title: Anti-CHMP5 single chain variable fragment antibody retrovirus infection induces programmed cell death of AML leukemic cells in vitro.
abstract: OBJECTIVE
  Over-expressed CHMP5 was found to act as oncogene that probably participated in leukemogenesis. In this study, we constructed the CHMP5 single chain variable fragment antibody (CHMP5-scFv) retrovirus and studied the changes of programmed cell death (PCD) of AML leukemic cells after infection by the retrovirus.
  
  
  METHODS
  The anti-CHMP5 KC14 hybridoma cell line was constructed to generate monoclonal antibody of CHMP5. The protein expression of CHMP5 was studied using immunofluorescence analysis. pMIG-CHMP5 scFv antibody expressible retroviral vector was constructed to prepare CHMP5-scFv retrovirus. AML leukemic U937 cells were infected with the retrovirus, and programmed cell death was studied using confocal microscope, FCM and Western blot.
  
  
  RESULTS
  We obtained a monoclonal antibody of CHMP5, and found the expression of CHMP5 was up-regulated in the leukemic cells. After U937 cells were infected with CHMP5-scFv retrovirus, CHMP5 protein was neutralized. Moreover, the infection resulted in a significant increase in apoptosis and necrosis of U937 cells. In U937 cells infected with CHMP5-scFv retrovirus, apoptosis-inducing factor (AIF)-mediated caspase-independent necrotic PCD was activated, but autophagic programmed cell death was not observed. Neither the intrinsic nor extrinsic apoptotic PCD pathway was activated. The granzyme B/perforin-mediated caspase-dependent apoptotic PCD pathway was not activated.
  
  
  CONCLUSIONS
  CHMP5-scFv retrovirus can neutralize the abnormally high levels of the CHMP5 protein in the cytosol of AML leukemic U937 cells, thereby inducing the programmed cell death of the leukemic cells via AIF-mediated caspase-independent necrosis and apoptosis.
authors.affiliation: Emergency Department, Xin-Hua Hospital, Shanghai Jiao Tong University School of Medicine, China.
authors.forename: Hai-rong
authors.initials: HR
authors.lastname: Wang
authors.forename: Zhen-yu
authors.initials: ZY
authors.lastname: Xiao
authors.forename: Miao
authors.initials: M
authors.lastname: Chen
authors.forename: Fei-long
authors.initials: FL
authors.lastname: Wang
authors.forename: Jia
authors.initials: J
authors.lastname: Liu
authors.forename: Hua
authors.initials: H
authors.lastname: Zhong
authors.forename: Ji-hua
authors.initials: JH
authors.lastname: Zhong
authors.forename: Ren-rong
authors.initials: RR
authors.lastname: Ou-Yang
authors.forename: Yan-lin
authors.initials: YL
authors.lastname: Shen
authors.forename: Shu-ming
authors.initials: SM
authors.lastname: Pan
mesh_terms: D000818:Animals; D017209:Apoptosis; D056827:Endosomal Sorting Complexes Required for Transport / Q000235:genetics / Q000276:immunology*; D015972:Gene Expression Regulation, Neoplastic; D057809:HEK293 Cells; D006801:Humans; D006825:Hybridomas; D015470:Leukemia, Myeloid, Acute / Q000235:genetics / Q000276:immunology* / Q000821:virology*; D051379:Mice; D008807:Mice, Inbred BALB C; D012190:Retroviridae / Q000235:genetics / Q000276:immunology*; D012192:Retroviridae Infections / Q000150:complications / Q000276:immunology; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology*; D020298:U937 Cells
chemical_list: C499838:CHMP5 protein, human; D056827:Endosomal Sorting Complexes Required for Transport; D057127:Single-Chain Antibodies