Ceramide induces apoptosis in neuroblastoma cell cultures resistant to CD95 (Fas/APO-1)-mediated apoptosis

Background/Purpose: Spontaneous tumor regression is a well-known characteri stic in neuroblastomas. Because preliminary reports have shown that regress ion may be caused by apoptosis (a lethal cascade mediated by the CD95 (APO- 1/ Fas)-receptor), we analyzed the expression of CD95-receptors in 5 human neuroblastoma cell lines, Ceramides (known stimuli of apoptosis downstream from the CD95-receptor complex) also were used to test whether apoptosis wo uld be induced in neuroblastoma cell cultures resistant to CD95-mediated pr ogrammed cell death. Methods: The expression of the CD95-receptor was assessed by flow cytometry after incubation with either fluorisothiocyanate-conjugated (FITC) anti-CD 95-antibody (UB2) or CD95-ligand for 16 hours. Apoptotic cell death was det ected via microscopy, cell viability testing (MTT, 3-[4,5 dimehylthiazole-2 -yl]-2,5 diphenyltetrazoliumbromide), and flow cytometric analysis after pr opidium iodide staining of the DNA. Results: CD95-receptor expression was found on all neuroblastoma cell lines . Stimulation of the CD95-receptor of the malignant glioblastoma cell line LN229 (positive control) with either anti-CD95-antibody or CD95-ligand indu ced apoptosis. Apoptosis was not seen, however, in any of the neuroblastoma cell lines when the CD95-receptor was stimulated with anti-CD95-antibody o r the CD95-ligand. Significant apoptosis was detected in all neuroblastoma cell lines after the addition of 25 mu mol/L C2- and C6-ceramide. Conclusions: CD95-receptors are present on neuroblastoma cell lines, and th ese cells are resistant to apoptosis stimulated by anti-CD95-antibody or CD 95-ligand. Apoptosis is induced, however, when these cells are treated with ceramide. A signal blockage downstream from the CD95-receptor complex and upstream of ceramide may account for this finding, and the "cellular FLICE inhibitory protein" (cFLIP) may be primarily responsible. Copyright (C) 200 0 by W.B. Saunders Company.