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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 *
1
Department of Molecular Biology Laboratory, Shanghai Sixth People’s Hospital Affiliated Shanghai Jiao Tong University School of Medicine,
Shanghai 200233, China
2
Department 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]
F1Fo-ATP synthase was originally thought to exclusively
locate in the inner membrane of the mitochondria.
However, recent studies prove the existence of ectopic
F1Fo-ATP synthase on the outside of the cell membrane.
Ectopic ATP synthase was proposed as a marker for
tumor target therapy. Nevertheless, the protein transport
mechanism of the ectopic ATP synthase is still unclear.
The specificity of the ectopic ATP synthase, with regard
to tumors, is questioned because of its widespread
expression. In the current study, we constructed green fluorescent protein-ATP5B fusion protein and introduced it
into HepG2 cells to study the localization of the ATP
synthase. The expression of ATP5B was analyzed in six
cell lines with different ‘malignancies’. These cells were
cultured in both normal and tumor-like acidic and
hypoxic conditions. The results suggested that the ectopic
expression of ATP synthase is a consequence of translocation from the mitochondria. The expression and catalytic
activity of ectopic ATP synthase were similar on the
surface of malignant cells as on the surface of less malignant cells. Interestingly, the expression of ectopic ATP
synthase was not up-regulated in tumor-like acidic and
hypoxic microenvironments. However, the catalytic
activity of ectopic ATP synthase was up-regulated in
tumor-like microenvironments. Therefore, the specificity
of ectopic ATP synthase for tumor target therapy relies
on the high level of catalytic activity that is observed in
acidic and hypoxic microenvironments in tumor tissues.
Keywords
tumor marker; ATP synthase; plasma
membrane; translocation; tumor-like microenvironment
Received: March 5, 2010
Accepted: May 10, 2010
Acta Biochim Biophys Sin (2010) | Volume 42 | Issue 8 | Page 530
Introduction
Using the electrochemical gradient across the inner membrane of the mitochondria that is generated during oxidative phosphorylation, F1Fo-ATP synthase generates
energy by coupling the transmembrane delivery of protons
to the synthesis of ATP [1]. For a long time, F1Fo-ATP
synthase was thought to exclusively locate in the inner
membrane of mitochondria. However, more and more evidence hints at the existence of F1Fo-ATP synthase on the
outside of the plasma membrane of tumor cells and some
types of normal cells, such as endothelial cells, hepatocytes, and adipocytes [2].
Ectopic ATP synthase always localizes on the lipid rafts
or caveolae of the cytoplasmic membrane [3–5]. The
mechanisms responsible for the protein transport have not
been established. Wang et al. [6] found that cholesterol
loading increased the level of ATP synthase of the cytoplasmic membrane. However, no increased overall
expression of the protein was observed. It was reported that
most of the proteins localized to the inner membrane of
mitochondria could also be found on the cytoplasmic membrane [7]. Therefore, Wang et al. hypothesized that ectopic
ATP synthase might translocate from the mitochondria.
However, no direct evidence has been observed to support
this hypothesis until now.
Ectopic ATP synthase not only functions as energy generators but also as proton channels and receptors for
various ligands, which are involved in numerous biological
processes including the mediation of intracellular pH,
cholesterol homeostasis, the regulation of the proliferation
and differentiation of endothelial cells, and the recognition
of immune responses of tumor cells [2]. Some proteomic
Ectopic ATP synthase – translocation and the existence in tumor microenvironments
analysis of membrane fractions indicate that the expression
of ectopic ATP synthase in certain ‘malignant’ cells tends
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
anti-tumor and anti-angiogenesis therapies. Some in vitro
and in vivo research suggested that the proliferation of vascular endothelial cells and certain tumor cells could be
inhibited by anti-ATP synthase antibodies or inhibitors of
ATP synthase [12,13]. For anti-tumor and anti-angiogenesis
therapy, specificity for tumor tissues should be considered.
