Related Experiment Videos
Ectopic p16(ink4) expression enhances CPT-11-induced apoptosis through increased delay in S-phase progression in
K Fukuoka1, K Nishio, H Fukumoto
1Pharmacology Division, National Cancer Center Research Institute, Tokyo, Japan.
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.
Insights
The tumor suppressor p16INK4 enhances sensitivity of non-small-cell lung cancer cells to CPT-11 chemotherapy. This involves increased apoptosis via caspase-3 activation and cyclin A down-regulation, leading to cell death.
Area of Science:
- Oncology
- Molecular Biology
- Cell Cycle Regulation
Background:
- p16INK4 is a tumor suppressor gene regulating cell-cycle progression.
- CPT-11 is a topoisomerase-I inhibitor used in cancer therapy.
- Understanding p16INK4's role in drug sensitivity is crucial for NSCLC treatment.
Purpose of the Study:
- To investigate the effect of ectopic p16INK4 expression on non-small-cell lung cancer (NSCLC) cell sensitivity to CPT-11.
- To elucidate the molecular mechanisms underlying CPT-11-induced apoptosis in p16INK4-expressing NSCLC cells.
Main Methods:
- Transfection of A549 NSCLC cells with p16INK4 cDNA.
- Treatment with CPT-11 and apoptosis inhibitors (e.g., caspase inhibitors).
- Analysis of apoptosis markers (DNA fragmentation, PARP cleavage), caspase activity, cell cycle progression (DNA histograms), and protein levels (cyclin A, B, cdk2, cdc2).
Main Results:
- Ectopic p16INK4 expression sensitized A549 cells to CPT-11, enhancing apoptosis.
- CPT-11-induced apoptosis was mediated by caspase-3 activation and suppressed by caspase inhibitors.
- p16INK4 expression led to decreased cyclin A levels and altered cell cycle progression (G2/M arrest), promoting apoptosis.
Conclusions:
- p16INK4 plays a positive role in CPT-11-induced apoptosis pathway activation, specifically involving caspase-3.
- Down-regulation of cyclin A by p16INK4 contributes to CPT-11-induced apoptosis in NSCLC cells.
- Targeting p16INK4 or pathways it modulates may enhance chemotherapy efficacy in NSCLC.