Involvement of p70S6K in Cisplatin-Induced Cell Death

Dhar, Rohini
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Rohini Dhar, Involvement of p70S6K in cisplatin-induced cell death. Doctor of Philosophy (Biochemistry and Molecular Biology) May 2008, 151 pp., 23 illustration, 3 tables, 153 references. Cisplatin is used for the treatment of solid tumors; however its success is often compromised due to relapse and chemoresistance. The purpose of this dissertation is to delineate the role of p70S6K in cisplatin-induced apoptosis. A comparison of p70S6K levels in H69 cells that acquired resistance to cisplatin (H69/CP) compared to parental H69 cells revealed that levels of phosphorylated p70S6K and S6 were elevated. Cisplatin treatment resulted in the activation of p70S6K and downregulation of total p70S6K. Inhibition of the phosphoinositide 3-kinase (P13K) pathway by itself augmented cisplatin-induced PARP cleavage and also blocked the phosphorylation of p70S6K. Inhibition of extracellular signal-regulated kinase (ERK) pathway however attenuated cisplatin-induced PARP cleavage. These results reveal that phosphorylation of p70S6K is associated with increased cisplatin resistance, and inhibition of P13K/ p70S6K pathway could reverse cisplatin resistance. We have found that cisplatin caused a time- and concentration-dependent downregulation of p70S6K. While the calpain and proteasome inhibitors had no effect on the downregulation of p70S6K, the broad specificity caspase inhibitor z-VAD-fmk (z-VAD) reversed p70S6K downregulation by cisplatin. Furthermore, the caspase-3 inhibitor and knockdown of caspase-3 prevented cisplatin-induced proteolytic cleavage of p70S6K. While, cisplatin failed to induce cleavage of p70S6K in MCF-7 cells that lack functional caspase-3, overexpression of caspase-3 in these cells resulted in cisplatin-induced cleavage of p70S6K. Thus, these results demonstrate that p70S6K is a novel substrate for caspase-3. Examination of the role of p70S6K in cisplatin-induced death shows that rapamycin in a pharmacological inhibitor of p70S6K, enhanced cisplatin-induced apoptosis in A549 cells. However, knockdown of p70S6K by siRNA resulted in a decrease in cisplatin-iduced apoptosis. In addition, caspase-3 mediated cleavage of p70S6K at the aspartic acid residue at the 393 position and site-directed mutagenesis of Asp393 to Ala resulted in protection against cisplating-mediated apoptosis. Interestingly, introduction of the N-terminal cleaved fragment [∆(394-525)] resulted in potentiation of cisplatin-induced apoptosis. These results suggest that the proteolytic cleavage of p70S6K by caspase-3 is important for cisplatin-induced apoptosis.