First, one should consider whether or not the expression and
activity of ectopic ATP synthase in ‘malignant’ cells are
always higher than in ‘less malignant’ cells. Second, one
should also consider whether ATP synthase could be regulated by tumor-like acidic and hypoxic microenvironments.
To visualize the protein transport of the ectopic ATP
synthase which located on the cell surface, the b-subunit of
the ATP synthase gene (ATP5B) was cloned and
co-expressed with the green fluorescent protein (GFP) in
human hepatocarcinoma cells (HepG2). The fusion protein
was visualized by immunofluorescence and confocal
microscopy. To verify the specificity of ATP synthase to
tumor tissues, we measured the expression and enzymatic
activity of ATP5B in six different cell lines by flow cytometry
in both normal and tumor-like acidic and hypoxic conditions.
Materials and Methods
Cell culture and treatments
Human umbilical vein endothelial cells (HUVECs) were
obtained from the Sciencell Research Laboratories
(Carlsbad, USA). Human hepatocellular liver carcinoma
cell line HepG2, hepatic cell line L-02, human highly
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
Collection of the Chinese Academy of Sciences (Shanghai,
China). The cells were cultured in RPMI 1640 medium
supplemented with FCS, glutamine and antibiotics except
for HepG2 (DMEM), A549 (F12-K), and HUVECs
(EGMTM-2). Except EGMTM-2 medium (LONZA,
Walkersville, USA), all media are from Gibco (Carlsbad,
USA). The cells were incubated at 378C under normal (5%
CO2, 20% O2) or tumor-like low pH and hypoxic (17%
CO2, 0.5% O2, and N2 balanced) conditions. The gaseous
environment was maintained by a ProOxC system with
ProCO2 controller (Biospherix, Lacona, USA). The pH and
partial pressure of oxygen (PO2) of the medium were monitored by a pH/blood gas analyzer (Ciba Corning,
Cambridge, USA). For culture medium, normal condition
was defined as pH 7.2, PO2 150–170 mmHg, and low pH
and hypoxic condition was defined as pH 6.7, PO2 30–
40 mmHg. For all experiments, the cells were dissociated
by incubation with PBS containing 2 mM EDTA ( pH 7.4).
Plasmids
All kits for gene cloning were from Takara (Dalian,
China), except for TrizolTM (Invitrogen, Carlsbad, USA).
The expression plasmid for the GFP-ATP5B fusion protein
was prepared as follows: total mRNA was isolated from
HUVECs using the TrizolTM reagent according to the manufacturer’s instructions, and then was reverse-transcribed
into single-stranded cDNA using AMV Reverse
Transcriptase (Takara). The ATP5B precursor (ATP5Bp)
coding sequence (with mitochondrial signal sequence) was
PCR-amplified using the primers 50 -AACAAGCTTGCCA
CCATGTTGGGGTTTG-30 and 50 -GCACGGCGAATTCT
CGATGAATGCTCTT-30 . The mature ATP5B (ATP5Bm)
coding sequence (without mitochondrial signal sequence)
was then PCR-amplified using primers 50 -TCGAAGCTTG
CCACCATGGCGCAAACATCTC-30 and 50 -GCGGCGGG
AACTTAAGACGATGAATGCTC-30 . The resulting PCR
fragments were subsequently digested with EcoRI and
HindIII, and ligated to the corresponding sites in the
pEGFP-N1 vector (Clontech, Mountain View, USA). The
reading frame was confirmed by restriction enzyme digestion and DNA sequencing.
Transfection and microscopy
HepG2 cells were transiently transfected with
LipofectamineTM 2000 (Invitrogen). Microscopy of GFP
fusion protein was performed using a Nikon A1 confocal
microscope (Nikon, Tokyo, Japan) at an interval of 8 h for
a total duration of 48 h. The mitochondria were visualized
by MitoTrackerTM Red (Invitrogen) staining.
Immunofluorescence microscopy
Transfected HepG2 were plated at 2 105 cells/ml on
glass coverslips and allowed to adhere overnight. The coverslips were then washed with PBS and fixed in 2% paraformaldehyde. A control slide was permeabilized in 0.5%
Triton X-100 for 15 min at room temperature after fixation.
