ABSTRACT
p21(Cip1/WAF1) is the principle mediator of cell cycle arrest in response to DNA damage. p21 primarily mediates G(1) cell cycle arrest by inactivating G(1)-associated cyclin A- and cyclin E-containing cyclin/cdk complexes. In the present study we investigate the role of p21 in DNA damage-induced G(2) cell cycle arrest, particularly with respect to the G(2)-associated cyclin, cyclin B1. We demonstrate that cells lacking p21 or deficient in their ability to upregulate p21 are unable to mediate the downregulation of cyclin B1 in response to DNA damage as compared to wild-type cells. Decreased levels of cyclin B1 in response to DNA damage seen in wild-type cells is due to p21-mediated degradation of cyclin B1 as this can be inhibited by a proteasomal inhibitor. Cell cycle analysis reveals that p21-null cells are unable sustain G(2) cell cycle arrest and accumulate at greater than 4N DNA content. These results indicate that p21-mediated degradation of cyclin B1 in response to DNA damage is necessary for the maintenance of G(2) cell cycle arrest.
Subject(s)
Cyclin B/metabolism , Cyclin-Dependent Kinase Inhibitor p21/metabolism , DNA Damage/physiology , Cell Line, Tumor , Cyclin B/genetics , Cyclin B1 , Cyclin-Dependent Kinase Inhibitor p21/genetics , Flow Cytometry , G2 Phase , Humans , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
Cellular stress and DNA damage up-regulate and activate p53, fundamental for cell cycle control, senescence, DNA repair and apoptosis. The specific mechanism(s) that determine whether p53-dependent cell cycle arrest or p53-dependent apoptosis prevails in response to specific DNA damage are poorly understood. In this study, we investigated two types of DNA damage, chromium treatment and gamma irradiation (IR) that induced similar levels of p53, but that mediated two distinct p53-dependent cell fates. Chromium exposure induced a robust DNA-dependent protein kinase (DNA-PK)-mediated apoptotic response that was accompanied by the rapid loss of the cyclin-dependent kinase inhibitor 1A (p21) protein, whereas IR treatment-induced cell cycle arrests that was supported by the rapid induction of p21. Inhibition of DNA-PK effectively blocked chromium-, but not IR-induced p53 stabilization and activation. In contrast, inhibition of ATM and ATR by caffeine had the inverse effect of blocking IR-, but not chromium-induced p53 stabilization and activation. Chromium exposure ablated p21 transcription but PUMA and Bax transcription was significantly enhanced compared to non-damaged cells. In contrast, IR treatment triggered significant p21 mRNA synthesis in addition to PUMA and Bax mRNA production. While chromium treatment enhanced the binding of p53 and RNA polymerase II (RNA Pol II) to both the p21 and PUMA promoters, RNA Pol II elongation was only observed along the PUMA gene and not the p21 gene. In contrast, following IR treatment, RNA Pol II elongation was observed on both p21 and PUMA. Chromium-induced apoptosis therefore involves DNA-PK-mediated p53 activation followed by preferential transcription of pro-apoptotic PUMA over anti-apoptotic p21 genes.
Subject(s)
Apoptosis/drug effects , Cell Cycle Proteins/metabolism , Chromium/pharmacology , DNA Damage/drug effects , DNA-Activated Protein Kinase/physiology , DNA-Binding Proteins/metabolism , Genes, p53 , Protein Serine-Threonine Kinases/metabolism , Tumor Suppressor Proteins/metabolism , Ataxia Telangiectasia Mutated Proteins , Cell Cycle/drug effects , Cell Cycle/radiation effects , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p21/genetics , DNA Damage/radiation effects , Gamma Rays , Humans , Models, Biological , Signal TransductionABSTRACT
The cyclin-dependent kinase inhibitor p21(CIP1/WAF1) is a key component in cell cycle control and apoptosis, directing an anti-apoptotic response following DNA damage. Chromium exposure resulted in a 500-1000 fold increase in apoptosis-induced cell death in p21-/- HCT116 cells compared to wild-type or p53-/- cells. p53 shRNA (or transient p53 siRNA) into p21-/- HCT116 cells reduced Cr(VI) sensitivity, suggesting the enhanced apoptosis in p21-/- cells is p53-dependent. Under non-DNA damage conditions, the p53 level in p21-/- cells was significantly higher than in wild-type cells, due to enhanced p53 phosphorylation and stabilization rather than elevated p53 transcription. Wild-type cells showed significant p53 protein induction upon DNA damage whereas p21-/- cells showed no p53 increase. p21-/- cells display the constitutive activation of upstream p53 kinases (ATM, DNA-PK, ATR, AKT and p38). 2D gel analysis revealed p53 patterns in p21-/- cells were distinct from those in wild-type cells before and after chromium exposure. Our results suggest that p21 has an important role in the cellular response to normal replicative stress and its absence leads to a "chronic DNA damage" state that primes the cell for p53-dependent apoptosis.
