Sensitization of DNA damage–induced apoptosis by the proteasome inhibitor PS-341 is p53 dependent and involves target proteins 14-3-3S and survivin

Proteasome inhibition following DNA damage results in the synergistic induction of apoptosis via a nuclear factor-KB– independent mechanism. In this study, we identify the role of p53 in mediating apoptosis by the sequence-specific treatment involving the DNA-damaging, topoisomerase I–targeting drug SN-38 followed by the proteasome inhibitor PS-341 (SN-38!PS-341). The p53-dependent sensitization of DNA damage– induced apoptosis by PS-341 is accompanied by persistent inhibition of proteasome activity and increased cytosolic accumulation of p53, including higher molecular weight forms likely representing ubiquitinated species. In contrast, pretreatment with PS-341 followed by treatment with SN-38 (PS-341!SN-38), which leads to an antagonistic interaction, results in transient inhibition of proteasome activity and accumulation of significantly lower levels of p53 localized primarily to the nucleus. Whereas cells treated with PS-341!SN-38 undergo G2 + M cell cycle arrest, cells treated with SN-38!PS-341 exhibit a decreased G2 + M block with a concomitant increase in the sub-G1 population. Decreased accumulation of cells in the G2 +M phase of the cell cycle in SN-38!PS-341–treated cells compared with PS-341!SN-38–treated cells correlates with enhanced apoptosis and reduced expression of two p53-modulated proteins, 14-3-3S and survivin, both of which play critical roles in regulating G2 + M progression and apoptosis. The functional role of 14-3-3S or survivin in regulating the divergent function of p53 in response to SN-38!PS-341 and PS-341!SN-38 treatment in inducing apoptosis versus G2 + M arrest/DNA repair, respectively, was confirmed by targeted down-regulation of these proteins. These results provide insights into the mechanisms bywhich inhibition of proteasome activity modulates DNA damage–induced apoptosis via a p53-dependent pathway. [Mol Cancer Ther 2005;4(12):1880–90]