DNA hypomethylation and gene expression of p16/CDKN2 in breast carcinoma
p16/CDKN2, a cell cycle regulator residing on chromosome band 9p21, has been demonstrated to contain CpG islands within and surrounding the first and second exons of the gene. Methylation of these regions has been implicated in the regulation of gene expression. We hypothesized that abnormal DNA methylation and the resultant alteration in p16 gene expression may be a mechanism of gene inactivation in breast carcinoma. We examined 23 samples (10 primary breast carcinomas (Ca), 7 corresponding normal breast tissue (Nl), and 6 corresponding metastatic carcinoma specimens (MCa)), as well as 3 breast cancer cell lines (CL) (ZR75-1, T47-D and MDA-MB-468) for p16/CDKN2 methylation by Southern blotting techniques. In addition, all cell lines, 1 Nl, 7 Ca, and 5 MCa were subjected to RT-PCR to evaluate p16 expression. We found that p16/CDKN2 was methylated in 5/7 Nl specimens, in p16/COKN2 methylation status Proportion expressing p16 Methylated Nonmethyl. Partially methyl. Methylated Nonmethyl. Partially methyl. Nl (n=7) 5 0 2 Nl 0/1 Ca (n=10) 0 5 5 Ca 4/4 1/3 MCa (n=6) 0 3 3 MCa 3/3 2/2 CL (n=3) 1 1 1 CL 0/1 1/1 1/1 contrast to 0/10 Ca and 0/6 MCa specimens. In all cases evaluated, nonmethylated p16/CDKN2 was associated with the presence of p16 expression. We conclude that hypomethylation of p16/CDKN2 and the associated p16 expression may be a significant event in the development of breast carcinoma.