title: Characterisation of FAP-1 expression and CD95 mediated apoptosis in the A818-6 pancreatic adenocarcinoma differentiation system.
abstract: The present study investigated the expression and localisation of FAP-1 (Fas associated phosphatase-1) and CD95 in a 3D differentiation model in comparison to 2D monolayers of the pancreatic adenocarcinoma cell line A818-6. Under non-adherent growth conditions, A818-6 cells differentiate into 3D highly organised polarised epithelial hollow spheres, resembling duct-like structures. A818-6 cells showed a differentiation-dependent FAP-1 localisation. Cells grown as 2D monolayers revealed FAP-1 staining in a juxtanuclear cisternal position, as well as localisation in the nucleus. After differentiation into hollow spheres, FAP-1 was relocated towards the actin cytoskeleton beneath the outer plasma membrane of polarised cells and no further nuclear localisation was observed. CD95 surface staining was found only in a subset of A818-6 monolayer cells, while differentiated hollow spheres appeared to express CD95 in all cells of a given sphere. We rarely observed co-localisation of CD95 and FAP-1 in A818-6 monolayer cells, but strong co-localisation beneath the outer plasma membrane in polarised cells. Analysis of surface expression by flow cytometry revealed that only a subset (36%) of monolayer cells showed CD95 surface expression, and after induction of hollow spheres, CD95 presentation at the outer plasma membrane was reduced to 13% of hollow spheres. Induction of apoptosis by stimulation with agonistic anti-CD95 antibodies, resulted in increased caspase activity in both, monolayer cells and hollow spheres. Knock down of FAP-1 mRNA in A818-6 monolayer cells did not alter resposiveness to CD95 agonistic antibodies. These data suggested that CD95 signal transduction was not affected by FAP-1 expression in A818-6 monolayer cells. In differentiated 3D hollow spheres, we found a polarisation-induced co-localisation of CD95 and FAP-1. A tight control of receptor surface representation and signalling induced apoptosis ensures controlled removal of individual cells instead of a "snowball effect" of apoptotic events.
authors.affiliation: Institute for Experimental Cancer Research, Division Molecular Oncology, University Hospital Schleswig-Holstein Campus Kiel, Germany.
authors.forename: Boris J N
authors.initials: BJ
authors.lastname: Winterhoff
authors.forename: Alexander
authors.initials: A
authors.lastname: Arlt
authors.forename: Angelika
authors.initials: A
authors.lastname: Duttmann
authors.forename: Hendrik
authors.initials: H
authors.lastname: Ungefroren
authors.forename: Heiner
authors.initials: H
authors.lastname: Schäfer
authors.forename: Holger
authors.initials: H
authors.lastname: Kalthoff
authors.forename: Marie-Luise
authors.initials: ML
authors.lastname: Kruse
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism; D000230:Adenocarcinoma / Q000378:metabolism; D017209:Apoptosis / Q000235:genetics*; D002454:Cell Differentiation / Q000235:genetics*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000648:ultrastructure; D016764:Cell Polarity; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D010179:Pancreas / Q000166:cytology / Q000254:growth & development / Q000378:metabolism; D054595:Protein Tyrosine Phosphatase, Non-Receptor Type 13 / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D015398:Signal Transduction; D019014:fas Receptor / Q000235:genetics / Q000378:metabolism*
chemical_list: C491901:FAS protein, human; D034741:RNA, Small Interfering; D019014:fas Receptor; C516742:PTPN13 protein, human; D054595:Protein Tyrosine Phosphatase, Non-Receptor Type 13