Cells were washed by PBS and incubated at 48C overnight
in PBS ( pH 7.0) containing 1% bovine serum albumin
(BSA) with murine monoclonal anti-GFP IgG (Beyotime,
Nantong, China) or 10% goat serum (Biostar, Wuhan,
China). After that, cells were washed three times and incubated at 48C for 1 h with goat anti-mouse IgG conjugated
to biotin (Biostar). All cells were then washed three times
and incubated for 1 h in the dark at 48C with avidin conjugated to indocarbocyanine (Cy3) (Biostar). Nuclei were
stained by DAPI. After a final wash, the cells were visualized 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
As described by Chi et al. [14], cells in 24-well plates
were incubated in normal or tumor-like acidic and hypoxic
conditions until they were 80% confluent. Then, the cells
were washed and equilibrated into serum-free medium for
30 min with or without oligomycin (10 mg/ml) which was
pre-incubated at 378C with the corresponding gaseous
environment for 1 h. The cells were then incubated with
0.05 mM ADP (gaseous balanced) for 20 s. The supernatants were harvested and assayed for ATP production by
CellTiterGloTM luminescence assay (Promega). Recordings
were made on the multifunction microplate reader (BioTek,
Winooski, USA). Data are expressed in moles of ATP per
cell based on standards determined for each independent
experiment.
Flow cytometry
According to Zhang et al. [13], cells were harvested and
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
ice to prevent the internalization of surface antigens. Cells
were incubated for 45 min with anti-b ATP synthase IgG
(Invitrogen). After washing twice with ice-cold staining
buffer, the cells were incubated with goat anti-mouse
IgG-FITC (SouthernBiotech, Birmingham, USA) or isotypic IgG for 30 min. The cells were then washed twice, and
non-viable cells were identified by propidium iodide staining prior to the final wash. The mean relative fluorescence
excited at 488 nm was determined on a flow cytometer
(Beckman-Coulter, Brea, USA).
Results
Tracing the expression of GFP fusion protein in HepG2
by confocal microscopy
To trace the location of ATP synthase in cells, the ATP5B
gene with or without the mitochondrial signal sequence
was cloned. The ATP5B-GFP fusion protein, with or
without the transit peptide, was subsequently generated in
HepG2 cells. The translocation of fusion protein in HepG2
cells was visualized by confocal microscopy. Mature
ATP5B-GFP fusion protein without the mitochondrial
transit peptide (ATP5Bm-GFP) was translated and localized
in the cell plasma. Otherwise, the ATP5B precursor-GFP
fusion protein with the mitochondrial transit peptide
(ATP5Bp-GFP) was translated in the cytoplasm and translocated to the mitochondria (Fig. 1). Due to a weak signal,
relative to those in the cytoplasm, we cannot confirm the
existence of fusion protein on the cell surface.
Detection of GFP fusion protein on the outside of the
plasma membrane by immunofluorescence
To detect trace quantities of ATP5B-GFP fusion protein on
the outside of the plasma membrane, immunofluorescence
with anti-GFP antibody and biotin–avidin system was
employed. We observed positive signals of the ATP5BGFP fusion protein with punctuate appearance on the
surface of non-permeabilized HepG2 cells transfected by
ATP5Bp-GFP instead of ATP5Bm-GFP. As a control, positive signals with a different staining pattern were observed
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.
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Ectopic ATP synthase – translocation and the existence in tumor microenvironments
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.
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
cytometry
In this study, the expression of ectopic ATP5B in six cell
lines from various tissues and with different ‘malignancies’
was determined by flow cytometry. To avoid the detection
of ATP5B in the cytoplasm, all flow cytometry experiments were performed on intact cells (cells without propidium iodide staining). As shown in Fig. 3, ectopic ATP5B
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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
production was determined by luciferin-luciferase assay.
