INACTIVATION OF P16 IN HUMAN MAMMARY EPITHELIAL-CELLS BY CPG ISLAND METHYLATION

Proliferation of human mammary epithelial cells (HMEC) is limited to a few passages in culture due to an arrest in G(1) termed selection or mortality stage 0, M0. A small number of cells spontaneously escape M0 , continue to proliferate in culture, and then enter a second mortalit y stage, M1, at which they senesce. Evidence that M0 involves the Rb p athway comes from the observation that expression of human papillomavi rus type 16 E7 alleviates the M0 proliferation block, and we further s haw that the Rb-binding region of E7 is required to allow cells to byp ass M0. In contrast, E6 does not prevent HMEC from entering M0 but, ra ther, is involved in M1 bypass, Here we show that inactivation of the D-type cyclin-dependent kinase inhibitor p16(INK4A) is associated,vith escape from the M0 proliferation block. Early-passage HMEC express re adily detectable amounts of p16 protein, whereas normal or E6-expressi ng HMEC that escaped M0 expressed markedly reduced amounts of p16 mRNA and protein. This initial reduction of p16 expression was associated with limited methylation of the p16 promoter region CpG island, At lat er passages, a further reduction in p16 expression occurred, accompani ed by increased CpG island methylation. In contrast, reduction of p16 expression did not occur in E7-expressing HMEC that bypassed M0, due t o inactivation of Rb. These observations in the E6-expressing HMEC cor relate well with the finding that CpG island methylation is a mechanis m of p16 inactivation in the development of human tumors, including br east cancer.