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Introduction
HMGB1 (high-mobility group box-1, amphoterin, formerly
named high-mobility group (HMG)1, belonging to the HMG
protein family, is a cytokine-like, 215 amino acid nuclear
protein, which is an important mediator of the body's
inflammation, ischemia, and injury[1,2]. HMGB1 is structured
into 3 domains: 2 basic HMG boxes (HMG domains A and B)
and a highly acidic C-terminal domain, which confer an
overall dipolar appearance to this protein. Each of the HMG
boxes is formed by 2 short and 1 long a-helix, that upon
folding produce an L-or V-shaped 3-D domain structure. The
concave surface of the L-or V-shaped HMG box domain
contacts the DNA in the minor groove in 2 slightly different
ways introducing important modifications in the structure of
DNA, in particular a strong bend[1,2]. Presumably, these
features are of relevance for the biological functions in which
HMGB1 has been involved (DNA repair, recombination,
replication, and transcription)[1,2]. HMGB1 can interact
through its HMG box domains with a broad range of
proteins ranging from nuclear cell proteins to viral proteins,
including the recombination activation gene protein, several
transcription factors, including the cellular tumor
suppressor p53, octamer transcription factors Oct1, Oct2, Oct4, and
Oct6, some homeotic HOX proteins, the steroid receptors,
the general initiation factor human TATA-binding protein
(hTBP), and the viral replication proteins Rep78 and
Rep68[1,2]. HMG box A is important for binding to hTBP and
p53, whereas the binding to Oct factors, HOX factors, and
hormone receptors can take place through boxes A or B.
Additionally, it has been found that HMGB1 was abundantly
expressed in breast cancer tissues compared to normal breast
tissues by a breast tissue
microarray[3]. However, the critical roles of HMGB1 in breast cancer still need to be explored.
The retinoblastoma (Rb) gene, encoding a 928 amino acid
phosphoprotein, is a tumor suppressor, and the loss or
inactivation of Rb gene activity is seen as contributing to a broad
range of tumors, including breast
cancer[4,5]. RB plays important roles in the regulation of cell proliferation, cell cycle
progression, apoptosis, telomerase activity, and so
on[5_9]. At least 4 distinct protein-binding domains of RB have been
identified and extensively characterized, including the A/B
pocket, the large A/B pocket, the C-pocket, and the
N-terminal domain[5,6]. The activated (hypo-phosphorylated) RB
protein inhibits cell cycle progression from
G1→S, in part via an interaction between the large A/B pocket of RB and
the activation domains of the E2F family transcription factors,
resulting in the repression of E2F target
genes[7]. The cell cycle inhibitory activity of RB is regulated via interactions
of the standard A/B-binding pocket domain of RB with the
LXCXE ((where L=leucine, C=cysteine, E=glutamic acid and
X=any amino acid) motif of target proteins. For example,
interactions between RB and cell cycle regulatory proteins
(G1/S cyclins and Cyclin-dependent kinases) and viral
oncoproteins (Simian virus 40 large T antigen, adenovirus
E1A, and human papillomavirus E7) inactivate the cell cycle
inhibitory activity of RB and mediate its transcriptional
repression[8]. Thus, the A/B pocket is likely to play an
important role in RB tumor suppressor
functions[5,6]. The A/B and C domains are conserved in 2 other
Rb gene family proteins, p107 and p130, which also bind to LXCXE and regulate cell
cycle-dependent transcription[10]. The activities of p107 and
p130 overlap with, but are not identical to RB, and these
proteins may partially substitute for RB functions. The
standard A/B-binding pocket, which regulates the
phosphorylation state and cell cycle regulatory activity of RB, is the site
of most tumor-associated Rb
mutations[4,5].
Sequence analysis suggests that the HMGB1 protein
contains a consensus RB-binding LXCXE motif (amino acid
104_108). Therefore, in this study we have studied the potential
association of HMGB1 and RB and the in vitro
and in vivo activities of HMGB1 in human breast cancer cells, and
demonstrated that the HMGB1 protein associated with the RB
protein, the LXCXE motif was required for the interaction.
We found that enhanced expression of HMGB1 suppressed
in vitro cell growth and reduced in vivo
tumor growth in a RB-dependent fashion. Finally, we found that enhanced
expression of HMGB1increased sensitization of breast
cancer cells to ionizing radiation, which was via a
RB-independent mechanism.
