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in Human Breast Cancer Cells by Histone Deacetylase Inhibition1
The Johns Hopkins Oncology Center, Johns Hopkins University, Baltimore, Maryland 21231 [X. Y., A. T. F., S. J. N., D. L. P., K. A. B., J. G. H., N. E. D.], and Section of Hematology/Oncology, University of Chicago Medical Center, Chicago, Illinois 60637 [S. M. W.]
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ABSTRACT |
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 Top ABSTRACT IntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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(ER) gene expression has been associated with
aberrant methylation of its CpG island in a significant fraction of
breast cancers, we tested whether histone deacetylase activity
contributes to the transcriptional inactivation of the methylated
ER gene in a panel of ER-negative human breast cancer
cells. Treatment of these cells with trichostatin A, a specific histone
deacetylase inhibitor, led to dose- and time-dependent re-expression of
ER mRNA as detected by reverse transcription-PCR without alteration in
ER CpG island methylation. Trichostatin A-induced ER
re-expression was associated with increased sensitivity to DNase I at
the ER locus in MDA-MB-231 cells. These data implicate
inactive chromatin mediated by histone deacetylation as a critical
component of ER gene silencing in human breast cancer
cells. Therefore, histone deacetylation may be a potential target for
therapeutic intervention in the treatment of a subset of ER-negative
breast cancers. |
Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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3
plays an
important role in breast carcinogenesis and breast cancer treatment.
That estrogen stimulates the growth of certain breast cancers is well
established, and hormonal therapy via estrogen depletion or
antiestrogen administration is widely used to block the action of
estrogen in women with breast cancer. However, patients whose breast
cancers lack ER seldom respond to endocrine therapy; therefore, a
potential mechanism for hormone resistance is de novo or
acquired loss of ER gene expression at the transcriptional
level during disease progression (1
, 2)
.
One possible mechanism for loss of ER in ER-negative breast cancers is
cytosine methylation of the ER CpG island in the 5'
regulatory region of the gene (3)
. Indeed the
ER gene CpG island is extensively methylated in ER-negative
breast cancer cells, and 
50% of unselected primary breast tumors
but remains unmethylated in normal breast tissue and many ER-positive
tumors and ER-positive cancer cell lines (4
, 5)
. The
functional importance of this finding is demonstrated by the fact that
treatment of ER-negative human breast cancer cells with the
demethylating agent, 5-aza-dC, led to reactivation of expression of ER
mRNA and functional ER protein (6)
. Recent studies
indicate that silencing of a gene by methylation involves the
generation of an inactive chromatin structure characterized by
deacetylated histones. An abundant chromosomal methyl CpG-binding
protein, MeCP2, was the first protein identified to link methylated DNA
and a HDAC-containing transcriptionally repressive complex for gene
silencing. Subsequently, several MBD proteins have been identified
that, similar to MeCP2, couple methylated DNA to HDAC (7
, 8)
. More recently, the well-known maintenance methyltransferase,
Dnmt1, was found to interact physically with HDAC through its N
terminus, thereby forming a transcriptionally inactive chromatin
structure that represses transcription (9
, 10)
. All of
these findings demonstrate the important role of HDAC in
transcriptional regulation. The HDACs deacetylate lysine groups of
histones H3 and H4, allowing ionic interactions between positively
charged lysines and negatively charged DNA, which result in a more
compact nucleosome structure that limits transcription. The
availability of specific HDAC inhibitors such as TSA (11)
permits the study of the role of HDAC in silencing a variety of
tissue-specific methylated genes (7
, 12)
.
Here, we have tested whether loss of ER expression in some breast cancers is associated with transcriptional repression through HDAC activity on the methylated ER gene. Our data demonstrate that specific HDAC inhibition via TSA treatment can reactivate ER transcription in the presence of the methylated DNA. The activated gene transcription is associated with increased sensitivity of the ER promoter to DNase I treatment. These data suggest that inactive chromatin mediated by HDAC is critical to ER gene silencing.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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RNA Isolation and RT-PCR Analysis of ER Expression.
