Ectopic p16(INK4) expression enhances CPT-11-induced apoptosis through increased delay in S-phase progression in human non-small-cell-lung-cancer cells

A tumor-suppressor gene, p16(INK4) which is deleted or mutated in tumors, r egulates cell cycle progression through a G(1)-S restriction point by inhib iting CDK4(CDK6)/cyclin-D-mediated phosphorylation of pRb. We have found th at ectopic p16(INK4) expression increased cellular sensitivity of human non -small-cell-lung-cancer (NSCLC) A549 cells to a selective growth-inhibitory effect induced by the topoisomerase-1 inhibitor 11,7-ethyl-10-[4-(1-piperi dino)-1-piperidino] carbonyloxy camptothecin (CPT-11) in vitro. In this stu dy, we observed enhanced apoptosis characterized by DNA fragmentation in A5 49 cells transfected with p16(INK4) cDNA (A549/p16-1) and treated with CPT- 11. This apoptosis was suppressed by the inhibitor of interleukin-1 beta-co nverting enzyme (ICE/caspase-1) or ICE-like proteases, Z-Asp-CH2-DCB, as de termined by DNA fragmentation and proteolytic cleavage of poly(ADP-ribose) polymerase, a natural substrate for CPP32/caspase-3, In A549/p16-1 cells, c ytosolic peptidase activities that cleaved Z-DEVD-7-amino-4-trifluoromethyl coumarin increased during CPT-11-induced apoptosis and were suppressed by a highly specific caspase-3 and caspase-3-like inhibitor, Z-DEVD-fluoromethy lketone. These findings indicate that p16(INK) is positively involved in th e activation pathway of the caspase-3 induced by CPT-11. The increased dela y in S-phase progression and subsequent induction of apoptosis were observe d in CPT-11-treated A549/ p16-1 cells on the basis of DNA histograms. Speci fic downregulation of the cyclin-A protein level in A549/p16-1 cells was ob served after CPT-11-treatment, whereas cyclin B, cdk2, and cdc2 protein lev els were unaffected. These results suggest that ectopic p16(INK4) expressio n inappropriately decreases cyclin A and thereby terminates CPT-11-induced G(2)/M accumulation, which is followed by increased apoptosis in p16(INK4)- expressing A549 cells. Int. J. Cancer 86:197-203, 2000, (C) 2000 Wiley-Liss , Inc.