Materials and methods
Cell culture and irradiation Human breast cancer cell
lines MCF-7 (containing wild-type RB) and BT-549 (containing RB deletion mutation), and human osteoblastic
cell line SAOS-2 (containing negative RB) were originally
purchased from the American Type Culture Collection
(Manassas, VA, USA) and maintained in Dulbecco's
modified Eagle's medium (Invitrogen, Carlsbad, CA, USA)
supplemented with 5% fetal bovine serum, 100 unit/mL
penicillin, 100 µg/mL streptomycin, and a mixture of
non-essential amino acids (Sigma-Aldrich, St Louis, MO, USA).
The cell lines were incubated in a humidified atmosphere
of 95% air and 5% CO2. RB+/+ and RB_/_ MEF
(mouse embryonic fibroblasts) were maintained in Dulbecco's
modified Eagle's medium (DMEM) containing 10% fetal
calf serum (FCS), and all experiments were conducted
using early passage (< 6 passages) MEF. Irradiation was
performed with γ-radiation (JL Shepherd Mark I Radiator)
using a 137Cs source emitting at a fixed dose rate of 3.5 Gy/min.
Transfection To establish stable transfections, the cells
in 100 mm Petri tissue culture dishes at about 50%_60%
confluence, were incubated overnight with 5 µg plasmid
DNA, using Lipofectamine 2000 (Invitrogen, USA),
according to the manufacturer's instructions. The transfected cells
were incubated in medium containing G418 (0.5 mg/mL),
the G418-resistant colonies were observed approximately
3 weeks after transfection.
HMGB1 and RB vectors The wild-type HMGB1
expression plasmid (wtHMGB1) was created by cloning the
full-length HMGB1 cDNA into a mammalian expression vector
pCMV-Tag2B (Invitrogen, USA). LXCXE-defective HMGB1
(HMGB1-RXRXH, in which the LXCXE changed into the RXRXH) and LXCXE-truncated HMGB1
(HMGB1ΔLXCXE, in which the LXCXE was deleted) expression vectors were
created by modification of the wtHMGB1 cDNA in the
pCMV-Tag2B vector by a MORPH site-directed plasmid DNA
mutagenesis kit (Stratagene, La Jolla, CA, USA).
pGEX5X-RB was described in
elsewhere[11]. The cyclin A reporter plasmid was obtained by cloning into the
KpnI and HindIII restriction sites of the pGL2Basic vector with a 213 bp
fragment of the human cyclin A promoter (from
-165 to +48 bp, relative to the most 3' transcription initiation
site), which was generated by PCR with oligonucleotides
5'-CTCCGGTACCAGCCAGTTTGTTTCTC-3' and
5'-TGGCAAGCTTAAGACGCCCAGAGATG-3'.
Generation of recombinant HMGB1 adenoviruses
The E1-deleted adenovirus-β-galactosidase
(Ad-β-gal) was obtained from Introgen Therapeutics (Houston, TX, USA). A
recombinant adenovirus (pAd/CMV/V5-DEST, Invitrogen,
USA) containing a DNA fragment encoding the complete
amino acid sequence of human HMGB1 (Ad5-HMGB1), HMGB1-RXRXH (Ad5-HMGB1-RXRXH) or
HMGB1DLXCXE (Ad5-HMGB1ΔLXCXE) between the CMV promoter (pCMV) and the polyadenylation signal (TK pA)
was prepared. These adenoviral vectors were propagated in
293 human embryonic kidney cells (Invitrogen, USA)
using the Stratagene MBS mammalian transfection kit
(USA) with a modified calcium phosphate transfection protocol.
The transfected cells were incubated at 37 °C for 7 d, then
harvested and subjected to 4 freeze (liquid nitrogen)/thaw
(a 37 °C water bath) cycles. The cell lysates were
centrifuged at 12 000×g for 10 min at 4 °C, and the supernatant
(primary virus stock) was transferred to
a fresh screw-cap mini-centrifuge tube and stored at -80 °C. Recombinant
adenoviruses were further amplified using the same
proce-dure; the cell lysates were centrifuged on cesium chloride
step gradients at 60 000×g at 4 °C for 2 h to separate viruses
from defective particles and empty capsids. Recovered
virus bands were dialyzed 3 times against
phosphate-buffered saline (PBS). The viruses were aliquoted in a buffer
containing 10 mmol/L Tris, pH 7.4, 10 mmol/L
MgCl2, and 10% v/v glycerol) and stored at -80 °C. Under these
condi-tions, there was no precipitation of virus particles or loss of
virus infectivity due to inactivation or aggregation. To
control the biological effect of the virus per second, the vector,
Ad5.CMV.Null, expressing no transgene (Ad5), was
constructed in a similar manner, but without subcloned gene
sequences.