Total cellular RNA was isolated from cell lines with TRIzol reagent
according to the recommendations of the supplier (Life Technologies,
Inc., Rockville, MD). RNA (3 µg) was reversibly transcribed by
Moloney murine leukemia virus reverse transcriptase (Life Technologies)
using OligoDT15 primer (Promega Corp., Madison,
WI) in a final volume of 50 µl. Four % of synthesized cDNA (2 µl,
derived from 150 ng of initial RNA) was used for PCR amplification of
ER and the constitutively expressed housekeeping gene
ß-actin (13)
. Specific sense and
antisense PCR primers used for the amplifications across the seventh
intron of ER and the first intron of ß-actin
genes, yielding 470 and 400 bp of PCR products respectively, were
described previously (6)
. PCR products were resolved by
2% agarose gel electrophoresis and visualized by ethidium bromide
staining.
Quantitative Competitive PCR Analysis of ER Reactivation.
A quantitative assay was performed to determine the level of ER mRNA in
TSA- and vehicle-treated MDA-MB-231 cells as compared with the
expression levels in the known ER-positive MCF-7 and T-47D cells using
the method of Wang and Rowley (14)
. This assay involves
coamplification of a wild-type target cDNA (wER) of unknown amount and
a competitive template (cER) in known amounts. A truncated competitive
template was generated with a sense primer containing a 22-bp deletion
(primer 2) and the same antisense primer (primer 3) as the wild-type
(Fig. 2A)
. After amplification, the competitive template was
separated, gel purified, and quantified by Spectrophotometer DU 640
(Beckman, CA). Thus, target wild-type and known amounts of
competitive templates can be coamplified with the pair of wild-type
primers (primers 1 and 3) and differentiated by size. Because the most
accurate results are obtained when wild-type and competitive templates
are amplified at nearly equivalent concentrations, resulting in the
signal ratio of wER:cER equivalent to 1, we first performed an initial
titration in log and then in 2-fold dilutions to determine the
approximate concentration of the wild-type ER cDNA in our experimental
samples. RNAs under comparison were simultaneously reversibly
transcribed to achieve equal efficiency for reverse transcription. The
PCR reactions were carried out with 0.5 µM of
wild-type sense and antisense primers for 35 cycles. The wild-type and
competitive PCR products were fractionated on 2.5% agarose gel,
stained with ethidium bromide, and scanned by Densitometer (EagleSight
Software of Eagle Eye II Imaging System; Stratagene, La Jolla, CA). The
ratio between wild-type and control templates was determined and used
to calculate the amount of target wild-type cDNA because the input of
competitive template is known.
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DNase I Sensitivity Assay.
This assay was performed according to the method of Keshet et
al. (16)
using cells from TSA- or vehicle-treated
MDA-MB-231 and MCF 7 cells (1 x 107 cells/each) with the following modifications.
The isolated DNA was digested with EcoRI, the recognition
sites of which flank the ER promoter region to yield a 3.1-kb fragment
that was separated by 1% agarose gel electrophoresis. DNAs blotted on
nylon membrane were probed with a PCR-amplified, 561-bp DNA fragment
corresponding to the ER CpG island (Fig. 4A)
. The
sense and antisense oligonucleotides used to amplify the fragment are
5'-AGACCAGTACTTAAAGTTGGAGGCC-3' and 5'-GGGAAACCCCCCAGG-3'. The
amplified DNA was cloned into pCR2.1-TOPO vector (Invitrogen,
CA) according to the manufacturer’s protocol. Colonies containing
amplified DNA sequence, determined by Mini-Prep (Promega Wizard
Mini-Prep kits), were grown and purified. The purified plasmids were
sequenced via automated sequencing (Johns Hopkins Sequencing Core
Facility). The specific ER CpG probe was prepared from
sequence-confirmed plasmid and labeled with bio-16-dUTP (Boehringer
Mannheim, IN) by PCR using the above-mentioned primer set. The
3.1-kb DNA band containing the ER promoter region was
visualized by chemiluminescence using a streptavidin-conjugated,
alkaline phosphatase-catalyzed substrate, CDP-star. The band signals
that reflect the resistance to DNase I were quantified by densitometry
(Stratagene), and the cumulative DNase I sensitivity was calculated as
follows. The band density at each DNase I concentration was divided by
the density of the control band and then multiplied by 100%. This
value was subtracted from 100% to yield the percentage of DNase I
sensitivity. The sum of the percentage of DNase I sensitivity at each
dose was considered to be the cumulative DNase I sensitivity.
