CD95(APO-1/Fas) is a cell surface receptor that, when oligomerized by natural ligand, CD95L, or antibody, confers an apoptotic signal to apoptosis-sensitive cells. Whereas CD95 is expressed in every colonocyte of normal colon mucosa, CD95 is down-regulated or lost in the majority of colon carcinomas.
To investigate the sensitivity to CD95-mediated apoptosis of normal and neoplastic colonocytes, we applied cross-linking CD95(anti-APO-1) monoclonal antibody to freshly isolated colon crypts and colon carcinoma cell lines and monitored apoptosis by DNA fragmentation and morphology.
Normal colonocytes were constitutively sensitive to CD95-mediated apoptosis. All carcinoma lines were constitutively resistant but were sensitized upon pretreatment with IFN-γ. Transcription blocking, protein synthesis, and export in carcinoma cells indicated that even low surface levels of CD95 were sufficient to efficiently transmit the signal. Despite low CD95 surface levels of non-IFNγ-treated cells, actinomycin D, cycloheximide, and brefeldin A each sensitized all cell lines, but at different rates and kinetics. In this context, it was observed that a greatly delayed apoptotic response of SW620 cells was associated with the absence of antibody-induced CD95 capping. Phorbol 12-myristate 13-acetate inhibited CD95-mediated apoptosis by counteracting the IFNγ-, actinomycin D-, and cycloheximide-mediated but not the brefeldin A-mediated sensitization. This phorbol 12-myristate 13-acetate-induced protection against apoptosis was completely abolished by staurosporine and by a selective protein kinase C inhibitor, Goe 6983.
We conclude that, during malignant transformation, colonocytes acquire different mechanisms to escape CD95-mediated apoptosis. These include abrogation of CD95, inhibition of CD95 capping, and activation of antiapoptotic programs, both governed by and independent of protein kinase C.
This study was supported by grants from the Medizinische Fakultät der Universität Ulm, the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (Gesundheit 2000), the Tumorzentrum Heidelberg/Mannheim, and the Deutsche Krebshilfe.