In vitro cell growth kinetics To assess
in vitro cell proliferation, the infected cells were inoculated into 6-well
dishes at 3×104 cells/well in 5.0 mL complete growth medium
(DMEM plus 5% FCS) on d 0. For each clone tested,
duplicate wells were counted by a hemocytometer on d 1_8. The
duplicate cell counts were within ±5% SEM of the mean
values.
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl
tetrazolium bromide) assay of cell viability MTT assays are based
on the ability of viable cells to convert MTT dye
(3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide), a
soluble tetrazolium salt (thioazyl blue) into an insoluble
formazan precipitate, which is quantitated by
spectrophotometry following solubilization in dimethyl
sulfoxide[12]. Briefly, subconfluent proliferating cells in 96-well dishes were
treated with a cytotoxic drug in standard growth medium,
washed vigorously to remove the drug, and then
postincu-bated for 48 h in fresh, drug-free culture medium. At this
time, the cells were solubilized and absorbance readings
were taken using a Dynatech 96-well spectrophotometer
(Billingshurst, UK). The amount of MTT dye reduction
was calculated based on the difference between absorbance
at 570 and at 630 nm. Cell viability was expressed as the
amount of dye reduction relative to that of the untreated
control cells. Ten replicate wells were tested per assay
condition, and each experiment was repeated at least 3
times.
Clonogenic assay The cells were trypsinized
immediately after irradiation and counted. Known numbers were
subcultured in 100 mm culture dishes in 2 sets of triplicates
for each dose of radiation; sufficient numbers were seeded
to ensure that about 50_100 macroscopic colonies would
appear in each plate of untreated and uninfected control
cells at the end of the 15 d. Colonies were then fixed, stained,
and counted. Surviving fractions were normalized by the
plating efficiency of unirradiated controls (30%_50% for
MCF-7 and 40%_60% for BT-549).
Flow cytometry assay of cell cycle and apoptosis
Cell cycle progression and apoptosis analysis was performed by
flow cytometry. The culture medium was collected into
centrifuge tubes. The cells removed by trypsin were poured
into the same tubes. The cells were centrifuged for 5 min
at 900×g. The supernatant were poured out, washed once
with 1×PBS and centrifuged for another 5 min. The cells
were finally fixed by 5 mL precooled 70% ethanol for at
least 4 h. The fixed cells were centrifuged and washed with
1×PBS. After being centrifuged, the cell pellets were
resuspended in 500 μL propidium iodine (10 μg/mL) containing
300 μg/mL RNase (Sigma-Aldrich, USA). Then the cells were
incubated on ice for 30 min and filtered with a 53
μm nylon mesh. Cell cycle distribution was calculated from
10 000 cells with ModFit LT software (Becton Dickinson, San
Joes, CA, USA) using FACSCaliber (Becton Dickinson,
USA).
Immunoprecipitation Subconfluent proliferating cells in
150 cm2 dishes were harvested, and nuclear extracts were
prepared, as described earlier[11,13]. Each
immunoprecipitation (IP) was carried out using 6 μg antibody or antibody
combination and 500 μg nuclear extract protein. The
precipitated proteins were collected using protein G beads, washed,
eluted in boiling Laemmli sample buffer, and subjected to
Western blotting. The HMGB1 IP antibody was antibody
T32, kindly provided by Dr Kimitoshi KOHNO, University of
Occupational and Environmental Health, Fukuoka, Japan.
The control IP antibody was a normal mouse
immunoglobulin G (IgG, Santa Cruz Biotechnology, Santa Cruz, CA, USA).
Western blotting Western blotting assays were
performed as described earlier[11,12]. Equal aliquots of protein
extract (100 μg/lane) were electrophoresed on SDS-PAGE,
transferred to nitrocellulose membranes (Millipore, Bedford,
MA, USA), and blotted with primary antibodies: a mouse
monoclonal anti-HMGB1 antibody (R&D Systems, Minneapolis, MN, USA); a rabbit polyclonal anti-RB
antibody (C-15, Santa Cruz Biotechnology, USA) or a mixture of
a rabbit polyclonal anti-PRAP antibody (P7607, Sigma, USA)
plus a rabbit polyclonal anti-PRAP antibody (214/215,
cleavage site specific antibody, Sigma, USA). The appropriate
secondary antibodies were used at a dilution of 1:2000.
Blotted proteins were visualized using the enhanced
chemiluminescence system (Amersham Life Sciences, Arlington
Heights, IL, USA), with colored markers (Bio-Rad
Labora-tories, Hercules, CA, USA) as molecular size standards. As
an internal control for the amount of protein loaded, the same
filter was also immunoblotted with a polyclonal α-actin
antibody (I-19, Santa Cruz Biotechnology, USA).
Glutathione-S-transferase capture assays
Glutathione-S-transferase (GST) capture assays were performed as
described in our previous
studies[11,13]. 35S-methionine-labeled
proteins were prepared by in vitro transcription and
translation, using the T3 promoter of the pCMV-Tag2B vector.
The GST fusion proteins were generated from cDNA cloned
into the GST vector (pGEX), expressed in Escheria
coli, and purified by affinity chromatography.
In vitro translated labeled proteins were incubated with either GST alone
(negative control) or GST fusion proteins for 4 h at 48
oC, recovered using GSH gluathione agarose beads, eluted in
boiling Laemmli sample buffer, and analyzed by SDS-PAGE
autoradiography. To confirm their expression, the GST
fusion proteins were visualized by Western blotting, using an
anti-GST mouse monoclonal antibody (B-14, 1:5000 dilution,
Santa Cruz Biotechnology, USA).
Assays of transcriptional activity Proliferating cells at
50%±70% confluency in 24-well dishes were incubated
overnight with 0.25 µg of each vector (unless otherwise indicated)
in serum-free DMEM containing Lipofectamine 2000 (Invitrogen, USA). The total transfected DNA was kept
constant by the addition of the control vector. The cells were
washed, incubated in serum-free, phenolphthalein-free
DMEM (0.2 µL/well) for 24 h, and harvested for luciferase
assays. To control transfection efficiency, plasmid
pRSV-β-gal was cotransfected to allow normalization of luciferase
values to β-gal activity in the same sample. Values were
mean±SEM of 4 replicate wells and were representative of 3
independent experiments.
Measurement of caspase-3 activity The caspase-3
activity assay kits purchased from BioVison
(Mountain View, CA, USA) were used for the detection of caspase-3
activity following the protocol recommended by the
manufac-turer. In brief, proteolytic reactions were done containing
cytosolic extracts, 2×reaction buffer containing
DL-dithio-threitol (DTT), and caspase-3
colorimetric substrate (DEVD-p-nitroanilide). The reaction mixture was incubated at 37 °C
for 1_2 h, and then the formation of
p-nitroanilide was measured at 405 nm using an ELISA reader.
Tumor studies In the studies of MCF-7 cells, female
nu/nu BALB/c mice were implanted subcutaneously with
17β-estradiol-sustained release pellets (60 d release
pellets; 0.72 mg estradiol; Innovative Research of America,
Sarasota, FL, USA). The mice were inoculated
subcutaneously with approximately
5×106 infected MCF-7 or BT-549 cells suspended in 0.3 mL Matrigel-DMEM. Inoculum size
was selected to produce <100% tumor incidence under
optimal conditions, based on our experience with these
cells, so that we could detect either an increase or decrease
in tumor incidence. The in vivo studies were independently
performed twice. Tumor volumes were monitored weekly
by caliper measurement of the length, width, and height,
and were calculated using the formula for a semiellipsoid
(4/3πr3/2) up to 3 weeks. Each group contained 10 mice;
the data from 2 independent experiments were combined
in the analysis.
Statistical analysis Cell viability and luciferase activity
data were analyzed using Student's paired t-test. Animal
data were subjected to ANOVA with Statistica software
(StatSoft, Berkeley USA) to identify individual differences.
Comparisons were considered to be statistically significant
when P<0.05.
Results
HMGB1 interacts with RB The sequence analysis shows
that the central region of the HMGB1 protein contains a
consensus RB-binding motif, LXCXE
(aa104LFCSE108), thus we first assessed if the HMGB1 protein interacts with the RB
protein by IP, followed by Western blotting. As shown in
Figure 1A, the RB protein was easily detected in the HMGB1
IP of MCF-7 cells containing the wild-type Rb
gene; but no RB protein was found in the HMGB1 IP of BT-549 cells that
have Rb gene deletion. The HMGB1 protein was also
detected in the RB IP in MCF-7 cells. The RB or HMGB1
protein was not found in the normal mouse IgG IP (as a negative
control). These data indicate that endogenous HMGB1 and
RB physically associate with each other in MCF-7 cells.
