DIFFERENTIAL SENSITIVITY TO LOSS OF CYTOSINE METHYL-GROUPS WITHIN THEHEPATIC P53 GENE OF FOLATE METHYL DEFICIENT RATS/

Dietary folate/methyl deficiency provides a unique model of endogenous hepatocarcinogenesis in which to study progressive alterations in DNA methylation patterns during tumor progression in vivo. Weanling male F344 rats were given a semi-purified diet deficient in the methyl dono rs choline, methionine and folic acid for a period of 9 weeks, Using a genomic sequencing procedure based on the PCR amplification of bisulf ite-modified DNA, the methylation status of individual CpG sites withi n exons 6 and 7 of the p53 gene in liver samples from control and defi cient rats was determined, Treatment of denatured nuclear DNA with sod ium bisulfite quantitatively converts all cytosine residues to uracil which are then amplified as thymine in the PCR reaction, In contrast, 5-methylcytosine is resistant to bisulfite deamination under the react ion conditions and is amplified as cytosine, Automated sequencing of b isulfite-modified DNA will then elucidate the methylation status of ea ch cytosine residue within a defined gene sequence, In addition to eva luation of the methylation status of the p53 gene, the relative activi ty of the DNA methyltransferase was also quantified in nuclear extract s from control and folate/methyl deficient rats, The results indicated that specific 5-methyl cytosines within the hepatic p53 gene from met hyl deficient rats are resistant to demethylation despite the diet-ind uced decrease in S-adenosylmethionine and the increase in cell prolife ration associated with this dietary intervention, Progressive demethyl ation was observed at other methylated cytosine residues in folate/met hyl deficient rats after 9 weeks despite a paradoxical increase in DNA methyltransferase activity, The application of this sequence-specific technology will allow the definition of the methylation status of eve ry CpG site within a coding sequence or promoter region and should pro vide new insights into mechanisms and consequences of methylation dysr egulation during progressive multistage carcinogenesis.