title: Latent sensitivity to Fas-mediated apoptosis after CD40 ligation may explain activity of CD154 gene therapy in chronic lymphocytic leukemia.
abstract: Patients with chronic lymphocytic leukemia (CLL) treated with adenovirus (Ad)-CD154 (CD40L) gene therapy experience reductions in leukemia cell counts and lymph node size associated with induction of the death receptor Fas (CD95). CD4 T cell lines can induce apoptosis of CD40-activated CLL cells via a CD95 ligand (CD95-L)-dependent mechanism. To examine whether CD95-L was sufficient to induce cytolysis of CD40-activated CLL cells, we used Chinese hamster ovary cells transfected with CD95-L as cytotoxic effector cells. CD40-activated CLL cells were initially resistant to CD95-mediated apoptosis despite high-level expression of CD95. However, after 72 h, CLL cells from seven of seven patients became increasingly sensitive to CD95-mediated apoptosis. This sensitivity correlated with a progressive decline in Flice-inhibitory protein (FLIP), which was induced within 24 h of CD40 ligation. Down-regulation of FLIP with an antisense oligonucleotide or a pharmacologic agent, however, was not sufficient to render CLL cells sensitive to CD95-mediated apoptosis in the 24-72 h after CD40 activation. Although the levels of pro-Caspase-8 appeared sufficient, inadequate levels of Fas-associated death domain protein (FADD) and DAP3 may preclude assembly of the death-inducing signaling complex. Seventy-two hours after CD40 ligation, sensitivity to CD95 and a progressive increase in FADD and DAP3 were associated with the acquired ability of FADD and FLIP to coimmunoprecipitate with the death-inducing signaling complex after CD95 ligation. Collectively, these studies reveal that CD40 ligation on CLL B cells induces a programmed series of events in which the cells initially are protected and then sensitized to CD95-mediated apoptosis through shifts in the balance of the anti- and proapoptotic proteins FLIP and FADD.
authors.affiliation: The Chronic Lymphocytic Leukemia Research Consortium, Biomedical Sciences Graduate Program, Division of Hematology/Oncology, Stein Institute for Research on Aging, School of Medicine, University of California at San Diego, La Jolla, CA 92093-0663, USA.
authors.forename: Peter
authors.initials: P
authors.lastname: Chu
authors.forename: Dieter
authors.initials: D
authors.lastname: Deforce
authors.forename: Irene M
authors.initials: IM
authors.lastname: Pedersen
authors.forename: Youngsoo
authors.initials: Y
authors.lastname: Kim
authors.forename: Shinichi
authors.initials: S
authors.lastname: Kitada
authors.forename: John C
authors.initials: JC
authors.lastname: Reed
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Kipps
mesh_terms: D000818:Animals; D017209:Apoptosis; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism; D019013:CD40 Antigens / Q000378:metabolism*; D023201:CD40 Ligand / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism* / Q000627:therapeutic use; D016466:CHO Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D006224:Cricetinae; D015536:Down-Regulation / Q000187:drug effects; D053222:Fas Ligand Protein; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D015316:Genetic Therapy; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000235:genetics / Q000378:metabolism* / Q000473:pathology* / Q000628:therapy; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D009828:Oleanolic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D011233:Precipitin Tests; D015398:Signal Transduction; D013997:Time Factors; D014407:Tumor Cells, Cultured; D019014:fas Receptor / Q000378:metabolism*
chemical_list: C116817:2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; D019013:CD40 Antigens; C506386:CFLAR protein, human; D002352:Carrier Proteins; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; D019014:fas Receptor; D023201:CD40 Ligand; D009828:Oleanolic Acid; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases