title: Fanconi's anemia cell lines show distinct mechanisms of cell death in response to mitomycin C or agonistic anti-Fas antibodies.
abstract: OBJECTIVE
  Fanconi anemia (FA) cells are characteristically hypersensitive to bifunctional alkylating agents, notably mitomycin C (MMC), causing increased programmed cell death (PCD). FA cells also have abnormalities in mitochondrial function. We hypothesized that the abnormalities in PCD are mitochondrially mediated. We examined mitochondrial function in FA cells, comparing the intrinsic death pathway induced by MMC with the extrinsic pathway via Fas antibody, which can bypass the mitochondria.
  
  
  METHODS
  Normal and FA lymphoblastoid cell lines were treated with MMC or agonistic anti-Fas antibody. PCD was assessed using flow cytometry, Western blot analysis, and DNA gel electrophoresis.
  
  
  RESULTS
  FA cells showed hypersensitivity to MMC, but slight resistance to Fas-mediated PCD. MMC induced chromatin condensation, but not apoptotic body formation. Fas induced classical apoptosis. MMC failed to induce mitochondrial depolarization, while some depolarization occurred with anti-Fas. These results suggested that MMC failed to induce caspase activity in FA cells. No cleavage of caspase 3 was observable and PCD was not inhibited by the caspase inhibitor zVAD-fmk. Fas-induced caspase 3 cleavage, and cell death was inhibited by zVAD-fmk. There were common downstream abnormalities in the execution phase of PCD, as both agonists failed to cleave PARP, or to induce nucleosomal fragmentation.
  
  
  CONCLUSIONS
  Our results suggest that mitochondrial function in FA cells is abnormal, resulting in necrotic or caspase independent PCD, but that further abnormalities may exist downstream of the mitochondria. This may have implications in explaining in vivo aspects of FA.
authors.affiliation: St George's Hospital Medical School, Department of Cellular and Molecular Medicine, Division of Hematology, Cranmer Terrace, London, UK. a.clarke@sghms.ac.uk
authors.forename: Alan A
authors.initials: AA
authors.lastname: Clarke
authors.forename: Frances M
authors.initials: FM
authors.lastname: Gibson
authors.forename: Julie
authors.initials: J
authors.lastname: Scott
authors.forename: Nyree
authors.initials: N
authors.lastname: Myatt
authors.forename: Tim R
authors.initials: TR
authors.lastname: Rutherford
mesh_terms: D000911:Antibodies, Monoclonal / Q000494:pharmacology / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism / Q000502:physiology; D018797:Cell Cycle Proteins / Q000235:genetics; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000172:deficiency / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D005199:Fanconi Anemia / Q000188:drug therapy* / Q000235:genetics; D051856:Fanconi Anemia Complementation Group Proteins; D006801:Humans; D019169:Jurkat Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D008214:Lymphocytes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D008297:Male; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D016685:Mitomycin / Q000494:pharmacology / Q000627:therapeutic use*; D009687:Nuclear Proteins / Q000172:deficiency / Q000235:genetics; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D014407:Tumor Cells, Cultured; D019014:fas Receptor / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D051856:Fanconi Anemia Complementation Group Proteins; D009687:Nuclear Proteins; D019014:fas Receptor; D016685:Mitomycin; D011065:Poly(ADP-ribose) Polymerases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases