The role of mitochondrial dysregulation in cellular death-resistance: Survival following a single genotoxin exposure Open Access
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Death-resistance and mitochondrial dysfunction may be two of the earliest events in carcinogenesis. It is well known that tumor cells utilize the glycolytic pathway to produce energy, even in the presence of sufficient oxygen levels. This has been termed the Warburg effect, and has been correlated with damaged or dysregulated mitochondria. In contrast, studies have demonstrated that mitochondria remain functional in tumor cells. Despite the controversy surrounding the role of mitochondria in carcinogenesis, a growing body of evidence has emerged focusing on mitochondrial function as a mechanism, marker, and/or mediator of cancer. Therefore, we hypothesize that selected survivors of genotoxin exposure may gain mitochondrial-mediated apoptosis-resistance, acquire a growth advantaged phenotype associated with mitochondrial dysregulation, and become predisposed to neoplastic progression. We have reported on the generation of sub-populations of BJ-hTERT human diploid foreskin fibroblasts, which have acquired resistance to hexavalent chromium [Cr(VI)]-induced death. Certain forms of Cr(VI) are known human respiratory carcinogens. Here, we report on subclones from clonogenic survivors of BJ-hTERT cells treated with 5 µM Cr(VI) (DR1, DR2, DR3, and DR4) or without (CC1, CC2). We investigated caspase 3 cleavage in these cell lines after 24 h exposure to Cr(VI). The CC cells exhibited a >70 fold increase in cleaved caspase 3 expression after Cr(VI) treatment, accompanied by substantial release of mitochondrial cytochrome c, consistent with induction of the mitochondrial apoptosis pathway. In sharp contrast, DR1 and DR2 cells showed significant resistance to Cr(VI)-induced caspase 3 cleavage, which was ~10-fold increase as compared to control, and Cr(VI)-induced cytochrome c release was negligible. Apoptotic resistance was not related to differences in hTERT, p53, or Bax (anti-apoptotic protein) protein levels, which were similar among the cell lines before and after Cr(VI) treatment. Moreover, the DR cells showed an increase in the expression of the pro-survival protein Bcl-2, after treatment with Cr(VI), which was significant in the DR1 cells, with no change in the death sensitive cells studied. We also investigated potential intrinsic mitochondrial alterations that may contribute to the observed apoptosis resistant phenotype. We measured mitochondrial (mt) DNA copy number by RT-PCR of the mtND1 gene, and found no difference among the cell lines, before or after Cr(VI) treatment. Notably, mtDNA is highly susceptible to oxidative damage. Amplification of an 8.9 kb mtDNA product has been shown to be inversely proportional to mtDNA damage. Intriguingly, our data show that the CC1 cells display 10-20% less amplifiable mtDNA, as compared to the DR1 and DR2 cells (reaching statistical significance in DR1), with no effect of Cr(VI) treatment. Mitochondrial spare respiratory capacity (SRC) has been associated with maintenance of a cellular energy reserve in the face of oxidative stress. We measured basal SRC in the CC and DR cells after injection of 1.5 µM FCCP, a respiratory chain uncoupler. The DR cells showed a significant, ~2-fold increase in oxygen consumption rate (OCR) as compared to CC cells, with no difference in basal OCR. Taken together, these data indicate that DR cells have both decreased mtDNA damage and increased SRC, which may contribute to their intrinsic death-resistance. In conclusion, our data suggest that cell survival after a single genotoxic insult involves the selection of cells with intrinsic mitochondrial dysregulation, leading to death-resistance, which may play a role in neoplastic progression.