ALTERATIONS IN HEPATIC P53 GENE METHYLATION PATTERNS DURING TUMOR PROGRESSION WITH FOLATE METHYL DEFICIENCY IN THE RAT/

Chronic dietary methyl deficiency in F344 rats was used as an in vivo mammalian model in which to evaluate the gene-specific alterations in DNA methylation patterns during multistage hepatocarcinogenesis. Using bisulfite mapping, the site-specific methylation profile within exons 6-7 of the 53 gene was determined in control liver, preneoplastic nod ules (after 36 weeks of folate/methyl deficiency) and in hepatocellula r carcinoma (after 54 weeks of deficiency). A progressive loss of meth yl groups was observed at most CpG sites on both coding and non-coding strands during the first 36 weeks of folate/methyl deficiency, with t he greatest loss occurring on the coding strand. When the same sequenc e was evaluated in tumor DNA after 54 weeks of deficiency, the majorit y of cytosines were unexpectedly found to have-become remethylated. Cp G sites that had previously lost methyl groups on both strands during preneoplasia as well as CpG sites that had been constitutively non-met hylated, had undergone de novo methylation in tumor DNA. Maintenance m ethyltransferase and de novo methyltransferase activity in nuclear ext racts were assessed using hemimethylated and non-methylated DNA substr ates, respectively. In tumor, de novo methyltransferase capacity was i ncreased similar to 4-fold relative to control or preneoplastic Liver and associated with a relative increase in both p53 and genome-wide me thylation density. In the preneoplastic nodules, the level p53 mRNA wa s increased and associated with hypomethylation in the coding region o f the gene, whereas in tumor tissue, p53 mRNA was decreased and associ ated with relative hypermethylation. Taken together, these results pro vide additional insights into the dysregulation and instability in DNA methylation that accompanies the transition to tumor. (C) 1997 Elsevi er Science Ireland Ltd.