Involvement of p21Waf1/Cip1 cleavage during roscovitine-induced apoptosis in non-small cell lung cancer cells.
Roscovitine, a cyclin-dependent kinases (CDKs) inhibitor, has been reported to have anti-tumor effects in some cancer cell lines by inducing apoptosis. However, the exact underlying mechanisms are not fully understood. Here, we report that roscovitine induces expression and cleavage of the universal CDK inhibitor p21Waf1/Cip1 in non-small cell lung cancer (NSCLC) A549 cells in a dose-dependent manner. Western blots of roscovitine-treated cells undergoing apoptosis consistently demonstrated a 15 kDa band that was not detected in control cultures. CDK2 activity and PCNA expression were repressed with increasing dose of roscovitine. Accompanying these molecular changes was a progressive arrest of G2 phase and decreasing of 5-bromo-2-deoxyuridine (Brdu) incorporation of S phase cells. Caspase-3 inhibitor z-DEVD-fmk almost completely abolished roscovitine-induced apoptosis, as well as the appearance of 15 kDa band, indicating that p21Waf1/Cip1 cleavage was mediated by caspase-3 activity. Furthermore, this band was predominant in the floating apoptotic cells, while weakened in the adherent cells which were vital and pre-apoptotic. We also showed that roscovitine induced an enhanced expression of gamma-H2AX, which was blocked by caspase-3 inhibition, suggesting that p21Waf1/Cip1 cleavage may interfere with DNA repair, leading to increased frequency of double strand breaks (DSBs) and enhanced apoptosis. Here we show, for the first time, that p21Waf1/Cip1 cleavage, which is mediated by caspase-3 activity, is involved in roscovitine-induced apoptosis.
Zhang
Tao
T
Department of Thoracic Surgery, Tangdu Hospital, The Fourth Military Medical University, Xi'an, P.R. China.
Jiang
Tao
T
Zhang
Feng
F
Li
Chen
C
Zhou
Yong-An
YA
Zhu
Yi-Fang
YF
Li
Xiao-Fei
XF
D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy / Q000378:metabolism*; D061945:Caspase Inhibitors; D002453:Cell Cycle; D045744:Cell Line, Tumor; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism / Q000502:physiology*; D004260:DNA Repair; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic; D006657:Histones / Q000378:metabolism; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy / Q000378:metabolism*; D011687:Purines / Q000494:pharmacology*; D000077546:Roscovitine
D000970:Antineoplastic Agents; C493956:CDKN1A protein, human; D061945:Caspase Inhibitors; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004791:Enzyme Inhibitors; C468783:H2AX protein, human; D006657:Histones; D011687:Purines; D000077546:Roscovitine