From the Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Received for publication, October 15, 2002
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ABSTRACT |
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The study presented here investigates the
effect of HMGB1 knockout on the sensitivity of mouse embryonic
fibroblasts treated with the anticancer drug cisplatin. We evaluated
both the growth inhibition by cisplatin and cisplatin-induced cell
death in the Hmgb1 cis-Diamminedichloroplatinum(II)
(cisplatin)1 is one of the
most widely used anticancer drugs for the treatment of a variety of human malignancies (1). Whereas cisplatin is extremely effective in
treating testicular cancer, the cure rate being >90% when tumors are
promptly diagnosed (2), the curative potential of the drug against
other tumors, such as ovarian, breast, and lung cancers, is
significantly undermined by intrinsic and acquired resistance (3).
Determining the factors that influence cellular sensitivity to
cisplatin is thus important for understanding the anticancer activity
of cisplatin and for developing a more effective platinum-based chemotherapy.
The cytotoxicity of cisplatin arises from its ability to react with DNA
and form covalent DNA adducts (4). The major adducts, 1,2-intrastrand
d(GpG) and d(ApG) platinum-DNA cross-links, are formed by
coordination of the
{Pt(NH3)2}2+ moiety to the
N7 atoms of adjacent purines in double-stranded DNA. The
cisplatin modification produces distinct changes in the architecture of the DNA duplex that inhibit replication and transcription and stimulate
nucleotide excision repair (1). Cisplatin damage throughout the genome
leads to cell cycle arrest and apoptosis (5). Moreover, cisplatin-DNA
adducts are recognized by a variety of cellular proteins, a process
that may affect the fate of platinum-DNA lesions and the responsiveness
of tumor cells to cisplatin treatment (6, 7). Recognition of
cisplatin-modified DNA by damage recognition proteins in the nucleotide
excision repair pathway leads to the removal of platinum lesions and
restoration of genomic integrity. Studies have suggested that increase
in the repair of platinum-DNA adducts is key to cisplatin resistance
(3). Several proteins not involved in repair, including HMGB1,
TATA-binding protein, and other structure-specific recognition
proteins, also bind tightly to the major platinum-DNA adducts (1). The
role that these proteins might play in mediating the cytotoxicity of cisplatin is a subject of much current interest. One hypothesis, which
has been proposed in various studies, suggests that the binding of
these proteins to platinum-DNA adducts blocks the removal of DNA
lesions, thereby enhancing the sensitivity of cells to cisplatin
(8-14). This model is termed repair shielding. HMGB1, an abundant
chromosomal protein in mammalian cells, interacts with platinum-DNA
intrastrand d(GpG) and d(ApG) cross-links and interferes with their
repair in vitro. It is thus one of the candidate proteins
for participation in the repair shielding mechanism (11, 15).
HMGB1 belongs to the family of proteins that contain at least one HMG
box domain, an 80-amino acid DNA-binding motif that recognizes bent DNA
structures (16, 17). HMGB1 is highly conserved in mammals with >95%
amino acid identity between rodent and human forms (16). HMGB1 is an
extremely versatile protein. It increases transcription
activation involving steroid hormone receptors, p53, Hox, and Pou
protein (18-20). The regulation of transcription activation by HMGB1
is attributed to its DNA binding and bending activity. Protein-protein
interactions between HMGB1 and the transcription factors presumably
facilitate the formation of high order nucleoprotein complexes required
for transcription initiation (for a review, see Ref. 21). Hmgb1
knockout mice die shortly after birth due to hypoglycemia resulting
from a defect in transcription activation by the glucocorticoid
receptor (22). Beside its intranuclear roles, HMGB1 was also discovered
recently to function as an extracellular signaling molecule during
inflammation, cell differentiation, cell migration, and tumor
metastasis (23-25). HMGB1 is secreted by certain cells, including
macrophages and monocytes (21). The secretion and signaling mechanism
by which HMGB1 activates cells during these processes is largely unknown.
