RAPID CYTOCHROME-C RELEASE, ACTIVATION OF CASPASE-3, CASPASE-6, CASPASE-7 AND CASPASE-8 FOLLOWED BY BAP31 CLEAVAGE IN HELA-CELLS TREATED WITH PHOTODYNAMIC THERAPY

Photodynamic therapy (PDT) is a clinical approach that utilizes light- activated drugs for the treatment of a variety of pathologic condition s. The initiating events of PDT-induced apoptosis are poorly defined. It has been shown for other pro-apoptotic stimuli that the integral en doplasmic reticulum protein Bap31 is cleaved by caspases 1 and 8, but not by caspase-3, Further, a 20 kDa Bap31 cleavage fragment is generat ed which can induce apoptosis, In the current report, we sought to det ermine whether Bap31 cleavage and generation of p20 is an early event in PDT-induced apoptosis, The mitochondrial release of cytochrome c, i nvolvement of caspases 1, 2, 3, 4, 6, 7, 8, and 10 and the status of s everal known caspase substrates, including Bap31, were evaluated in PD T-treated HeLa cells. Cytochrome c appeared in the cytosol immediately following light activation of the photosensitizer benzoporphyrin deri vative monoacid ring A. Activation of caspases 3, 6, 7, and 8 was evid ent within 1-2 h post PDT, Processing of caspases 1, 2, 4, and 10 was not observed, Cleavage of Bap31 was observed at 2-3 h post PDT. The ca spase-3 inhibitor DEVD-fmk blocked caspase-8 and Bap31 cleavage sugges ting that caspase-8 and Bap31 processing occur downstream of caspase-3 activation in PDT-induced apoptosis, These results demonstrate that r elease of mitochondrial cytochrome c into the cytoplasm is a primary e vent following PDT, preceding caspase activation and cleavage of Bap31 , To our knowledge, this is the first example of a chemotherapeutic ag ent inducing caspase-8 activation and demonstrates that caspase-8 acti vation can occur after cytochrome c release, (C) 1998 Federation of Eu ropean Biochemical Societies.