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Introduction
Osteosarcoma (OS) is the most common
non-hematological malignant bone tumor appearing in the first 2 decades of
life with an annual incidence rate of 5.6 per
million[1]. It is characterized by osteoid material produced by malignant
osteoblastic cells. After an initial diagnosis, patients
usually receive standard treatment, such as neoadjuvant
chemotherapy and surgical resection. Unfortunately, only
60%_70% of the adolescents with non-metastatic OS are cured by
standard treatments[2,3]. Although given more intensive
upfront systemic treatment using more powerful drugs and
maximally aggressive surgery of the primary tumor and all
metastatic foci, one-third of the patients ultimately deceased
due to respiratory failure because of a poor response to
multi-agent chemotherapy and progressive pulmonary
metastasis[2]. The challenge is that patients exhibiting a poor histological
response to presurgical chemotherapy (tumor necrosis rate
<90%) will have a worse prognosis[3]. It is thus important to
identify tumor cell-specific biomarkers for the early
detection and prognosis, which will hasten appropriate diagnoses
and the initiation of therapeutic intervention.
Understanding the underlying molecular mechanisms that lead to
OS will contribute to the identification of such candidate
biomarkers.
Comparative proteomic studies using 2-D gel
electrophoresis coupled to mass spectrometric identification has
been applied in studies of various cancers, including
hepatomas[4], lung cancer[5],
prostate[6], and ovarian
cancers[7]. The search for markers and vaccines at the protein level is more
reliable than at the transcriptional level, as the translation
process is often unpredictable and the
spatiotemporal in situ localization of a given protein is not often correlated with its
in situ expression pattern[8]. Although several genomic
studies have been performed using pediatric OS primary tumor
tissues or cells[9,10], comparative proteomic studies on OS
are rare. Recently, a preliminary investigation on the
difference between SaOS-2 and primary osteoblastic cell cultures
was performed[11].
Molecular studies of OS face technical challenges of
obtaining adequate material after presurgical necrotizing
chemotherapy and the need for decalcification of
specimens[1]. In addition, the overall rarity of OS significantly contributes
to the difficulty in studying OS. The human immortalized
osteoblastic cell line carries more similar biological aspects
and phenotypes than primary osteoblastic cell cultures
compared with OS cell lines. In the present study, we selected 3
human OS cell lines (U2OS, SaOS-2, and IOR/OS9) and the
SV40-immortalized normal osteoblastic cell line (hFOB1.19)
as a model system. To investigate differences in the protein
levels between OS cells and control cells, we performed
differential protein level analyses using 2-D gel
electrophoresis and matrix-assisted laser desorption/ionization-time of
flight (MALDI_TOF) mass spectrometry. We identified 26
proteins, the levels of which were either enhanced or
decreased in OS cells relative to the control cells. The
overproduction of activator of 90 kDa shock protein ATPase
homolog 1 (AHA1) and stomatin-like protein 2 (SLP-2) was
further evaluated by Western blot analyses. We
demonstrate for the first time that AHA1 is overproduced in OS
tumor cells and confirm the overproduction of SLP-2 in OS
cells, which was also observed in other tumor
cells[12]. The latter proteins and others may be useful in identifying novel
biomarkers for OS.
Materials and methods
Reagents and materials Electrophoresis reagents
including acrylamide, methylenebisacrylamide,
N,N,N',N'-tetramethylethyl-diamide, hydroxymethyl aminomethane
(Tris), glycine, SDS, dithiothreitol (DTT), iodoacetamide,
α-cyano-4-hydroxy-cinnamic acid, urea, and thiourea were
obtained from Sigma-Aldrich (St Louis, MO, USA).
3-(3-[Cholamidopropyl] dimethyl-ammonio)-1-propane-sulfonate
(CHAPS), Immobiline DryStrips, Immobilized pH gradient
(IPG) buffer, the 2D Clean-up Kit, the 2D Quant Kit, and the
Deep Purple Kit were purchased from Amersham Pharmacia
(Uppsala, Sweden). Trypsin was obtained from Boehringer
Mannheim (Mannheim, Germany).
Dulbecco's modified Eagle's medium (DMEM) and fetal
bovine serum (FBS) were purchased from Gibco (Carlsbad,
CA, USA). Culture plates were obtained from Corning Costar
(Corning, NY, USA).
