Wen-Li et al. Journal of Experimental & Clinical Cancer Research 2012, 31:92 http://www.jeccr.com/content/31/1/92 Page 2 of 9 Background F1F0 ATP synthase is a complex molecular motor re- sponsible for the majority of ATP synthesis in all organ- isms [1,2]. The ATP synthase β subunit is mostly expressed in the inner mitochondrial membrane of nor- mal cells [3-9]. Over the last few years, reports by sev- eral independent groups have described the presence of various subunits of ATP synthase at the cell surface of mammalian cells, which have been termed ecto-F1F0- ATPase [5,10-13]. Recent studies have shown that the β- subunits of F1F0 ATPase are located on the plasma membrane, as well as within the mitochondrial mem- brane of human vascular endothelial cells and tumor cells [5,6,10,14]. Most of the cell lines which are reported to express ecto-F1F0-ATPase β-subunits are leukemia cell lines, including K562, Raji [15], Daudi, U937 [11], Jurkat [16], ST-Emo and Rma-S [17]. In endothelial cells, the ecto-F1F0-ATPase β subunit has been identified as a receptor for angiostatin, a naturally oc- curring inhibitor of angiogenesis [5,14] which inhibits endothelial cell proliferation, tube formation and migration. Several conflicting reports have debated whether ecto- F1F0-ATPase is functional in tumor cells [3,10,15,17-20]. Recent data has shown that the mitochondrial F1- ATPase is expressed on tumor cell surface and promotes tumor recognition by Vgamma9Vdelta2 T cells. [11]. T lymphocytes are known to participate in the immune re- sponse against various intracellular pathogens, including tumor cells. Additionally, other research has demon- strated that inhibition of the ecto-F1F0-ATPase β- subunit is directly cytotoxic to tumor cells [3,18,21]. This data indicates that identification of novel ecto-F1F0- ATPase β subunit inhibitors, with both anti-angiogenic and anti-tumorigenic activities, may confer a greater therapeutic advantage by affecting cancer cells via by multiple mechanisms with potentially additive effects. In this study, we analyzed expression of the ecto-F1F0- ATPase β subunit in eleven cell lines derived from hematological malignancies and HUVECs, a positive control human vascular endothelial cell line. Most of cell lines derived from hematological malignancies expressed the ecto-F1F0-ATPase β subunit. We produced a monoclonal antibody 7E10 (McAb7E10) specific to the human F1F0 ATPase β subunit, which inhibited proliferation and induced significant apoptosis in the acute myeloid leukemia (AML) cell lines, MV4-11 and HL-60. These results suggest that the abnormal cell surface expression of the ecto-F1F0-ATPase β subunit may provide a potential target for cancer immuno- therapy in hematological malignancies, particularly AML. Methods Cell culture Cell lines derived from hematological malignancies (HL- 60, MV4-11, U937, K562, Raji, and Jurkat) were obtained from the American Type Culture Collection (ATCC). SHI-1, MOLT4, DAMI, CCRF and 697 cell lines (gifts from Professor Wang Jian-Rong, The Cyrus Tang Hematology center of Soochow University). The entire cell lines were maintained at 37°C in the RPMI 1640 supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin [22]. HUVEC cells were a gift from Professor Yang Zhi-Hua (Depart- ment of Cell and Molecular Biology, Cancer Institute, Chinese Academy of Medical Sciences, Beijing, China) and were cultured in M200 basal culture media supple- mented with low serum growth supplement (Cascade Biologics, PL, USA), 100 U/ml penicillin and 100 mg/ml streptomycin [23-25]. All cells were cultured at 37°C in a 5% CO2 humidified atmosphere. Flow cytometric assay Cells were collected, washed twice with phosphate buf- fered saline (PBS), adjusted to 1 × 106 cells/ml, and incu- bated with ATP synthase subunit beta monoclonal antibody (1:300; MitoScience MS503, EA, USA) for 30 min at 4°C. After washing three times with PBS, fluorescein-isothiocyanate (FITC)-labeled goat anti- mouse IgG (Jackson,WG, PA) diluted in PBS was added, incubated for 20 min at 4°C, then cells were washed three times with PBS, 1 ug/ml PI(Propidium Iodide, Sigma, St. Louis, MO, USA)) was added to exclude the dead cells and membrane antigen expression was ana- lyzed using a fluorescence-activated cell sorter (ESP Elite, Beckman Coulter, Fullerton, CA, USA). All experi- ments were performed three times. Production of functional F1F0 ATPase β subunit antibody Six to eight weeks old female BALB/c mice were sub- cutaneously immunized with hATP5B (F1F0 ATPase β subunit) which had been expressed using a prokaryotic system, as previously described [3], and mixed with Freund’s complete adjuvant (Sigma, St. Louis, MO, USA). The antibody valences in peripheral blood were determined using an ELISA as Gou, L. T. described [21], and three days after the last boost, 5 × 108 sensitized spleen cells were harvested, mixed and fused with 1 × 108 SP2/0 myeloma cells, in 50% polyethylene glycol 1500 in a proportion of 8:1. The fused cells were plated in 96-well plates (6 × 105/well) and cultured for two weeks in RPMI 1640 with 10% fetal calf serum contain- ing hypoxanthine, aminopterin, and thymidine to select for positive hybrid cells. The positive hybridoma cells were subcloned by limiting dilution, and 10–12 week old female BALB/c mice were inoculated with 3 × 106 hybri- doma cells [3,26]. The antibodies were further purified from the ascites via Protein-A affinity chromatography [3]. The antibody with the highest valence against the