The direct interaction of HMGB1 and RB was also
observed by GST capture assays. As shown in Figure 1B,
the in vitro-translated (IVT) wtHMGB1 protein bound to
beads coated with the GST-RB protein, but not to beads
coated with the GST protein alone. However,
IVT-HMGB1-RXRXH, in which
104LXCXE108 was mutated into
104RXRXH108 using a site-directed mutagenesis kit (Stratagene, USA), failed
to bind to the GST-RB protein. These results suggest that
the LXCXE motif of the HMGB1 protein is critical and
essential for HMGB1 binding to RB.
HMGB1 enhances RB-mediated transcription
repression The ability to block cell cycle progression is intimately
linked to the ability of RB to repress E2F-regulated
transcription[13]. We next examined if HMGB1 showed any effects on
RB-meditated E2F transcription repression. As shown in
Figure 2A, the activation of an E2F reporter by E2F1 in
RB-negative SAOS-2 cells was repressed by RB (69% reduction,
P<0.01). The ability of RB in the transcription repression
was enhanced by cotransfection of wtHMGB1 (about 97%
reduction, P<0.005), but not affected by HMGB1-RXRXH or
"empty" control pCMV-Tag2B vector alone; wtHMGB1 by
itself did not result in any effects on E2F transcription activity.
Similar results were obtained with HMGB1-mediated RB
repression of cyclin A (Figure 2B). These studies suggest
that HMGB1 can act as a cofactor for RB-mediated
transcription repression, at least for E2F and cyclin A.
HMGB1 causes RB-dependent growth inhibition
To further study the roles of HMGB1 in breast cancer cells, we
tried to stably overexpress HMGB1 in 2 breast cancer cell
lines, MCF-7 (containing wild-type RB) and BT-549 (containing mutant-RB) by transfection with
pCMV-Tag2B/wtHMGB1) or pCMV-Tag2B/HMGB1-RXRXH. The
transfected cells were postcultured in medium containing 500
µg/mL neomycin (G418), and the G418-resistant clones
were viewed about 21 d following transfection. The
formation of G418-resistant clones could be seen in the BT-549 cell
line transfected with wtHMGB1, but no significant clones
were viewed in plates with MCF-7 cells transfected with
wtHMGB1. These findings suggest that the
ectopic expression of HMGB1 inhibits the proliferation of
functional RB-containing breast cancer cells, not mutant
RB-containing cells. However, G418-resistant clones, formed in
the culture plates of both MCF-7 and BT-549 cell lines and
transfected with pCMV-Tag2B-HMGB1-RXRXH, indicating
that the suppression of cell growth by HMGB1 requires
HMGB1-RB interaction. In other words, the LXCXE motif is
necessary for HMGB1 anticell growth.
To further identify the antigrowth properties of
HMGB1-expressing cells, we next produced an adenovirus
expression system to achieve a high expression of HMGB1 levels.
The cDNA encoding wtHMGB1 or HMGB1-RXRXH under the CMV promoter were inserted into the E1 region of E1-
and E3-deleted adenovirus type 5 DNA encoding bacterial
β-gal, Ad5CMV-lacZ. The replication-defective recombinant
viruses, designated Ad5-wtHMGB1 or Ad5-HMGB1-RXRXH,
were isolated by plaque purification, and high-titer virus
stocks were produced. Preparations with a higher ratio
exhibited increased cytotoxicity to breast cancer cells using
a MOI of >100 pfu/cell (data not shown). As shown in
Figure 3A, the time course of HMGB1 expression was
determined in MCF-7 cells by immunoblotting after infection with
Ad5-wtHMGB1 at a MOI of 100 pfu/cell. An increased
expression of the HMGB1 protein was readily detected after
24 h infection. Similar results were observed by infection
with Ad5-HMGB1-RXRXH and obtained with the employment of BT-549 cells (data not shown). The number of
infected cells was then counted every day up to 8 d. The
proliferation of the MCF-7 cells was inhibited by
Ad5-wtHMGB1, starting at 72 h following infection, whereas the
proliferation of BT-549 cells was not affected by
Ad5-wtHMGB1 (Figure 3B). In contrast, Ad5-HMGB1-RXRXH
caused no significant effects of MCF-7 cell growth. Similar
results were also obtained in another 2 human breast cancer
cell lines, T-47D containing wild-type RB (data not shown).
