Mechanisms of human epithelial cell immortalization and p16NK4a induced telomere-independent sencescence

As human epithelial cells age in culture, protein levels of the tumor suppressor protein p16^INK4a continue to increase resulting in growth arrest independent of telomere length. Telomere-independent senescence induced by the p16^INK4a/Rb tumor suppressor pathway prevents many epithelial cells from becoming immortalized by telomerase alone. Differences in culture conditions have been hypothesized to modulate both p16^INK4a expression and replicative capacity of human epithelial cells; however, the mechanism(s) of p16^INK4a regulation under these conditions is unknown.

We have demonstrated that p16^INK4a expression is delayed and replicative capacity increased in human keratinocytes grown in co-culture with post-mitotic, fibroblast feeder cells as compared to keratinocytes grown on tissue culture plastic alone. We have found that p16^INK4a induction in human keratinocytes cultured on plastic alone is associated with a migratory phenotype and that p16^INK4a expression is selectively induced in cells possessing markers of keratinocyte migration. Furthermore, we have identified that tyrosine kinase activity and proper functioning of the urokinase plasminogen activation system are required for p16^INK4a induction during keratinocyte migration whereas specific signaling through either Src-PTKs or FAK does not appear to regulate this phenomenon.

We have shown that human keratinocytes possessing telomerase activity and co-cultured with feeder cells do become immortal without any apparent cellular crisis. In contrast to previous reports, however, we demonstrate that telomerase immortalized keratinocytes co-cultured with feeders do not consistently growth arrest upon transfer to the plastic culture condition and display an increased frequency of p16^INK4a promoter methylation.

In summary, p16^INK4a-induced, telomere-independent senescence is associated with an epithelial migration response and provides a significant proliferation barrier to epithelial cell immortalization regardless of culture conditions. These results provide new insights into p16^INK4a regulation and have significant implications for the study of epithelial tumor cell invasion and telomerase reactivation therapies.