Effects of demethylating agent 5-aza-2′-deoxycytidine and histone deacetylase inhibitor FR901228 on maspin gene expression in oral cancer cell lines

Murakami, Jun; Asaumi, Jun Ichi; Maki, Yuu; Tsujigiwa, Hidetsugu; Kuroda, Masahiro; Nagai, Noriyuki; Yanagi, Yoshinobu; Inoue, Tetsuyoshi; Kawasaki, Shoji; Tanaka, Noriaki; Matsubara, Nagahide; Kishi, Kanji

doi:10.1016/j.oraloncology.2003.12.008

Cited by 42 publications

(24 citation statements)

References 29 publications

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“…This is consistent with the function of DNA methylation which is important in X-chromosome-inactivation, development, and tumorigenesis (Holliday and Pugh, 1975;Riggs, 1975;Tycko, 2000). Recently, promoter methylation of maspin was shown to regulate tissue-specific gene expression and changes in gene expression upon transformation of normal cells to carcinoma cells (Domann et al, 2000;Domann and Futscher, 2003;Fujisawa et al, 2005;Murakami et al, 2004;Ohike et al, 2003;Sato et al, 2004;Yatabe et al, 2004). Conversion of the maspin promoter from the hypomethylated state observed in freshly isolated (non-cultured) stromal cells to a hypermethylated state occurred between the time corneal keratocytes were released from the matrix of the cornea and when the non-dividing P0 stromal cells in SFDM were harvested at three days or the P0 cells cultured in the presence of 5% FBS became confluent.…”

Section: Discussionsupporting

confidence: 78%

“…Even though the primary function of 5-Aza-dC at 10 μM or less is to inhibit the DNA methyltransferase, DNMT-1, it could have another role as a histone methyltransferase inhibitor (Kondo et al, 2003;Murakami et al, 2004;Wozniak et al, 2007;Zhu et al, 2001). This role has been demonstrated for histone H3 associated with the maspin gene.…”

Section: Discussionmentioning

confidence: 99%

“…Two μg genomic DNA was denatured and sodium bisulfite (Sigma-Aldrich) treated at 55°C overnight in dark (Murakami et al, 2004). The treated DNA was desalted using Wizard DNA Clean Up System (Promega, Madison, WI) and desulfonated with NaOH.…”

Section: Sodium Bisulfite Sequencingmentioning

confidence: 99%

“…The maspin promoter was amplified in the region that includes CpG sites from −254 to + 152 relative to the transcription start site under conditions used by Murakami et al (Murakami et al, 2004). This region was chosen based on the region most often studied in previous papers and is methylated under the same as two downstream CpG islands (Futscher et al, 2002).…”

Section: Sodium Bisulfite Sequencingmentioning

confidence: 99%

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Epigenetic silencing of maspin expression occurs early in the conversion of keratocytes to fibroblasts

Horswill

Narayan

Warejcka

et al. 2008

Experimental Eye Research

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Maspin, a 42 kDa non-classical serpin (serine protease inhibitor) that controls cell migration and invasion, is mainly expressed by epithelial-derived cells but is also expressed in corneal stromal keratocytes. Upon culture of stromal keratocytes in the presence of FBS, maspin is down regulated to nearly undetectable levels by passage two. DNA methylation is one of several processes that controls gene expression during cell differentiation, development, genetic imprinting, and carcinogenesis but has not been studied in corneal stromal cells. The purpose of this study was to determine whether DNA methylation of the maspin promoter and histone H3 dimethylation are involved in the mechanism of down regulation of maspin synthesis in human corneal stromal fibroblasts and myofibroblasts. Human donor corneal stroma cells were immediately placed into serum-free defined medium or cultured in the presence of FBS and passed into serum-free medium or medium containing FBS or FGF-2 to induce the fibroblast phenotype or TGF-β1 for the myofibroblast phenotype. These cell types are found in wounded corneas. The cells were used to prepare RNA for semi-quantitative or quantitative RT-PCR or to extract protein for western analysis. In addition, P4 FBS cultured fibroblasts were treated with the DNA demethylating agent, 5-aza-2′-deoxycytidine (5-Aza-dC), and the histone deacetylase inhibitor, trichostatin A (TSA). Cells with and without treatment were harvested and assayed for DNA methylation using sodium bisulfite sequencing. The methylation state of histone H3 associated with the maspin gene in the P4 fibroblast cells was determined using a ChIP assay. Freshly harvested corneal stromal cells expressed maspin but upon phenotypic differentiation, maspin mRNA and protein were dramatically down-regulated. Sodium bisulfite sequencing revealed that the maspin promoter in the freshly isolated stromal keratocytes was hypomethylated while both the P0 stromal cells and the P1 cells cultured in the presence of serum free defined medium, FGF-2 and TGF-β1 were hypermethylated. Down regulation of maspin synthesis was also associated with histone H3 dimethylation at Lysine 9. Both maspin mRNA and protein were reexpressed at low levels with 5-Aza-dC but not TSA treatment. Addition of TSA to 5-Aza-dC treated cells did not increase maspin expression. Treatment with 5-Aza-dC did not significantly alter demethylation of the maspin promoter but did demethylate histone H3. These results show maspin promoter hypermethylation and histone methylation occur with down regulation *Corresponding Author: Sally S. Twining, PhD, Department of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, Phone: 414-456-8431, Fax: 414-456-6510, e-mail: stwining@mcw.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of...

