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Introduction
The hepatitis B X-interacting protein (HBXIP), encoding
a 9.6 kDa protein, was originally identified by its interaction
with the C-terminus of the hepatitis B virus (HBV) X protein
(HBX) and located at human chromosome
1p13.3[1]. It contained a putative leucine zipper motif and 2 consensus
phosphorylation sites at threonines 12 and 36 for protein kinase C
and casein kinase II. Investigation for the role of HBXIP in
the HBV replication in hepatoma cells revealed that HBXIP
reduced the replication of the wild-type HBV following
transfection with a HBX-minus virus[1]. The transactivation
effect of HBX on an activating protein-1 (AP-1) binding site as
well as on the HBV enhancers was abolished by
co-expression with the HBXIP. HBXIP could form complex with
survivin, an anti-apoptotic protein that is overexpressed in
most human cancers[2]. The complex of HBXIP and survivin
binded pro-caspase-9 to prevent its recruitment to Apaf I,
and thereby selectively suppressing apoptosis initiated via
the mitochondrial/cytochrome c pathway. HBXIP was
required for bipolar spindle formation and was a regulator of
centrosome dynamics and cytokinesis in
cells[3]. Recently, an interaction factor of HBXIP was identified, which was a
human ATP-dependent RNA/DNA helicase hSuv3p. The nucleotide triphosphate (NTP)-dependent DNA/RNA DExH
box helicase was predominantly localized in the
mitochondria. It was found that the HBXIP-binding domain was important
for mitochondrial import and the stability of the Suv3
protein[4]. Moreover, we found that HBXIP inhibited apoptosis induced
by HBX in hepatoma cells[5]. We previously demonstrated
that 8 types of the HBXIP gene were homologous (data not
shown), such as (i) human fetal tissues of skeletal muscle,
cardiac muscle and uterus muscle; (ii) human adult uterus
muscle; (iii) mouse tissues of skeletal muscle, cardiac muscle
and uterus muscle; and (iv) SM3 cells (a derivation of rabbit
vascular smooth muscle), suggesting that HBXIP was a
conserved protein in evolution.
Although some binding proteins of HBXIP have been
identified in the last few years, the basic biological functions
of HBXIP are still unclear. In the present study, we are
interested in the basic biological functions of HBXIP and
address whether HBXIP is able to influence cell proliferation
by transfection with HBXIP gene in cells, such as breast
cancer MCF-7 cells and hepatoma H7402 cells, and in normal
liver L-O2 cells.
Materials and methods
Cancer cells and cell culture MCF-7 cells, H7402
cells[6], and L-O2 cells[6] were cultured in RPMI 1640 medium
supplemented with 10% fetal calf serum, 2 mmol/L glutamine and
100 U/mL penicillin, and 100 µg/mL streptomycin in
humidified 5% CO2 at 37 oC.
Construction of the HBXIP gene in vectors
Total RNA was extracted from H7402 cells using Trizol (Invitrogen,
Carlsbad, CA, USA) reagent following the manufacturer's
recommendations. Prior to the first cDNA strand synthesis,
total RNA was digested with RNase-free DNase I (TaKaRa,
Tokyo, Japan) at 37 oC for 20 min and inactivated at 60
oC for 10 min. With 2 µg of total RNA as the template and oligo (dT)
as the primers, the first cDNA strand was synthesized in a 20
μL reaction system with M-MLV reverse transcriptase
(TaKaRa). One µL of cDNA template was used in a 50 µL
reaction volume with rTaq DNA polymerase (TaKaRa) and
oligonucleotide primers as follows:
5'-GACGAATTCATGG-AGGCGACCTTGGAGCA-3' (forward) and
5'-GATCTC-GAGTCAAGAGGCCATTTTGTGCA-3' (reverse). The
resultant cDNA fragments were ligated into pET30a vector
(termed pET30a-hbxip) and pGEX-4T-1 encoding glutathione
S-transferase (GST) vector (termed pGEX-4T-hbxip),
respec-tively. The pET30a-hbxip plasmids and pGEX-4T-hbxip
plasmids were induced by 0.4 mmol/L isopropyl
β-D-thiogalacto-pyranoside (IPTG) in Escherichia coli
BL21 (DE3) at 37 oC for 4 h. The purified fusion protein of GST-HBXIP (35.6 kDa)
expressed by pGEX-4T-hbxip plasmids was used to identify
the specificity of rabbit anti-HBXIP antibody as the antigen.
Generation of antibody of rabbit
anti-HBXIP pET30a-hbxip was induced by 0.4 mmol/L IPTG in
Escherichia coli BL21 (DE3) at 37
oC for 4 h, and the HBXIP band was collected from the gel after SDS-PAGE electrophoresis, which
was used as the immunogen. Equal amounts of antigen
solution and adjuvant were mixed thoroughly. HBXIP
antigen (0.1 mg) was administered by an intradermal injection of
rabbit antibody. First immunizations were done with
complete Freund's adjuvant (Sigma, St Louis, MO, USA) and
subsequent immunizations were done employing incomplete
Freund's adjuvant (Sigma) weekly for 5 times. The serum
was harvested by bleeding at the sixth week. The rabbit
antibody against HBXIP was purified from the immune sera
by affinity purification.
Construction of RNA interference (RNAi) targeting the
HBXIP gene in the vector According to a
report[2], a synthesized 63 mer oligonucleotide containing a specific sequence
for a targeting region of the HBXIP open reading frame was
inserted into the pSilencer-3.0-H1 of the RNAi vector. The
sequence of oligonucleotide 1 was as follows: 5'-GATCCGC-
AGCTAAGCTAACCTCTGTTCAAGAGACAGAGGTTAG-CTTAGCTGCTTTTTTGGAAA-3'; the sequence of
oligonucleotide 2 was as follows:
5'-AGCTTTTCCAAAAAAG-CAGCTAAGCTAACCTCTGTCTCTTGAACAGAGGTTA-
GCTTAGCTGCG-3'. The 2 annealed complementary
oligonucleotides were inserted into the
BamH I/Hind III site of
the pSilencer 3.0 H1 vector. After PCR and enzyme digestion,
identification of the RNAi fragment of HBXIP inserted in the
vector was performed by sequencing.
Transfection One day before transfection, MCF-7 cells,
H7402 cells, and L-O2 cells were collected, and seeded into
6-well plates at 1×105 cells per well
(n=3, each group). MCF-7, H7402, or L-O2 cells were transfected with 2
μg plasmids such as pcDNA3 empty vector, pcDNA3-hbxip encoding
HBXIP[5], and pSilencer-hbxip respectively, using
Lipofecta-mine 2000 (Invitrogen) according to the manufacturer's
instruction. The transfection mixture was removed after
6 h. Transfection efficiency in the cells was monitored by
co-transfection of 0.2 µg pEGFP-C2 plasmid, which expresses
green fluorescence protein (GFP). After 24 h transfection,
5-bromo-2-deoxyuridine (BrdU) incorporation assay was
performed, and after 48 h transfection,
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, flow
cytometry analysis, and Western blot analysis were followed
to detect cell proliferation, cell cycle, and expression of
proteins associated with proliferation.
MTT assay Cell proliferation was measured by MTT
assay[7]. Briefly, an amount of 200 µL cell suspensions
(5×103 cells/mL) was added to each well of 96-well plates and
incubated at 37 oC for 48 h. After 48 h transfection (as described
earlier), an amount of 20 µL MTT (5 mg/mL,
Genview, Houston, TX, USA) was added and incubated at 37
oC for 4 h. After removing the supernatant, 200 µL dimethyl sulfoxide
was added to resolve formazan crystals, and the value of the
optical density was detected at 570 nm. The results are based
on the cleavage of the tetrazolium salt by viable cells that
were proportional to the number of living cells in the wells.
BrdU labeling and immunofluorescent
staining The detailed procedures were followed
accordingly[8]. In brief, the cells were seeded into 6-well plates and were grown
overnight prior to transfection. All groups
(n=3 in every group) were incubated with fresh medium containing 10 µmol/L BrdU
(Sigma) for 4 h prior to immunofluorescence staining with
mouse anti-BrdU antibody. The cells were fixed for 15 min
with 4% paraformaldehyde in phosphate buffered saline
(PBS). After 1 h incubation with PBS containing 2 mol/L HCl
to denature DNA, cover slips were washed 3 times with 0.5%
bovine serum albumin (BSA) and 0.5% Tween 20 in PBS, and
incubated overnight (4 oC) with a mouse anti-BrdU antibody
(NeoMarkers, Fremont, CA, USA) at 1:300 dilution.
Reactions were developed using fluorescein isothiocyanate
(FITC)-conjugated goat anti-mouse IgG (Dako, Glostrup,
Denmark) at 1:100 dilution for BrdU staining. The BrdU
labeling index was assessed by point counting through a
Nikon TE200 inverted microscope (Nikon, Tokyo, Japan)
using a 40×objective lens. A total of 700_800 nuclei were
counted in 6_8 representative fields. The labeling index was
expressed as the number of positively-labeled nuclei/total
number of nuclei. Propidium iodine (PI) (Sigma) staining for
nuclei in 50 µg/mL was used as the control to all cells in each
group.
Flow cytometry analysis After 48 h transfection as
earlier described, the cells
(1×106) were harvested by
trypsinization and washed twice with PBS. Washed cells were
resuspended in 0.6 mL PBS (pH 7.4), and fixed by the addition of
1.4 mL 100% ethanol at 4 oC overnight. The fixed cells
were rinsed twice with PBS, and resuspended in propidium iodine
(PI) solution, including 50 µg/mL PI and 50
µg/mL RNaseA (Sigma) in PBS without calcium and magnesium, and
incubated at 37 oC for 30 min in the dark. Stained cells were
passed through a nylon-mesh sieve to remove cell clumps
and analyzed by a FACScan flow cytometer and Cell Quest
analysis software (Becton Dickinson, San Jose, CA, USA).
Flow cytometry analysis was repeated 3 times.
Western blot analysis For the identification of the
generated rabbit anti-HBXIP antibody, cell lysates from MCF-7
and purified GST-HBXIP from BL21 (DE3) cells were
examined by Western blot analysis. After washing twice in cold
PBS, the cells were lysed with ice-cold lysis buffer (150 mmol/L
NaCl, 20 mmol/L Tris-HCl, pH 7.4, 0.1% SDS, 1.0% Nonidet
P-40, 0.5% Na-deoxycholate, 0.2 mmol/L phenylme-thylsulfonyl fluoride, and protein inhibitor cocktails).
Lysates were centrifuged at 12 000×g for 20 min, and the
supernatants were used as total cell lysates. The protein
concentration was determined by Bradford protein assay (Bio-Rad,
Hercules, CA, USA). A quantity of 30 µg total protein per
lane was separated by SDS-PAGE and transferred onto
polyvinylidene fluoride membranes (Millipore, Bedford, MA,
USA). Membranes were blocked with 5.0% milk powder in
0.05% Tween-PBS, incubated with the specific antibodies
such as rabbit anti-HBXIP (1:1000 dilution) or anti-GST
antibody (Tiangen, Beijing, China, 1:800 dilution), at room
temperature for 2 h, followed by a peroxidase-conjugated
secondary antibody diluted in 0.3% BSA/Tween-PBS at room
temperature for 1 h. Detection of the target proteins on the
membranes was performed using the enhanced
chemiluminescence (ECL) system Western Blotting Detection Reagents
(Amersham Biosciences, Buckinghamshire, UK). All
experiments were repeated at least 3 times. After 48 h transfection,
Western blot analysis was performed as above, the primary
antibodies were mouse anti-p27 (NeoMarkers, Fremont, CA,
USA, 1:500 dilution), mouse anti-c-Myc (Santa Cruz
Biotechnology, Santa Cruz, CA, USA, 1:500 dilution), and
mouse anti-Bcl-2 (NeoMarkers, Fremont, CA, USA, 1:500
dilution), rabbit anti-proliferating cell nuclear antigen (PCNA)
(NeoMarkers, Fremont, CA, USA, 1:1000 dilution), mouse
anti-survivin (Chemicon, Temecula, CA, USA, 1:1000 dilution)
and mouse anti-β-actin (Sigma, 1:20000 dilution).
Statistical analysis All data were expressed as
mean±SD. Statistical analysis was performed by the
Student's t-test. P<0.05 was indicated to be statistical significant.
Results
Identification of antibody of rabbit
anti-HBXIP To examine HBXIP, we generated rabbit anti-HBXIP antibody
recognizing HBXIP. Western blot analysis showed that this
anti-HBXIP antibody specifically recognized HBXIP in MCF-7
cells, and the GST-HBXIP fusion protein expressed
in Escherichia coli BL21 (DE3) and GST in the fusion protein
GST-HBXIP from BL21 (DE3) cells could also be detectable
using antibody of anti-GST (Figure 1).
Promotion of cell proliferation
by HBXIP overexpression Transfection efficiency revealed that approximately
70%_80% of cells showed green fluorescence (Figure 2). To
evaluate whether HBXIP expression correlated with fundamental
cellular processes, we investigated the cell proliferation by
MTT assay, BrdU incorporation assay, flow cytometry
analysis, and Western blot analysis after transfection. MTT
assay showed that transfection with pcDNA3-hbxip
plasmid promoted cell proliferation
(P<0.05 vs control), whereas transfection with the pSilencer-hbxip plasmid decreased cell
proliferation by MTT assay in MCF-7 cells
(P<0.05 vs control, data not shown). To further confirm the results, we repeated
the same experiments in hepatoma H7402 cells and L-O2 cells.
BrdU incorporation analysis showed that the induction of
DNA synthesis by HBXIP was assessed in MCF-7, H7402,
and L-O2 cells. The results showed that the percentage of
cells in the S phase increased significantly in MCF-7, H7402,
and L-O2 cells transfected with pcDNA3-hbxip compared
with the cells transfected with pcDNA3 (P<0.05), and was
reduced significantly in cells transfected with pSilencer-hbxip
plasmids (Figure 3). No statistically significant difference
was observed between the control cells and cells transfected
with pcDNA3 empty vector.
HBXIP overexpression significantly accelerated the cell
proliferation compared with untransfected and pcDNA3
transfected cells. Cell proliferation was quantified by PI. PI
is the sum of the S and G2/M phase activities of the cell cycle
expressed as a fraction of the total cell population, that is,
PI=[(S+G2/M)/(G0/G1
+S+G2/M)]×100%. Flow cytometry analysis showed that overexpression of HBXIP, transfected
with pcDNA3-hbxip plasmids, led to an increased cell PI from
46.25% to 58.28% in MCF-7 cells. However, the
downregula-tion of HBXIP, transfected with pSilencer-hbxip plasmid,
resulted in a decreased cell PI (from 46.25% to 31.67%) and
increased percentage of cells in the G1
phase (from 53.75% to 68.33%, P<0.01; Figure 4A). Flow cytometry analysis was
also repeated in L-O2 cells (Figure 4B), and the PI of the L-O2
cells transfected with pcDNA3-hbxip plasmids increased
significantly compared with the pcDNA3 control (from 29.62%
to 35.54%, P<0.01). The PI in pSilencer-hbxip transient
transfection cells was only 22.72%, so HBXIP also accelerated
the cell proliferation of L-O2 cells. Flow cytometry was
repeated 3 times.
Involvement of proteins in the promotion of cell
proliferation To investigate the mechanism, we examined some
proteins related to cell proliferation and cell-cycle regulation,
such as c-Myc, Bcl-2, PCNA, and p27. Western blot
analysis showed that transient transfection with pcDNA3-hbxip
plasmid upregulated expression of c-Myc, Bcl-2, and PCNA,
but downregulated p27 in MCF-7, H7402, and L-O2 cells.
However, RNAi targeting HBXIP mRNA with the
pSilencer-hbxip plasmid reduced the expression levels of c-Myc, Bcl-2,
PCNA, whereas the p27 expression level was upregulated
with the downexpression of HBXIP in these cells. Since
HBXIP was a cofactor for survivin, we also detected the
expression of survivin in the same condition. The
expression level of survivin was not affected by the overexpression
or depression of HBXIP, which was consistent with the
previous report[2]. We further confirmed this finding by
applying Glyco Band-Scan software (PROZYME, San Leandro,
CA, USA; Figure 5).
