Loss of p16INK4 Expression by Methylation Is Associated with Lifespan Extension of Human Mammary Epithelial Cells

Inactivation of pl6INK4 tumor suppressor gene function is frequently observed in breast cancer. We examined pl6INK4 expression in human mammary epithelial cell (HMEC) cultures established from four normal donors. Normal HMECs divide a limited number of times before prolif eration ceases in a state referred to as selection (or M,,). The cell subpopu lation that emerges spontaneously from selection undergoes a further limited period of proliferation before senescence. By Âimmunofluorescence and Western blot analysis of four independent cultures, we have shown loss of pl6INK4 expression in postselection HMECs. In contrast, pl6INK4 was present in both early and late passage fibroblasts from the same individuals. Bisulfite genomic sequencing revealed extensive methylation of the pl6INK4 CpG island in post- but not preselection cells. Thus, the extended period of growth observed in postselection HMECs is associated with hypermethylation of the pl6INK4 CpG island and loss of pl6INK4