Correlates with Gene Silencing Suppressor Gene in Normal and Transformed Human Tissues

Loss of heterozygosity on 9p21, where thepl6/CDKN2 tumor suppres sor and the pJ5INK4Bcell cycle regulator genes are located, is a common genetic alteration in bladder cancer. However, it has been difficult to demonstrate homozygous deletions and intragenic mutations in either of these two genes in primary transitional cell carcinomas (TCC) of the bladder. Similarly, colon cancer-derived cell lines have shown no homozy gous deletions of the p16/CDKN2 locus in contrast to a wide variety of tumor-derived cell lines. We have investigated abnormal methylation of the5' CpGislandsofthepl6/CDKN2andp15@4'@genesasanalternative mechanism of inactivation of these genes in bladder and colon cancers. De novo methylation of the 5' CpG island ofpl6/CDKN2 was observed in 12 of 18 (67%) uncultured bladder TCCS and in 2 of 3 (67%) bladder cell lines. In contrast, only 1 of 10 (10%) colon cardnomas showed methyla don of the 5' CpG lsland ofpl6/CDKN2. It was striking to fInd that this region was extensively methylated and the gene not expressed in the normal colonic mucosa of 6 of 10 (60%) patients with colon cancer, whereas 5 of the corresponding colon tumors showed no methylation and high levels ofpl6/CDKN2 expression. Our data show a significant corre latlon (P = 0.00001, two-sided) between the absence of p16/CDKN2 ex presslon and methylation ofits 5' CpG island in bladder tumors, cell lines, and normal colon mucosa. In contrast, no association was observed be tween expression and methylation status ofthe 5' CpG lsland 0f@J5@4B• Our resultssuggesthat thep16/CDKN2tumor suppressor genemay be inactivated by methylation of its 5' CpG lsland in TCCs of the bladder. We also present evidence of methylation of the 5' CpG island in this autosomal gene in normal colonic tissue.