title: Alemtuzumab induces caspase-independent cell death in human chronic lymphocytic leukemia cells through a lipid raft-dependent mechanism.
abstract: Alemtuzumab is a humanized IgG1 kappa antibody directed against CD52, a glycosyl-phosphatidylinositol linked cell-membrane protein of unknown function. Herein, we demonstrate that alemtuzumab promotes rapid death of chronic lymphocytic leukemia (CLL) cells in vitro, in a complement and accessory cell free system. Using minimal detergent solubilization of CLL membranes, we found that CD52 colocalizes with ganglioside GM-1, a marker of membrane rafts. Fluorescence microscopy revealed that upon crosslinking CD52 with alemtuzumab+anti-Fc IgG, large patches, and in many cases caps, enriched in CD52 and GM-1 formed upon the CLL cell plasma membrane. Depletion of membrane cholesterol or inhibition of actin polymerization significantly diminished the formation of alemtuzumab-induced caps and reduced alemtuzumab-mediated CLL cell death. We compared alemtuzumab-induced direct cytotoxicity, effector cell-mediated toxicity and complement-mediated cytotoxicity of CLL cells to normal T cells. The direct cytotoxicity and observed capping was significantly greater for CLL cells as compared to normal T cells. Cell-mediated and complement-mediated cytotoxicity did not significantly differ between the two cell types. In summary, our data support the hypothesis that alemtuzumab can initiate CLL cell death by crosslinking CD52-enriched lipid rafts. Furthermore, the differential direct cytotoxic effect suggests that CD52 directed antibodies could possibly be engineered to more specifically target CLL cells.
authors.affiliation: Division of Hematology-Oncology, Department of Medicine, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: A P
authors.initials: AP
authors.lastname: Mone
authors.forename: C
authors.initials: C
authors.lastname: Cheney
authors.forename: A L
authors.initials: AL
authors.lastname: Banks
authors.forename: S
authors.initials: S
authors.lastname: Tridandapani
authors.forename: N
authors.initials: N
authors.lastname: Mehter
authors.forename: S
authors.initials: S
authors.lastname: Guster
authors.forename: T
authors.initials: T
authors.lastname: Lin
authors.forename: C F
authors.initials: CF
authors.lastname: Eisenbeis
authors.forename: D C
authors.initials: DC
authors.lastname: Young
authors.forename: J C
authors.initials: JC
authors.lastname: Byrd
mesh_terms: D000199:Actins / Q000187:drug effects / Q000378:metabolism; D000074323:Alemtuzumab; D000911:Antibodies, Monoclonal / Q000187:drug effects / Q000494:pharmacology*; D061067:Antibodies, Monoclonal, Humanized; D000912:Antibodies, Neoplasm / Q000187:drug effects / Q000494:pharmacology*; D015703:Antigens, CD / Q000096:biosynthesis / Q000378:metabolism; D000951:Antigens, Neoplasm / Q000096:biosynthesis / Q000378:metabolism; D000074301:CD52 Antigen; D020169:Caspases / Q000187:drug effects*; D016923:Cell Death / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D005677:G(M1) Ganglioside / Q000096:biosynthesis; D006023:Glycoproteins / Q000096:biosynthesis / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000188:drug therapy* / Q000473:pathology*; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism*; D047392:beta-Cyclodextrins / Q000494:pharmacology
chemical_list: D000199:Actins; D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D000912:Antibodies, Neoplasm; D015703:Antigens, CD; D000951:Antigens, Neoplasm; D000074301:CD52 Antigen; C000613862:CD52 protein, human; D006023:Glycoproteins; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D005677:G(M1) Ganglioside; D000074323:Alemtuzumab; D020169:Caspases