Cell culture Human osteogenic sarcoma cell lines U2OS
and SaOS-2, and the normal osteoblastic cell line hFOB1.19
(expressing SV40 large T antigen), were originally obtained
from the American Type Culture Collection (Manassas, VA,
USA). The IOR/OS9 cell line was a generous gift from Dr
Massimo SERRA (Rizzoli Institute, Bologna, Italy). All of
the cells were adherent and epithelial. IOR/OS9 was
established from a bone metastasis of a high-grade
OS[9]. The patient had received chemotherapy with adriamycin, cisplatin,
and methotrexate before establishment of the cell line. The
OS cell lines were cultured in DMEM, and hFOB1.19 was
cultured in a 1:1 mixture of Ham's F12 medium and DMEM
without phenol red and with 2.5 mmol/L L-glutamine and
0.3 mg/mL G418, both containing 10% FBS, 100 units/mL
penicillin, and 100 μg/mL streptomycin. All cultures were
maintained in 10 cm diameter plates in a humidified
atmosphere of 5% CO2 at 37 oC. U2OS, IOR/OS9, and hFOB1.19
cells were passaged every 2 to 3 d, and SaOS-2 cells were
passaged every 4 to 5 d. The cells were collected when
con-fluence was obtained.
Sample preparation Monolayers were washed 3 times
with 10 mL ice-cold Tris-buffered sucrose (10 mmol/L Tris,
250 mmol/L sucrose; pH 7) and then lysed by incubation for
10 min on ice at room temperature with 300 µL extraction
buffer containing 7 mol/L urea, 4% CHAPS, 2 mol/L thiourea,
and 30 mmol/L Tris (pH 8.5). The cells were harvested using
a cell scraper and sonicated on ice. The cytosolic fraction
was separated by centrifugation at 12 000×g
for 30 min at
4 oC. The supernatant was collected and stored in aliquots
of 500 µL in microcentrifuge tubes at -80
oC. Prior to use, the samples were desalted using the 2D Clean-up Kit, and the
protein concentration was determined using the 2D Quant
Kit, according to the manufacturer's instructions.
2-D gel electrophoresis Total cytosolic protein
fractions (400 µg) were mixed with 225 µL loading buffer and
rehydration buffer containing 7 mmol/L Urea, 2 mmol/L
thiourea and 4% CHAPS to be separated using IPG strips and to
obtain a final volume of 450 µL. The samples were applied to
the gel for re-swelling with a dry IPG of 24 cm, pH 3_10
non-linear gradient strips on an IPGPhor II system (Amersham
Pharmacia, Uppsala, Sweden). Complete sample uptake into
the strips was achieved after electrophoresis for 12 h at 30 V.
Focusing was performed at 500 V for 1 h and at 1000 V for
1 h, and then at 8000 V up to total 85 000 V·h. The current
was limited to 50 µA/strip and the temperature was kept at
20 oC for all IEF steps. For SDS-PAGE, the IPG strips were
incubated for 15 min twice, once in equilibration buffer A
containing 50 mmol/L Tris-HCl (pH 8.8), 6 mol/L urea, 2%
SDS, 30% glycerol, and 1% DTT, and once in equilibration
buffer B, containing 50 mmol/L Tris-HCl (pH 8.8), 6 mol/L
urea, 2% SDS, 30% glycerol, and 2.5% iodoacetamide.
Electrophoresis was carried out at 20 oC using an Ettan Daltsix
system (Amersham Pharmacia, Uppsala, Sweden) with 25
mmol/L Tris added to the running buffer containing 192
mmol/L glycine and 0.1% SDS, at 2 W/gel for 50 min and at 17 W/gel
for almost 5 h, until the bromophenol blue front had reached
the bottom of the gel. Samples were run 3 times for 3
independent cell culture experiments.
Analysis of gel images Gels were stained with Deep
Purple according to the manufacturer's instructions. Stained
2-D gels were scanned using Image scanner Typhoon 9400
(Amersham Pharmacia, Uppsala, Sweden) and analyzed
using ImageMaster 2D Platinum software 5.0 (Amersham
Pharmacia, Uppsala, Sweden). After automatic spot detection,
the background was removed from each gel and the images
were manually edited (eg adding, splitting, and removing
spots). One gel was chosen as the master gel and used for
the automatic matching of spots in the other 2-D gel
electro-phoresis. The average volume of each spot was calculated
using 3 gels. 2-D images of the OS cell lines were compared
to those obtained using hFOB1.19 cells. Proteins, the levels
of which were at least enhanced or decreased by 2.0-fold,
were considered as significant (P<0.05 according to Student's
t-test).
In-gel trypsin digestion Spots corresponding to the
proteins of interest were excised from the prepared 2-D
gels and subjected to fully automated spot handling in an
Ettan Spot Handling Workstation (Amersham Pharmacia,
Uppsala, Sweden). The gel plugs were removed using a 1.4
µm picking head and destained at room temperature, twice
in 50% methanol/50 mmol/L ammonium bicarbonate and
once in 75% acetonitrile before drying. The dried gel
pieces were rehydrated with 20 µL of 50 mmol/L
ammonium bicarbonate (pH 8), containing 20 µg/mL trypsin and
allowing protein digestion at 37 oC overnight. The samples
were then dried in a vacuum centrifuge and dissolved in
3 µL matrix containing 5 mg/mL recrystallized
α-cyano-4-hydroxy-cinnamic acid. In the last step, 0.3 µL dissolved
sample was spotted on the target slides.
MALDI-TOF mass-mediated identification and
database searching The samples were then analyzed to
generate peptide mass fingerprinting (PMF) by an Ettan
MALDI-TOF mass spectrometer (Amersham Pharmacia, Uppsala,
Sweden) equipped with a 337.1 nm nitrogen laser and the
delayed extraction facility. All spectra were acquired in the
positive ion reflector mode and an average of 200 laser shots
were recorded per sample. Tryptic monoisotopic peptide
masses were searched for in the SwissProt database, using
Mascot software (http://www.matrixscience.com) with a mass
tolerance setting of 100 ppm, one missed cleavage site as
fixed parameters, and cysteine carbamidomethylation and
methionine oxidation as variable modification.
Western blotting Thirty-microgram aliquots of cell
lysates were separated on denaturing 10% polyacrylamide
gels, electroblotted onto nitrocellulose membranes
(Amer-sham Pharmacia, Uppsala, Sweden), and blocked with 4%
skim milk powder in TBS. Subsequently, the membranes
were incubated at 4 oC overnight with monoclonal
antibodies against AHA1 (1:500 dilution; Santa Cruz
Biotechnology, CA, USA) or SLP-2 (1:200 dilution; BD, Bedford, MA, USA)
followed by incubation with horseradish
peroxidase-conjugated anti-goat or anti-mouse IgG for 1 h, respectively.
Thereafter, the membranes were washed with TBS containing
0.05% Tween-20. Signals were developed using an enhanced
chemi-luminescence system (Amersham Pharmacia, Uppsala,
Sweden).
Results
Evaluation of protein patterns of OS and control
cells using 2-D gel electrophoresis 2-D gel electrophoresis was
performed 3 times for each cell line (U2OS, SaOS-2, and
IOR/OS9) to ensure reproducibility. Approximately 2000 protein
spots were detected on the Deep Purple-stained gel by
ImageMaster (1885±82 spots in hFOB1.19, 1826±121 spots
in U2OS, 1892±81 spots in SaOS-2, and 1870±90 spots in
IOR/OS9 protein samples; Figure 1). After matching the
patterns obtained by the 3 independent experiments, the
patterns of the U2OS, SaOS-2, and IOR/OS9 protein samples
were compared with that of hFOB1.19 cells. Several series of
proteins, the levels of which appeared to be at least enhanced
or decreased by 2-fold in the OS cell lines as compared to the
control cells were determined. Among those, we identified
AHA1, the level of which increased 12.4-, 24.1-, and
23.8-fold in SaOS-2, IOR/OS9, and U2OS cells, respectively with
respect to normal osteoblastic cells (Figure 2, #1732), SLP-2,
the level of which increased 10.4- and 7.8-fold in IOR/OS9,
and U2OS cells, respectively, but no obvious changes were
observed in SaOS-2 cells (Figure 2, #1758). On the
contrary, the level of glutathione transferase omega-1 (GSTO-1)
decreased 74.1-, 47.2-, and 35.1-fold (Figure 2, #1215), the
level of the 52 kDa Ro protein (SS-A) decreased 12.1-, 12.6-,
and 45.5-fold (Figure 2, #1459), the level of
phosphoacetylglucosamine mutase (PAGM) decreased 9.2-, 11.2-, and
10.8-fold (Figure 2, #1129), and the level of
interferon-induced GTP-binding protein Mx (IFI-78K) decreased 24.6-,
22.9-, and 18.2-fold (Figure 2, #892) in SaOS-2, IOR/OS9, and
U2OS cells, respectively for hFOB1.19. Fifty-eight
differentially-produced proteins of interest were selected for
subsequent analysis by mass spectrometry.
MALDI-TOF mass spectrometry-mediated protein
identification and peptide mass fingerprinting
The selected protein spots from the Deep Purple-stained gels were excised
and subjected to in-gel tryptic digestion. The extracted
peptides were analyzed by MALDI-TOF mass spectrometry to
generate PMF. In total, we successfully identified 29
differentially produced protein spots from the 58 spots that
were selected. Although some corresponded to the same
protein, for example spots #892 and #926 identified as the
same protein (interferon-induced GTP-binding protein Mx1),
26 proteins, the levels of which were either enhanced or
decreased, were identified by PMF searching against the
SwissProt database (Table 1). Spot #1732 was identified
as the activator of 90 kDa heat shock protein ATPase
homolog 1 (AHA1) by database searching using PMF
(Figure 3).
Western blot analyses Two over-produced proteins of
interest, AHA1 and SLP-2, were selected for further
validation by Western blot analyses. The Western blot results
confirmed that the level of AHA1 was significantly increased
in U2OS, IOR/OS9, and SaOS-2 for hFOB1.19 cells (Figure
4A). Similarly, the level of SLP-2 was significantly increased
in U2OS and IOR/OS9, but no obvious changes were
observed in SaOS-2 cells (Figure 4B). The results obtained by
the Western blots are in accordance with the observations
based on the comparative proteomic analyses.
Discussion
The comparative proteomic analysis has been widely
accepted as a tool in gaining insight into different protein
expressions reflecting complex cellular states, such as tumor
versus normal tissue[5]. In this study, we identified 26
proteins, the levels of which were either enhanced or
decreased in human OS cells as compared to the SV40-
immortalized normal human immortalized osteoblastic cells
using 2-D gel electrophoresis coupled to MALDI-TOF mass
spectrometry.
We identified 9 up-regulated proteins, among which
heterogeneous nuclear ribonucleoprotein K and glycolytic
isoenzyme pyruvate kinase type M2 have already been
reported as tumor markers contributing to neoplastic
transformation[13,14]. The up-regulation of pyruvate kinase in SaOS-2
cells with respect to the primary osteoblastic cultures
reported recently[11] supports our observations.
Among the up-regulated proteins, AHA1 increased most
significantly in all 3 OS cell lines when compared to
hFOB1.19. It is known that AHA1 stimulates the inherent ATPase cycle
of Hsp90 and plays a determining role in Hsp90 client
protein binding and activation cycle in
vivo[15_17]. AHA1 and its yeast homolog, Hch1, stimulate the ATPase activity and
are the first Hsp90 co-chaperones identified with such
activity[18], whereas some other co-chaperones of Hsp90
(Hop, Cdc37, p23) has been reported to inhibit ATPase
activity[19]. AHA1 appears to be a general upregulator of Hsp90
function rather than a stage-specific co-chaperone due to its
ability to coexist in complexes with early but also with late
co-chaperones without interfering with binding or their
activity[20]. The presence of Hsp90 with high ATPase
activity may facilitate malignant tumor
progression[21]. Interestingly, we report here for the first time that AHA1 is
up-regulated in OS cells compared to normal osteoblastic
cells based on comparative proteomics and further
verification using Western blot analyses. Because of the crucial
role in the activation and stimulation of Hsp90 ATPase which
may be essential for tumor cell growth and/or survival under
non-optimal environment through activating multiple and
overlapping signaling pathways for Hsp90 facilitating the
proper folding, maturation, and activity of a number of
oncogenic proteins[22], AHA1 may obviously serve as a novel
and effective target for OS therapy.