Neither Ad5-wtHMGB1 nor Ad5-HMGB1-RXRXH influenced
BT-549 cell proliferation. The similar studies were performed
with mouse embryonic fibroblasts (MEF). As illustrated in
Figure 4, Ad5-wtHMGB1 inhibited cell growth in
Rb+/+ MEF, not in Rb_/_ MEF derived from embryos isolated from the
mating of mice with Rb+/_. Again, neither RB+/+ nor Rb_/_
MEF responded to Ad5-HMGB1-RXRXH. Thus, these
results indicate that the adenovirus-mediated transfer of the
HMGB1 gene causes cell growth suppression in a RB-dependent manner.
HMGB1 causes RB-dependent G1 arrest and apoptosis
induction To examine the mechanism(s) by which HMGB1
mediates growth suppression, we studied the impacts of
HMGB1 on cell cycle progression and apoptosis induction.
Increasing G1 cell cycle arrest and
sub-G1 population (indicat-ing apoptosis) were observed in the MCF-7 cells infected
with Ad-wtHMGB1 at a MOI of 100 pfu/cell for 72 and 96 h,
as determined by flow cytometry assays (Figure 5). The
sub-G1 population was accumulated with the increased time
of Ad5-wtHMGB1 infection in the MCF-7 cells, that is, 5.4%
and 13.5% for 72 and 96 h infection, respectively. In contrast,
infection with Ad5-HMGB1-RXRXH or Ad5-lacZ caused no
significant changes in the cell cycle profile of MCF-7 cells.
No G1 arrest and sub-G1 population accumulation was
induced in BT-549 cells infected with Ad5-wtHMGB1,
Ad5-HMGB1-RXRXH or Ad5-lacZ (data not shown). Therefore,
these findings indicate that RB is a determinant in the
HMGB1-mediated G1 arrest and apoptosis, which may be
important mechanisms contributing to the HMGB1
inhibition of cell proliferation.
To examine the molecule mechanisms which may
contribute to HMGB1-induced apoptosis, we determined if HMGB1
exhibited any affect on poly (ADP-ribose) polymerase
(PARP) cleavage and caspase 3 activity. It has known that
the proteolytic cleavage of PARP is 1 characteristic event of
apoptosis. PARP is a nuclear enzyme involved in DNA repair,
DNA stability, and transcriptional regulation. Caspases, in
particular caspases-3 and -7, cleave the 116 kDa form of
PARP-1 at the DEVD site to generate an 85 and 24 kDa
fragment[14,15]. The cleavage of PARP-1 between Asp214 and
Gly215 results in the separation of the 2 zinc-finger,
DNA-binding motifs from the automodification and catalytic
domains. Thus, PARP-1 cleavage has been
considered a hallmark of apoptosis. As shown in Figure 5B, Ad5-HMGB1
caused a clear cleavage of PARP, while neither
Ad5-HMGB1-RXRXH nor Ad5-LacZ affected PARP in the MCF-7 cells.
Moreover, Ad5-HMGB1 increased caspase-3 activity in a
time-dependent manner (Figure 5C). Therefore, although
further studies are needed, these results indicate that the
HMGB1↑→caspase-3↑→
PARP↑→apoptosis may be an important pathway for HMGB1-mediated apoptosis
induction.
HMGB1 increases cell sensitivity to ionizing radia-tion
To determine the effects of HMGB1 on the radiosensitivity
of breast cancer cells, a MTT survival analysis was
performed with the 2 human breast cancer cell lines MCF-7 and
BT-549. Exponentially growing cells cultured in 6-well tissue
culture dishes were uninfected or infected with
Ad5-wtHMGB1, Ad5-HMGB1-RXRXH or Ad5-lacZ (a control) at
a MOI of 100 pfu/cell only for 24 h, and then irradiated with
various doses of γ-rays. The cells were subjected to MTT
assay for cell survival 24 h following irradiation. As shown
in Figure 6A, the sensitivity to γ-ray irradiation in the
MCF-7 cells infected with Ad5-wtHMGB1 or Ad5-HMGB1_RXRXH
was significantly higher than the uninfected cells or the cells
infected with Ad5-lacZ. For example, 4 Gy of γ-ray
irradiation reduced the cell viability to about 38%_40% in both the
uninfected and Ad5-LacZ-infected control cells, while in the
cells infected with Ad5-wtHMGB1 or Ad5-HMGB1-RXRXH,
the cell viability was reduced to approximately 13%_15%
(>3-fold, P<0.05, Student's t-test). Twenty four hours of
infection with Ad5-wtHMGB1, Ad5-HMGB1-RXRXH, or Ad5-lacZ
at a MOI of 100 pfu/cell did not cause any loss of cell viability,
consistent with no significant inhibition of MCF-7 cell growth
by <48 h infection of Ad5-wtHMGB1 (Figure 3), therefore,
the HMGB1 radiosensitivity was not just due to the
addition of HMGB1 and irradiation cytotoxicity. Similar
results were also observed with BT-549 cells infected with
Ad5_wtHMGB1, although the BT-549 cells were slightly more
resistant to γ-ray irradiation compared to MCF-7 cells.
