Resistance to CD95-mediated apoptosis of CD40-activated chronic lymphocytic leukemia B cells is not related to lack of DISC molecules expression.
In B-cell chronic lymphocytic leukemia (CLL), accumulation of neoplastic B cells may be the result of dysregulated apoptosis. One of the major molecules triggering apoptosis, CD95 (FAS), is not expressed on CLL B cells at resting conditions. However, CD40 triggering of CLL B cells upregulates receptors belonging to the tumor necrosis factor (TNF) superfamily, like CD95. In the present study, we analyzed in B cells from 20 CLL patients the effect of CD40/CD40L interaction on: (i) CD95 modulation; (ii) CD95-mediated apoptosis and (iii) mRNA and protein expression of the death-inducing signaling complex (DISC) molecules.CD40 activation of CLL B cells was carried out by coculture with CD40L-transfected cells and cytofluorimetric analyses were performed to study CD95 modulation and apoptosis induction by an anti-CD95 moAb. Despite strong CD95 upregulation on the membrane of all the cases studied, only a minority of cases analyzed (3/20) proved weakly responsive to CD95-mediated apoptosis. Multiplex RT-PCR was used to analyze FLICE, FAS, FADD and TRADD mRNAs before and after CD40 triggering. In agreement with the cytofluorimetric data, FAS mRNA appeared significantly increased after CD40 triggering; the other molecules involved in DISC formation and in CD95-mediated apoptosis were also expressed without relevant differences between resting and activated conditions. Western blot analyses further confirmed FLICE and FADD protein expression by resting and activated CLL cells. Our findings demonstrate that, following CD40 triggering, CLL B cells are resistant to CD95-mediated apoptosis despite a strong CD95 upregulation on the membrane and a normal mRNA or protein expression of the DISC components.
de Totero
Daniela
D
Immunopharmacology Lab, National Institute for Cancer Research, Genoa, Italy. detotero@yahoo.com
Montera
Mariapina
M
Rosso
Ombretta
O
Clavio
Marino
M
Balleari
Enrico
E
Foa
Robin
R
Gobbi
Marco
M
D048868:Adaptor Proteins, Signal Transducing; D000368:Aged; D000369:Aged, 80 and over; D017209:Apoptosis / Q000235:genetics*; D019013:CD40 Antigens / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics; D053181:Caspase 8; D020169:Caspases / Q000235:genetics; D018920:Coculture Techniques; D053200:Fas-Associated Death Domain Protein; D005260:Female; D015973:Gene Expression Regulation, Leukemic / Q000235:genetics*; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000235:genetics* / Q000473:pathology; D008213:Lymphocyte Activation / Q000235:genetics / Q000502:physiology; D008297:Male; D008875:Middle Aged; D011506:Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology; D047990:TNF Receptor-Associated Factor 1; D014162:Transfection; D015854:Up-Regulation / Q000235:genetics / Q000502:physiology; D019014:fas Receptor / Q000235:genetics / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D019013:CD40 Antigens; D002352:Carrier Proteins; C505533:FADD protein, human; D053200:Fas-Associated Death Domain Protein; D011506:Proteins; D012333:RNA, Messenger; D047990:TNF Receptor-Associated Factor 1; D019014:fas Receptor; C505530:CASP8 protein, human; D053181:Caspase 8; D020169:Caspases