Here we examined the role of cellular vitamin C in genotoxicity of carcinogenic chromium(VI) that requires reduction to induce DNA damage. In the presence of ascorbate (Asc), low 0.2–2 μM doses of Cr(VI) caused 10–15 times more chromosomal breakage in primary human bronchial epithelial cells or lung fibroblasts. DNA double-strand breaks (DSB) were preferentially generated in G2 phase as detected by colocalization of γH2AX and 53BP1 foci in cyclin B1-expressing cells. Asc dramatically increased the formation of centromere-negative micronuclei, demonstrating that induced DSB were inefficiently repaired. DSB in G2 cells were caused by aberrant mismatch repair of Cr damage in replicated DNA, as DNA polymerase inhibitor aphidicolin and silencing of MSH2 or MLH1 by shRNA suppressed induction of γH2AX and micronuclei. Cr(VI) was also up to 10 times more mutagenic in cells containing Asc. Increasing Asc concentrations generated progressively more mutations and DSB, revealing the genotoxic potential of otherwise nontoxic Cr(VI) doses. Asc amplified genotoxicity of Cr(VI) by altering the spectrum of DNA damage, as total Cr-DNA binding was unchanged and post-Cr loading of Asc exhibited no effects. Collectively, these studies demonstrated that Asc-dependent metabolism is the main source of genotoxic and mutagenic damage in Cr(VI)-exposed cells.

Acquired or inherent drug resistance is the major problem in achieving successful cancer treatment. However, the mechanism(s) of pleiotropic drug resistance remains obscure. We have identified and characterized a cellular metabolic strategy that differentiates drug-resistant cells from drug-sensitive cells. This strategy may serve to protect drug-resistant cells from damage caused by chemotherapeutic agents and radiation. We show that drug-resistant cells have low mitochondrial membrane potential, use nonglucose carbon sources (fatty acids) for mitochondrial oxygen consumption when glucose becomes limited, and are protected from exogenous stress such as radiation. In addition, drug-resistant cells express high levels of mitochondrial uncoupling protein 2 (UCP2). The discovery of this metabolic strategy potentially facilitates the design of novel therapeutic approaches to drug resistance.

Mismatch repair (MMR) strongly enhances cyto- and genotoxicity of several chemotherapeutic agents and environmental carcinogens. DNA-double-strand breaks (DSB) formed after two replication cycles play a major role in MMR-dependent cell death by DNA alkylating drugs. Here we examined DNA damage detection and the mechanisms of the unusually rapid induction of DSB by MMR proteins in response to carcinogenic chromium(VI). We found that MSH2-MSH6 (MutSα) dimer effectively bound DNA probes containing ascorbate-Cr-DNA and cysteine-Cr-DNA crosslinks. Binary Cr-DNA adducts, the most abundant form of Cr-DNA damage, were poor substrates for MSH2-MSH6 and their toxicity in cells was weak and MMR-independent. Although not involved in the initial recognition of Cr-DNA damage, MSH2-MSH3 (MutSβ) complex was essential for the induction of DSB, micronuclei and apoptosis in human cells by chromate. In situ fractionation of Cr-treated cells revealed MSH6 and MSH3 chromatin foci that originated in late S phase and did not require replication of damaged DNA. Formation of MSH3 foci was MSH6- and MLH1-dependent whereas MSH6 foci were unaffected by MSH3 status. DSB production was associated with progression of cells from S into G2 phase and was completely blocked by the DNA synthesis inhibitor aphidicolin. Interestingly, chromosome 3 transfer into MSH3-null HCT116 cells activated an alternative, MSH3-like activity that restored dinucleotide repeat stability and sensitivity to chromate. Thus, sequential recruitment and unprecedented cooperation of MutSα and MutSβ branches of MMR in processing of Cr-DNA crosslinks is the main cause of DSB and chromosomal breakage at low and moderate Cr(VI) doses.