Hypersensitivity to chromium-induced DNA damage correlates with constitutive deregulation of upstream p53 kinases in p21−/− HCT116 colon cancer cells

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.