title: CD95-related apoptotic machinery is functional in pancreatic cancer cells.
abstract: The CD95 (FAS, Apo-1) system is a major death pathway in normal and tumor cells. Recent evidence indicates that pancreatic cancer cells express CD95R and CD95L but are insensitive to CD95-mediated apoptosis. Here we show that treatment of human pancreatic cancer cells with RNA synthesis inhibitor actinomycin D (ActD) converted the phenotype of cancer cells from CD95 resistant to CD95 sensitive. Flow cytometric analysis demonstrated that all pancreatic cancer cell lines studied responded with cell surface CD95R and CD95L upregulation to bleomycin treatment, and PANC1 (mt p53) cells demonstrated a dose-dependent response to interferon gamma and bleomycin treatment with CD95R and CD95L up-regulation. However, only bleomycin sensitized PANC1 cells to CD95-mediated apoptosis. Taxol sensitized PANC1 and HPAC cells to CD95-mediated apoptosis without surface up-regulation of CD95R. These data suggest that pancreatic cancer cells possess a p53-independent mechanism of CD95R and CD95L surface upregulation and that surface expression of CD95R is not predictive of apoptotic function. Protein extracts of HPAC and PANC1 cells treated for 24 hours with a combination of ActD/agonist anti-CD95 antibodies demonstrated significantly higher Acetyl-Asp-Glu-Val-Asp-ase (DEVDase) cleavage activity (caspase 3-like activity) than extracts from cells treated with ActD only. In the present study, we also investigated the time kinetics of DEVDase (caspase 3-like) activation in PANC1 (mt p53) and HPAC (wt p53) pancreatic cancer cell lines. We found that DEVDase activity in PANC1 cells responds to ActD and ActD/anti-CD95 antibodies earlier than in HPAC cells; however, at 24 hours HPAC cells demonstrated much stronger activation. Cytosolic protein extracts from untreated cells did not influence caspase 3-like activity when added to extracts from the ActD/anti-CD95 antibody-treated cells. Collectively, these data suggest that pancreatic cancer cells have functional CD95-related apoptotic machinery with preserved apoptotic signal transduction, CD95R upregulation. and caspase activation. However, this system is blocked by some unknown protein(s) that is either located in the organelle fraction of the cell and/or requires an intact cell for manifestation of its activity.
authors.affiliation: Department of Pathology, Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI 48201, USA.
authors.forename: A L
authors.initials: AL
authors.lastname: Glazyrin
authors.forename: V N
authors.initials: VN
authors.lastname: Adsay
authors.forename: V K
authors.initials: VK
authors.lastname: Vaitkevicius
authors.forename: F H
authors.initials: FH
authors.lastname: Sarkar
mesh_terms: D000230:Adenocarcinoma / Q000276:immunology* / Q000473:pathology*; D017209:Apoptosis; D001761:Bleomycin / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D004789:Enzyme Activation; D005434:Flow Cytometry; D016158:Genes, p53 / Q000235:genetics; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D007700:Kinetics; D009154:Mutation; D017239:Paclitaxel / Q000494:pharmacology; D010190:Pancreatic Neoplasms / Q000276:immunology* / Q000473:pathology*; D010447:Peptide Hydrolases / Q000378:metabolism; D015398:Signal Transduction; D014407:Tumor Cells, Cultured; D019014:fas Receptor / Q000032:analysis / Q000502:physiology*
chemical_list: D019014:fas Receptor; D001761:Bleomycin; D003609:Dactinomycin; D007371:Interferon-gamma; D010447:Peptide Hydrolases; C108711:DEVDase; D017239:Paclitaxel