Mitochondrial F1fo-ATP Synthase Translocates to Cell Surface in Hepatocytes and Has High Activity in Tumor-Like Acidic and Hypoxic Environment

Acta Biochim Biophys Sin 2010, 42: 530–537 | ª The Author 2010. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DOI: 10.1093/abbs/gmq063.

Original Article

Mitochondrial F1Fo-ATP synthase translocates to cell surface in hepatocytes and has high activity in tumor-like acidic and hypoxic environment

Zhan Ma 1†, Manlin Cao 2†, Yiwen Liu 1, Yiqing He 1, Yingzhi Wang 1, Cuixia Yang 1, Wenjuan Wang 1, Yan Du 1, Muqing Zhou 1, and Feng Gao 1*

1Department of Molecular Biology Laboratory, Shanghai Sixth People’s Hospital Affiliated Shanghai Jiao Tong University School of Medicine, Shanghai 200233, China Downloaded from 2Department of Rehabilitation Medicine, Shanghai Sixth People’s Hospital Affiliated Shanghai Jiao Tong University School of Medicine, Shanghai 200233, China †These authors contributed equally to this work. *Correspondence address. Tel: þ86-21-64369181; Fax: þ86-21-63701361; E-mail: [email protected] abbs.oxfordjournals.org F1Fo-ATP synthase was originally thought to exclusively Introduction locate in the inner membrane of the mitochondria. However, recent studies prove the existence of ectopic Using the electrochemical gradient across the inner mem- F1Fo-ATP synthase on the outside of the cell membrane. brane of the mitochondria that is generated during oxi- Ectopic ATP synthase was proposed as a marker for dative phosphorylation, F1Fo-ATP synthase generates energy by coupling the transmembrane delivery of protons tumor target therapy. Nevertheless, the protein transport at Shanghai Jiao Tong University on October 20, 2010 mechanism of the ectopic ATP synthase is still unclear. to the synthesis of ATP [1]. For a long time, F1Fo-ATP The specificity of the ectopic ATP synthase, with regard synthase was thought to exclusively locate in the inner to tumors, is questioned because of its widespread membrane of mitochondria. However, more and more evi- expression. In the current study, we constructed green flu- dence hints at the existence of F1Fo-ATP synthase on the orescent protein-ATP5B fusion protein and introduced it outside of the plasma membrane of tumor cells and some into HepG2 cells to study the localization of the ATP types of normal cells, such as endothelial cells, hepato- synthase. The expression of ATP5B was analyzed in six cytes, and adipocytes [2]. cell lines with different ‘malignancies’. These cells were Ectopic ATP synthase always localizes on the lipid rafts cultured in both normal and tumor-like acidic and or caveolae of the cytoplasmic membrane [3–5]. The hypoxic conditions. The results suggested that the ectopic mechanisms responsible for the protein transport have not expression of ATP synthase is a consequence of transloca- been established. Wang et al.[6] found that cholesterol tion from the mitochondria. The expression and catalytic loading increased the level of ATP synthase of the cyto- activity of ectopic ATP synthase were similar on the plasmic membrane. However, no increased overall surface of malignant cells as on the surface of less malig- expression of the protein was observed. It was reported that nant cells. Interestingly, the expression of ectopic ATP most of the proteins localized to the inner membrane of synthase was not up-regulated in tumor-like acidic and mitochondria could also be found on the cytoplasmic mem- hypoxic microenvironments. However, the catalytic brane [7]. Therefore, Wang et al. hypothesized that ectopic activity of ectopic ATP synthase was up-regulated in ATP synthase might translocate from the mitochondria. tumor-like microenvironments. Therefore, the specificity However, no direct evidence has been observed to support of ectopic ATP synthase for tumor target therapy relies this hypothesis until now. on the high level of catalytic activity that is observed in Ectopic ATP synthase not only functions as energy gen- acidic and hypoxic microenvironments in tumor tissues. erators but also as proton channels and receptors for various ligands, which are involved in numerous biological Keywords tumor marker; ATP synthase; plasma processes including the mediation of intracellular pH, membrane; translocation; tumor-like microenvironment cholesterol homeostasis, the regulation of the proliferation and differentiation of endothelial cells, and the recognition Received: March 5, 2010 Accepted: May 10, 2010 of immune responses of tumor cells [2]. Some proteomic

