In gliomas and other tumors, expression of the drug metabolizing and stress response signaling protein, GSTP1, has been associated with poor patient response to therapy and with poor survival. Although, the mechanisms underlying GSTP1 expression in tumors are still not fully understood, methylation of CpGs in the 5′-region of the GSTP1 gene has been established as a key mechanism in this process. In this study, we examined the methylation status of CpGs in a 458 bp region of the GSTP1 gene covering nucleotides +58 to -400 in 22 primary gliomas and 15 early passage glioma cell lines of different levels of GSTP1 gene expression. Genomic DNA from each tumor was bisulfite-treated and PCR amplified with three sets of primers spanning the three main CpG clusters in the region, the PCR amplified products were subcloned into plasmid vectors. Following transformation in E. coli and plating on agar, up to twelve positive colonies from each tumor were sequenced to determine the specific CpGs that were methylated. GSTP1 transcript levels were determined by quantitative PCR and by immunohistochemistry. A methylation map of the 458-nucleotide region was created for each tumor showing the methylation status of each CpG. From the results, we identified two main CpG clusters at -371 to -260 and -214 to -130 to be most frequently methylated in tumors that had little to no GSTP1 expression, as well as, the most commonly methylated CpGs in these clusters. We then designed methylation-specific primers covering these regions and used them to develop methylation-specific PCR (MSP) assays for these methylation hotspots. The results of screening of tumors with the MSP assay were consistent with those of the bisulfite sequencing. We also observed that tumors with heterogeneous intra-cellular GSTP1 expression were characterized by significant heterogeneity among the clones with respect to methylation of the hotspot CpG regions. Treatment of glioma cells with 5-azacytidine reversed the methylation of these hotspots and increased GSTP1 expression. Together, these results demonstrate significant differences between individual CpGs and CpG clusters in the GSTP1 5′-region with respect to transcriptional regulation of the GSTP1 gene and that the methylation status of the CpG clusters I and III are most critical in determining GSTP1 expression in gliomas. The results further indicate that differential methylation of these two hotspot CpG clusters between the tumor cells account for the intercellular heterogeneity in GSTP1 expression observed in gliomas and that methylation inhibitors may cause increased resistance of low GSTP1 expressing gliomas to alkylating agent therapy. Supported by Grants RO1 CA 91438, P50 CA108786, 5P30 CA 114236 from the National Cancer Institute and by a grant from the Pediatric Brain Tumor Foundation.
[Proc Amer Assoc Cancer Res, Volume 47, 2006]