Impaired expression and promotor hypermethylation of O6-methylguanine-DNA methyltransferase in retinoblastoma tissues.

PURPOSE To investigate the role of epigenetic changes in the promoter region of tumor-suppressor genes in the retinoblastoma genome and to study the disruption of expression of O6-methylguanine-DNA Methyltransferase (MGMT) due to aberrant methylation and its association with retinoblastoma. METHODS A series of 23 retinoblastoma tissue specimens and 2 retinoblastoma cell lines (Y79 and WERI-Rb1) were subjected to methylation-specific PCR (MSP) analysis of hypermethylated genes identified in human cancers, including p14(ARF), p15(INK4b), p16(INK4a), VHL, and MGMT. Further, the expression of MGMT was studied by immunohistochemistry and, when fresh tissue was available, by Western blot analysis and RT-PCR. RESULTS Aberrant methylation of at least one MGMT locus was detected in 8 of the 23 tumors (35%), all of which (100%) had impaired or absent expression of MGMT. The remaining 15 tumor specimens were nonmethylated, and, among them, 7 (43%) showed defective expression. No methylation of tumor DNA was found on the p14(ARF), p15(INK4b), p16(INK4a), and VHL genes. Hypermethylation in the MGMT promoter was found to be prominently present in retinoblastoma with poor tissue differentiation, and was more frequently detected among patients with bilateral disease. Production of MGMT was consistent with expression of mRNA. No methylation of MGMT promoter was detected in the two retinoblastoma cell lines (Y79, WERI-Rb1). CONCLUSIONS The data show a clear association between impaired production of MGMT and hypermethylation of the MGMT promoter, which appeared to relate to early onset and poor differentiation, suggesting that epigenetic silencing of MGMT by methylation of the promoter and reduced expression of MGMT may play an important role in the development and progression of retinoblastoma.