Discussion
The functions of HBXIP were investigated in breast
cancer MCF-7 cells, hepatoma H7402 cells, and the normal
hepatic cell line, L-O2. We overexpressed HBXIP in the above
cells by transfection with the pcDNA3-hbxip plasmid and
followed with an investigation of cell proliferation by MTT,
BrdU incorporation assay, and flow cytometry analysis. The
findings showed that the overexpression of HBXIP was able
to promote cell proliferation. The depression of cell
proliferation was found by RNAi targeting HBXIP mRNA in the
cells. As we know, cell proliferation is functionally linked to
the expression of genes associated with growth control. The
maintenance of normal cell function and tissue homeostasis
is depend on the precise regulation of multiple signaling
pathways that control cellular decisions to either proliferate,
differentiate, arrest cell growth, or apoptosis. Previous
studies indicated that HBXIP was a necessary cofactor of survivin
in the process of suppression of apoptosis in cancer cells,
and increased HBXIP was found in both cancerous and
non-malignant liver tissue of humans with chronic HBV
infection[2], so we supposed that the function of HBXIP might relate to
the cell proliferation.
Marusawa et al reported that HBXIP was a link in
bridging HBX and survivin[2]. HBX plays an important role in the
development of liver disease and exhibits effects on gene
transcription, cell proliferation, survival, and
apoptosis[9,10]. It stimulates cell-cycle progression, shortening the emergence
of cells from quiescence (G0) and entry into the S phase by
stimulating the Ras-signaling pathway, and accelerating
transition through checkpoint controls at the
G0/G1 and G2/M
phases[11]. Survivin is a member of IAP (inhibitor of apoptosis
protein) family and has been implicated in anti-apoptosis,
cell division, and cell-cycle
control[12_15]. Under normal physiological conditions, survivin is involved in coordinating
chromosomes and mitosis[16].
In the present study, our data indicated that overexpression
of HBXIP resulted in more MCF-7 cells going into the S phase
of the cell cycle. p27 is a member of the Cip/Kip family of
cyclin-dependent kinase inhibitor (CDKI) that regulate
cell-cycle progression, thus inhibiting various cycle-CDK
complexes. Physiologically, p27 is believed to primarily
regulate the progression of cells from late
G1 into the S phase by interacting with the cyclin E-CDK2
complex[17,18]. We found that transient transfection with the pcDNA3-hbxip plasmid
upregulated the expression of c-Myc, Bcl-2, and PCNA, but
downregulated the expression of p27 in these cell lines.
c-Myc is a nuclear phosphoprotein that functions as a
transcription factor stimulating both cell-cycle progression and
apoptosis. It plays a critical role in normal cell-cycle
progres-sion, especially during transition from
G1 to the S phase[19]. c-Myc is also an early response gene, which responds
directly to mitogenic signals to push cells in the
G1 phase of the cell
cycle[20]. The proto-oncoprotein Bcl-2 is a powerful
antagonist of the mitochondrial pathway of apoptosis
initiated by a variety of extra- and
intra-cellular stresses. As the Bcl-2
family members reside upstream of irreversible cellular
damage and focus much of their efforts on the level of
mitochondria, they play a pivotal role in deciding whether a
cell will live or die[21]. PCNA is a highly conserved 36 kDa
acidic nuclear protein that is expressed during cell
replication and DNA repair, and is correlated with cell
proliferation[22]. The same results, that is, upregulation of c-Myc, Bcl-2, and
PCNA, were also observed in H7402 cells and L-O2 cells by
overexpression of HBXIP.
Protein-protein interactions are crucial for all biological
processes. Using systematic, automated, large-scale Y2H
matrix interaction mating, Ulrich Stelzl et
al[23] screened other interacting proteins of HBXIP, such as eukaryotic
translation elongation factor 1 alpha 1 (EEF1A1),
glyceraldehyde-3-phosphate dehydrogenase (GAPD), G protein-coupled
receptor kinase interactor 1 (GIT1), KIAA1377, and
rap1-interacting factor 1 (RIF1). These proteins in cellular functions
are different. Therefore, HBXIP may also contribute more
besides its effects on apoptosis and cell proliferation.
Additionally, the expression of HBXIP could be regulated
by some factors, such as garlic[24], gonadotropins, and
forskolin[25].
Taken together, our findings indicate that one of the
functions of HBXIP is its involvement in proliferation regulation
in cancer cell lines and the normal liver cell line, which is
related to cell-cycle transition through checkpoint controls
at the G0/G1 or
G2/M phases and the downregulation of p27.
In addition, the upregulation of c-Myc by HBXIP may also
play an important role in cell-cycle transition.
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