We also employed clonogenic assay to confirm radiosensitivity. Similar results with HMGB1-mediated
radiosensitivity were observed, as shown in Figure 6B. The
sensitivity enhancement ratio was 3.2
(ID50) and 1.6 (ID90) for MCF-7, and 2.6
(ID50) and 1.4 (ID90) for BT-549, respec-tively.
These results indicate that the enhanced expression of
HMGB1 results in a significant increase in radiosensitivity
of human breast cancer cells. Moreover, such
HMGB1-mediated radiosensitivity is independent on RB gene status,
since radiosensitization by HMGB1 was observed in both
the MCF-7 and BT-549 cell lines. The further investigation
of the underlying mechanism(s) of HMGB1 radiosensitivity
is ongoing in our laboratory.
HMGB1 suppresses in vivo tumor
growth To determine the in vivo antitumor activities of HMGB1, we investigated
the tumorigenic potential of Ad5-HMGB1-infected MCF-7
and BT-549 cells compared with infections of
Ad5-HMGB1-RXRXH, and Ad5-HMGB1ΔLXCXE was deleted using the
MORPH site-directed plasmid DNA mutagenesis kit (Invitrogen, USA). As illustrated in Figure 7, tumor growth
and size were markedly suppressed in mice inoculated with
Ad5-wtHMGB1-infected MCF-7 cells (P<0.001). In contrast,
Ad5-wtHMGB1-infected BT-549 tumor growth and size showed no significant difference from those in the tumors
formed with uninfected cells or Ad5-lacZ-infected control cells
(P>0.05). Ad5-HMGB1-RXRXH or
Ad5-HMGB1ΔLXCXE
tumors versus uninfected or Ad5-LacZ-infected control
tumors were not statistically significant in both the MCF-7
and BT-549 tumor models. These data, consistent with the
in vitro antitumor activity described earlier, suggest that
HMGB1 may be a breast cancer growth suppressor through
a mechanism that depends on the Rb gene status and
HMGB1-RB interaction, that is, RB is an important
determinant of HMGB1-mediated tumor suppression. The LXCXE
motif of HMGB1 is essential for HMGB1 antitumor growth.
Discussion
In a series of experiments, we studied the in
vitro and in vivo activities of HMGB1, a nuclear protein containing a
consensus RB-binding LXCXE motif in breast cancer cell lines
with different Rb gene status. HMGB1 as a
pro-inflammatory cytokine has been implicated in the pathogenesis of a
broad range of immune-mediated diseases, including
arthritis[1,2]. The importance of this study demonstrated for the
first time that HMGB1 can function as a tumor suppressor in
breast cancer cells via a RB-dependent pathway. First, we
discovered a physical interaction between the HMGB1 and
RB protein; this association is via a strong
LXCXE-dependent mechanism (Figure 1). It is known that a number of
endogenous proteins that interact with RB also contain a
LXCXE or LXCXE-like sequence, such as histone deacetylase
(HDAC)-1 and HDAC2, and ATPase and BRM/SWI2-related
gene (BRG1) from the SWI/SNF nucleosome remodeling
complex. The oncoproteins of several DNA tumor viruses,
including E1A from adenovirus E7 from human
papillo-mavirus and T antigen from SV40, inactivate the ability of RB
to suppress cell growth via directly binding to the LXCXE
binding site in the RB protein. Moreover, the mutation of
the LXCXE sequence in these proteins prevents their
inhibitory effect on Rb and their ability to transform cells,
indicating the importance of the LXCXE binding site. Therefore,
further studies in our laboratory are ongoing to determine if
HMGB1 blocks or reduces the binding capability of these
DNA tumor virus proteins to RB by competing for the LXCXE
binding site.
