CD95/Fas downregulation in lymphoma cells through acquired alkyl-lysophospholipid resistance; partial role of associated sphingomyelin deficiency
Résumé
The alkyl-lysophospholipid (ALP) 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 previously found impaired in ALP uptake via lipid raft-mediated endocytosis. Here, we report that these cells display cross-resistance to Fas/CD95 ligation (FasL), 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, cFLIP or the caspase 8 inhibitor IETD. ALP-resistant cells showed decreased Fas expression, both at the mRNA and protein level, 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 sphingomyelin (SM) synthesis, which seems 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 acquired resistance of S49 cells to ALP is associated with downregulated SM synthesis and Fas gene transcription and that SM in lipid rafts stabilizes Fas expression at the cell-surface.
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