A diet lacking
folic acid and
choline and low in
methionine (
folate/methyl deficient diet, FMD diet) fed to rats is known to produce preneoplastic nodules (PNNs) after 36 weeks and
hepatocellular carcinomas (
tumors) after 54 weeks. FMD diet-induced
tumors exhibit global hypomethylation and regional hypermethylation. Restriction landmark genome scanning analysis with methylation-sensitive
enzyme NotI (RLGS-M) of genomic
DNA isolated from control livers, PNNs and
tumor tissues was performed to identify the genes that are differentially methylated or amplified during multistage hepatocarcinogenesis. Out of the 1250 genes analysed, 2 to 5 genes were methylated in the PNNs, whereas 5 to 45 genes were partially or completely methylated in the
tumors. This analysis also showed amplification of 3 to 12 genes in the primary
tumors. As a first step towards identifying the genes methylated in the PNNs and primary
hepatomas, we generated a rat NotI-EcoRV genomic library in the pBluescriptKS vector. Here, we describe identification of one methylated and downregulated gene as the rat
protein tyrosine phosphatase receptor type O (PTPRO) and one amplified gene as rat C-MYC. Methylation of PTPRO at the NotI site located immediate upstream of the trancription start site in the PNNs and
tumors, and amplification of C-MYC gene in the
tumors were confirmed by Southern blot analyses.
Bisulfite genomic sequencing of the CpG island encompassing exon 1 of the PTPRO gene revealed dense methylation in the PNNs and
tumors, whereas it was methylation free in the livers of animals on normal diet. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed significant decrease in the expression of PTPRO in the
tumors and in a transplanted rat
hepatoma. The expression of PTPRO
mRNA in the transplanted
hepatoma after demethylation with
5-azacytidine, a potent inhibitor of
DNA methyltransferases, further confirmed the role of methylation in PTPRO gene expression. These results demonstrate alteration in methylation profile and expression of specific genes during
tumor progression in the livers of rats in response to
folate/methyl deficiency, and further implicate the potential role of PTPRO as a novel growth regulatory gene at least in the
hepatocellular carcinomas.