The p16 gene is silenced epigenetically in both unirradiated and irradiated human mammary epithelial cells. (A) Immunoblot analysis shows that p16 protein expression is very low in the eighth passage (8p) variant human mammary epithelial cells (vHMEC) cultures derived from both unirradiated and irradiated cells, but that it is readily induced to similar extents in both cultures treated with 5-aza-deoxycytidine (Aza). Lysates of HMEC undergoing stasis and MCF-7 cells were used as positive and negative controls, respectively. (B) p16 immunohistochemistry indicates that 8p vHMEC cultures derived from both unirradiated and irradiated cells contain primarily p16(-) cells, and that p16 protein can be induced in virtually all cells in both cultures by 72 hr treatment with 3.3 μM 5-aza-deoxycytidine. (C) DNA sequence of the CpG island region and a map of the -159 to +233 region of the p16 gene indicate the 35 CpG sites (CG or vertical lines) analyzed for methylation status, and their positions relative to the translational start site (ATG) and exon 1α. Primers used for PCR amplification are underlined. (D) The percent methylation of specific CpG sites (indicated by degree of shading) in the -159 to +233 region of the p16 gene was determined by sequencing of individual bisulfite-treated DNAs obtained from the indicated pre-stasis HMEC and vHMEC cultures from specimens B1450 and B1400.