title: p21 blocks irradiation-induced apoptosis downstream of mitochondria by inhibition of cyclin-dependent kinase-mediated caspase-9 activation.
abstract: The role of the cyclin-dependent kinase (CDK) inhibitor p21 as a mediator of p53-induced growth arrest is well established. In addition, recent data provide strong evidence for new emerging functions of p21, including a role as a modulator of apoptosis. The mechanisms, however, by which p21 interferes with the death machinery, especially following ionizing radiation (IR), are largely unknown. Here, we report that IR induced caspase-9 and caspase-3 activation and subsequent apoptosis only in p21-deficient colon carcinoma cells, whereas similar treated wild-type cells were permanently arrested in the G(2)-M phase, correlating with the induction of cellular senescence. Interestingly, activation of the mitochondrial pathway, including caspase-2 processing, depolarization of the outer mitochondrial membrane, and cytochrome c release, was achieved by IR in both cell lines, indicating that p21 inhibits an event downstream of mitochondria but preceding caspase-9 activation. IR-induced p21 protein expression was restricted to the nucleus, and no evidence for a mitochondrial or cytoplasmic association was found. In addition, p21 did neither interact with caspase-3 or caspase-9, suggesting that these events are not required for the observed protection. Consistent with this assumption, we found that CDK inhibitors potently abrogated IR-induced caspase processing and activation without affecting mitochondrial events. In addition, in vitro caspase activation assays yielded higher caspase-3 activities in extracts of irradiated p21-deficient cells compared with extracts of similar treated wild-type cells. Thus, our results strongly indicate that p21 protects cells from IR-induced apoptosis by suppression of CDK activity that seems to be required for activation of the caspase cascade downstream of the mitochondria.
authors.affiliation: Institute of Molecular Medicine, University of Düsseldorf, Düsseldorf, Germany.
authors.forename: Dennis
authors.initials: D
authors.lastname: Sohn
authors.forename: Frank
authors.initials: F
authors.lastname: Essmann
authors.forename: Klaus
authors.initials: K
authors.lastname: Schulze-Osthoff
authors.forename: Reiner U
authors.initials: RU
authors.lastname: Jänicke
mesh_terms: D017209:Apoptosis / Q000528:radiation effects*; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism*; D061945:Caspase Inhibitors; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects / Q000528:radiation effects; D005838:Genotype; D045325:HCT116 Cells; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000378:metabolism / Q000528:radiation effects*; D051336:Mitochondrial Membranes / Q000187:drug effects / Q000502:physiology; D009154:Mutation / Q000235:genetics; D009842:Oligopeptides / Q000494:pharmacology; D011485:Protein Binding / Q000528:radiation effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011687:Purines / Q000494:pharmacology; D011839:Radiation, Ionizing; D000077546:Roscovitine; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism
chemical_list: D061945:Caspase Inhibitors; D050759:Cyclin-Dependent Kinase Inhibitor p21; D009842:Oligopeptides; D047428:Protein Kinase Inhibitors; D011687:Purines; D016159:Tumor Suppressor Protein p53; C110772:benzoylcarbonyl-aspartyl-glutamyl-valyl-aspartyl-fluoromethyl ketone; C427308:benzoylcarbonyl-valyl-aspartyl-valyl-alanyl-aspartyl-fluoromethyl ketone; D000077546:Roscovitine; D018844:Cyclin-Dependent Kinases; D053148:Caspase 3; D053453:Caspase 9