Acta Biochim Biophys Sin (2010) | Volume 42 | Issue 8 | Page 530 Ectopic ATP synthase–translocation and the existence in tumor microenvironments analysis of membrane fractions indicate that the expression 40 mmHg. For all experiments, the cells were dissociated of ectopic ATP synthase in certain ‘malignant’ cells tends by incubation with PBS containing 2 mM EDTA (pH 7.4). to be higher than in ‘less malignant’ cells [8–11]. Since this molecule is involved in the regulation of endothelial cells, ectopic ATP synthase may serve as a marker for both Plasmids All kits for gene cloning were from Takara (Dalian, anti-tumor and anti-angiogenesis therapies. Some in vitro TM and in vivo research suggested that the proliferation of vas- China), except for Trizol (Invitrogen, Carlsbad, USA). cular endothelial cells and certain tumor cells could be The expression plasmid for the GFP-ATP5B fusion protein was prepared as follows: total mRNA was isolated from inhibited by anti-ATP synthase antibodies or inhibitors of TM ATP synthase [12,13]. For anti-tumor and anti-angiogenesis HUVECs using the Trizol reagent according to the man- therapy, specificity for tumor tissues should be considered. ufacturer’s instructions, and then was reverse-transcribed First, one should consider whether or not the expression and into single-stranded cDNA using AMV Reverse Transcriptase (Takara). The ATP5B precursor (ATP5Bp)

activity of ectopic ATP synthase in ‘malignant’ cells are Downloaded from always higher than in ‘less malignant’ cells. Second, one coding sequence (with mitochondrial signal sequence) was 0 should also consider whether ATP synthase could be regu- PCR-amplified using the primers 5 -AACAAGCTTGCCA 0 0 lated by tumor-like acidic and hypoxic microenvironments. CCATGTTGGGGTTTG-3 and 5 -GCACGGCGAATTCT 0 To visualize the protein transport of the ectopic ATP CGATGAATGCTCTT-3 . The mature ATP5B (ATP5Bm) coding sequence (without mitochondrial signal sequence)

synthase which located on the cell surface, the b-subunit of abbs.oxfordjournals.org 0 the ATP synthase gene (ATP5B) was cloned and was then PCR-amplified using primers 5 -TCGAAGCTTG 0 0 co-expressed with the green fluorescent protein (GFP) in CCACCATGGCGCAAACATCTC-3 and 5 -GCGGCGGG 0 human hepatocarcinoma cells (HepG2). The fusion protein AACTTAAGACGATGAATGCTC-3 . The resulting PCR was visualized by immunofluorescence and confocal fragments were subsequently digested with EcoRI and microscopy. To verify the specificity of ATP synthase to HindIII, and ligated to the corresponding sites in the tumor tissues, we measured the expression and enzymatic pEGFP-N1 vector (Clontech, Mountain View, USA). The activity of ATP5B in six different cell lines by flow cytometry reading frame was conﬁrmed by restriction enzyme diges- at Shanghai Jiao Tong University on October 20, 2010 in both normal and tumor-like acidic and hypoxic conditions. tion and DNA sequencing.

Materials and Methods Transfection and microscopy HepG2 cells were transiently transfected with Cell culture and treatments LipofectamineTM 2000 (Invitrogen). Microscopy of GFP Human umbilical vein endothelial cells (HUVECs) were fusion protein was performed using a Nikon A1 confocal obtained from the Sciencell Research Laboratories microscope (Nikon, Tokyo, Japan) at an interval of 8 h for (Carlsbad, USA). Human hepatocellular liver carcinoma a total duration of 48 h. The mitochondria were visualized cell line HepG2, hepatic cell line L-02, human highly by MitoTrackerTM Red (Invitrogen) staining. metastatic lung cancer cell line 95-D, human lung cancer cell line A549, and human embryonic kidney cell line 293 were obtained from the Cell Bank of Type Culture Immunofluorescence microscopy Collection of the Chinese Academy of Sciences (Shanghai, Transfected HepG2 were plated at 2 105 cells/ml on China). The cells were cultured in RPMI 1640 medium glass coverslips and allowed to adhere overnight. The cov- supplemented with FCS, glutamine and antibiotics except erslips were then washed with PBS and fixed in 2% paraf- for HepG2 (DMEM), A549 (F12-K), and HUVECs ormaldehyde. A control slide was permeabilized in 0.5% (EGMTM-2). Except EGMTM-2 medium (LONZA, Triton X-100 for 15 min at room temperature after fixation. Walkersville, USA), all media are from Gibco (Carlsbad, Cells were washed by PBS and incubated at 48C overnight USA). The cells were incubated at 378C under normal (5% in PBS (pH 7.0) containing 1% bovine serum albumin CO2, 20% O2) or tumor-like low pH and hypoxic (17% (BSA) with murine monoclonal anti-GFP IgG (Beyotime, CO2, 0.5% O2, and N2 balanced) conditions. The gaseous Nantong, China) or 10% goat serum (Biostar, Wuhan, environment was maintained by a ProOxC system with China). After that, cells were washed three times and incu- ProCO2 controller (Biospherix, Lacona, USA). The pH and bated at 48C for 1 h with goat anti-mouse IgG conjugated partial pressure of oxygen (PO2) of the medium were moni- to biotin (Biostar). All cells were then washed three times tored by a pH/blood gas analyzer (Ciba Corning, and incubated for 1 h in the dark at 48C with avidin conju- Cambridge, USA). For culture medium, normal condition gated to indocarbocyanine (Cy3) (Biostar). Nuclei were was defined as pH 7.2, PO2 150–170 mmHg, and low pH stained by DAPI. After a final wash, the cells were visual- and hypoxic condition was defined as pH 6.7, PO2 30– ized using Nikon A1 confocal microscope.

Acta Biochim Biophys Sin (2010) | Volume 42 | Issue 8 | Page 531 Ectopic ATP synthase–translocation and the existence in tumor microenvironments

Cell surface ATP generation assay Results As described by Chi et al.[14], cells in 24-well plates were incubated in normal or tumor-like acidic and hypoxic Tracing the expression of GFP fusion protein in HepG2 conditions until they were 80% confluent. Then, the cells by confocal microscopy were washed and equilibrated into serum-free medium for To trace the location of ATP synthase in cells, the ATP5B 30 min with or without oligomycin (10 mg/ml) which was gene with or without the mitochondrial signal sequence pre-incubated at 378C with the corresponding gaseous was cloned. The ATP5B-GFP fusion protein, with or environment for 1 h. The cells were then incubated with without the transit peptide, was subsequently generated in 0.05 mM ADP (gaseous balanced) for 20 s. The super- HepG2 cells. The translocation of fusion protein in HepG2 natants were harvested and assayed for ATP production by cells was visualized by confocal microscopy. Mature CellTiterGloTM luminescence assay (Promega). Recordings ATP5B-GFP fusion protein without the mitochondrial were made on the multifunction microplate reader (BioTek, transit peptide (ATP5Bm-GFP) was translated and localized

Winooski, USA). Data are expressed in moles of ATP per in the cell plasma. Otherwise, the ATP5B precursor-GFP Downloaded from cell based on standards determined for each independent fusion protein with the mitochondrial transit peptide experiment. (ATP5Bp-GFP) was translated in the cytoplasm and translocated to the mitochondria (Fig. 1). Due to a weak signal, Flow cytometry relative to those in the cytoplasm, we cannot confirm the

