INCREASED P16 EXPRESSION WITH FIRST SENESCENCE ARREST IN HUMAN MAMMARY EPITHELIAL-CELLS AND EXTENDED GROWTH CAPACITY WITH P16 INACTIVATION

Aberrations affecting the tumor suppressor gene p16(INK4a) have been d escribed for a variety of tumors. In breast cancer, approximately 50% of tumors show low or lack p16 expression. While evidence provided by some studies has implicated a possible role for p16 in normal replicat ive senescence, other studies have suggested that the Rb, pathway thro ugh which p16 functions, may not be involved in senescence control. Pr eviously we observed that all immortal lines derived from normal mamma ry epithelium which were analysed for p16 displayed inactivation of th is gene through distinct mechanisms, supporting p16 inactivation as a possible necessary event in escape from senescence. To further clarify this issue, we have analysed p16 expression in a panel of normal fini te lifespan human mammary epithelial cells (HMEC) from initial propaga tion through growth arrest, using media which confer different replica tive capacity. Approximately 10-25-fold increase in pld expression was observed for all normal HMEC with initial onset of a senescence pheno type following 15-25 population doublings in culture. These cells also displayed expression of the senescence associated beta-galactosidase, Interestingly, HMEC with additional long term replicative capacity (a pproximately 80 population doublings) arose from these growth arrested cultures, showing lack of pld expression. This extended growth capaci ty appears to be associated with a methylation phenomenon since treatm ent of these cells with the methylation inhibitor 5-aza-2-deoxycytidin e resulted in growth arrest concurrent with reacquisition of p16 expre ssion and senescence associated beta-galactosidase. Analysis of p21waf 1 expression revealed no change in expression during growth in vitro. These results support p16(INK4a) as the 9p senescence gene and suggest a role for p16 loss in the escape from initial onset of senescence an d in acquisition of an extended life span of human mammary epithelial cells.