HMGB1 has been linked to cisplatin activity in a number of
studies. HMGB1 binds specifically to cisplatin-modified 1,2-intrastrand d(GpG) and d(ApG) cross-links but not to the DNA adducts formed by the
clinically inactive isomer,
trans-diamminedichloroplatinum(II) (15, 26). Addition of
HMGB1 to an in vitro nucleotide excision repair assay
inhibited the repair of cisplatin-DNA damage (11, 14). Given the
abundance of HMGB1 in nuclei, one copy per kb of the human genome, and
its roles in various biological processes, it was hypothesized that the
cellular HMGB1 level might modulate the cisplatin sensitivity of cancer
cells. Consistent with this notion, hormone-induced HMGB1 up-regulation
in MCF-7 breast cancer cells correlates with enhanced cisplatin
sensitivity (27). Increased cisplatin sensitivity was also observed in
a lung adenocarcinoma cell line transfected with a plasmid expressing
HMGB2, a protein with more than 80% identity to HMGB1 (28). A recent
study, however, showed that loss of NHP6A, an abundant HMG box protein
in Saccharomyces cerevisiae, sensitized yeast to cisplatin,
suggesting that a mechanism other than repair shielding was in effect
in the yeast system (29).
To investigate further how HMGB1 might affect cisplatin sensitivity in
mammalian cells, we studied the cisplatin sensitivity and
cisplatin-induced apoptosis in the mouse Hmgb1 Tissue Culture--
Mouse embryonic fibroblast cell lines,
Hmgb1+/+ and Hmgb1 Western Blotting--
Cell extracts were prepared from near
confluent cells on 100-mm plates. Cells were washed with
phosphate-buffered saline and lysed in 250 µl of buffer containing
2% SDS, 10 µg/ml of pepstatin, leupeptin and aprotinin, and 0.5 mg/ml Pefabloc in phosphate-buffered saline. The lysate was pulled
through a 25G5/8 syringe needle to shear genomic DNA and
was cleared by centrifugation. The protein concentrations of extracts
were determined by a bicinchoninic acid protein assay kit (Sigma). The
proteins in cell extracts were resolved on 12% SDS-PAGE gels
and then electroblotted onto polyvinylidene difluoride membranes
(Bio-Rad). Following blocking with a 5% bovine serum albumin and
0.01% Tween 20 solution in Tris-buffered saline at pH 7.4, membranes
were incubated in the primary antibody, rabbit Growth Inhibition Assay--
Cells were seeded on 96-well plates
at a density of 1,000 cells/100 µl/well. Treatment with cisplatin
began the next day after cells had attached to the plates. For
continuous treatment, cells were incubated in growth medium containing
various concentrations of cisplatin for 72 h. For short time
treatment, cells were treated with cisplatin-containing medium for
4 h followed by incubation in drug-free medium for an additional
90 h. After incubation, the cell density of each sample was
determined by using WST-1, a tetrazolium salt (Chemicon
International Inc.). The assay was carried out according to the
manufacturer's protocol. Briefly, 10 µl of WST-1 dissolved in
Electro Coupling Solution (Chemicon International Inc.) was added to
each well of 96-well plates that contained cells at the end of the
incubation. Plates were incubated under standard growth conditions for
1.5 h, and the absorbance at 440 nm was recorded by using an
absorbance microplate reader, SpectroMax 340pc (Molecular Devices).
Percentages of surviving cells were calculated by the ratio of
absorbance of cisplatin-treated cultures over that of untreated control
culture. Cisplatin kill curves were obtained by plotting the percentage
of survival against the concentration of cisplatin. In addition, a
second set of cultures was analyzed by using sulforhodamine B (SRB).
First, 10% cold trichloroacetate solution was added to the cells.
Plates were incubated for 30 min at 4 °C to allow fixation to come
to completion and then washed five times with distilled water. Plates
were air-dried at room temperature in the hood. Trichloroacetate-fixed
cells in each well were then stained with 100 µl of 0.4% (w/v) SRB
in 1% acetic acid for 30 min. Excess SRB was removed by washing the plates four times with 1% acetic acid. Plates were air-dried until no
remaining moisture was visible. The bound SRB was solubilized by adding
100 µl of 10 mM Tris base (pH 10.5) to each well and shaking for 5 min on a shaker platform. The absorbance at 564 nm was
recorded by using the microplate reader. Cisplatin kill curves were
obtained as described above.
Annexin V Assay--
A portion (1.5 × 105) of
cells was plated on 60-mm plates and allowed to attach overnight. Cells
were then incubated in cisplatin-containing media for 34 h. After
incubation, the attached cells in each plate were collected by
trypsinization and combined with the detached cells of the same sample.
Cells were washed twice with cold phosphate-buffered saline and
resuspended in the binding buffer (Pharmingen). Cell suspensions were
counted with a hemocytometer and diluted to a final concentration of
1 × 106 cells/ml. An aliquot of cells (100 µl) was
labeled with propidium iodide (PI) and fluorescein
isothiocyanate-conjugated annexin V (annexin V-FITC) according
to the manufacturer's instructions. The labeled cell suspensions were
analyzed by a flow cytometer, FACScan, equipped with a 488-nm argon
laser light source (BD Biosciences). The emission filter used was
515-545 nm for FITC and 563-607 nm for DNA·PI complexes. Data were
analyzed with CellQuest software. Cell debris was excluded from data analysis.