According to Zhang et al.[13], cells were harvested and existence of fusion protein on the cell surface. abbs.oxfordjournals.org resuspended (5 106 cells/ml) in ice-cold staining buffer (Hanks’ balanced salt solution containing 1% BSA and 0.1% sodium azide). All subsequent steps were done on Detection of GFP fusion protein on the outside of the ice to prevent the internalization of surface antigens. Cells plasma membrane by immunofluorescence were incubated for 45 min with anti-b ATP synthase IgG To detect trace quantities of ATP5B-GFP fusion protein on (Invitrogen). After washing twice with ice-cold staining the outside of the plasma membrane, immunofluorescence at Shanghai Jiao Tong University on October 20, 2010 buffer, the cells were incubated with goat anti-mouse with anti-GFP antibody and biotin–avidin system was IgG-FITC (SouthernBiotech, Birmingham, USA) or isoty- employed. We observed positive signals of the ATP5B- pic IgG for 30 min. The cells were then washed twice, and GFP fusion protein with punctuate appearance on the non-viable cells were identified by propidium iodide stain- surface of non-permeabilized HepG2 cells transfected by ing prior to the final wash. The mean relative fluorescence ATP5Bp-GFP instead of ATP5Bm-GFP. As a control, posi- excited at 488 nm was determined on a flow cytometer tive signals with a different staining pattern were observed (Beckman-Coulter, Brea, USA). when the cells were permeabilized (Fig. 2).

Figure 1 ATP5Bp-GFP fusion protein located on mitochondria (A–C) HepG2 cells were transfected by ATP5Bp-GFP. (D–F) HepG2 cells were transfected by ATP5Bm-GFP. Green channel shows the fluorescence of GFP (A,D). Mitochondria were visualized by MitoTrackerTM Red staining (B,E). Merged images show that fusion protein ATP5Bp-GFP located on mitochondria whereas ATP5Bm-GFP located in cell plasma (C,F). Scale bar ¼ 50 mm.

Acta Biochim Biophys Sin (2010) | Volume 42 | Issue 8 | Page 532 Ectopic ATP synthase–translocation and the existence in tumor microenvironments Downloaded from abbs.oxfordjournals.org

Figure 2 ATP5Bp-GFP fusion protein locate on the out surface of cell membrane HepG2 cells were transfected by ATP5Bp-GFP and immunostained with anti-GFP antibody. (A) Red channel shows ATP5B-GFP fusion protein with punctate appearance located on the surface of non-permeabilized cells. (B) Green channel (GFP) of same cells. (C) Nuclei were visualized by DAPI staining. (D) Merged image. (E) Same cells permeabilized show different staining pattern. (F) No positive signals observed on the surface of HepG2 cells which were transfect by ATP5Bm-GFP. Representative images of three independent experiments were shown. Scale bar ¼ 50 mm. at Shanghai Jiao Tong University on October 20, 2010

Figure 3 b-subunit of ATP synthase on cell surface was detected by flow cytometry Cells were cultured in acid and hypoxia environment for 24 h, and then incubated with isotypic IgG (1) or antibody against the b-subunit of ATP synthase (3). Cells were cultured in normal environment, and then incubated with an isotypic IgG (2) or antibody against the b-subunit of ATP synthase (4). Expression of ectopic ATP synthase in malignant cells was not always higher than that in less malignant cells (HepG2.L-02 but 95-D,A549). After acid and hypoxia treatment, expression of ectopic ATP synthase in 95-D and 293 was up-regulated, whereas in other cells it was not.

