Abstract

Our earlier studies have shown that in normal hepatocytes, orotic acid (OA) inhibits DNA synthesis induced by several growth factors in vitro and after two-thirds partial hepatectomy (PH) in vivo. As in the normal liver OA induces an imbalance in nucleotide pools (specifically, an increase in uridine nucleotides, including deoxyuridine nucleotides, and a decrease in adenosine nucleotides, including ATP) and creation of this imbalance is crucial for the mitoinhibitory effects of OA, we hypothesized that ribonucleoside diphosphate reductase (RNR), a key enzyme in DNA synthesis that is regulated by nucleotide/deoxynucleotide levels, might be one of the targets for the inhibition of DNA synthesis by OA. To test this hypothesis, we subjected male Fischer 344 rats (130-150 g) to two-thirds PH in the absence or in the presence of OA (a 300-mg tablet of OA methyl ester implanted intraperitoneally at the time of two-thirds PH). The rats were killed at different times later, and their livers were processed for analysis of levels of RNR enzyme activity, 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 transcripts for both the M1 and M2 subunits of RNR. Nuclear run-off assays indicated that this decrease in the levels of mRNA transcripts could not be attributed to an effect on transcription. However, administration of OA 20 h after two-thirds PH, when RNR mRNA transcripts were maximally induced, resulted in increased degradation of the RNR M1 and M2 subunits. Taken together, these results indicate that OA treatment decreases RNR levels induced by two-thirds PH, at the levels of enzyme activity, protein, and mRNA transcripts, and the decreased levels of mRNA transcripts appeared to be due to increased degradation of the transcripts.

Characterization of the Rodent Genes for Arylacetamide Deacetylase, a Putative Microsomal Lipase, and Evidence for Transcriptional Regulation

“…Nuclear run-on was performed essentially as described (24,25). 32 P-Labeled RNA was hybridized to AADA or GAPDH cDNA that had been spotted onto Hybond N membranes.…”

Role of eIF3 p170 in controlling synthesis of ribonucleotide reductase M2 and cell growth

“…It has also been shown that the activity of ribonucleotide reductase, and therefore DNA synthesis and cell proliferation, is controlled during cell cycle by the synthesis and degradation of the M2 subunit (Eriksson et al, 1984). Several antiproliferation agents such as hydroquinone, orotic acid, and hydroxyurea were reported to inhibit DNA synthesis by inhibiting the expression or activity of M2 and consequently reducing the deoxyribonucleotide pool (Yarbro, 1992;Zhou et al, 1995;Li et al, 1998;Manjeshwar et al, 1999).…”

Simultaneous Determination of Different Anions in Milk Samples Using Ion Chromatography with Conductivity Detection

“…Recent experimental studies have demonstrated that it is involved in carcinogenesis, hepatic lipid storage and renal toxicity [14]. Several studies have shown that repeated dosing of orotic acid promotes the formation of tumors initiated by various known carcinogenic substances [15]. Sample preparation is an important part of the analysis of anions in a complex matrix especially in milk to manage a good analytical procedure.…”