Biochemical and Biophysical Research Communications
TSA-induced DNMT1 down-regulation represses hTERT expression via recruiting CTCF into demethylated core promoter region of hTERT in HCT116
Introduction
Modulation of chromatin structure by histone acetylation/deacetylation is well known to be one of the major mechanisms involved in the regulation of gene expression [1]. Two opposing enzyme activities determine the acetylation state of histones: histone acetyltransferase (HATs) and histone deacetylase (HDACs), which acetylate and deacetylate the epsilon-amino groups of lysine residues located in the amino-terminal tails of the histones, respectively [2], [3]. A number of structurally divergent classes of HDAC inhibitors have been identified [4]. They have been shown to induce cell-cycle arrest, terminal differentiation, and apoptosis in various cancer cell lines as well as inhibit tumor growth [5]. In particular, the reversible HDAC inhibitor trichostatin A (TSA) can effectively and selectively induce tumor growth arrest at very low concentrations [6], [7]. Therefore, an understanding of the events in TSA-induced apoptosis may be valuable for improving the efficacy of cancer therapy.
Recently, it was reported that the human telomerase reverse transcriptase (hTERT) gene that encodes the catalytic subunit of telomerase holoenzyme may be a primary target of TSA for induction of apoptosis in various cancer cells [8], [9], [10]. hTERT is activated in a cancer cell-specific manner and is well-known as an important target for the diagnosis of malignancy and a potential candidate for the development of cancer therapy [11]. Numerous researchers have been interested in studying the role of hTERT in TSA-induced apoptosis [10], [12], [13]; however, these reports have only focused on the down-stream mechanisms of hTERT expression in TSA-induced apoptosis rather than how the expression of hTERT can be regulated by TSA. In addition, it has been reported that TSA acts differently in cancer cells compared with normal cells. Specifically, TSA induces transcriptional activation of hTERT expression in normal cells, but significantly represses the expression of hTERT in cancer cells [8], [9]. In particular, inhibition of HDACs by TSA generally increases histone acetylation on the promoter of their target genes, which consequently results in gene activation. Nevertheless, histone acetylation induced by TSA in cancer cells does not induce increased hTERT expression, suggesting that there may be another epigenetic regulation mechanism controlling hTERT expression by TSA in a cancer cell-specific manner.
Recently, it has been reported that treatment with TSA is associated with a significant decrease in global DNA methylation [14], suggesting that DNA methylation of hTERT may be a new target of epigenetic regulation by TSA. The majority of DNA methylation results in the modification of cytosine at CpG sites, and this phenomenon is associated with repression of gene expression [15]. However, in the case of hTERT, increased DNA methylation of the hTERT promoter has been observed in hTERT-positive cancer cells, while lack of methylation has been found in normal hTERT negative cells [16]. This correlation is opposite of the general model of regulation by DNA methylation, in which the presence of methylated cytosine in a promoter typically inhibits gene transcription. Although DNA methylation of the hTERT promoter is an important factor for hTERT expression and hTERT is a strong candidate as a target gene of TSA, the correlation between TSA and DNA methylation of hTERT promoter has not yet been elucidated.
In the present study, we evaluated the changes in hTERT expression in the apoptosis process induced by TSA and explored DNA methylation of the hTERT promoter as one of the possible mechanisms of regulation of hTERT expression. Our results showed that TSA-induced demethylation of CpGs on the hTERT promoter via down-regulation of DNA methyltransferase 1 (DNMT1) after which the demethylation of CpGs promoted CTCF binding, an inhibitor of hTERT transcription, on hTERT promoter for the inhibition of hTERT expression. These findings suggest the possibility that demethylation of CpGs by TSA can epigenetically regulate the expression of tumor related genes and may provide important clues on how to approach cancer-specific therapies using TSA.
Section snippets
Materials and methods
Cell culture and drug treatments. The HCT116 human colon cancer cell line was obtained from the Korean Cell Line Bank (KCLB No. 10237) and cultured at 37 °C under a 5% CO2 atmosphere in RPMI-1640 medium (WelGENE, Deagu, Korea) supplemented with 10% fetal bovine serum (WelGENE). Twenty-four hours after seeding, TSA was added to the culture medium to a final concentration of 1 μM. Afterwards cells were incubated with TSA for various time points as indicated in the figure legends.
Quantification of
Induction of cell-cycle arrest and apoptosis in colon cancer cell line by TSA
To analyze the induction of cell-cycle arrest and apoptosis by TSA in HCT116, we performed flow cytometric analysis using PI staining and Western blot analysis with p53, p21 and PARP antibodies. Flow cytometric analysis showed that 1 μM TSA-induced arrest of cell cycle at the G2/M phase and resulted in the appearance of sub-G1 populations after 48 h (Fig. 1A). In addition, the expression of p53 and p21 in cells increased in response to TSA treatment and the cleaved form of PARP was observed after
Conclusions
TSA was well known drug that it generally involved in up-regulation of gene expression through induction of histone acetylation on the promoter of target gene. However, in HCT116, TSA-induced the down-regulation of hTERT expression via demethylation of the site-specific CpGs on the hTERT promoter rather than induction of histone acetylation. Especially, the demethylation of the 31st–33rd CpGs by TSA resulted in the binding of CTCF on the hTERT promoter. The down-regulation of DNMT1 for the
Acknowledgments
This research was supported by the Chung-Ang University Research Scholarship Grants in 2009.
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