Our earlier studies had demonstrated that inhibition of DNA methylation following carcinogen treatment potentiated initiation of the carcinogenic process in the rat liver system. The hepatic nodules developed by initiation-promotion protocols showed a characteristic hypomethylation in the cell-cycle-related genes c-fos, c-myc and c-Ha-ras. In the present study we have found that the gene for beta-hydroxy-beta-methyl glutaryl coenzyme A reductase, a major rate-limiting enzyme in the biogenesis of mevalonate, is also hypomethylated at both CCGG and GCGC sites and expressed in hepatic nodules. This gene, however, did not exhibit hypomethylation in CCGG sequences in non-nodular surrounding liver, livers from rats subjected to two-thirds partial hepatectomy, or exposed to initiator alone (1,2-dimethylhydrazine given 18 h after partial hepatectomy) or to diets containing 1% orotic acid alone (promoting regimen). The activity of the enzyme and mevalonate formation are positively correlated with DNA synthesis and cell proliferation--two key components of the carcinogenic process. Taken together, the results suggest that hypomethylation of specific genes occurs in the carcinogenic process and this altered pattern of DNA methylation may play a role in the growth of the nodules.
Orotic acid (OA), a promoter of liver carcinogenesis, inhibited proliferation of primary hepatocytes in culture as monitored by labelling index, mitotic index and total DNA content. The mitoinhibitory effect of OA was seen even in the presence of a strong mitogen such as epidermal growth factor (EGF). The growth inhibitory effect of OA was not due to cell killing. Upon exposure to OA the hepatocytes exhibited an increase in the ratio of uridine nucleotides to adenosine nucleotides, and as this ratio increased the response of hepatocytes to proliferate in the presence or absence of EGF decreased. Washing the hepatocytes free of added OA resulted in a gradual decrease in the ratio of uridine nucleotides to adenosine nucleotides, paralleled by an increase in hepatocytic proliferation. Adenine, an agent that inhibits the metabolism of OA to uridine nucleotides, not only inhibited the increase in the ratio of uridine nucleotides to adenosine nucleotides but also counteracted the OA-induced mitoinhibitory effect. These results, together with our earlier observations, suggest that an imbalance in nucleotide pools composed of an increase in uridine nucleotides and a decrease in adenosine nucleotides appears to be important for OA-induced mitoinhibition.
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