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Molecular Biology and Genetics |
Departments of Molecular Biology [G. N., M. N., H. T., T. Is., K. Ka., T. Im., H. I., K. Ko.] and Otolaryngology [K. M.], School of Medicine, University of Occupational and Environmental Health, Fukuoka 807-8555, Japan
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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15 kb of contiguous DNA, include
5 kb of the 5' flanking region as well as the entire coding sequence.
We sequenced 1500 bp upstream of the first exon. The segment proximal
to the transcription initiation site did not contain a TATA box but did
possess an activating transcription factor site, an activator protein-2
site, one CCAAT box, and two CCAAT-binding transcription
factor/nuclear factor-1 (CTF/NF-1) sites. HMG1 promoter activity was
3–10-fold higher in cisplatin-resistant KB-CP20 cells than in parental
KB cells. An in vivo footprint experiment showed several
differences of dimethyl sulfate modifications between KB and KB-CP20
cells in the area around the CTF/NF-1 sites. In addition,
electrophoretic gel mobility shift assays showed that binding of a
nuclear factor from cisplatin-resistant cells to the CTF/NF-1 site was
significantly higher than the binding of the same factor from parental
cells. Semiquantitative reverse transcription-PCR and Western blot
analysis also showed that expression of CTF/NF-1 was 3–20-fold higher
in the resistant cell line than in its parental counterpart. These
findings suggest that, in cisplatin-resistant cells, the expression of
HMG1 gene product is enhanced at the transcriptional level and that
this probably occurs through the enhanced expression of the CCAAT
binding factor, CTF/NF-1. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present work, we demonstrated that HMG1 is overexpressed in three cisplatin-resistant cell lines. To better understand the mechanism of HMG1 overexpression in cisplatin-resistant cells, we isolated and characterized the HMG1 gene promoter. We compared the activity of this promoter in cisplatin-resistant cells and their drug-sensitive counterparts. Finally, we identified the specific factor that regulates HMG1 gene expression.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-32P]dCTP and
[
-32P]ATP were obtained from Amersham
Pharmacia Biotech. Restriction enzymes, Taq polymerase, and
DNA-modifying enzymes were from Takara Shuzo (Kyoto, Japan). Synthetic
oligonucleotides were obtained from Hokkaido System Science Co. Ltd.
(Hokkaido, Japan). Sepasol for RNA extraction was from Nakarai Tesque,
Inc. (Kyoto, Japan).
Cell Culture.
All cell lines were cultured in either Eagle’s minimal essential
medium or RPMI 1640, both containing 10% fetal bovine serum, 0.292
mg/ml L-glutamine, 100 units/ml penicillin, and 100 µg/ml
kanamycin. KB/VJ300 and KB/VM-4 cells were obtained from human
epidermoid cancer KB cells by sequential selection with increasing
doses of vincristine or teniposide, respectively (19)
. KB
and KB-CP4 cells were kindly provided by Dr. Akiyama (Kagoshima,
Japan; Ref. 20
); A2780, A2780-E80, KB, and KB-CP20
cells were donated by Dr. Fojo (National Cancer Institute,
Bethesda, MD; Ref. 21
); and PC3 and PC5 cells were kindly
provided by Dr. Nakagawa (Ohita, Japan; Ref. 3
).
Cloning and Sequencing.
Starting with RNA from KB cells, human HMG1 cDNA was cloned by RT-PCR,
using the primers 5'-AACATGGGCAAAGGAGATCC-3' and
5'-TACCAGGCAAGGTTAGTGGC-3'. The resulting cDNA was subsequently
sequenced.
Genomic HMG1 DNA clones were isolated by screening a human placenta library in EMBL3 with the HMG1 cDNA probe. Positive phages were mapped with EcoRI and SalI. Several restriction fragments around the first exon were subcloned into pUC18 and sequenced. All plasmids were sequenced from both ends using an ABI 373 automated sequencer. Primer extension analysis was performed as described, using the primers, 5'-CCTCAGCGAGGCACAGAGTCG-3', derived from the sequence of the first exon of the HMG1 gene (22) .