Analysis of ectopic b-ATP synthase expression by flow was determined by flow cytometry. To avoid the detection cytometry of ATP5B in the cytoplasm, all flow cytometry exper- In this study, the expression of ectopic ATP5B in six cell iments were performed on intact cells (cells without propi- lines from various tissues and with different ‘malignancies’ dium iodide staining). As shown in Fig. 3, ectopic ATP5B

Acta Biochim Biophys Sin (2010) | Volume 42 | Issue 8 | Page 533 Ectopic ATP synthase–translocation and the existence in tumor microenvironments can be found on the surface of all cells. There was no evidence to indicate that the expression of ectopic ATP5B was higher in ‘malignant’ cells (95-D, HepG2) than in ‘less malignant’ cells (A549, L-02, 293, HUVECs). Similarly, there was no evidence to indicate that the expression of ectopic ATP5B could be up-regulated after 24-h incubation in acidic and hypoxic conditions.

Cell surface ATP generation assay Cell surface ATP synthesis was evaluated in each cell line by determining the production of ATP 20 s after the exposure of cells to ADP and inorganic phosphate. ATP Figure 5 The increase of extracellular ATP production in acid and hypoxia environment Compared with that in the normal environment, production was determined by luciferin-luciferase assay. Downloaded from in acidic and hypoxia conditions, extracellular ATP production was The results indicate that ATP synthesis activity is not increased by from 80% to 800%. When ATP synthase was inhibited by always higher in ‘malignant’ cells than in ‘less malignant’ oligomycin, the increase was not observed. cells in both normal and tumor-like acid and hypoxia conditions. For all cell lines, the activity of cell surface ATP synthesis was up-regulated remarkably after acid and ectopic ATP synthase in cells is still unclear. Compared abbs.oxfordjournals.org hypoxia treatment (Fig. 4), whereas this tendency had not with those in mitochondria, the expression of ectopic ATP been observed in cells which had been treated by ATP synthase is so low that it is difficult to isolate them for synthase inhibitor oligomycin (Fig. 5). further analysis. Thus, there was still no evidence to indicate that ectopic ATP synthase is different from those in the mitochondria. To explore the protein transport mechan- Discussion ism of the ectopic ATP synthase, in this study, we focused on the b-subunit of ATP synthase (ATP5B). The ATP5B at Shanghai Jiao Tong University on October 20, 2010 F1Fo-ATP synthase comprises a soluble F1 portion and a gene is located on chromosome 12 in the p13 region, membrane-spanning Fo portion. The catalytic active site, which has 10 exons and encodes a mitochondrial transit which catalyzes the synthesis of ATP from ADP and inor- peptide of 49 amino acids and a mature protein of 480 ganic phosphate, is the b-subunit of F1 portion. For every amino acids [15,16]. The sequence of the translation three to four protons that are released into the matrix of product was analyzed by the signal prediction software mitochondria from the intermembrane space, one ATP is SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP/). No synthesized. Besides mitochondria, in the last few years, putative cellular membrane target signal was predicted. researchers observed the existence and activities of Then, how does ectopic ATP synthase locate to the cellular F1Fo-ATP synthase on the surface of many cell lines membrane? To visualize the localization and translocation (Fig. 6). Ectopic F1Fo-ATP synthase was thought to be a of ATP5B in the cell, recombinants encoding ATP5B-GFP potential marker for tumor target therapy. The origin of fusion protein were constructed and introduced into the hepatocarcinoma cell line HepG2, which expresses high levels of ectopic ATP synthase. Results suggested that with the help of transit peptides, ATP5B was translated in the cytoplasm and localized to the mitochondria. As a consequence of the bright background in the cytoplasm, weak signals in the cellular membrane cannot be excluded. Upon biotin–avidin amplification, ATP5B-GFP fusion protein was clustered into puncta when the cells were transfected with pATP5Bp-GFP. However, the cells were negative for cell membrane-localized ATP synthase when the cells were transfected with pATP5Bm-GFP. Importantly, when cells were permeabilized, the GFP staining pattern (Fig. 2)was Figure 4 Quantification of extracellular ATP production by luciferin- totally different from the non-permeabilized cells, which luciferase assay After 20 s of ADP and phosphate exposure, suggested that mature ATP5B could not translocate to cel- extracellular ATP production of intact cells was determined by luciferin-luciferase assay. Extracellular ATP production in acid and lular membrane alone. Mitochondrial transit peptide is hypoxia environment was higher than that in normal environment. *P , essential for ATP5B translocation to the cellular membrane 0.05, **P , 0.01, n ¼ 3. and the mitochondria. The mitochondrial transit peptide is

