Involvement of FKHR-Dependent TRADD Expression in Chemotherapeutic Drug-Induced Apoptosis

Abstract

Chemotherapeutic drugs exhibit their cytotoxic effect by inducing apoptosis in tumor cells. Because the serine/threonine kinase Akt is involved in apoptosis suppression, we investigated the relationship between Akt activity and drug sensitivity. We discovered that certain chemotherapeutic drugs induced apoptosis with caspase activation only when Akt was inactivated after drug treatment, while inactivation of Akt was not observed when tumor cells showed resistance to the drug-induced caspase activation. So, turn-off of the Akt-mediated survival signal is correlated with the sensitivity of the cells to chemotherapy. With a cDNA microarray, we revealed that tumor necrosis factor receptor-associated death domain (tradd) gene expression was elevated in response to Akt inactivation. Reportedly, Forkhead family transcription factors are phosphorylated by Akt, which results in their nuclear exit and inactivation. Analysis of the tradd promoter revealed that it contains at least one potential Forkhead family transcription factor-responsive element, and we confirmed that this element was involved in chemotherapeutic drug-induced TRADD expression. Overexpression of mutant TRADD proteins to block its apoptosis-inducing capability attenuated chemotherapeutic drug-induced apoptosis. Thus, chemotherapeutic drugs exhibited their cytotoxic effects in part by down-regulating Akt signaling following TRADD expression. These results indicate that Akt kinase activity after drug treatment is a hallmark of sensitivity of the cells to chemotherapeutic drugs.Many growth factors and cytokines have been reported to promote cell survival. The characterization of survival signal transduction pathways stimulated by these factors has revealed that phosphatidylinositide-3-OH kinase (PI3K) is involved in apoptosis suppression (16). PI3K is a heterodimeric lipid kinase consisting of an 85-kDa regulatory subunit and a 110-kDa catalytic subunit. After growth factor stimulation, PI3K is activated and generates phospholipid second-messenger molecules, phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P 3 ] and phosphatidylinositol-3,4-bisphosphate [PtdIns(3,4)P 2 ], that raise a diverse set of cellular responses. The major targets of PtdIns(3,4,5)P 3 and PtdIns(3,4)P 2 are pleckstrin homology domain-containing proteins.One target of PI3K is the pleckstrin homology domaincontaining serine/threonine kinase Akt (also known as protein kinase B) (16), and some pharmacological PI3K inhibitors, such as wortmannin and LY294002, suppress Akt kinase activity. Akt was originally identified as the cellular homolog of the viral oncogene v-akt (45). Akt mediates many PI3K-regulated biological responses, including glucose uptake, protein synthesis, cell cycle progression, and inhibition of apoptosis. Catalytically inactive Akt resides within the cytosol. By stimulation with growth factors and cytokines, the PtdIns(3,4,5)P 3 and PtdIns(3,4)P 2 proteins generated lead to the recruitment of Akt to the plasma membrane. Akt is then phosphorylated at two specific ph...

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