The results indicate that ATP synthesis activity is not
always higher in ‘malignant’ cells than in ‘less malignant’
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
hypoxia treatment (Fig. 4), whereas this tendency had not
been observed in cells which had been treated by ATP
synthase inhibitor oligomycin (Fig. 5).
Discussion
F1Fo-ATP synthase comprises a soluble F1 portion and a
membrane-spanning Fo portion. The catalytic active site,
which catalyzes the synthesis of ATP from ADP and inorganic phosphate, is the b-subunit of F1 portion. For every
three to four protons that are released into the matrix of
mitochondria from the intermembrane space, one ATP is
synthesized. Besides mitochondria, in the last few years,
researchers observed the existence and activities of
F1Fo-ATP synthase on the surface of many cell lines
(Fig. 6). Ectopic F1Fo-ATP synthase was thought to be a
potential marker for tumor target therapy. The origin of
Figure 4 Quantification of extracellular ATP production by luciferinluciferase assay After 20 s of ADP and phosphate exposure,
extracellular ATP production of intact cells was determined by
luciferin-luciferase assay. Extracellular ATP production in acid and
hypoxia environment was higher than that in normal environment. *P ,
0.05, **P , 0.01, n ¼ 3.
Acta Biochim Biophys Sin (2010) | Volume 42 | Issue 8 | Page 534
Figure 5 The increase of extracellular ATP production in acid and
hypoxia environment Compared with that in the normal environment,
in acidic and hypoxia conditions, extracellular ATP production was
increased by from 80% to 800%. When ATP synthase was inhibited by
oligomycin, the increase was not observed.
ectopic ATP synthase in cells is still unclear. Compared
with those in mitochondria, the expression of ectopic ATP
synthase is so low that it is difficult to isolate them for
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 mechanism of the ectopic ATP synthase, in this study, we focused
on the b-subunit of ATP synthase (ATP5B). The ATP5B
gene is located on chromosome 12 in the p13 region,
which has 10 exons and encodes a mitochondrial transit
peptide of 49 amino acids and a mature protein of 480
amino acids [15,16]. The sequence of the translation
product was analyzed by the signal prediction software
SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP/). No
putative cellular membrane target signal was predicted.
Then, how does ectopic ATP synthase locate to the cellular
membrane? To visualize the localization and translocation
of ATP5B in the cell, recombinants encoding ATP5B-GFP
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
totally different from the non-permeabilized cells, which
suggested that mature ATP5B could not translocate to cellular membrane alone. Mitochondrial transit peptide is
essential for ATP5B translocation to the cellular membrane
and the mitochondria. The mitochondrial transit peptide is
Ectopic ATP synthase – translocation and the existence in tumor microenvironments
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
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.
Thus, ectopic ATP5B may be translocated from the mitochondria by an unknown underlying mechanism. This
result agrees with the hypothesis of Wang et al. [6]. In
addition, some researchers reported that most of proteins
that localize to the inner membrane of mitochondria could
be found on the surface of the plasma membrane [17],
suggesting that translocation is possible.
Proteomic research indicated that for certain tumor cells,
the expression of ectopic-ATP synthase in ‘malignant’ cells
was higher than in ‘less malignant’ cells [10,11]. Earlier
reports indicated that ectopic ATP synthase was observed
on tumor cells and vascular endothelial cells. Because vascular endothelial cells are involved in angiogenesis,
researchers originally thought that ectopic ATP synthase
might exist as a bifunctional marker for anti-tumor therapy
and anti-tumor angiogenesis therapy [12]. Some antibodies
or inhibitors of ATP synthase were demonstrated to be
effective both in vitro and in vivo [9–12]. However, for
target therapy, specificity is critically important. Since ATP
synthase was expressed on the surface of many cells, the
tumor specificity of ectopic ATP synthase was naturally
questioned. Hence, ‘tumor specificity’ can be defined as:
(i) expression of the molecule is different in tumor cells
and normal cells; and (ii) for the same cell, the expression
or activity of the molecule in tumor tissue or tumor-like
microenvironments is different from normal tissue or
normal microenvironments.
