Attenuated AKT1 knockdown in BJ-hTERT fibroblast “survivors” of Cr(VI) genotoxic insult that are resistant to genotoxin-induced clonogenic death Free

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Certain hexavalent chromium [(Cr(VI)] compounds are human respiratory carcinogens and genotoxins. Inappropriate response of key cell survival signaling pathways following genotoxic insult can contribute to autonomous cell growth and neoplastic transformation. Constitutive activation of AKT plays an important role in lung carcinogenesis and cells with activated AKT can overcome a G2/M checkpoint arrest induced by DNA damage. Our previous studies in human lung fibroblasts suggest that maintenance of protein tyrosine phosphorylation, which correlates with AKT activation, overrides Cr(VI)-induced growth arrest and enhances clonogenic survival. We have generated a subclonal population of BJ-hTERT foreskin fibroblasts that survived 24 hour Cr(VI) exposure and have previously shown that these DP 5.14 cells have acquired resistance to Cr-induced clonogenic lethality that is not related to altered uptake. Moreover, DP 5.14 cells are dramatically impaired in their ability to cleave pro-caspase 3 after Cr(VI) exposure. Our preliminary studies suggested that the enhanced survival of DP 5.14 cells in the face of genotoxic insult may be due to a deregulated AKT pathway. The aim of the present study was to determine the role of Akt1 in death resistance of DP 5.14 cells. BJh-TERT and DP 5.14 cells were transfected with increasing amounts of Akt1 siRNA and AKT1 and pro-caspase 3 protein expression were determined following 24 h Cr(VI) treatment. We found that 30nM of Akt1 siRNA was the maximum concentration necessary to knockdown AKT1 in BJ-hTERT cells by greater than 75%. In contrast, a 3- fold greater concentration of Akt1 siRNA was needed to achieve a similar level of knockdown in DP 5.14 cells. These data support preliminary studies suggesting that AKT was constitutively activated in DP 5.14 cells. Therefore, we decreased the basal AKT activity in both BJh-TERT and DP 5.14 cells prior to siRNA transfection by 4 h serum starvation. Under this condition we achieved at least a 70% knockdown of AKT1 in both BJh-TERT and DP 5.14 cells. Finally, AKT1 knockdown in DP 5.14 cells by siRNA resulted in pro-caspase 3 cleavage following Cr(VI) exposure suggesting restoration of sensitivity to Cr-induced cell death. Taken together, these results support a key role for Akt1 in resistance to Cr-induced cell death which in turn may contribute to genomic instability and lead to carcinogenesis. Supported by NIH grants ES05304 and ES09961 to SRP and CA107972 to SMC.