Latent Sensitivity to Fas-Mediated Apoptosis after CD40 Ligation May Explain Activity of CD154 Gene Therapy in Chronic Lymphocytic Leukemia

• Published in: Proceedings of the National Academy of Sciences - PNAS 2002-03, Vol.99 (6), p.3854-3859
• Main Authors: Chu, Peter, Deforce, Dieter, Pedersen, Irene M., Kim, Youngsoo, Kitada, Shinichi, Reed, John C., Kipps, Thomas J.
• Format: Article
• Language: English
• Subjects: Animals; Apoptosis; B lymphocytes; Biological Sciences; Blood; Carrier Proteins - genetics; Carrier Proteins - metabolism; CASP8 and FADD-Like Apoptosis Regulating Protein; Caspase 8; Caspase 9; Caspases - metabolism; CD154 antigen; CD4-Positive T-Lymphocytes - immunology; CD4-Positive T-Lymphocytes - metabolism; CD40 antigen; CD40 Antigens - metabolism; CD40 Ligand - administration & dosage; CD40 Ligand - genetics; CD40 Ligand - metabolism; CD40 Ligand - therapeutic use; CD95L protein; Cell lines; Cells; CHO Cells; Chronic lymphocytic leukemia; Cricetinae; Cytokines; Down-Regulation - drug effects; FADD protein; Fas antigen; Fas Ligand Protein; fas Receptor - metabolism; FLIP protein; Gene Expression Regulation, Neoplastic - drug effects; Gene therapy; Genetic Therapy; Humans; Intracellular Signaling Peptides and Proteins; Leukemia; Leukemia, Lymphocytic, Chronic, B-Cell - genetics; Leukemia, Lymphocytic, Chronic, B-Cell - metabolism; Leukemia, Lymphocytic, Chronic, B-Cell - pathology; Leukemia, Lymphocytic, Chronic, B-Cell - therapy; Ligation; Membrane Glycoproteins - genetics; Membrane Glycoproteins - metabolism; Oleanolic Acid - analogs & derivatives; Oleanolic Acid - pharmacology; Precipitin Tests; Signal Transduction; T lymphocytes; Time Factors; Tumor Cells, Cultured
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.022604399

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.