title: Ectopic p16(ink4) expression enhances CPT-11-induced apoptosis through increased delay in S-phase progression in human non-small-cell-lung-cancer cells.
abstract: A tumor-suppressor gene, p16(INK4), which is deleted or mutated in tumors, regulates cell-cycle progression through a G(1)-S restriction point by inhibiting CDK4(CDK6)/cyclin-D-mediated phosphorylation of pRb. We have found that 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-I inhibitor 11, 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxy camptothecin (CPT-11) in vitro. In this study, we observed enhanced apoptosis characterized by DNA fragmentation in A549 cells transfected with p16(INK4) cDNA (A549/p16-1) and treated with CPT-11. This apoptosis was suppressed by the inhibitor of interleukin-1beta-converting enzyme (ICE/caspase-1) or ICE-like proteases, Z-Asp-CH2-DCB, as determined by DNA fragmentation and proteolytic cleavage of poly(ADP-ribose) polymerase, a natural substrate for CPP32/caspase-3. In A549/p16-1 cells, cytosolic peptidase activities that cleaved Z-DEVD-7-amino-4-trifluoromethylcoumarin increased during CPT-11-induced apoptosis and were suppressed by a highly specific caspase-3 and caspase-3-like inhibitor, Z-DEVD-fluoromethylketone. These findings indicate that p16(INK) is positively involved in the activation pathway of the caspase-3 induced by CPT-11. The increased delay in S-phase progression and subsequent induction of apoptosis were observed in CPT-11-treated A549/p16-1 cells on the basis of DNA histograms. Specific down-regulation of the cyclin-A protein level in A549/p16-1 cells was observed after CPT-11-treatment, whereas cyclin B, cdk2, and cdc2 protein levels were unaffected. These results suggest that ectopic p16(INK4) expression 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.
authors.affiliation: Pharmacology Division, National Cancer Center Research Institute, Tokyo, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Fukuoka
authors.forename: K
authors.initials: K
authors.lastname: Nishio
authors.forename: H
authors.initials: H
authors.lastname: Fukumoto
authors.forename: H
authors.initials: H
authors.lastname: Arioka
authors.forename: H
authors.initials: H
authors.lastname: Kurokawa
authors.forename: T
authors.initials: T
authors.lastname: Ishida
authors.forename: Y
authors.initials: Y
authors.lastname: Iwamoto
authors.forename: A
authors.initials: A
authors.lastname: Tomonari
authors.forename: T
authors.initials: T
authors.lastname: Suzuki
authors.forename: J
authors.initials: J
authors.lastname: Usuda
authors.forename: N
authors.initials: N
authors.lastname: Narita
authors.forename: N
authors.initials: N
authors.lastname: Saijo
mesh_terms: D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D002166:Camptothecin / Q000031:analogs & derivatives* / Q000494:pharmacology; D002289:Carcinoma, Non-Small-Cell Lung / Q000473:pathology*; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D019925:Cyclin A / Q000378:metabolism; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics*; D053938:DNA Fragmentation; D018076:DNA, Complementary; D004789:Enzyme Activation; D015870:Gene Expression; D006801:Humans; D000077146:Irinotecan; D008175:Lung Neoplasms / Q000473:pathology*; D011480:Protease Inhibitors / Q000494:pharmacology; D016196:S Phase; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D061945:Caspase Inhibitors; D019925:Cyclin A; D019941:Cyclin-Dependent Kinase Inhibitor p16; D018076:DNA, Complementary; D011480:Protease Inhibitors; C093260:benzyloxycarbonyl-Asp-CH2OC(O)-2,6-dichlorobenzene; D001224:Aspartic Acid; D000077146:Irinotecan; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D002166:Camptothecin