Abstract 122: Mitochondrial dysregulation and cellular death resistance in response to genotoxic stress
Authors: Nickens Kristen P. , Han Ying , Shandilya Harini , Gerard Gary F. , Kaldjian Eric P. , Wikstrom Jakob , Shirihai Orian , Patierno Steven , Ceryak Susan 
Published: February 2011

Cellular death resistance is at the foundation of neoplastic progression. Our objective is to identify mechanisms by which genotoxin exposure of normal human cells may lead to intrinsic alterations allowing for selection of survivors with a growth-advantaged phenotype. Certain forms of hexavalent chromium [(Cr(VI)] are human respiratory carcinogens/oxidizing agents. We have generated populations of BJ-hTERT fibroblasts (stably transfected with the telomerase gene), which have acquired resistance to Cr(VI)-induced death, that was not related to DNA adduct formation. In the present study, subclones were derived from clonogenic survivors of BJ-hTERT cells treated with either 0 µM (CC) or a single exposure to 5 µM (DR1 and DR2) Cr(VI).

We investigated caspase 3 cleavage in these lines after 24 h exposure to Cr(VI). CC cells exhibited a &gt;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 either hTERT or p53 protein levels, which were similar among the cell lines before and after Cr(VI) treatment.

We next 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 CC cells display 10-20% less amplifiable mtDNA, as compared to the DR 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. Of note, DR1 and DR2 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 be related 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. Supported by NIH grants CA107972 to SC and supplement to KW, ES05304 and ES09961 to SRP.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 122.