Northern Blot Analysis.
Total RNA from parental and cisplatin-resistant cells was isolated
using Sepasol reagent. RNA samples (20 µg/lane) were separated on a
1% formaldehyde-agarose gel and transferred to a Hybond N+ filter
(Amersham Pharmacia Biotech) with 10x SSC. Prehybridization and
hybridization were performed as described previously (19)
.
Construction of Luciferase Reporter Plasmid.
The Pica gene vectors PGV-P2 and PGV-B2 were purchased from Nippongene
(Tokyo, Japan), and pCH110 (pSV-ß-gal) was obtained from Amersham
Pharmacia Biotech. Different deletions of the 5' regions of the HMG1
gene were ligated into the HindIII site of PGV-B2. Details
of constructions are available upon request. Plasmid DNAs were isolated
using Qiagen-tip columns (QIAGEN) according to the manufacturer’s
instructions.
DNA Transient Transfection and Luciferase Assay.
Using 8 µl of SuperFect (QIAGEN), we transfected DNA (2 µg of
plasmid) into KB and KB-CP20 cells when the cells were 60–80%
confluent. After incubation for 8 h at 37°C, the cells were
washed and incubated for another 36 h in culture medium. The cells
were lysed in 200 µl of 25 mM Tris phosphate (pH 7.5)
containing 1% Triton X-100 and centrifuged at 14,000 x g for 15 s. Luciferase activity in the resulting
supernatants was assayed using a Picagene kit (Toyoinki, Tokyo, Japan)
as described previously (23)
. Light intensity was measured
for 15 s with a luminometer (Dynatech ML 1500; JEOL,
Japan).
All cells were cotransfected with pSV-ß-gal as a control for transfection efficiency, and ß-galactosidase activity was measured using a GAL-XE kit (Aurora, Costa Mesa, CA).
In Vivo Footprint Analysis.
DNA was extracted from cells treated in vivo with DMS
(24)
, and incubated with 1 M
piperidine at 90°C for 30 min. As a control guanine ladder, genomic
DNA from KB cells was treated in vitro with DMS and
incubated with piperidine. Ligation-mediated PCR was performed
as described (25
, 26)
. The nucleotide sequences of the
HMG1 upper strand primers were as follows:
Primer 1: 5'-GGAGCCAGACGCAGCC-3'
Primer 2: 5'-GCTCTGTAACATTACTCTCCAGCCAGCG-3'
Primer 3: 5'-GCTCTGTAACATTACTCTCCAGCCAGCGCGGCTC-3'
The HMG1 lower strand primers were as follows:
Primer 1': 5'-CTCTGCTTTTCCCGTAGCAGC-3'
Primer 2': 5'-GCTTCTGCCTCTGCCTCTCCCG-3'
Primer 3': 5'-GCCTCTGCCTCTCCCGCCTCAGCC-3'
Primers 1 and 1' were used for first-strand synthesis, whereas primers
2 and 2' were used for PCR amplification. Primers 3 and 3' were labeled
at their 5' ends with [-32P]ATP and used for
detection of the ladder. Samples were analyzed on a 6% polyacrylamide
sequencing gel.
Preparation of Nuclear Extracts and EMSA.
Nuclear extracts were prepared as described (23)
. Briefly,
2 x 107 cells were collected with
PBS; resuspended in 2 ml of ice-cold 10 mM HEPES-KOH (pH
7.9), 10 mM KCl, 0.1 mM EDTA, 0.1
mM EGTA, 1 mM DTT, and 0.5 mM
phenylmethylsulfonyl fluoride; and incubated on ice for 15 min. The
cells were lysed with dropping 0.6% NP-40, and the lysate was
centrifuged at 400 x g for 10 min. The
resulting nuclear pellet was resuspended in 300 µl of ice-cold 20
mM HEPES-KOH (pH 7.9), 0.4
M NaCl, 1 mM EDTA, 1
mM EGTA, 1 mM DTT, 1
mM phenylmethylsulfonyl fluoride; incubated for
15 min on ice with frequent gentle mixing; and centrifuged for 5 min at
4°C in a microcentrifuge to remove insoluble material. The resulting
supernatant (nuclear extract) was stored at -70°C, and its protein
concentration was determined (27)
.
EMSAs were performed as described (13) . Briefly, 6 µg of nuclear extract protein was incubated for 30 min at room temperature in 20 µl of 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM MgCl2, 1 mM EDTA, 8% glycerol, 1 mM DTT, 0.1 µg of poly(dIdC), and 1 x 104 cpm of 32P-labeled oligonucleotide probe (1 ng) in the absence or presence of various nucleotide competitors (data not shown). The reaction mixtures were applied to a nondenaturing 4% polyacrylamide gel and subjected to electrophoresis at 7 W for 1.5 h in 44.5 mM Tris-borate, 1 mM EDTA. The gels were subsequently exposed to X-ray film with intensifying screens. The sequence of oligonucleotides used for EMSAs is followed.
Nc: 5'-GGCTTTGGATTGAAGCCAATATGAGG-3' and 3'-CGAAACCTAACTTCGGTTATACTCCG-5'
N1: 5'-CCGTGAGTTGGCGGTAGCCAATAGGAGC-3' and 3'-GCACTCAACCGCCATCGGTTATCCTCGG-5'
N2: 5'-GGACAATGGGAGGTATCGGGCCAATGAGC-3' and 3'-CTGTTACCCTCCATAGCCCGGTTACTCGC-5'
ATF: 5'-CAATCCTTGATGACGTGTCC-3' and 3'-TTAGGAACTACTGCACAGGG-5'
The anti-CTF1 antibody for supershift assay was kindly provided by Dr. Tanese, New York Medical Center (28) .
Western Blotting.
Nuclear extract containing 100 µg of protein was separated on 10%
SDS-PAGE. Proteins were transferred to polyvinylidene difluoride
membrane (Millipore) using a semidry blotter. Prestained protein marker
(New England Biolabs) was used as a molecular weight standard. The
membrane was immunoblotted with antiserum to CTF/NF-1 used in the super
shift assay.
RT-PCR.
Total RNA was isolated from KB and KB-CP20 cells using Sepasol reagent,
and first-strand cDNA was synthesized by reverse transcription
of 1 µg of RNA in 20 µl, using 5 µM random primers
and mouse mammary tumor virus reverse transcriptase (Life Technologies,
Inc.).
PCR amplification was performed in a 10-µl volume containing various amount of cDNA together with 10x PCR buffer, 250 µM each deoxynucleotide triphosphate, Ex Taq polymerase, and 1 µM each primer.
The human CTF/NF-1 primers were 5'-GCTCACGCACGAAGTAGGCCA-3' and 5'-GGCGAGGGAAGAAGACCTTTGCTATCCC-3'. The human ß2-microglobulin primers were 5'-ACCCCCACTGAAAAAGATGA-3' and 5'-ATCTTCAAACCTCCATGATG-3'.
Amplification was performed for a predetermined optimal number of cycles. PCR products were separated by electrophoresis on 2% agarose gels, which were stained with ethidium bromide. To quantify the amount of DNA, each gel was analyzed on a FLA2500 densitometer (Fuji, Japan).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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.
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Sequence Analysis of the Promoter Region.
A 4.7-kb HindIII fragment of EMBL-H1G2 was subcloned into
pUC18. This fragment, designated pH1-5, was mapped (data not shown).
The nucleotide sequences of the first exon and first intron as well as
that of the 5' flanking region of the gene were determined (Fig. 2)
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Up-Regulation of HMG1 Gene Expression in Cisplatin-resistant Cells.
To determine whether the region upstream of the putative
transcriptional start sites can activate transcription, available
restriction sites were used to construct a series of deletion plasmids
(Fig. 3)
. The basal transcriptional activity of the HMG1 gene promoter was
measured in KB cells transiently transfected with various luciferase
reporter plasmids. We observed maximal luciferase activity with
pH1Luc2. In addition, the promoter activity of pH1Luc3 was almost as
high as that of PGV-P2, indicating that the region between nt -273 and
-45 is essential for basal promoter activity (Fig. 3)
.
