Down-regulation of bcl-2 is associated with p16(INK4)-mediated apoptosis in non-small cell lung cancer cells

We introduced a functional p16 cDNA into non-small cell lung cancer (NSCLC) cell lines expressing different combinations of normal and mutated p16, p5 3, and Rb genes via a recombinant adenovirus to determine the effect of exo genous p16 expression on cell growth. Analysis of p16-deficient cells infec ted with Adv/p16 identified growth arrest of the cells in the G(0)-G(1) pha se early on, Apoptosis was identified to occur by the 5th day after infecti on which corresponded with increased p16 expression, reduced Rb expression, and increased Rb hypophosphorylation, but only occurred in cells expressin g functional p53. Further analysis indicated that the expression of the ant i-apoptotic protein bcl-2 was greatly reduced in the NSCLC cell lines H460 and A549 (both -p16, +p53, +Rb), again only by the 5th day after Adv/p16 in fection, but no affect on Bar expression was observed, H1299 cells (-p16, - p53, +Rb) infected with Adv/p16 only exhibited apoptosis by an additional i nfection with Adv/p53 which also corresponded with a down-regulation of bcl -2, In addition, the infection of A549 cells with Adv/p16 followed by a sub sequent infection with Adv/Rb lead to a significant decrease in apoptosis w hich correlated with an increase in bcl-2 expression. These studies suggest that p16 is capable of mediating apoptosis in NSCLC cell lines expressing wild-type p53, through a direct down-regulation of Rb and an indirect down- regulation of the anti-apoptotic protein bcl-2.