Ceramide induces apoptosis in neuroblastoma cell cultures resistant to CD95 (Fas/APO-1)-mediated apoptosis.
OBJECTIVE
Spontaneous tumor regression is a well-known characteristic in neuroblastomas. Because preliminary reports have shown that regression 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 would be induced in neuroblastoma cell cultures resistant to CD95-mediated programmed cell death.


METHODS
The expression of the CD95-receptor was assessed by flow cytometry after incubation with either fluorisothiocyanate-conjugated (FITC) anti-CD95-antibody (UB2) or CD95-ligand for 16 hours. Apoptotic cell death was detected via microscopy, cell viability testing (MTT, 3-[4,5 dimehylthiazole-2-yl]-2,5 diphenyltetrazoliumbromide), and flow cytometric analysis after propidium 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 induced apoptosis. Apoptosis was not seen, however, in any of the neuroblastoma cell lines when the CD95-receptor was stimulated with anti-CD95-antibody or the CD95-ligand. Significant apoptosis was detected in all neuroblastoma cell lines after the addition of 25 micromol/L C2- and C6-ceramide.


CONCLUSIONS
CD95-receptors are present on neuroblastoma cell lines, and these cells are resistant to apoptosis stimulated by anti-CD95-antibody or CD95-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.
Schaefer
J T
JT
Department of Surgery, University of Tuebingen School of Medicine, Germany.
Barthlen
W
W
Schweizer
P
P
D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D001932:Brain Neoplasms / Q000378:metabolism* / Q000473:pathology; D002518:Ceramides / Q000494:pharmacology*; D053222:Fas Ligand Protein; D005434:Flow Cytometry; D006801:Humans; D008562:Membrane Glycoproteins / Q000494:pharmacology; D009365:Neoplasm Regression, Spontaneous; D009447:Neuroblastoma / Q000378:metabolism* / Q000473:pathology; D019014:fas Receptor / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*
D002518:Ceramides; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D008562:Membrane Glycoproteins; D019014:fas Receptor