title: Acquisition of Fas resistance by Fas receptor mutation in a childhood B-precursor acute lymphoblastic leukemia cell line, MML-1.
abstract: Programmed cell death, or apoptosis, is a physiological means of eliminating unwanted cells and maintaining immune homeostasis. One of the primary mechanisms is the Fas (CD95)/Fas ligand system. Its inactivation in normal cells and malignant cells may be involved in malignant trans-formation and refractory clinical course, respectively. We established a Fas resistant clone and evaluated the molecular basis for its mechanism of resistance. The Fas-sensitive leukemia cell line, MML-1, was established from a child with B-precursor acute lymphoblastic leukemia. A Fas resistant clone, MML-1R, was obtained by co-culture selection with anti-Fas antibody CH-11. Flow cytometry analysis showed both cell lines had equivalent expression of cell surface CD13, 15, 19, 22 and Fas receptor. Western blot analysis revealed equal expression of FADD (Fas-associated death domain protein), caspase-3 and -8. MML-1 was quite sensitive to both CH-11 and etoposide-induced apoptotis. By contrast, MML-1R had similar sensitivity to etoposide but no response to CH-11. Fas receptor mutation analysis showed a heterozygous death domain A --> G point mutation at 1009 bp, causing a switch from glutamine to glycine at amino acid 256. Immunoprecipitation assay showed decreased binding of Fas to FADD. We also found that etoposide bypassed Fas-FADD interaction in MML-1R by activating caspase-8 and caspase-3. These results indicate that Fas resistance can result from mutations of the gene encoding the Fas receptor which result in decreased FADD binding, thereby blocking formation of the death inducing signaling complex. Screening for similar Fas mutations in therapy resistant malignancies would lead to a better understanding of tumorigenesis and recurrence.
authors: Department of Pediatrics, Mie University School of Medicine, Tsu, Mie 514-8507, Japan. hiroto.inaba@stjude.org
authors: Hiroto
authors: H
authors: Inaba
authors: Keiji
authors: K
authors: Shimada
authors: Yan-Wen
authors: YW
authors: Zhou
authors: Masaru
authors: M
authors: Ido
authors: Steven
authors: S
authors: Buck
authors: Shin
authors: S
authors: Yonehara
authors: Joseph
authors: J
authors: Kaplan
authors: Yoshihiro
authors: Y
authors: Komada
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000276:immunology / Q000378:metabolism; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D018941:Antigens, CD19 / Q000378:metabolism; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D001483:Base Sequence; D015153:Blotting, Western; D053148:Caspase 3; D053181:Caspase 8; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D002648:Child; D004252:DNA Mutational Analysis; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D005047:Etoposide / Q000494:pharmacology; D053200:Fas-Associated Death Domain Protein; D005434:Flow Cytometry; D006801:Humans; D016256:Lewis X Antigen / Q000378:metabolism; D009154:Mutation; D015452:Precursor B-Cell Lymphoblastic Leukemia-Lymphoma / Q000235:genetics / Q000473:pathology; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000235:genetics / Q000473:pathology; D011485:Protein Binding / Q000187:drug effects / Q000276:immunology; D019014:fas Receptor / Q000235:genetics* / Q000276:immunology / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000911:Antibodies, Monoclonal; D018941:Antigens, CD19; D000972:Antineoplastic Agents, Phytogenic; C505533:FADD protein, human; D053200:Fas-Associated Death Domain Protein; D016256:Lewis X Antigen; D019014:fas Receptor; D005047:Etoposide; C505407:CASP3 protein, human; C505530:CASP8 protein, human; D053148:Caspase 3; D053181:Caspase 8; D020169:Caspases