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1 August 2002 Promotion of Photodynamic Therapy-Induced Apoptosis by the Mitochondrial Protein Smac/DIABLO: Dependence on Bax
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Photodynamic therapy (PDT) using the second-generation photosensitizer phthalocyanine (Pc) 4 causes mitochondrial damage and induces apoptosis through the release of cytochrome c to the cytosol. Another protein of the mitochondrial intermembrane space, Smac/DIABLO (second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI), is also released to the cytosol in response to apoptotic stimuli and promotes caspase activation by binding IAP. To investigate the possible role of Smac/DIABLO in apoptosis induced by Pc 4-PDT, we transfected Smac/DIABLO (tagged at its C-terminus with green fluorescent protein [GFP]) into MCF-7c3 cells (human breast cancer MCF-7 cells stably transfected with procaspase-3) and DU-145 cells (human prostate cancer cells that express no Bax because of a frameshift insertion mutation). Confocal microscopy showed that recombinant Smac/DIABLO, like cytochrome c, localized to mitochondria and colocalized with MitoTracker Red. Three hours after exposure of MCF-7c3 cells to PDT (200 nM Pc 4 and 150 mJ/cm2 red light), Smac/DIABLO–GFP, as well as cytochrome c, was found largely in the cytosol. In contrast, for DU-145 cells, both Smac/DIABLO–GFP and cytochrome c remained in the mitochondria after PDT. By staining with Hoechst 33342, typical apoptotic nuclei were observed in MCF-7c3 cells, but not in DU-145 cells, after Pc 4-PDT. These results suggest that the release of Smac/DIABLO from mitochondria may be regulated by a Bax-mediated mechanism and that Smac/DIABLO may cooperate with the cytochrome c–dependent apoptosis pathway. In addition, in MCF-7c3 cells transfected by Smac/DIABLO–GFP, apoptosis induced by Pc 4-PDT was greater than in cells transfected with the GFP vector alone or in untransfected cells, as determined by flow cytometry. Thus, Smac/DIABLO promotes apoptosis after Pc 4-PDT in a Bax-dependent manner and may facilitate the passage of PDT-treated cells through the late steps of apoptosis.

Jitsuo Usuda, Song-mao Chiu, Kashif Azizuddin, Liang-yan Xue, Minh Lam, Anna-Liisa Nieminen, and Nancy L. Oleinick "Promotion of Photodynamic Therapy-Induced Apoptosis by the Mitochondrial Protein Smac/DIABLO: Dependence on Bax," Photochemistry and Photobiology 76(2), 217-223, (1 August 2002).<0217:POPTIA>2.0.CO;2
Received: 19 March 2002; Accepted: 1 May 2002; Published: 1 August 2002

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