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In Vivo Footprint Analysis of HMG1 Promoter.
As shown above, the present sequence analysis revealed three CCAAT
boxes and AP-2 and ATF sites in the promoter region of the HMG1 gene,
all of which may be binding sites for transcription factors.
Protein-DNA interactions in the proximal promoter region may be
detected by in vivo DMS footprint experiments. When we
assayed DMS modifications of the HMG1 gene promoter in KB and KB-CP20
cells, several structural alterations were observed (Fig. 4)
. The most distal ATF binding site was clearly protected in both
parental and cisplatin-resistant cells (Fig. 4A)
. Protection
of the ATF site was also observed in the lower strand (Fig. 4B)
as well as concomitant hypersensitive signals (guanines
at nt -99, -98, and -97) in the vicinity of the ATF site. These
observations strongly suggest that this ATF binding site constitutively
binds ATF or related factor(s).
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The AP-2 site is located between two proximal CCAAT boxes (Fig. 4)
.
Among the three CCAAT boxes detected in the HMG1 promoter region, two
resembled the CTF/NF-1 binding site,
TGGA/C(N)5GCCAA. Moreover, a family of binding
factors has been shown to bind to this palindromic element. Although no
clear protection in the factor contact sites at either end of this
consensus sequence was observed, a significant number of hypersensitive
signals in the spacer region were detected, e.g., at
guanines -16 and -17 in the most proximal CTF/NF-1 site, and at
guanines -45, -46, and -51 in the second CTF/NF-1 site (Fig. 4A)
. Similarly, the adenines at -50 and -48 and the
guanine at -47 showed hypersensitive signals in the lower strand (Fig. 4B)
. Because hypersensitive sites in the vicinity of the
consensus sequence were routinely observed (factor contact
nucleotides), the presence of hypersensitive signals in the spacer
regions of the CTF/NF-1 sites suggests that these CTF/NF-1 sites may be
occupied by binding factors. Hypersensitive signals in the AP-2 site
situated between two CTF/NF-1 binding sites could also be interpreted
as evidence of the binding of factors to the CTF/NF-1 sites. With
respect to the distal CCAAT box, slightly hypersensitive nucleotides
were detected in both strands (guanines -88 and -81 in the upper
strand, and guanines -76 and -90 in the lower strand), suggesting
that this distal CCAAT box is occupied by a type of CCAAT binding
factor, at least in vivo.
Characterization of Nuclear Factor Binding to CCAAT Boxes.
To gain insight into the transcription factors that may regulate
HMG1 gene expression, EMSAs were performed using nuclear extracts
prepared from various drug-resistant cell lines. We designed four
oligonucleotides probes as described in "Materials and Methods."
The N1, N2, and ATF probes were based on the HMG1 promoter sequence,
whereas Nc contained the CTF/NF-1-binding consensus sequence. We found
that although the ATF probe formed multiple complexes, it formed equal
numbers of complexes with nuclear extracts from parental and
drug-resistant cells (data not shown). Probes N1, N2, and Nc each
formed a single smear complex, suggesting that the protein that binds
both N1 and N2 is CTF/NF-1. Alterations of the DNA-binding activities
toward Nc, N1, and N2 were not observed in cell lines resistant to
teniposide (KB-VM4) or vincristine (KB-VJ300; Fig. 5
, left). However, we observed enhanced DNA-binding activities
in all cisplatin-resistant cell lines compared with parental cells.
Cross-competition experiments showed that a 20-fold molar excess of
unlabeled N1 and N2 could disrupt interaction with either N1 or N2
(data not shown).
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. These findings suggest that the nuclear factors binding to probes N1
and N2 are identical.
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. This is consistent with the findings obtained from EMSAs.