Acta Biochim Biophys Sin (2010) | Volume 42 | Issue 8 | Page 534 Ectopic ATP synthase–translocation and the existence in tumor microenvironments Downloaded from

Figure 6 Ectopic ATP synthase located on the caveolae of cell membrane Ectopic ATP synthase locates on the caveolae of cell membrane with the

F1 portion directed into the extracellular space. With the synthesis of ATP, protons are pumped out of the cell. As autocrine agents, in the caveolae, ATP abbs.oxfordjournals.org synthesis products may excite the purinoceptor (P2Y) to promote the proliferation of the cell, and then lead to tumor growth or angiogenesis. (based on Chi et al.[2]). then removed by signal peptidases in the mitochondria. tumor-like acidic and hypoxic conditions. As shown in Thus, ectopic ATP5B may be translocated from the mito- Fig. 3, among the six cell lines with different ‘malignan- chondria by an unknown underlying mechanism. This cies’, ectopic ATP synthase was detected on the cell result agrees with the hypothesis of Wang et al.[6]. In surface of all cells. The expression of ectopic ATP synthase at Shanghai Jiao Tong University on October 20, 2010 addition, some researchers reported that most of proteins in ‘malignant’ cells was not higher than that in ‘less malig- that localize to the inner membrane of mitochondria could nant’ cells. Acid and hypoxia treatment did not change the be found on the surface of the plasma membrane [17], expression. In additional experiments, the expression of suggesting that translocation is possible. ectopic ATP synthase in mesenchymal stem cells (MSCs) Proteomic research indicated that for certain tumor cells, and malignant MSCs, which were induced by carcinogen the expression of ectopic-ATP synthase in ‘malignant’ cells 3-methycholanthrene treatment [18], was measured by was higher than in ‘less malignant’ cells [10,11]. Earlier FACS. In this case, the expression of ectopic ATP synthase reports indicated that ectopic ATP synthase was observed in the MSCs after malignant transformation was also not on tumor cells and vascular endothelial cells. Because vas- higher than in the original MSCs (data not shown, see cular endothelial cells are involved in angiogenesis, Supplementary Figures). The prevailing dogma that ‘malig- researchers originally thought that ectopic ATP synthase nant cells express more ectopic ATP synthase than less might exist as a bifunctional marker for anti-tumor therapy malignant one [11]’ is not necessarily true. This might be and anti-tumor angiogenesis therapy [12]. Some antibodies true for some cells, but certainly not for all. or inhibitors of ATP synthase were demonstrated to be Ectopic ATP synthase is not only a structural element effective both in vitro and in vivo [9–12]. However, for but also an enzyme involved in ATP synthesis and proton target therapy, specificity is critically important. Since ATP transport. These functions may be critical for cell survival synthase was expressed on the surface of many cells, the in tumor-like microenvironments. Thus, the important tumor specificity of ectopic ATP synthase was naturally question is whether or not the activity of ectopic ATP questioned. Hence, ‘tumor specificity’ can be defined as: synthase on the surface of malignant cells is higher than in (i) expression of the molecule is different in tumor cells less malignant cells; or whether the activity of ectopic ATP and normal cells; and (ii) for the same cell, the expression synthase in tumor-like microenvironments is higher than in or activity of the molecule in tumor tissue or tumor-like normal conditions. If the answer is ‘yes’ to either of the microenvironments is different from normal tissue or above questions, we can conclude that ATP synthase is a normal microenvironments. tumor-specific target. To identify the possibility and specificity of ectopic ATP In the current study, the ATP synthesis activity across synthase as a target for tumor therapy, here, the expression six different cell lines was examined in both normal and of ectopic ATP synthase across six cell lines with different tumor-like (acidic and hypoxic) conditions. The results malignancies was measured by FACS in both normal and suggested that the activity of ATP synthesis is independent

