Colon carcinoma cells use different mechanisms to escape CD95-mediated apoptosis.

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-gamma. 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-IFNgamma-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 IFNgamma-, 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.

Early detachment of colon carcinoma cells during CD95(APO-1/Fas)-mediated apoptosis. I. De-adhesion from hyaluronate by shedding of CD44.

Ligation of CD95 (APO-1/Fas) cell surface receptors induces death in apoptosis-sensitive cells. Induction of apoptosis in adherent gamma interferon-stimulated HT-29 and COLO 205 colon carcinoma cells by cross-linking CD95 with anti-APO-1 monoclonal antibody resulted in detachment of the cells from hyaluronate starting about 1 h after antibody exposure. Loss of adhesion was paralleled by a substantial reduction of the multifunctional cell surface adhesion molecule CD44. As evidenced by cycloheximide treatment, this effect was not caused by impaired protein synthesis. Depletion of surface CD44 was also not due to membrane blebbing, since cytochalasin B failed to inhibit ascension from hyaluronate. Instead, ELISA and time kinetics showed increasing amounts of soluble CD44 in the supernatant of CD95-triggered cells. SDS-PAGE revealed that soluble CD44 had an apparent molecular mass of about 20 kD less than CD44 immunoprecipitated from intact cells. Thus, CD95-triggering induced shedding of CD44. Shedding is a novel mechanism operative in early steps of CD95-mediated apoptosis. Shedding surface molecules like CD44 might contribute to the active disintegration of dying epithelial cells in vivo.

[CD95 (APO-1/Fas) and CD95-ligand (CD95L). Implications of these apoptosis mediating receptor/ligand systems in the pathogenesis of autoimmune diseases]. / CD95 (APO-1/Fas) and CD95-Ligand (CD95 L). Implikationen dieses Apoptose-vermittelnden Rezeptor/Ligand-Systems in der Pathogenese von Autoimmunerkrankungen.

The CD95 (APO-1/Fas) receptor mediates programmed cell death in apoptosis sensitive cells upon oligomerization either by CD95 antibody or by its natural ligand, CD95 ligand (CD95 L). Studies on tissue distribution have shown that CD95 is broadly expressed in normal adult tissues. Furthermore, the spectrum of CD95-expressing cells in inducibly enlarged in the context of chronic inflammation. In contrast, the number of cells capable of expressing CD95 L is strikingly limited to small subsets of lymphocytes and histiocytes and to some specialized normal epithelia. This suggests that cell death via this receptor/ligand system, although possible in almost every tissue and cell type, is limited to very special scenarios one of which is chronic lymphohistiocytic inflammation. The lpr/lpr mouse and the gld/gld mouse are well-known models for autoimmune disorders. Both mutants have abnormal B and T lymphocytes and high titers of autoantibodies. Recently, these mice have been discovered to have functional defects in the murine equivalents of human CD95 and CD95 L, respectively. This suggests that the CD95/CD95 L system might act by preventing autoimmune disease, e.g., by preventing emergence of autoreactive T and B lymphocytes. A human homologue of the lpr mutation has been described as autoimmune lymphoproliferative syndrome. We show that, in mice, CD95/ CD95 L might be operative in experimental graft versus host disease. Further, we suggest a role of this system in early steps of ulcerative colitis. Considering our observations against the background of current literature, CD95/CD95 L is likely to play a dual role in induction and maintenance of peripheral tolerance on the one hand and in tissue damage by chronic inflammation on the other.