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Results and Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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. A detectable level of ER
mRNA, as demonstrated by a RT-PCR product with predicted 470-bp size,
was noted after treatment with 50 ng/ml (160 nM)
TSA for 48 h, and ER transcript was clearly present after 100
ng/ml (330 nM) TSA for the same duration. A time
course analysis showed that a weak signal could be seen after 24 h
of 100 ng/ml TSA treatment, whereas ER transcript was readily observed
after 48 or 72 h (Fig. 2B)
. Multiple experiments were
done to achieve optimal conditions for TSA induction of ER mRNA in
MDA-MB-231 cells. These showed that maximal ER reactivation was
achieved with 100 ng/ml TSA for 48 h using an initial seeding
density of 8 x 103
cells/cm2
. Higher inoculating cell densities
reduced ER transcript signal (data not shown).
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C, TSA treatment led to re-expression of ER mRNA in all
three ER-negative cell lines tested. Optimal ER gene re-expression was
observed after treatment of MDA-MB-231 cells with 100 ng/ml (330
nM), Hs578t cells with 400 ng/ml (1.32
µM), and MCF-7/AdrR with
25 ng/ml (82.5 nM) TSA for 48 h. Therefore,
TSA treatment consistently induced ER re-expression in the panel of
ER-negative cell lines, supporting a role for HDAC in ER
gene silencing.
A quantitative competitive PCR assay was used to assess the magnitude
of TSA-induced ER mRNA transcript in MDA-MB-231 cells. Fig. 2A
shows the design of the primer sets used, and validation
of the quantitative competitive PCR is shown in Fig. 2B
. As
shown in Fig. 2
C, a 5-fold increase in ER transcript was
obtained after TSA exposure in MDA-MB-231 cells (100 ng/ml for 48 h). This effect was specific for the methylated ER promoter because TSA
treatment (50 or 100 ng/ml for 48 h) of ER-positive, unmethylated
MCF-7 cells had no effect on the level of ER mRNA expression using the
same quantitative assay (data not shown). However, TSA treatment of
MDA-MB-231 cells did not restore ER mRNA expression to the levels seen
in cell lines with endogenous ER expression, as shown in Fig. 2C
. Quantitative assay suggested that the level of ER
transcript seen with TSA treatment of MDA-MB-231 cells represented
about 1 and 10% of that seen in the ER-positive MCF-7 and T-47D cell
lines, respectively. Several possibilities might account for this:
(a) It is possible that only partial reactivation is seen because only a fraction of cells responded to the treatment. Indeed, a similar pattern of partial reactivation was seen in MDA-MB-231 cells exposed to a demethylating agent, 5-aza-dC, in our previous study (6) .
(b) It has been shown that a component of the repression mediated by MeCP2 transcriptional repression domain is partially HDAC independent; mSin3A could retain some ability to repress transcription, even in the absence of associated HDACs (7) .
(c) Simultaneous inhibition of several components in the methylation-associated repressive complexes might be necessary to achieve maximal reactivation of the repressed genes (12) .
The ability of TSA to reactivate ER expression raised the question of
whether the ER CpG island remained methylated. Our previous
studies with 5-aza-dC demonstrated that ER re-expression was associated
with demethylation of the ER CpG island. However, a parallel
study of the progesterone receptor in progesterone receptor-negative
human breast cancer cells showed that the ligand-bound ER could
overcome methylation-related repression of the progesterone receptor,
even in the continuing presence of a methylated progesterone receptor
CpG island (17)
. We therefore examined the methylation
status of the ER CpG island in TSA-treated MDA-MB-231 cells
using a sensitive MSP assay that allowed examination of methylation
status across the ER CpG island (Fig. 3A)
. As shown in Fig. 3
B, the ER CpG island was completely methylated
across the entire CpG island in MDA-MB-231 cells treated with vehicle
or TSA (100 ng/ml for 48 h). A single primer set, ER 5, was used
to confirm this finding in the other TSA-treated, ER-negative human
breast cancer cells, Hs578t and MCF-7/AdrR (Fig. 3C)
. As expected, the unmethylated ER-positive, MCF-7 cells
demonstrated an unmethylated pattern using all four primer sets that
span the ER CpG island. In summary, TSA treatment of
ER-negative breast cancer cells can lead to re-expression of ER mRNA
without an apparent alteration in the methylation status of the
ER CpG island.
