title: Fas/CD95 down-regulation in lymphoma cells through acquired alkyllysophospholipid resistance: partial role of associated sphingomyelin deficiency.
abstract: The ALP (alkyl-lysophospholipid) edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) induces apoptosis in S49 mouse lymphoma cells. A variant cell line, S49AR, made resistant to ALP, was found previously to be impaired in ALP uptake via lipid-raft-mediated endocytosis. In the present paper, we report that these cells display cross-resistance to Fas/CD95 ligation [FasL (Fas ligand)], and can be gradually resensitized by prolonged culturing in the absence of ALP. Fas and ALP activate distinct apoptotic pathways, since ALP-induced apoptosis was not abrogated by dominant-negative FADD (Fas-associated protein with death domain), cFLIP(L) [cellular FLICE (FADD-like interleukin 1beta-converting enzyme)-inhibitory protein long form] or the caspase 8 inhibitor Z-IETD-FMK (benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethylketone). ALP-resistant cells showed decreased Fas expression, at both the mRNA and protein levels, in a proteasome-dependent fashion. The proteasome inhibitor MG132 partially restored Fas expression and resensitized the cells to FasL, but not to ALP. Resistant cells completely lacked SM (sphingomyelin) synthesis, which seems to be a unique feature of the S49 cell system, having very low SM levels in parental cells. Lack of SM synthesis did not affect cell growth in serum-containing medium, but retarded growth under serum-free (SM-free) conditions. SM deficiency determined in part the resistance to ALP and FasL. Exogenous short-chain (C12-) SM partially restored cell-surface expression of Fas in lipid rafts and FasL sensitivity, but did not affect Fas mRNA levels or ALP sensitivity. We conclude that the acquired resistance of S49 cells to ALP is associated with down-regulated SM synthesis and Fas gene transcription and that SM in lipid rafts stabilizes Fas expression at the cell surface.
authors.affiliation: Division of Cell Biology (B5), The Netherlands Cancer Institute/Antoni van Leeuwenhoek Hospital, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. w.v.blitterswijk@nki.nl
authors.forename: Wim J
authors.initials: WJ
authors.lastname: van Blitterswijk
authors.forename: Jeffrey B
authors.initials: JB
authors.lastname: Klarenbeek
authors.forename: Arnold H
authors.initials: AH
authors.lastname: van der Luit
authors.forename: Maaike C
authors.initials: MC
authors.lastname: Alderliesten
authors.forename: Menno
authors.initials: M
authors.lastname: van Lummel
authors.forename: Marcel
authors.initials: M
authors.lastname: Verheij
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D015536:Down-Regulation; D019008:Drug Resistance, Neoplasm; D053222:Fas Ligand Protein / Q000494:pharmacology; D053200:Fas-Associated Death Domain Protein / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D015151:Immunoblotting; D007976:Leupeptins / Q000494:pharmacology; D008223:Lymphoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008246:Lysophospholipids / Q000494:pharmacology*; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013109:Sphingomyelins / Q000172:deficiency / Q000378:metabolism*; D014162:Transfection; D019014:fas Receptor / Q000235:genetics / Q000378:metabolism*
chemical_list: D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; C506387:Cflar protein, mouse; D053222:Fas Ligand Protein; D053200:Fas-Associated Death Domain Protein; D007976:Leupeptins; D008246:Lysophospholipids; D061988:Proteasome Inhibitors; D034741:RNA, Small Interfering; D013109:Sphingomyelins; D019014:fas Receptor; D049452:Green Fluorescent Proteins; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde