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.
Hill
Richard
R
Department of Microbiology & Immunology, Dalhousie University, Halifax, NS, Canada.
Leidal
Andrew M
AM
Madureira
Patricia A
PA
Gillis
Laura D
LD
Cochrane
Haley K
HK
Waisman
David M
DM
Chiu
Arthur
A
Lee
Patrick W K
PW
D017304:Annexin A5; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002453:Cell Cycle / Q000187:drug effects*; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D002857:Chromium / Q000633:toxicity; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics* / Q000378:metabolism; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D015180:Electrophoresis, Gel, Two-Dimensional; D005786:Gene Expression Regulation / Q000187:drug effects* / Q000502:physiology; D006801:Humans; D034741:RNA, Small Interfering; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
D017304:Annexin A5; D050759:Cyclin-Dependent Kinase Inhibitor p21; D034741:RNA, Small Interfering; D016159:Tumor Suppressor Protein p53; D002857:Chromium