Furthermore, our studies demonstrate that SLP-2 is
up-regulated in U2OS and IOR/OS9 cells, but not in SaOS-2
cells. SLP-2 is a novel and unusual stomatin homologue
with unknown functions and is ubiquitously expressed at
low levels in human organisms[23]. Thus far, the primary
structure of SLP-2 is still not completely elucidated and the
etiology of increased SLP-2 expression in human cancers is
unknown[12,24]. Based on its homology to stomatin, SLP-2
was predicted to be located in the
cytoplasm[25]. Zhang et
al[12] detected that SLP-2 was overproduced in human esophageal
squamous cell carcinoma, lung cancer, laryngeal cancer, and
endometrial adenocarcinoma. Transfection with SLP-2
antisense oligonucleotides inhibited tumor cell growth and
proliferation. Our findings suggest that SLP-2 might be a
potential candidate biomarker of OS, but more information
regarding the role of SLP-2 in tumorigenesis needs to be
obtained. Other proteins, the levels of which were
significantly increased in the 3 selected OS cell lines, also appear
to be candidate biomarkers for OS. We also detected 5 other
increased proteins, at least in 1 of the 3 OS cell lines selected,
including the Fk506-binding protein 4, calponin-3, 60S acidic
ribosomal protein P0, adenosylhomocysteinase, and hydroxymethylglutaryl-CoA synthase.
The comparative proteomic analysis also revealed 17
proteins, the production of which was inhibited in OS cells,
which may be valuable for understanding the biochemical
mechanisms associated with OS tumorigenesis. This group
includes plasminogen activator inhibitor 2 precursor, which
was detected as being under-expressed, and has been
already reported as tumor marker[26].
We detected a remarkable decrease in the level of
GSTO-1, SS-A, PAGM, and IFI-78K in all 3 OS cell lines. GSTO-1
exhibits glutathione-dependent thiol transferase and
dehydroascorbate reductase activity and influences the age
of onset of both Alzheimer's and Parkinson's
disease[27]. GSTO-1 also plays a
role in regulating the intracellular
Ca2+ concentration, potentially by protecting cells from
radiation damage and apoptosis induced by
Ca2+ mobilization from intracellular
stores[28]. Marahatta et
al[29] demonstrated that polymorphisms within the
GSTO gene act as a risk in some cancers. SS-A presents as an independent serum marker in
connective tissue disease[30]. SS-A is detected in some
patients with esophageal squamous cell
carcinoma[31], but its function remains unknown. The level of IFI-78K was
significantly decreased in the 3 OS cell lines, and the level of
tryptophanyl-tRNA synthetase was decreased in 2 of the
detected cell lines, which may be explained by the fact that
hFOB1.19 was SV40-transfected cell line. Although this
observation has been reported
previously[32], it illustrates the usefulness of differential 2-D gel electrophoresis-based
proteomics validity.
The level of several proteins belonging to the
intermediate filament family (eg cytokeratin-8, cytokeratin-18, and
desin) decreased in all 3 OS cell lines. Loss of CK8, CK18,
and CK19 is involved in epithelial-mesenchymal transition
in human micrometastatic and primary breast carcinoma
cells[33]. Furthermore, we observed that the level of some
other proteins that are closely related in the intermediate
filament family decreased in at least 1_2 of the OS cell lines
selected, including macrophage capping protein, ezrin, WD
repeat protein 1, plastin-3, and coronin-1B.
In conclusion, a differential proteomic analysis offers the
possibility of directly identifying relative protein levels in
OS cells with respect to normal osteoblastic cells. Besides
the identification of proteins that may play a role in OS tumor
development, maintenance, or metastasis, this method is
useful in identifying effective biomarkers for OS which will
contribute to strategies for early detection, prognosis, and
effective intervention. Future studies with more cell lines
and primary tumors are required to confirm and supplement
these data and to better clarify the role of AHA1 in the Hsp90
system and the possible impact for use in OS therapy.
Acknowledgements
We thank Dr Massimo SERRA (Rizzoli Institute, Bologna,
Italy) for kindly providing OS cell lines and Shao-jun LIU,
Wei LIU, Li-na WANG, and Xiao-hui HUANG for technical
support.
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