Acta Biochim Biophys Sin (2010) | Volume 42 | Issue 8 | Page 535 Ectopic ATP synthase–translocation and the existence in tumor microenvironments of the malignant status of cells, but that it was significantly References up-regulated in tumor-like conditions. For ectopic ATP synthase, its specificity for tumors relies on its increased 1 Stock D, Leslie AG and Walker JE. Molecular architecture of the rotary catalytic activity in acidic and hypoxic microenvironments motor in ATP synthase. Science 1999, 286: 1700–1705. when compared with normal tissues. 2 Chi SL and Pizzo SV. Cell surface F1Fo ATP synthase: a new paradigm? This result agrees with the report by Chi et al.[14]. But Ann Med 2006, 38: 429–438. 3 Kim BW, Choo HJ, Lee JW, Kim JH and Ko YG. Extracellular ATP is opposite results were obtained by Mangiullo et al.[19] generated by ATP synthase complex in adipocyte lipid rafts. Exp Mol who found that cell surface ATP synthase activities of hep- Med 2004, 36: 476–485. tocyte were greater in high pH than in low pH. Different 4 Hong D, Jaron D, Buerk DG and Barbee KA. Transport-dependent results may be caused by different conditions of cell calcium signaling in spatially segregated cellular caveolar domains. Am J culture. For example, cells were cultured in normal con- Physiol Cell Physiol 2008, 294: C856–C866. 5 Bae TJ, Kim MS, Kim JW, Kim BW, Choo HJ, Lee JW and Kim KB, ditions and put into acidic conditions 4 min before ATP et al. Lipid raft proteome reveals ATP synthase complex in the cell

analysis in Mangiullo’s experiment. But in our present surface. Proteomics 2004, 4: 3536–3548. Downloaded from research, cells were cultured and analyzed in acidic and 6 Wang T, Chen Z, Wang X, Shyy JY and Zhu Y. Cholesterol loading hypoxia conditions all along. pH gradient across the cell increases the translocation of ATP synthase beta chain into membrane membrane is an important but not the only factor respon- caveolae in vascular endothelial cells. Biochim Biophys Acta 2006, 1761: sible for the activity ATP synthase on the cell membrane. 1182–1190. 7 Champagne E, Martinez LO, Collet X and Barbaras R. Ecto-F1Fo ATP

