Abstract
2723
Mitochondria are critical to homeostatic control, and are at the crux of cell growth and death. Certain forms of hexavalent chromium [Cr(VI)] are known respiratory carcinogens. We have generated populations of BJ-hTERT (death-sensitive, DS) fibroblasts subcloned from survivors of Cr(VI) exposure that acquired resistance to genotoxin-induced clonogenic lethality (death-resistant, DR). We found that after genotoxin exposure, DS cells displayed increased caspase-3 cleavage, mitochondrial membrane depolarization and VDAC mRNA expression, in sharp contrast to the DR cells. Moreover, DR cells exhibited genotoxin-induced Akt activation, while Akt was downregulated in treatment-matched DS cells. Our overarching hypothesis is that survival after genotoxic insult may involve selection of cells with mitochondrial dysregulation, leading to death resistance. Hexokinase (HK) catalyzes the first step in glucose metabolism. HKII has high affinity for both glucose and ATP, and is overexpressed in many tumors. Due to its co-localization to the outer mitochondrial membrane via VDAC, HKII can couple glycolysis to oxidative phosphorylation, therefore supporting the highly glycolytic phenotype of tumors. Notably, HKII-mitochondrial (HKII-mito) association is correlated with decreased apoptosis and enhanced by Akt activation. Our aim was to elucidate the role of mitochondrial HKII in cellular death resistance and genomic instability. Basally, HKII-mito association was 4.5-fold higher in DR versus DS cells. Moreover, following 24h exposure to 6μM Cr (VI), HKII-mito association was decreased, but DR cells still exhibited a 3-fold higher HKII-mito association, compared to DS cells. Total HKII protein expression was also 3-fold higher in the DR versus DS cells following Cr(VI) exposure. Mitochondrial morphology was monitored by immunofluorescence (IF) and electron microscopy (EM). The DR cells displayed a diffuse reticular network as evidenced by IF staining with Mitotracker-Green, while the DS cells showed peri-nuclear localization. However no changes were found in mitochondrial shape or size by EM. Additionally, FACS analysis with Mitotracker-CMXRos suggested increased mitochondrial activity in the DR cells, which we postulate may correlate with increased oxidative phosphorylation (i.e., ATP synthesis). Mitochondria are thought to be involved in the maintenance of genomic stability by regulating ATP-dependent pathways such as DNA repair and replication. We also found that DR cells were able to override the G2/M cell cycle checkpoint following 24h exposure to 1μM Cr (VI). The potential link between G2/M override and mitochondrial-mediated survival is currently under investigation. In conclusion, our data suggest a role for mitochondrial regulation in survival signaling and 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.
99th AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA