title: Inhibition of the ecto-beta subunit of F1F0-ATPase inhibits proliferation and induces apoptosis in acute myeloid leukemia cell lines.
abstract: BACKGROUND
  Leukemia, a heterogeneous clonal disorder of hematopoietic progenitor cells, presents a world-wide health problem, especially in childhood. F1F0 ATPase, an inner mitochondrial enzyme, is expressed on the plasma membrane of tumor cells, and its inhibition induces both anti-angiogenic and anti-tumorigenic activity.
  
  
  METHODS
  Monoclonal Antibody (McAb) against ATPase was produced by polyethylene glycol-mediated fusions and screened by ELISA. Proliferation, cell cycle and apoptosis of cells were analyzed when the surface ATPase of cells was blockaded with McAb.
  
  
  RESULTS
  We detected cell-membrane expression of the F1F0 ATPase β subunit on 0.1% to 56% of the 11 cell lines derived from leukemia, including acute myeloid leukemia (AML). We produced a monoclonal antibody, McAb7E10, which recognizes both the native and recombinant ATPase β subunit, with a dissociation constant (KD) of 3.26E-10. We demonstrate that McAb7E10 binds to ATPase at the cell surface, where it is able to inhibit ATP synthesis. McAb7E10 significantly inhibited proliferation of AML cell lines in vitro: the relative inhibitory rates of 50 μg/mL McAb7E10 treated MV4-11and HL-60 cells were 69.6% and 81.9% respectively. Cell cycle analysis indicated that McAb7E10 significantly induced apoptosis in MV4-11 and HL-60 cells: the relative rates of apoptosis in 5, 10 and 50ug/mL McAb7E10 treated MV4-11 cells was 3.6 ± 0.83%, 8.4 ± 1.69% and 17.3 ± 2.56% compared to 1.5% ± 0.85% in mouse IgG treated cells (p < 0.01). The relative rate of apoptosis in 5, 10 and 50ug/mL McAb7E10 treated HL-60 cells was 5.5 ± 2.37%, 11.3 ± 3.62% and 19.9 ± 3.31% compared to 1.56% ± 0.97% in mouse IgG treated cells (p < 0.01). Annexin V staining demonstrated that the relative apoptotic rates in 50 μg/mL McAb7E10 treated MV4-11 and HL-60 cells were 50.5% ± 7.04% and 32.9% ± 4.52%, respectively, significantly higher than IgG control antibody treated cells were 21.9% ± 3.11% and 15.3% ± 3.95%, p < 0.01.
  
  
  CONCLUSIONS
  These findings indicate that ectopic expression of ATPase β subunit may be a tumor-associated antigen in hematological malignancies. The F1F0 ATPase β subunit provides a potential target for immunotherapy in AML and hematological malignancies.
authors.affiliation: Department of Hematology and Oncology, Children's Hospital of Soochow University, Suzhou, China.
authors.forename: Zhao
authors.initials: Z
authors.lastname: Wen-Li
authors.forename: Wang
authors.initials: W
authors.lastname: Jian
authors.forename: Tao
authors.initials: T
authors.lastname: Yan-Fang
authors.forename: Feng
authors.initials: F
authors.lastname: Xing
authors.forename: Li
authors.initials: L
authors.lastname: Yan-Hong
authors.forename: Zhu
authors.initials: Z
authors.lastname: Xue-Ming
authors.forename: Zhang
authors.initials: Z
authors.lastname: Min
authors.forename: Ni
authors.initials: N
authors.lastname: Jian
authors.forename: Pan
authors.initials: P
authors.lastname: Jian
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000008:administration & dosage*; D017209:Apoptosis / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000276:immunology; D018922:HL-60 Cells; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism; D025261:Mitochondrial Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000276:immunology; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D021122:Protein Subunits; C474451:F1F0-ATP synthase; D025261:Mitochondrial Proton-Translocating ATPases