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Advances in Brief |
Laboratory of Cell Regulation and Carcinogenesis [B. I. L., S. H. P., H. T. K., S-J. K.], Developmental Therapeutics Program [E. A. S.], and the Medicine Branch [J. W. K., J. B. T.], National Cancer Institute, Bethesda, Maryland 20892, and Mitsui Pharmaceuticals, Chiba 297-0017, Japan [O. N.]
| ABSTRACT |
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| Introduction |
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-amino group of lysine side chains of histone H2A, H2B, H3, and
H4 and thereby reconstitute the positive charge on lysine. Inhibitors
of HDACs have been shown to induce differentiation and/or apoptosis of
transformed cells, suggesting that alterations in chromatin structure
may also be important in the process of neoplasia (1
, 2)
. HDAC inhibitors such as sodium butyrate, suberoylanilide hydroxamic acid, and trichostatin A appear to arrest human tumor cells at G1 and G2-M phase and induce expression of the cell cycle kinase inhibitor p21WAF1 (4, 5, 6, 7, 8) . Increased expression of p21WAF1 may be one of the critical factors in the growth arrest induced in transformed cells by these agents.
TGF-ß, the prototypic multifunctional cytokine, participates in the regulation of vital cellular activities such as proliferation and differentiation (9 , 10) . Another essential function of TGF-ß is its tumor suppressor activity in a variety of different human cell types. Human cancer cells frequently demonstrate resistance to the normal growth-inhibitory effects of TGF-ß, and it has been proposed that the development of such TGF-ß resistance represents a significant step during carcinogenesis (11, 12, 13) . Many human cancer cell lines including breast and prostate cancer cell lines have TGF-ß resistance without detectable alterations in the TßRII gene (13) . These cells express low or undetectable levels of TßRI and TßRII genes, suggesting that abnormalities of transcriptional regulation, altered mRNA processing, or decreased mRNA stability might be involved.
In this study, we have examined the effect of a synthetic inhibitor of HDAC, MS-275, on the growth of human breast cancer cells and expression of TßRII (14) . We found that MS-275 induces the accumulation of acetylated histones in the chromatin of the TßRII gene and that this increase is associated with an increase in TßRII expression in human breast cancer cell lines. In addition, MS-275 treatment enhanced TGF-ß1-induced PAI-1 expression. These findings indicate that the growth inhibition of human breast cancer cells by MS-275 may be due, at least in part, to the restoration of TGF-ß signaling by inducing TßRII.
| Materials and Methods |
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Northern Blot Analysis.
Total RNA was isolated using Trizol (Life Technologies, Inc.,
Rockville, MD) according to the manufacturers instructions,
transferred onto Zeta-Probe blotting membrane (Bio-Rad Laboratories,
Hercules, CA), and hybridized with 32P-labeled
cDNA for human TßRII, TßRI, PAI-1, or ß-actin.
Acid-soluble Nuclear Protein Preparation and Western Blot
Analysis.
Acid-soluble proteins were prepared as described previously (8
, 15)
. Cells (5 x 106) were
cultured with and without MS-275 (0.5 µM). Protein
concentrations of histone preparations were determined using the
Bio-Rad Protein Assay Kit with BSA as the standard. Proteins (20 µg
protein) were denatured and electrophoresed in 15% polyacrylamide gels
for histones. After electrophoresis, samples were transferred onto
nitrocellulose. To verify equal protein loading, a parallel protein gel
was run and stained with Coomassie Blue. The blots were probed with
rabbit antiacetylated histone H3 and rabbit antiacetylated histone H4
(Upstate Biotechnology, Lake Placid, NY) and detected by using the
enhanced chemiluminescence method (Pierce).
ChIP Assay.
Cells were plated at a density of 5 x 106 cells/15-cm dish and incubated overnight. The
next day, cells were cultured with 0 or 1 µM MS-275 for
24 h. ChIP assay was performed using the method described by
Richon et al. (8)
. TßRI- and TßRII-specific
primers were used to carry out PCR from DNA isolated from ChIP
experiments and Input samples. The optimal reaction conditions for PCR
were determined for each primer pair. Parameters were denaturation at
95°C for 1 min and annealing at 58°C for 1 min, followed by
elongation at 72°C for 1 min. PCR products were analyzed by 2.5%
agarose/ethidium bromide gel electrophoresis. The primers of the TßRI
promoter (16)
for ChIP analysis were
5'-GTGGGGCGTGGCCAGAAAC-3' (-26/-44; uP1) and
5'-GCCCTTTGTAACTGCTCGGAGGAC-3' (+102/+125; dP2). The primers of the
TßRII promoter (17)
were 5'-CTGGTCTAGGAAACATGATTGG-3'
(-77/-98; uP1) and 5'-CGAGTGACTCACTCAACTTCAACTCAG-3' (+54/+33; dP2).
| Results |
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MS-275 Treatment Enhances TGF-ß Signaling.
Because an inhibitor of HDACs has profound effects on cell growth, we
could not examine the effect of the enhanced expression of
TßRII gene by the treatment of MS-275 on the
autocrine and paracrine growth-regulatory activities of TGF-ß in
these cells. Therefore, to determine whether reduced expression of
TßRII gene by histone deacetylation was
responsible for blocking TGF-ß signaling in human breast cancer
cells, we examined the expression of the PAI-1 in ZR-75 and HS-578t
cells cultured with MS-275 with or without TGF-ß1. MS-275 treatment
enhanced expression of PAI-1 mRNA (Fig. 5)
. However, treatment with both MS-275 and TGF-ß1 markedly enhanced
expression of PAI-1 mRNA (Fig. 5)
. These results suggest that treatment
with MS-275 enhances TGF-ß1 signaling through the induction of
TßRII gene expression.
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| Discussion |
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Gene expression can be suppressed by either a reduced expression of
positive transcription factors or an increased expression of
transcriptional suppressors. We have shown that the expression of
TßRII is regulated by the ets family of transcription
factors and that loss of expression of ERT/ESE-1/ESX/jen/Lef-3, a
member of the ets transcription factor family, leads to the
loss of TßRII expression (21, 22, 23)
. In this study, we
have shown that MS-275, an inhibitor of HDACs, induces the
transcription of TßRII and an accumulation of acetylated histones
associated with the promoter of that gene. The accumulation of
acetylated histones by MS-275 appears to be specific to TßRII (Fig. 4)
. The TßRI gene is not transcriptionally
activated, and there is no change in the level of acetylated histone in
chromatin associated with this gene in response to MS-275 (Fig. 4)
. We
have recently shown that TßRI expression is transcriptionally
repressed by DNA methylation and that the treatment with a
demethylating agent, 5-aza-2'-deoxycytidine, resulted in an increased
expression of the TßRI gene, but not the
TßRII gene (24)
. These results
suggest that the expression of TGF-ß receptors is actively
inactivated by two distinct epigenetic mechanisms, DNA methylation and
histone deacetylation.
HDAC inhibitors such as MS-275, sodium butyrate, and trichostatin induce the expression of p21WAF1 in a p53-independent manner (4, 5, 6, 7, 8) . The promoter region of the p21WAF1 gene responsive to sodium butyrate is required for the activation of the p21WAF1 promoter by TGF-ß (25 , 26) . However, Sp1, but not Sp3, mediates the p21WAF1 promoter activation by TGF-ß, whereas Sp3, but not Sp1, mediates the transcriptional activation of the p21WAF1 promoter by trichostatin A (7) . These findings suggest that HDAC inhibitors may induce the expression of the p21WAF1 gene through both TGF-ß-dependent and -independent mechanisms. The TGF-ß-independent pathway involves Sp3, whereas the TGF-ß-dependent pathway is mediated through the TGF-ß-responsive element in the p21WAF1 promoter by inducing TGF-ß signaling through the induction of TßRII expression.
Several HDAC inhibitors, including oxamflatin (27) , MS-275 (14) , and the hydroxamic acid-based hybrid polar compounds (4 , 5) , inhibit tumor growth in animal models. However, the detailed mechanisms of inhibition of tumor growth are not known. Our present study suggests that the restoration of TGF-ß signaling through the induction of TßRII is a major target of HDAC inhibitors in suppressing tumor growth.
| ACKNOWLEDGMENTS |
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| FOOTNOTES |
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1 These two authors equally contributed to this
work. ![]()
2 To whom requests for reprints should be
addressed, at Laboratory of Cell Regulation and Carcinogenesis,
National Cancer Institute, Bethesda, MD 20892-5055. Phone:
(301) 496-8350; Fax: (301) 496-8395; E-mail: kims{at}dce41.nci.nih.gov ![]()
3 The abbreviations used are: HDAC, histone
deacetylase; TGF, transforming growth factor; TßRII, TGF-ß type II
receptor; TßRI, TGF-ß type I receptor; PAI, plasminogen activator
inhibitor; ChIP, chromatin immunoprecipitation. ![]()
Received 11/14/00. Accepted 12/ 4/00.
| REFERENCES |
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