Primers were then designed to quantify the steady-state levels of
CTF/NF-1 mRNA, and the levels of endogenous CTF/NF-1 mRNA expression
were assayed using RT-PCR. The level of CTF/NF-1 mRNA, normalized
relative to that of ß2-microglobulin mRNA, was
found to be 3-fold higher in KB-CP20 cells than in KB cells (Fig. 7B)
.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, we demonstrated that HMG1 gene expression is
up-regulated in all cisplatin-resistant cell lines. Identification of
the promoter and transcription factors involved is essential for
understanding the molecular mechanism of HMG1 up-regulation by
cisplatin. In the present study, the isolation and characterization of the human HMG1 gene were demonstrated. Although the genomic cloning of HMG1 was reported previously (29) , the structure of the promoter was not shown. The overlapping clones, which cover the entire HMG1 gene, also contain the 5' flanking region of this gene (data not shown).
The 5' upstream sequence of the HMG1 gene was determined (Fig. 2)
. HMG1
gene was found to have three CCAAT boxes, but the sequences of these
CCAAT boxes were different.
Transient transfection of reporter constructs showed that the promoter activity of the HMG1 gene is 3–10-fold higher in cisplatin-resistant cells than in KB cells. This indicates that HMG1 gene expression is transcriptionally up-regulated in resistant cells. The present study showed that the proximal region, from nucleotide -273 to nucleotide -45 upstream from the transcription initiation site, functions as a core promoter in KB cells. This finding also suggests that the most proximal CCAAT box is necessary not only for the manifestation of core promoter activity, but also for up-regulation of expression in cisplatin resistant cells.
The three CCAAT boxes in the KB parental cell line showed slight
protection from DMS methylation. In contrast, the KB-CP20
cisplatin-resistant cells exhibited much less protection. However,
hypersensitive sites near these CCAAT boxes, at nt -99 to -96, -31
to -28, and -2 to +4, were constitutively observed, and the extent of
the hypersensitivity was higher in cisplatin-resistant cells than in
parental cells. Moreover, concomitant protection at nt -104 was also
constitutively observed within the ATF binding site. Taking the EMSA
results and the overexpression of CTF/NF-1 (Figs. 6
and 7)
into
account, the findings suggest that these CCAAT boxes could be key
elements in up-regulation of the HMG1 gene, whereas the surrounding
region (nt -104 to the transcription initiation site) may act as part
of the highly ordered regulatory complex (30)
.
It is important to characterize the nuclear factors that might mediate
the transcriptional activation of the HMG1 gene in cisplatin-resistant
cells. In the present EMSA experiments, the sequences of the two
proximal CCAAT boxes were similar to the consensus sequence for
CTF/NF-1, but that of the distal CCAAT box was not (28
, 31)
. The binding activity of two oligonucleotides (N1 and N2),
was 3–8-fold higher when nuclear extracts from three
cisplatin-resistant cells were tested than when extracts from parental
cells were assayed. In contrast, the binding activity of a third
oligonucleotide (ATF) to nuclear factors from KB-CP20 and KB cells were
similar (data not shown). We also found that this enhanced CTF/NF-1
site binding activity is specific for cisplatin-resistant cells because
the binding activity of nuclear extracts from vincristine- or
teniposide-resistant cells were similar to the activities of extracts
from parental cells (Fig. 5)
. A CTF/NF-1 dimer has been found to bind
to viral and cellular promoters recognizing
TGGA/C(N)5GCCAA sequences, primarily through the
two half-palindromes (32
, 33)
.
The two proximal CCAAT boxes in the HMG1 promoter are not completely
identical to those of the CTF/NF-1 consensus binding sites. The
canonical spacer length of the CTF/NF-1 consensus sequence has been
found to be 5–6 nt, and this spacer length has been reported to be an
important determinant of binding affinity (28
, 34
, 35) . We
found, however, that the spacer lengths of the first and second
CTF/NF-1 sites are 4 and 8 nt, respectively. Furthermore, these two
CTF/NF-1 binding sites (N1 and N2) were shown to have a lower binding
potential than the consensus CTF/NF-1 oligonucleotides (Nc). These
divergences of spacer length might therefore account for the weak
protection within factor contact sites. In addition, expression of the
CTF/NF-1 protein was significantly increased 3–20-fold in
cisplatin-resistant cells (Fig. 7A)
. Although expression of
CTF/NF-1 mRNA was not detected by Northern blotting, CTF/NF-1 mRNA was
3-fold higher in cisplatin-resistant KB-CP20 cells than in parental KB
cells by semiquantitative RT-PCR (Fig. 7B)
. Using the
CTF/NF-1 primers, we observed two RT-PCR bands of 857 and 785 bp. Human
CTF/NF-1 has been shown to encode multiple mRNA species for the
alternative coding regions CTF1, CTF2, and CTF3 (36
, 37)
,
and differences in both forms and the amount of CTF/NF-1 binding
activity among cell lines have been observed (38)
.
