p21 blocks irradiation-induced apoptosis downstream of mitochondria by inhibition of cyclin-dependent kinase-mediated caspase-9 activation

Cancer Res. 2006 Dec 1;66(23):11254-62. doi: 10.1158/0008-5472.CAN-06-1569.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apoptosis / radiation effects*
  • Blotting, Western
  • Caspase 3 / metabolism
  • Caspase 9 / metabolism*
  • Caspase Inhibitors
  • Cyclin-Dependent Kinase Inhibitor p21 / deficiency
  • Cyclin-Dependent Kinase Inhibitor p21 / genetics
  • Cyclin-Dependent Kinase Inhibitor p21 / metabolism*
  • Cyclin-Dependent Kinases / antagonists & inhibitors
  • Cyclin-Dependent Kinases / metabolism*
  • Dose-Response Relationship, Drug
  • Enzyme Activation / drug effects
  • Enzyme Activation / radiation effects
  • Genotype
  • HCT116 Cells
  • Humans
  • Membrane Potentials / drug effects
  • Mitochondria / metabolism
  • Mitochondria / radiation effects*
  • Mitochondrial Membranes / drug effects
  • Mitochondrial Membranes / physiology
  • Mutation / genetics
  • Oligopeptides / pharmacology
  • Protein Binding / radiation effects
  • Protein Kinase Inhibitors / pharmacology
  • Purines / pharmacology
  • Radiation, Ionizing
  • Roscovitine
  • Tumor Suppressor Protein p53 / genetics
  • Tumor Suppressor Protein p53 / metabolism

Substances

  • Caspase Inhibitors
  • Cyclin-Dependent Kinase Inhibitor p21
  • Oligopeptides
  • Protein Kinase Inhibitors
  • Purines
  • Tumor Suppressor Protein p53
  • benzoylcarbonyl-aspartyl-glutamyl-valyl-aspartyl-fluoromethyl ketone
  • benzoylcarbonyl-valyl-aspartyl-valyl-alanyl-aspartyl-fluoromethyl ketone
  • Roscovitine
  • Cyclin-Dependent Kinases
  • Caspase 3
  • Caspase 9