Contribution of death receptor and mitochondrial pathways to Fas-mediated apoptosis in the prostatic carcinoma cell line PC3.

BACKGROUND

Two main pathways of apoptosis in mammalian cells have been described: the death receptor pathway and the mitochondrial pathway. Two different cell types have been identified for Fas-mediated apoptosis, each using almost exclusively one of two different signaling pathways. Human prostatic carcinoma cell line, PC3 is sensitive to Fas-mediated apoptosis, but relation of receptor and mitochondrial pathways is not clear.

METHODS

Cell viability was estimated by calcein assay. Apoptosis was determined by preparation of DNA ladder. Expression of Fas-associated death domain-dominant negative (FADD-DN) and Bcl-2, activation of caspases, PARP, DFF45, Bid cleavage, and cytochrome c release were assessed using Western blotting techniques. [(35)S] Methionine-labeled caspase-3 was transcribed in vitro and translated using the TNT kit (Promega). A vector containing caspase-3 was prepared by the ligation of EcoR I/BamHI flanked PCR fragment of full size caspase-3 cDNA into pBlusckript II SK(+/-) (Stratagen).

RESULTS

Overexpression of both FADD-DN and Bcl-2 genes prevent Fas-mediated apoptosis in PC3. As predicted, overexpression of FADD-DN prevented activation of caspase-8 and Bid cleavage and attenuated the release of cytochrome c and activation of caspases -2, -7, and -9. Bcl-2 overexpression did not affect caspase-8 activation and cleavage of Bid but blocked the release of cytochrome c and activation of mitochondria localized caspases -2, -7, and-9. Overexpression of FADD-DN and Bcl-2 affected the activation of caspase-3 and PARP cleavage differently: FADD-DN attenuated the activation of caspase-3 and PARP cleavage whereas Bcl-2 overexpression prevented caspase-3 activation and completely blocked cleavage of PARP.

CONCLUSIONS

These data suggest that activation of caspase-8 is necessary but not sufficient to complete Fas-mediated apoptosis in PC3 cells without activation of the mitochondrial pathway. In addition, caspase-3 activation after Fas-receptor ligation involves two steps and is dependent on mitochondrial activation.