Cisplatin Sensitivity of Hmgb1+/+ and
Hmgb1 Cisplatin-Induced Apoptosis in Hmgb1+/+ and
Hmgb1
Fig. 2 shows the density plots of PI
fluorescence versus annexin V-FITC fluorescence obtained
from control (untreated) or cisplatin-treated Hmgb1+/+ and
Hmgb1 HMGB1 Expression Levels in the Hmgb1+/+ Mouse Cells and
MCF-7 Cells--
The expression levels of HMGB1 are similar in
different tissues of the same animal (40). It is not clear whether
different animals have significantly different levels of expressed
protein. Here, we used Western blotting to compare the levels of HMGB1 in the mouse fibroblasts with those in MCF-7 cells, a human breast cancer line. The same total amount of cellular proteins was subjected to SDS-PAGE separation and Western analysis using the HMGB1 antibody. The Western blot showed similar levels of HMGB1 expression in the mouse
fibroblasts and MCF-7 cells (Fig. 4).
The goal of this study was to explore the connection between HMGB1
levels and the sensitivity of mammalian cells to cisplatin. We
addressed this question by comparing the response of wild-type cells
with those of Hmgb1 knockout mouse cells established previously (22).
Hmgb1 Both the growth inhibition by cisplatin and cisplatin-induced apoptosis
in the Hmgb1 HMGB1 has been implicated in the cytotoxicity of cisplatin since
the discovery of its ability to bind cisplatin-modified DNA in vitro. A number of experiments (42-47) have discerned
the molecular interactions between HMGB1 and cisplatin-modified DNA.
Few studies, however, investigated the effect of HMGB1 on cisplatin
sensitivity at the cellular level. The present results suggest that
HMGB1 does not play a significant role in the mechanism of
cisplatin-induced cytotoxicity in the mouse embryonic cells. Given the
high binding affinity of HMGB1 for cisplatin-modified DNA and the
abundance of HMGB1 in mammalian cells, this observation seems
surprising. There are, however, a number of scenarios that might
prevent HMGB1 from interacting with cisplatin-modified DNA in the cells
and modulating their responses to cisplatin. One possibility is that the HMGB1 level in the mouse embryonic fibroblasts is considerably lower than that of cells in other tissues or animals, such as MCF-7
human breast cancer cells, the subject of a previous study (27). A
Western analysis for HMGB1 levels in both cell lines excluded this
possibility. More likely is that HMGB1 proteins engage in tight
interactions with other targets in the cells. HMGB1 contains two tandem
HMG boxes and an extremely acidic C-terminal domain (21), through which
it interacts with several other proteins (19, 20, 41). In the mouse
fibroblasts, HMGB1 proteins may be engaged in tight nucleoprotein
complexes and may not be available for ready binding to
cisplatin-modified DNA. A recent study discovered that during
apoptosis induced by tumor necrosis factor HMGB1 has been postulated to influence cellular sensitivity to
cisplatin through specific binding to cisplatin-DNA 1,2-intrastrand cross-links. Our results reveal little difference in the cisplatin sensitivity and cisplatin-induced apoptosis in embryonic native and
Hmgb1/
cells and its wild-type
counterpart. No significant differences were observed in the responses
of these cells to cisplatin, indicating that HMGB1 does not play a
significant role in modulating the cellular responses to cisplatin in
this context. Since HMGB1 significantly enhances the cytotoxicity of
cisplatin in other cells, these results illustrate the importance of
cell type in determining the ability of this and probably other
cisplatin-DNA-binding proteins to influence the efficacy of the drug.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES
/
cell
line. Here, we report the results of this work and discuss its
implications. Our studies reveal little difference in the cisplatin
sensitivity and cisplatin-induced apoptosis between the
Hmgb1
/
and its parental cell lines, suggesting that in
this mouse model, HMGB1 does not modulate cellular sensitivity to
cisplatin. To our knowledge, this is the first direct examination of
the impact of HMGB1 loss on cisplatin sensitivity in mammalian cells.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES
/
, were kindly provided
by M. E. Bianchi (22). Cells were maintained as monolayer cultures
in Dulbecco's modified Eagle's media containing 10%
heat-inactivated fetal bovine serum and 2 mM
L-glutamine at 37 °C in a humidified incubator with 5%
CO2.