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Section: Discussionsupporting

confidence: 78%

Section: Discussionmentioning

confidence: 99%

Section: Sodium Bisulfite Sequencingmentioning

confidence: 99%

Section: Sodium Bisulfite Sequencingmentioning

confidence: 99%

See 2 more Smart Citations

Epigenetic silencing of maspin expression occurs early in the conversion of keratocytes to fibroblasts

Horswill

Narayan

Warejcka

et al. 2008

Experimental Eye Research

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“…FR901288, a novel cyclic peptide inhibitor of HDAC, is isolated by the Fujisawa Pharmaceutical Co., Ltd. (Osaka, Japan) from a fermantation broth of Chromobacterium violaceum, and is currently in phase I of clinical trials. As FR901228 has a stronger cytotoxic activity than TSA, several numbers of genes involved are known to increase or decrease their transcriptional levels followed by hyperacetylation of histone [25].…”

Section: Introductionmentioning

confidence: 99%

Effects of histone deacetylase inhibitor FR901228 on the expression level of telomerase reverse transcriptase in oral cancer

Murakami

Asaumi

Kawai

et al. 2005

Cancer Chemother Pharmacol

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We speculated whether or not the expression level of telomerase reverse transcriptase (hTERT) would be modulated by agents targeting epigenetics in oral cancer cell lines. Although hTERT is known to be targeted by epigenetic changes, it remains unclear how chemoagents targeting epigenetics work on hTERT transcription. In the present study, the epigenetic effects of histone deacetylase (HDAC) inhibitor FR901228 on hTERT transcription were analysed by RT-PCR in oral cancer cell lines. The mRNA expression of hTERT was upregulated after exposure to FR901228 in hTERT-negative Hep2 cells, even in the hTERT highly expressed SAS and KB cells.Moreover, co-treatment of protein synthesis inhibitor cycloheximide (CHX) resulted in the induction of hTERT transcription by FR901228. This suggests that the induction of hTERT by FR901228 requires de novo protein synthesis to some extent and is more likely a direct than an indirect effect on epigenetic changes such as histone acetylation / deacetylation. We further examined the effect of FR901228 on c-myc protein, which is one of the main hTERT transcription activators. FR901228 repressed c-myc protein only in the absence of CHX, dependent of the enhancement of de novo protein synthesis. Our results indicate that c-myc protein is repressed indirectly by FR901228 but may not contribute FR901228-induced hTERT transcription. The present study showed that the HDAC inhibitor FR901228 induced the hTERT gene by a complex mechanism that involved other transcription factors except for c-myc, in addition to the inhibition of histone deacetylation.

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Epigenetic Dysregulation of Maspin (SerpinB5) in Cancer Invasion and Metastasis

Futscher

Domann

Cancer Metastasis — Biology and Treatment

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Effects of demethylating agent 5-aza-2′-deoxycytidine and histone deacetylase inhibitor FR901228 on maspin gene expression in oral cancer cell lines

Cited by 42 publications

References 29 publications

Epigenetic silencing of maspin expression occurs early in the conversion of keratocytes to fibroblasts

Epigenetic silencing of maspin expression occurs early in the conversion of keratocytes to fibroblasts

Effects of histone deacetylase inhibitor FR901228 on the expression level of telomerase reverse transcriptase in oral cancer

Epigenetic Dysregulation of Maspin (SerpinB5) in Cancer Invasion and Metastasis

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