Methylation changes in the human IGF2 P3 promoter parallel IGF2 expressionin the primary tumor, established cell line, and xenograft of a human hepatoblastoma

Hepatoblastoma (HB) is a rare malignant embryonal liver tumor. Its pathogen esis has been associated with altered regulation of the IGF2 and H19 genes, and previous studies have suggested a correlation between abnormal methyla tion and altered expression of these genes in hepatoblastoma. Upregulation of the activity of the IGF2 promoter P3 has previously been shown to be tig htly correlated with demethylation in hepatoblastoma. Here, we have used bi sulfite genomic sequencing to characterize the methylation pattern of the I GF2 promoter P3 in the hepatoblastoma-derived cell line Hep T1, in the orig inal tumor from which Hep TI is derived, and in nude mouse xenografts of th e Hep TI cell line. The results show a clear difference in methylation patt ern of the most proximal region of the IGF2 P3 promoter between the primary tumor, the cell line, and the xenografts. RNase protection and mRNA in sit u hybridization revealed that variations in methylation patterns was parall eled by the levels of IGF2 P3 mRNA, which was detectable in the primary tum or and xenografts, but not in the cell line. Furthermore, it was demonstrat ed that H19 was reactivated and demethylated in the HepT1 cell line by 5-az aCytidine, in contrast to IGF2 P3, which was not demethylated or reactivate d. We suggest that methylation of the proximal IGF2 P3 is important for its regulation. (C) 2001 Academic Press.