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Cell survival in the face of genotoxic stress, may result in the acquisition of a selective growth advantage that predisposes these cells to transformation. Dysregulation of the major survival pathway involving Akt is associated with neoplastic transformation. Certain forms of hexavalent chromium (Cr(VI)) are respiratory carcinogens and genotoxins. Certain DNA damaging agents have been shown to initially activate Akt but this activation is followed by downregulation of both phosphorylation state and total protein expression. We have shown in human lung fibroblasts (HLF), that 24h exposure to 1-6μM Cr(VI) results in a dose-dependent decrease in both phospho-ser473 Akt and total Akt expression that correlates with a decrease in Akt activity as measured by phosphorylation of the substrate GSK3β in vitro. The aim of this study was to determine the mechanism responsible for the Cr(VI) dose-dependent decrease in Akt expression in HLF cells. Involvement of the proteasome was ruled out since inhibition by MG132 (10-25μM) for 24h did not reverse the Cr(VI)-induced degradation of Akt in HLF cells. AKT has been reported to be downregulated by both caspase-dependent and independent mechanisms. We therefore studied caspase activation in response to 24h exposure to Cr(VI). Our results show an increase in caspase-9 cleavage in response to 24h exposure to 1-6μM Cr(VI) but no apparent change in caspase-8 expression as measured by cleaved caspase-8 expression by western blot. Furthermore, addition of the caspase-9 inhibitor Z-LEHD-FMK (50μM) prior to Cr(VI) exposure appeared to partially prevent Cr(VI)-induced Akt downregulation. We have also shown that maintenance of tyrosine phosphorylation through protein tyrosine phosphatase (PTP) inhibition activates Akt. Sodium orthovanadate (SOV, 10μM), a broad range protein tyrosine phosphatase inhibitor, when co-incubated with 1-6μM Cr(VI) resulted in the abrogation of the Cr(VI)-induced decrease in both phospho-ser473 and total Akt expression as well as Akt activity suggesting a role for protein tyrosine phosphatases in Akt downregulation. Therefore we studied the effect of PTP inhibition on caspase-9 cleavage. Our results show that 10μM SOV abrogated caspase-9 cleavage in response to 24h exposure to 1-6μM Cr(VI). These results suggest that the Cr(VI)-induced downregulation of phospho-ser473, total Akt, as well as Akt activity is caspase-dependent and that maintenance of tyrosine phosphorylation induces a survival response that most probably precedes caspase activation. Thus Akt appears to be at the nexus between death and survival after genotoxic exposure. Understanding the mechanisms involved in Akt down-regulation in response to genotoxic stress should provide a better understanding of why some cells survive genotoxic insult and eventually progress to a malignant phenotype. Supported by NIH grants ES05304 and ES09961 to SRP.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]