This suggests that the present 857 and 785-bp products were derived
from CTF2 and CTF3 mRNA, respectively. Although the intensities of the
two bands are similar in KB cells, the intensity of the upper band is
increased in cisplatin-resistant cells, suggesting that CTF2 is the
major CCAAT-binding species in the latter. We were unable, however, to
detect the 1010-bp PCR product, which is derived from CTF1 mRNA, in
either KB or KB-CP20 cells. Furthermore, it is of interest to examine
whether other CTF/NF-1 target genes are overexpressed in
cisplatin-resistant cells. Cisplatin-resistant cells were routinely
maintained in medium containing cisplatin. Regulation of the forms of
CTF/NF-1 has been shown to be sensitive to changes in cellular
environment (38)
. However, CTF/NF-1 activity, CTF
expression, and HMG1 expression were not induced by acute exposure of
cells to cisplatin (data not shown). These findings suggest that CTF2
overexpression in cisplatin-resistant cells may be attributable to
chronic exposure of the cells to cisplatin.
Chromatin remodeling, transcription factor binding, and transcription were inhibited by brief exposure of cells to cisplatin (39) . Chronic exposure of cells to cisplatin may affect gene expression through chromatin remodeling activity and the cellular levels of transcription factors. We previously have shown that KB-CP4 cells overexpressed a transcription factor, YB-1, with the extent of YB-1 expression correlating with cellular sensitivity to the cytotoxic effect of cisplatin (40) .
Interestingly, it has been shown that CTF/NF-1 can serve as an initiator of DNA replication (36) , suggesting that overexpression of CTF/NF-1 may facilitate DNA replication and repair and may play a role in both gene expression and maintenance of genomic stability (41) . Several different mechanisms for resistance to cisplatin have been demonstrated, including increased activity of DNA repair, increased levels of intracellular thiols, and reduced accumulation of cisplatin. In the same cisplatin-resistant cells used in this study, accumulation of cisplatin has been found to be decreased (4) . Alterations of certain proteins have been demonstrated in KB-CP20 cells (20) . Considering these findings together with our present results, cisplatin resistance appears to result from many different mechanisms. To clarify whether our observation is relevant to cisplatin resistance, isolation of stable transfectants that show overexpression of either the CTF/NF-1 or the HMG1 gene is under way in our laboratory.
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FOOTNOTES |
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1 This work was supported in part by a
grant-in-aid for Cancer Research from the Ministry of Education,
Science, Sports and Culture of Japan, by a research grant from the
Princess Takamatsu Cancer Research Fund, and by the Fukuoka Anticancer
Research Fund. 
2 To whom requests for reprints should be
addressed, at Department of Molecular Biology, Faculty of Medicine,
University of Occupational and Environmental Health, Japan,
Yahatanishi-ku Kitakyushu, Fukuoka 80-8555, Japan. Phone:
81-93-691-7423; Fax: 81-93-692-2766; E-mail: k-kohno{at}med.uoeh-u.ac.jp
3 The abbreviations used are: HMG, high-mobility
group; RT-PCR, reverse transcription-PCR; DMS, dimethyl sulfate; EMSA,
electrophoretic mobility shift assay; CTF/NF-1, CCAAT-binding
transcription factor/nuclear factor 1; ATF, activating transcription
factor; AP, activator protein; nt, nucleotide.
Received 2/14/00. Accepted 12/13/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gene by heat shock.. J. Biol. Chem., 273: 10550-10555, 1998. 1 subunit gene by in vivo footprinting.. Biochim. Biophys. Acta, 1264: 35-39, 1995.[Medline]
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