Cellular vitamin C increases chromate toxicity via a death program requiring mismatch repair but not p53.
Ascorbate (Asc) plays a key role in reductive activation of carcinogenic chromium(VI) in vivo. In addition to much higher rates (t(1/2) = 1 min for 1 mM Asc), its reactions at physiological conditions differ from other reducers by low yields of Cr(V) intermediates. Human cells in culture are severely Asc deficient, which results in distorted metabolism and potentially abnormal responses to Cr(VI). We found that restoration of physiological Asc levels in human lung cells (primary IMR90 fibroblasts and epithelial H460 cells) increased clonogenic lethality and apoptosis by Cr(VI). Enhanced cytotoxicity in mass cultures was more evident after normalization for lower Cr uptake caused by leakage of Asc into media. Asc did not change uptake-adjusted yields of Cr-DNA adducts and had no effect on cytotoxicity when delivered shortly after Cr(VI) exposure. Protein and Ser-15 phosphorylation levels of p53 did not show any association with the presence of Asc and there were no increases in p53-driven reporter activity in Cr-treated cells. Stable silencing of p53 expression by short hairpin RNA (shRNA) had no effect on toxicity of Cr(VI) in both -Asc and +Asc IMR90 and H460 cells. In contrast, shRNA-mediated depletion of essential components of MutS or MutL mismatch repair complexes greatly improved survival of all Cr-treated cells and eliminated Asc-potentiated effects on cell death. Thus, mismatch repair-mediated enhancement of Cr(VI) cytotoxicity by Asc should promote the selection of MSI+/wt-p53 phenotype found among chromate-induced human lung cancers. Our findings also indicate that Asc plays a dual role in Cr(VI) toxicity: protective outside and potentiating inside the cell.
Reynolds
Mindy
M
Department of Pathology and Laboratory Medicine, Brown University, 70 Ship Street, Room 507, Providence, RI 02912, USA.
Zhitkovich
Anatoly
A
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism; D017209:Apoptosis; D001205:Ascorbic Acid / Q000378:metabolism* / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002840:Chromates / Q000633:toxicity*; D018736:DNA Adducts / Q000378:metabolism*; D053843:DNA Mismatch Repair; D006801:Humans; D008168:Lung / Q000166:cytology / Q000378:metabolism; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D017680:Potassium Compounds / Q000633:toxicity*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D002840:Chromates; D018736:DNA Adducts; C501486:MLH1 protein, human; D009687:Nuclear Proteins; D017680:Potassium Compounds; D016159:Tumor Suppressor Protein p53; C027373:potassium chromate(VI); C497172:MSH2 protein, human; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein; D001205:Ascorbic Acid