Second, we observed that the increased expression of
HMGB1 enhanced the RB-mediated suppression of E2F and
cyclin A transcriptional activation (Figure 2); however,
HMGB1 by itself does not affect E2F and cyclin A
transcriptional activity. It is known that RB represses transcription
by at least 2 different mechanisms: it can bind transcription
factors, such as E2F, and cyclin A can block their ability to
activate transcription; and the Rb-E2F repressor complex that
forms at promoters can actively repress transcription. This
cooperativity of HMGB1 and RB is likely to be the result of
the direct interaction between HMGB1 and RB for 2 reasons:
the RB-binding LXCXE motif of the HMGB1 sequence is
required for the cooperation, and RB is able to contact both
HMGB1 and E2F1 simultaneously. It will be interesting to
know if HMGB1 is complexed with E2F1 or cyclin A.
Third, the high HMGB1 expression caused
G1 arrest and sub-G1 (apoptotic cell) accumulation in MCF-7 cells, not
BT-495 cells (Figure 5), suggesting that the anticell proliferation
of HMGB1 may have been a result of G1 cell cycle arrest and
apoptosis induction. HMGB1-mediated apop-tosis may be
due to caspase-3 induction and subsequent PARP cleavage.
It is known that the repression of E2F-containing promoters
by RB is considered to be one of the key mechanisms by
which RB induces G1 arrest. The ability of HMGB1 to
cooperate with RB in the repression of E2F1 may therefore be an
underlying mechanism for the observed cooperation between
RB and HMGB1 in the induction of G1 arrest. The LXCXE
motif, RB-binding region of HMGB1 is also important for
HMGB1 G1 arrest functions and apoptosis.
Fourth, we found that an increase of HMGB1 expression
by either transfection of the pCMV-Tag2/wtHMGB1
mammalian expression vector or the adenovirus-mediated HMGB1
gene (Ad5-wtHMGB1) expression significantly inhibits cell
proliferation of MCF-7 containing wild-type RB, rather than
BT-495 cells containing deletion mutant RB (Figures 3,4).
Ad5-wtHMGB1 significantly delays tumor growth in nud mice
(Figure 7). All of these activities of HMGB1 required the
interaction of HMGB1 and RB, since HMGB1-RXRXH (inactivating LXCXE mutant) or
HMGB1ΔLXCXE (LXCXE deletion mutant) failed to exhibit these antitumor activities.
In other words, the consensus RB-binding LXCXE motif is
critical and essential for the antitumor activities of HMGB1.
Therefore, a potential mechanism for HMGB1 antitumor
activity is proposed: HMGB1↑→HMGB1-RB
interaction→E2F or cyclin D1 transcription
activity↑→G1 arrest and apoptosis
induction of cell growth inhibition.
Finally, we demonstrated that the elevated expression of
HMGB1 by either Ad5-wtHMGB1 or Ad5-HMGB1-RXRXH significantly increased cellular sensitivity to ionizing
radiation in both wild-type RB MCF-7 cells and RB mutant BT-549
cells (Figure 6), indicating that HMGB1-mediated
radiosensitivity does not require the interaction of HMGB1 and RB,
that is, via a RB-independent mechanism. These exciting
results suggest that HMGB1, a well-known
pro-inflammatory cytokine, also functions as a tumor suppressor and a
radiosensitizer in human breast cancer, supporting additional
studies of the potential of the in vivo application of HMGB1
gene therapy in breast cancer patients. Further studies in
our laboratory are underway to determine if HMGB1 affects
DNA strand break and repair as a result of radiation, and the
expression of DNA damage repair genes.
Breast cancer is the second leading cause of death from
cancer in women worldwide, with more than 240 000 new
patients every year. RB has been reported to be aberrant in
approximately 20%_35% of breast
cancers[16,17] and has been associated with poor disease outcome. Furthermore, loss
of heterozygosity or other alterations at the
Rb locus are often observed in primary
breast cancer specimens[18_20]. The RB activities are regulated (activated or inactivated)
by a large number of RB-interacting proteins. For example,
the overproduction of cyclin D1 and cyclin E, which
mediate the inactivation of RB, are very common events in breast
cancer[21], and the deregulation of E2F target genes can be
associated with poor prognosis in certain breast cancer
cases[21]. These findings suggest that RB is an important
tumor suppressor in breast cancer. Therefore, based on
the observations in this study, further exploration of HMGB1
functions as a novel RB-associated protein will be
significant in understanding RB-related pathogenesis of tumor
progression and for identifying a new target for biological
therapy in breast cancer.
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