Inhibition of X-ray and Doxorubicin-induced Apoptosis by Butyrolactone I, a CDK-specific Inhibitor, in Human Tumor Cells

YANJUN LU HIRAKU TAKEBE TAKASHI YAGI apoptosis/CDK/doxorubicin/p21/X-ray Cell-cycle progression is coordinately regulated by cyclin-dependent kinases (CDKs). The inhibition of CDKs by p21Waf1/Cip1/Sdi1 prevents the apoptosis of cells treated with DNA-damaging agents. In this study, we found that butyrolactone I, a specific inhibitor of CDC2 family kinases, blocks the X-ray- or doxorubicin-induced apoptosis of DLD1 (p21+/+) human colorectal carcinoma cells in a dose-dependent manner. We also found that butyrolactone I inhibits the CDK2 activity and enhances cell survival after an X-ray irradiation or doxorubicin treatment in both DLD1 (p21-/-) and DLD1 (p21+/+) cells. These findings suggest that butyrolactone I prevents apoptosis by the direct inhibition of CDK and also, possibly, by CDKinhibition through p53-independent p21-induction. Our findings indicate that CDK activity is required for DNA-damaging agent-induced apoptosis. - Apoptosis is a physiological process of cell death that functions to control cell populations in the developing process of higher organisms. Apoptosis also occurs in certain types of cells when they suffer DNA damage. The signaling pathway that regulates apoptosis is not fully *Corresponding author: Phone; +8172254 9862, Fax; +8172254 9938, E-mail; Abbreviations: CDK; cyclin dependent kinase, DOX; doxorubicin, PBS; phosphate buffered saline understood. Evidence exists to suggest that the cell cycle and apoptosis may be interconnected. For example, in response to DNA damage, cells undergo cell-cycle arrest at both the G1/S and G2/M transitions before entry into subsequent stages to allow time for repair. However, some damaged cells still proceed with their cell cycles and thus escape from arrest to undergo apoptosis. Progression through G1 and entry into the S phase are regulated by the activation of cell cycledependent kinase 2 (CDK2), which makes a complex with cyclin A or cyclin E. The complexes phosphorylate pRb, thereby reversing Rb-mediated repression of those genes whose functions are required for DNA synthesis and entry into the S phase of the cycle. Apoptosis has been shown to be accompanied by changes in the CDK activity14). In a previous study, we demonstrated that DNA-damaging agents, doxorubicin (DOX) and X-rays, induce apoptosis, which is prevented by the ectopic expression of p21 (Waf1/Cip1/Sdi1) due to its CDK-binding or CDK-inhibitory activity in human colorectal carcinoma cells (DLD1)3). We also demonstrated that DOX- or X-ray-induced apoptosis was not prevented by mutated p21 lacking its CDK-binding or CDK-inhibitory activity, suggesting that the inhibition of CDK activity is required to prevent apoptosis. Butyrolactone I, -oxo--(p-hydroxyphenyl)--(p-hydroxy-m-3, 3-dimethylallyl benzyl)-methoxycarbonyl--butyrolactone, is a specific inhibitor of CDC2 family kinases which regulate the cell cycle by phosphorylation of the RB protein and H1 histone5,6). If the prevention of apoptosis by p21 is mediated by the inhibition of CDK activity, apoptosis should also be inhibited by butyrolactone I. To verify this postulation in the present study, we examined whether butyrolactone I inhibits CDK activity and prevents the DOX- or X-ray-induced apoptosis of p21 (+/+) and p21 (/) DLD1 cells. Cell culture DLD1 (p21/) and its parental DLD1 (p21+/+) cells were kindly supplied by Dr. B. Vogelstein. Both cells have a point mutation in the p53 gene7). The cells were cultured and maintained at 37C in Dulbeccos modified Eagles minimum essential medium (Nikken, Kyoto, Japan) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT). CDK2 kinase assay Cell extracts (100 g) obtained after the treatment of cells with butyrolactone I (0, 30, 40, 50, 60 M) for 24 h were incubated with an anti-CDK2 antibody (SC-163), and immune complexes were collected with 80 l protein G-plus-agarose (Santa Cruz Biotechnology, Santa Cruz, CA.) The cyclin-dependent kinase activity was measured using a CDK assay kit (Upstate Biotechnology, Lake Placid, NY). Briefly, the immune complexes (40 g) were mixed with [-32P] ATP (3000 Ci/mmol), histone H1 (2 g) as a substrate and a reaction buffer containing inhibitors of other kinases. After incubation for 10 min at 30C, phosphorylated histone H1 was resolved by 16% polyacrylamide-SDS gel electrophoresis and detected by autoradiography. Western blotting The cells at 90% confluence were washed with phosphate-buffered saline (PBS) and lysed at 4C for 10 min in a buffer consisting of 50 mM Tris-HCl, 150 mM NaCl, 0.05% SDS, 1% NP40, 1% trasylol and 0.2 mg/ml phenylmethylsulfonyl fluoride (Sigma, St. Louis, MO). Cellular debris was removed by centrifugation, and the protein content of the cell lysate was determined by the Bradford calorimetric assay method (Biorad, Richmond, CA). A 40 g aliquot of the cell lyate was resolved by 16% polyacrylamide-SDS gel electrophoresis and transferred to a Hybond-C Super membrane (Amersham, UK). The p21 protein was detected with an anti-p21 antibody (SC-187, Santa Cruz Biotechnology, Santa Cruz, CA.) and horseradish peroxidaseconjugated protein A using a Renaissance Chemiluminescence kit (Dupont, Boston, MA). Cell survival after X-ray irradiation or a doxorubicin treatment Details of the method were reported by Chen et al.8) In brief, exponentially growing cells were trypsinized, serially diluted and plated in 60 mm dishes, and then incubated in a medium supplemented with or without 60 M butyrolactone I for 24 h. After the cells were irradiated with X-rays or treated with DOX for 3 h, they were cultured in a medium with or without 60 M butyrolactone I for 24 h and then washed with PBS, followed by further incubation in a normal medium for 2 weeks. Colonies were stained with crystal violet and scored. Observation of apoptotic cells after X-ray irradiation or a doxorubicin treatment Cells were cultured in a medium containing butyrolactone I (0, 30, 40, 50, 60 M) after X-ray irradiation or a DOX (3 g/ml) treatment under the same conditions as mentioned above. They were then washed with PBS, stained with the DNA-binding dye Hoechst 33258 (0.5 g/ml in PBS), and observed under a fluorescence microscope. Cells showing condensed nuclear morphology and nuclear lobulations were regarded as being apoptotic. DNA fragmentation in cells after X-ray irradiation or doxorubicin treatment Cells were cultured in a medium with or without 60 M butyrolactone I after an X-ray irradiation (14 Gy) or DOX (3 g/ml) treatment, as mentioned above. DNA extracted with the Apoptosis Ladder Detection Kit (Wako, Osaka, Japan) was separated by agarose gel electrophoresis and stained with SYBR Green I (Molecular Probes, Eugene, OR). In a previous preliminary study, we demonstrated that butyrolactone I (50 M) markedly reduces a fraction of apoptotic cells that were produced by an X-ray or DOX treatment3). In the present study, we first treated DLD1 (p21+/+) cells with different concentrations of butyrolac (...truncated)