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. TSA treatment of
MDA-MB-231 cells resulted in an 2-fold increase in DNase I
sensitivity (Fig. 4C)
, suggesting that inhibition of HDAC
activity leads to a more open chromatin conformation, even in the
presence of CpG island methylation. Recently, the interaction between DNA methylation and histone deacetylation linked by methyl-binding proteins (MBDs), or the direct interaction of Dnmt1 with HDAC as well as other corepressors, has been an area of active study. More recently, a nucleosome-stimulated ATPase Mi2, a part of chromatin remodeling machinery, was also shown to bind the methylated DNA through MBD3 and deacetylase in Xenopus laevis and mammalian cells, further illustrating the role of HDAC on gene transcription regulation (8) . In addition to evaluating the role of HDAC in in vitro studies, it is of importance to study its role in silencing endogenous methylated genes. In some cases, HDAC inhibition alone seems to be sufficient to reactivate a methylated gene. For example, both sodium butyrate and TSA can restore transcription from methylated and silenced plant rRNA genes (20) . Also, reactivation of transcription of the methylated FMR1 gene was achieved by treatment with 4-phenylbutyrate, sodium butyrate or TSA (21) . However, in other cases, both demethylation and HDAC inhibition appear to be necessary. For example, certain hypermethylated genes like MLH1, TIMP3, CDKN2B, and CDKN2A can be transcriptionally activated in colon cancer cells by TSA only after Dnmt1 inhibition by 5-aza-dC, suggesting an important role of Dnmt1 in transcription repression although recruiting HDAC is essential (12) .
ER is a critical growth-regulatory gene in breast cancer, and its expression status is tightly linked to the prognosis and treatment outcome of breast cancer patients. Thus, it is important to understand its regulation. Our work suggests that histone deacetylation and DNA methylation may both play a role in ER transcription, and further studies will focus on the effects of TSA on ER protein expression. This is critical because it is possible that activation of the silenced ER by HDAC inhibition could open a new avenue for management of a subset of advanced breast cancer with hormonal resistance. Studies using primary breast cancers have shown that the antiestrogen, tamoxifen, confers a benefit to women whose breast cancer expresses ER by immunohistochemistry in as few as 1–10% of tumor cells. Thus, even partial re-expression of ER could be of clinical benefit (22) .
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FOOTNOTES |
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1 This work was supported by Grants CA78352 (to
N. E. D.) and 2-T32CA09110 (to X. Y. and S. J. N.) from the NIH,
Contracts DAMD17-00-1-0301 (to X. Y.) and DAMD17-98-1-8116 (to
S. J. N.) from the Department of Defense Breast Cancer Program, and
Grant PF4231 (to A. T. F.) from the American Cancer Society. 
2 To whom requests for reprints should be
addressed, at Johns Hopkins University School of Medicine, 1650 Orleans
Street, CRB Room 409, Baltimore, MD 21231. E-mail: davidna{at}jhmi.edu
3 The abbreviations used are: ER, estrogen
receptor ; 5-aza-dC, 5-aza-2'-deoxycytidine; MBD, methyl-CpG-binding
domain; Dnmt, DNA methyltransferase; HDAC, histone deacetylase; MeCP2,
methyl-CpG binding protein 2; MSP, methylation-specific PCR; TSA,
trichostatin A; RT-PCR, reverse transcription-PCR.
Received 7/10/00. Accepted 10/31/00.
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,
231C, cumulative DNase I sensitivity of 75) MDA-MB-231 cells (see
"Materials and Methods").