The mechanisms involved are still unclear. synthase/F1 ATPase: metabolic and immunological functions. Curr Opin abbs.oxfordjournals.org Considering the functions of the ectopic ATP synthase, Lipidol 2006, 17: 279–284. the above results are acceptable. The activity of ectopic ATP 8 Ruan Y and Wan M. An optimized procedure for solubilization, reduction, synthase in tumor tissues may provide additional extracellu- and transfer of human breast cancer membrane-enriched fraction by 2-DE. lar ATP to counteract the disadvantage of ischemic and Electrophoresis 2007, 28: 3333–3340. 9 Huang TC, Chang HY, Hsu CH, Kuo WH, Chang KJ and Juan HF. hypoxic conditions that are found in tumor tissues [20]. Targeting therapy for breast carcinoma by ATP synthase inhibitor aurover- With the synthesis of ATP, intracellular protons are pumped tin B. J Proteome Res 2008, 7: 1433–1444. out of the cell to prevent acidosis. When the activity of 10 Lu ZJ, Song QF, Jiang SS, Song Q, Wang W, Zhang GH and Kan B, at Shanghai Jiao Tong University on October 20, 2010 ectopic ATP synthase was blocked by angiostatin, the pro- et al. Identification of ATP synthase beta subunit (ATPB) on the cell liferation and migration of HUVECs could be inhibited, surface as a non-small cell lung cancer (NSCLC) associated antigen. BMC especially when the cells were cultured in low extracellular Cancer 2009, 9: 16. 11 Dowling P, Meleady P, Dowd A, Henry M, Glynn S and Clynes M. pH [14,21]. Moreover, ATP synthesis products may excite Proteomic analysis of isolated membrane fractions from superinvasive the purinoceptor, which has been reported to induce many cancer cells. Biochim Biophys Acta 2007, 1774: 93–101. biological effects like the proliferation of endothelial cells 12 Chi SL, Wahl ML, Mowery YM, Shan S, Mukhopadhyay S, Hilderbrand and the angiogenesis [22]. All the above functions indicated SC and Kenan DJ, et al. Angiostatin-like activity of a monoclonal anti- that activities of ectopic ATP synthase are critical for cell body to the catalytic subunit of F1F0 ATP synthase. Cancer Res 2007, 67: 4716–4724. survival in tumor-like microenvironments. Inhibiting the 13 Zhang X, Gao F, Yu LL, Peng Y, Liu HH, Liu JY and Yin M, et al. activities of ectopic ATP synthase can be a potential Dual functions of a monoclonal antibody against cell surface F1F0 ATP approach for tumor target therapy. synthase on both HUVEC and tumor cells. Acta Pharmacol Sinica 2008, 29: 942–950. 14 Chi SL and Pizzo SV. Angiostatin is directly cytotoxic to tumor cells at Supplementary Data low extracellular pH: a mechanism dependent on cell surface-associated ATP synthase. Cancer Res 2006, 66: 875–882. Supplementary Material is available at ABBS online. 15 Neckelmann N, Warner CK, Chung A, Kudoh J, Minoshima S, Fukuyama R and Maekawa M, et al. The human ATP synthase beta subunit gene: sequence analysis, chromosome assignment, and differential expression. Acknowledgements Genomics 1989, 5: 829–843. 16 Kane LA and Van Eyk JE. Post-translational modifications of ATP We thank Dr Chunfang Liu (Huashan hospital affiliated to synthase in the heart: biology and function. J Bioenerg Biomembr 2009, Fudan university, Shanghai, China) for kindly providing 41: 145–150. the malignant MSCs. 17 Kim KB, Lee JW, Lee CS, Kim BW, Choo HJ, Jung SY and Chi SG, et al. Oxidation-reduction respiratory chains and ATP synthase complex are localized in detergent-resistant lipid rafts. Proteomics 2006, 6: 2444–2453. Funding 18 Liu C, Chen Z, Chen Z, Zhang T and Lu Y. Multiple tumor types may originate from bone marrow-derived cells. Neoplasia 2006, 8: 716–724. 19 Mangiullo R, Gnoni A, Leone A, Gnoni GV, Papa S and Zanotti F. This work was supported by grants from the National High Structural and functional characterization of FoF1-ATP synthase on the Technology Researchand Development Program of extracellular surface of rat hepatocytes. Biochim Biophys Acta 2008, China(‘863’ Program) (2008AA02Z121). 1777: 1326–1335.

Acta Biochim Biophys Sin (2010) | Volume 42 | Issue 8 | Page 536 Ectopic ATP synthase–translocation and the existence in tumor microenvironments

20 Raghunand N, Gatenby RA and Gillies RJ. Microenvironmental and cellu- of human endothelial cells. Proc Natl Acad Sci USA 1999, 96: lar consequences of altered blood flow in tumours. Br J Radiol 2003, 76: 2811–2816. S11–S22. 22 Rumjahn SM, Yokdang N, Baldwin KA, Thai J and Buxton IL. Purinergic 21 Moser TL, Stack MS, Asplin I, Enghild JJ, Hojrup P, Everitt L and regulation of vascular endothelial growth factor signaling in angiogenesis. Hubchak S, et al. Angiostatin binds ATP synthase on the surface Br J Cancer 2009, 100: 1465–1470. Downloaded from abbs.oxfordjournals.org at Shanghai Jiao Tong University on October 20, 2010

Acta Biochim Biophys Sin (2010) | Volume 42 | Issue 8 | Page 537