HYPOMETHYLATION OF THE RAT GLUTATHIONE-S-TRANSFERASE PI(GSTP) PROMOTER REGION ISOLATED FROM METHYL-DEFICIENT LIVERS AND GSTP-POSITIVE LIVERNEOPLASMS

DNA methylation at the 5-position on the cytosine ring in CpG dinucleo tides (CpG sites) appears to play an important role in regulating gene expression. In general, there is an inverse relationship between prom oter CpG site methylation and the potential for transcription. Thus, c hanges in DNA methylation density may lead to altered levels of protei ns such as glutathione S-transferase pi (GSTP), which is frequently us ed as a marker to detect hepatocellular foci and neoplasms in the rat, In the present study, the level of CPG methylation in the rat GSTP pr omoter region was determined in bisulfite-treated DNA isolated from co ntrol (untreated) rat livers, chemically induced, GSTP-positive rat li ver neoplasms, and methyl-deficient rat livers that contained numerous GSTP-positive foci after administration of a defined diet deficient i n folate and choline and low in methionine (0.18%), Eight cytosines be tween -235 and +140 in the GSTP promoter region were methylated in a s ite-specific manner in GSTP-negative control liver, whereas these same sites were hypomethylated in all four chemically-induced, GSTP-positi ve neoplasms. Similarly, all CpG sites were unmethylated in methyl-def icient liver DNA within 3 weeks of the rats receiving the methyl-defic ient diet, and they remained unmethylated throughout the 36-week treat ment period. Five of the eight CpG sites are located within consensus sequences for the DNA binding proteins Spl and E2F. This indicates at least one possible mechanism that could potentially lead to transcript ional activation of GSTP in hepatocellular foci and neoplasms during r at hepatocarcinogenesis, These findings suggest that methylation of cr itical cytosines within the promoter region rather than all CpG-associ ated cytosines may be a determining factor in regulation of GSTP expre ssion.