-HMGB1 (1:2,000
dilution) (Pharmingen), in 0.01% Tween 20 at pH 7.4 with Tris-buffered
saline. Finally, blots were incubated in the secondary antibody,
1:2,000 dilution of
-rabbit whole antibody conjugated to horseradish
peroxidase (Amersham Biosciences). After washing, blots were
soaked for 1 min in chemiluminescent reagents (Sigma) and then exposed
to BioMax film (Eastman Kodak Co.).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES
/
Cells--
Two independent methods were used to
compare the cisplatin sensitivity of the Hmgb1
/
and
wild-type mouse cells. The WST-1 assay measures the live cell
population because WST-1 is quantitatively reduced to a colored formazan in viable cells (30, 31). The SRB assay measures cell density
by quantitating colored sulforhodamine B bound to cells fixed to the
plates by trichloroacetate (32, 33). Similar results were obtained by
using the two methods (Fig. 1). Cisplatin inhibited the growth of Hmgb1+/+ and Hmgb1
/
cells to a similar extent when added to growth medium for 72 h
(Fig. 1, A and B). Likewise, a 4-h cisplatin
treatment followed by an 84-h incubation in drug-free medium equally
inhibited the growth of both cell lines (Fig. 1, C and
D). No significant difference between the cisplatin
sensitivity of Hmgb1+/+ and Hmgb1
/
cells
was observed in repeated trials.
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Fig. 1.
Cisplatin kill curve of
Hmgb1+/+ and Hmgb1 /
cells. Cells were
plated in 96-well plates at a density of 1,000 cells/100 µl/well.
Cells were incubated with cisplatin-containing medium for 72 h
(A and B) or treated with cisplatin for 4 h
and incubated in fresh medium for 84 h (C and
D). Cell density was measured either by the SRB assay
(A and C) or by the WST-1 assay (B and
D).
/
Cells--
A common cellular response to
cisplatin exposure is activation of the apoptotic pathway (34). In the
present study, the apoptosis of Hmgb1+/+ and
Hmgb1
/
cells induced by cisplatin was probed by
labeling with PI and annexin V-FITC. Early apoptotic cells lose
membrane phospholipid asymmetry and expose phosphatidylserine on the
outer leaflet of the plasma membrane. Previous work has demonstrated
that phosphatidylserine externalization in apoptotic cells can be
labeled with annexin V-FITC and quantitated by flow cytometry (35-37).
The late apoptotic cells and dead cells can also be labeled with PI in
addition to the annexin V labeling because of damaged cell membrane.
The annexin V-FITC/PI assay has been successfully applied to study
cisplatin-induced apoptosis in a number of cell lines (35, 38, 39).
/
mouse cells. These cells were continuously
treated with cisplatin for 34 h before fluorescence-activated cell
sorter analysis. The plots illustrate four cell populations (live,
apoptotic, necrotic, and late apoptotic/dead) defined by their
fluorescence characteristics. Live cells are annexin V- and
PI-negative. Early apoptotic cells are annexin V-positive and
PI-negative; their membranes are not permeable. Necrotic cells are
annexin V-negative and PI-positive because of damaged cell membrane.
The late apoptotic and dead cells are both annexin V- and PI-positive.
The untreated Hmgb1+/+ and Hmgb1
/
cell
cultures contained very few apoptotic cells (<3%) but included 6-7% dead cells, which we assign as the background cell death in
these cultures. By comparison, significant percentages of apoptotic cells and dead cells were present in cisplatin-treated cultures. The
necrotic populations in all cultures were generally less than 4%,
suggesting that dead cells did not arise from necrosis following cisplatin treatment. The sums of apoptotic and dead cell populations in
cultures treated with various concentrations of cisplatin are plotted
in Fig. 3. Hmgb1
/
cells
exhibited similar levels of apoptosis as wild-type Hmgb1+/+
cells for all concentrations of cisplatin. Similar apoptotic responses
to cisplatin in the Hmgb1
/
and Hmgb1+/+
cells were consistently observed in repeated experiments.
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Fig. 2.
Density plots of PI
labeling versus annexin V-FITC binding in
Hmgb1+/+ (left panels) and
Hmgb1 /
(right panels) cells treated
with 0 (A), 2 (B), 5 (C), or 10 µM
(D) cisplatin for 34 h. FL1-H
represents PI fluorescence, and FL2-H represents annexin
V-FITC fluorescence. The four regions in each plot represent
vital (annexin V
/PI
), early apoptotic
(annexin V+/PI
), damaged (annexin
V
/PI+), and late apoptotic/dead (annexin
V+/PI+) cells, respectively.