To identify the possibility and specificity of ectopic ATP
synthase as a target for tumor therapy, here, the expression
of ectopic ATP synthase across six cell lines with different
malignancies was measured by FACS in both normal and
tumor-like acidic and hypoxic conditions. As shown in
Fig. 3, among the six cell lines with different ‘malignancies’, ectopic ATP synthase was detected on the cell
surface of all cells. The expression of ectopic ATP synthase
in ‘malignant’ cells was not higher than that in ‘less malignant’ cells. Acid and hypoxia treatment did not change the
expression. In additional experiments, the expression of
ectopic ATP synthase in mesenchymal stem cells (MSCs)
and malignant MSCs, which were induced by carcinogen
3-methycholanthrene treatment [18], was measured by
FACS. In this case, the expression of ectopic ATP synthase
in the MSCs after malignant transformation was also not
higher than in the original MSCs (data not shown, see
Supplementary Figures). The prevailing dogma that ‘malignant cells express more ectopic ATP synthase than less
malignant one [11]’ is not necessarily true. This might be
true for some cells, but certainly not for all.
Ectopic ATP synthase is not only a structural element
but also an enzyme involved in ATP synthesis and proton
transport. These functions may be critical for cell survival
in tumor-like microenvironments. Thus, the important
question is whether or not the activity of ectopic ATP
synthase on the surface of malignant cells is higher than in
less malignant cells; or whether the activity of ectopic ATP
synthase in tumor-like microenvironments is higher than in
normal conditions. If the answer is ‘yes’ to either of the
above questions, we can conclude that ATP synthase is a
tumor-specific target.
In the current study, the ATP synthesis activity across
six different cell lines was examined in both normal and
tumor-like (acidic and hypoxic) conditions. The results
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
up-regulated in tumor-like conditions. For ectopic ATP
synthase, its specificity for tumors relies on its increased
catalytic activity in acidic and hypoxic microenvironments
when compared with normal tissues.
This result agrees with the report by Chi et al. [14]. But
opposite results were obtained by Mangiullo et al. [19]
who found that cell surface ATP synthase activities of heptocyte were greater in high pH than in low pH. Different
results may be caused by different conditions of cell
culture. For example, cells were cultured in normal conditions and put into acidic conditions 4 min before ATP
analysis in Mangiullo’s experiment. But in our present
research, cells were cultured and analyzed in acidic and
hypoxia conditions all along. pH gradient across the cell
membrane is an important but not the only factor responsible for the activity ATP synthase on the cell membrane.
The mechanisms involved are still unclear.
Considering the functions of the ectopic ATP synthase,
the above results are acceptable. The activity of ectopic ATP
synthase in tumor tissues may provide additional extracellular ATP to counteract the disadvantage of ischemic and
hypoxic conditions that are found in tumor tissues [20].
With the synthesis of ATP, intracellular protons are pumped
out of the cell to prevent acidosis. When the activity of
ectopic ATP synthase was blocked by angiostatin, the proliferation and migration of HUVECs could be inhibited,
especially when the cells were cultured in low extracellular
pH [14,21]. Moreover, ATP synthesis products may excite
the purinoceptor, which has been reported to induce many
biological effects like the proliferation of endothelial cells
and the angiogenesis [22]. All the above functions indicated
that activities of ectopic ATP synthase are critical for cell
survival in tumor-like microenvironments. Inhibiting the
activities of ectopic ATP synthase can be a potential
approach for tumor target therapy.
Supplementary Data
Supplementary Material is available at ABBS online.
Acknowledgements
We thank Dr Chunfang Liu (Huashan hospital affiliated to
Fudan university, Shanghai, China) for kindly providing
the malignant MSCs.
Funding
This work was supported by grants from the National High
Technology Researchand Development Program of
China(‘863’ Program) (2008AA02Z121).
Acta Biochim Biophys Sin (2010) | Volume 42 | Issue 8 | Page 536
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