Fas/Fas ligand (FasL)-deregulated apoptosis and IL-6 insensitivity in highly malignant myeloma cells.
IL-6 is a growth factor which interferes in the apoptosis of malignant plasma cells. Here we explore its role in the spontaneous and Fas/FasL-regulated apoptosis of seven myeloma cell clones (MCC). MCC-2 and -7 were constitutively defective in Fas antigen in the presence of large membrane exposure of FasL, and showed a high rate of cell proliferation irrespective of the presence of IL-6. Cytofluorimetric analysis following propidium iodide (PI) staining revealed a minimal extent of spontaneous apoptosis, as in other IL-6-insensitive, though Fas-positive MCC, namely MCC-3 and -5. By contrast, a regular amplitude of apoptosis occurred in the remaining IL-6-dependent clones. Their propensity to cell death, as well as their FasL membrane expression, were promptly down-modulated by the cytokine, whereas no substantial effect was detected in IL-6-independent MCC. Furthermore, we investigated the quantitative secretion of FasL. Both [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT) cytotoxicity assay and PI staining of WC8 lymphoblasts from a Fas-transfected mouse lymphoma, incubated with supernatants from MCC, showed a variable cytocidal property, thus confirming the cellular release of FasL. However, a significant elevation of FasL secretion occurred in both Fas- MCC, whereas molecular cloning and sequencing of Fas revealed the presence of a splicing variant, namely Fas Exo4,6Del, in the cDNA from both MCC-3 and -5, which were previously demonstrated to be unresponsive to Fas stimulation. Taken together, these data provide evidence that concurrence of IL-6 insensitivity and deregulation of apoptosis in myeloma cells reflects a high malignancy grade. It is suggested that the secretion of Fas splicing variants in Fas+ plasma cells, as well as the over-production of FasL in Fas- myelomas, are differential mechanisms by which myeloma cells escape host immune surveillance.
Frassanito
M A
MA
Department of Biomedical Sciences and Human Oncology, University of Bari Medical School, Italy.
Silvestris
F
F
Silvestris
N
N
Cafforio
P
P
Camarda
G
G
Iodice
G
G
Dammacco
F
F
D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D015703:Antigens, CD; D000943:Antigens, Differentiation / Q000096:biosynthesis; D017209:Apoptosis; D002455:Cell Division; D003001:Cloning, Molecular; D018076:DNA, Complementary; D053222:Fas Ligand Protein; D006801:Humans; D015850:Interleukin-6 / Q000096:biosynthesis*; D008562:Membrane Glycoproteins / Q000096:biosynthesis*; D009101:Multiple Myeloma / Q000276:immunology* / Q000473:pathology; D009244:NAD+ Nucleosidase / Q000096:biosynthesis; D014407:Tumor Cells, Cultured; D019014:fas Receptor / Q000096:biosynthesis* / Q000235:genetics
D015703:Antigens, CD; D000943:Antigens, Differentiation; D018076:DNA, Complementary; C505661:FASLG protein, human; D053222:Fas Ligand Protein; C505658:Fasl protein, mouse; D015850:Interleukin-6; D008562:Membrane Glycoproteins; D019014:fas Receptor; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; D009244:NAD+ Nucleosidase; D051997:ADP-ribosyl Cyclase 1