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Fig. 3.
Cisplatin induces similar apoptotic responses
in Hmgb1+/+ and Hmgb1 /
cells. Each
bar represents the sum of percentages of the early apoptotic
(annexin V+/PI
) and late/dead cells (annexin
V+/PI
) in the given culture.
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Fig. 4.
Western analysis shows similar levels of
HMGB1 expression in MCF-7 (lane 1) and wild-type mouse
embryonic fibroblast cells (lane 2) as well as lack of
HMGB1 expression in Hmgb1 /
mouse cells (lane
3). A, Western blot using anti-HMGB1
antibodies. B, Western blot using anti-
-actin. *, a
protein band nonspecifically recognized by the anti-HMGB1 antibody
observed in the wild-type and Hmgb1
/
mouse cells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES
/
mice die shortly after birth due to a defect in
the transcriptional activation of the glucocorticoid receptor,
consistent with the notion that HMGB1 functions as a regulator of
transcription involving steroid hormone receptors. The HMGB1 deficiency
is also manifested at the cellular level. The cultured
Hmgb1
/
embryonic fibroblasts are defective in
transcription activation by the glucocorticoid and progesterone
receptors (22). The Hmgb1
/
cells have a morphology and
growth rate similar to those of the Hmgb1+/+ cells, and the
levels of Hmgb2 and Hmgb3 mRNA are comparable in both cell lines
(22). The Hmgb1
/
and the wild-type cell lines thus
provide an adequate system for investigating the involvement of HMGB1
in cellular responses to cisplatin.
/
and the wild-type cells were examined.
Two independent methods (see "Experimental Procedures") revealed
that continuous treatment with cisplatin inhibited the growth of
Hmgb1
/
cells to the same extent as that of
Hmgb1+/+ cells (Fig. 1). When cells were treated with
cisplatin for a short time (4 h), the survival rates of both cell lines
were also similar (Fig. 1). Furthermore, the cisplatin-induced
apoptosis in these cells was investigated by using annexin V-FITC/PI
labeling. In cisplatin-treated cell cultures of both lines, there was a significant apoptotic (annexin V-positive) but very little necrotic population (annexin V-negative/PI-positive), suggesting that the cells
die via the former pathway (Fig. 2). Increasing cisplatin concentration
in the cultures resulted in increasing apoptosis in both cell lines,
confirming that apoptosis of the cells was induced by cisplatin
(Figs. 2 and 3). Comparison of the apoptotic population in the
Hmgb1
/
and Hmgb1+/+ cultures treated with
the same concentrations of cisplatin showed essentially the same degree
of cell death in both cell lines (Fig. 3). Collectively, these results
indicate that the Hmgb1
/
cells are as sensitive to
cisplatin as the Hmgb1+/+ cells.
, HMGB1 became firmly
attached to the hypoacetylated chromatin. The binding of HMGB1 to
apoptotic chromatin may similarly block the interaction of HMGB1 to
platinum-DNA adducts (23). Finally, as yet unidentified proteins in
these embryonic cells may dominate the interaction with
cisplatin-modified DNA. A comprehensive analysis of the correlation between cisplatin sensitivity and protein expression levels in cells
may lead to the discovery of such protein candidates. In any event, our
results stress the importance of cell type in determining the ability
of this and possibly other platinated DNA-binding proteins to influence
the effectiveness of cisplatin as a cytotoxic agent.
CONCLUSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES
/
cell lines, suggesting that in this mouse model,
HMGB1 does not modulate the cellular sensitivity to cisplatin.
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ACKNOWLEDGEMENTS |
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We thank Dr. M. E. Bianchi for
the gift of Hmgb1/
and Hmgb1+/+ cell lines
and Glenn Paradis at the MIT flow cytometry core facility for
assistance on the fluorescence-activated cell sorter experiments.
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FOOTNOTES |
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* This work was supported by Grant CA34992 from the National Cancer Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Anna Fuller Fund postdoctoral fellow.
§ To whom correspondence should be addressed. Tel.: 617-253-1892; Fax: 617-258-8150; E-mail: lippard@lippard.mit.edu.
Published, JBC Papers in Press, November 11, 2002, DOI 10.1074/jbc.M210562200
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ABBREVIATIONS |
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The abbreviations used are: cisplatin, cis-diamminedichloroplatinum(II); HMG, high mobility group; HMGB1, HMG box protein 1; SRB, sulforhodamine B; PI, propidium iodide; FITC, fluorescein isothiocyanate; annexin V-FITC, FITC-conjugated annexin V.
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