Activation of the Fas pathway independently of Fas ligand during apoptosisinduced by camptothecin in p53 mutant human colon carcinoma cells

The present study explored the role of the cell surface receptor Fas (CD95/ APO-1) in apoptosis induced by camptothecin (CPT) in the HT29 colon carcino ma cell line. CPT-induced apoptosis was associated with high molecular weig ht DNA fragmentation as measured by filter elution, This fragmentation was inhibited by the caspase inhibitor, z-VAD-fmk and by cycloheximide, which a lso prevented proteolytic activation of caspase-3 and poly(ADP-ribose)polym erase cleavage. Under such conditions, Fas, Fas ligand, Bar, and p21 expres sion were increased and Fas recruited the FADD adaptor. Fas expression incr ease was blocked by cycloheximide but not by z-VAD-fmk, consistent with cas pase activation downstream from Fas, Treatment of HT29 cells with Fast or w ith the CH-11 agonistic anti-Fas antibody potentiated the apoptotic respons e of cells treated with CPT, The anti-Fas blocking antibody ZB4 and the Fas -ligand inhibitor failed to protect HT29 cells from CPT-induced apoptosis, Such a protection was obtained by transient expression of constructs encodi ng a dominant-negative mutant of FADD, FADD in an antisense orientation and E8 or MC159 viral proteins that inhibit Fas-induced apoptosis at the level of FADD and procaspase-8, respectively. Together, these data show that top oisomerase I-mediated DNA damage-induced apoptosis involves activation of t he Fas pathway without detectable Fas-ligand requirement in CPT-treated cel ls.