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03.30.08
Posted in research articles at 11:30 am by admin
Gao S, Mobley A, Miller C, Boklan J, Chandra J
Leuk Res (May 2008)
Epigenetic modifiers are currently in clinical use for various tumor types. Recently, numerous studies supporting the combination of histone deacetylase inhibitors (HDACi) and DNA methyltransferase inhibitors have emerged, encouraging early clinical trials of these agents together. Here we show that MS-275, an HDACi, and 5-azacytidine, a methyltransferase inhibitor, display synergistic cytotoxicity and apoptosis in AML and ALL cells. Intracellular production of reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, is a novel marker for this synergism in ALL cells. These data suggest that assessment of oxidative stress can serve as a marker of the concerted action of MS-275 and 5-azacytidine.
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Posted in research articles at 11:30 am by admin
Veerla S, Panagopoulos I, Jin Y, Lindgren D, Hglund M
Genes Chromosomes Cancer (May 2008)
DNA methylation is an important epigenetic modification that regulates several genes crucial for tumor development. To identify epigenetically regulated genes in bladder cancer, we performed genome wide expression analyses of eight-bladder cancer cell lines treated with the demethylating agents 5-aza-2′-cytidine and zebularine. To identify methylated C-residues, we sequenced cloned DNA fragments from bisulfite-treated genomic DNA. We identified a total of 1092 genes that showed > or =2-fold altered expression in at least one cell line; 710 showed up-regulation and 382 down-regulation. Extensive sequencing of promoters from 25 genes in eight cell lines showed an association between methylation pattern and expression in 13 genes, including both CpG island and non-CpG island genes. Overall, the methylation patterns showed a patchy appearance with short segments showing high level of methylation separated by larger segments with no methylation. This pattern was not associated with MeCP2 binding sites or with evolutionarily conserved sequences. The genes UBXD2, AQP11, and TIMP1 showed particular patchy methylation patterns. We found several high-scoring and evolutionarily conserved transcription factor binding sites affected by methylated C residues. Two of the genes, FGF18 and MMP11, that were down-regulated as response to 5-aza-2′-cytidine and zebularine treatment showed methylation at specific sites in the untreated cells indicating an activating result of methylation. Apart from identifying epigenetically regulated genes, including TGFBR1, NUPR1, FGF18, TIMP1, and MMP11, that may be of importance for bladder cancer development the presented data also highlight the organization of the modified segments in methylated promoters. This article contains supplementary material available via the Internet at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.
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Posted in research articles at 11:30 am by admin
Sekiguchi T, Hayashi N, Wang Y, Kobayashi H
Biochem Biophys Res Commun (Apr 2008)
Gtr1p and Gtr2p of Saccharomyces cerevisiae are members of the Ras-like GTP binding family and interact genetically with Prp20p (yeast RCC1), which is a guanine nucleotide exchange factor for Gsp1p (yeast homolog of Ran, involved in nuclear export). Recently, Gtr1p and Gtr2p were suggested to be molecular switches in the rapamycin-sensitive TOR signaling pathway. Here, we show that Gtr1p and Gtr2p genetically interact with the chromatin remodeling factor Ino80p. Gtr2p interacted physically with both Rvb1p and Rvb2p. Consistent with these results, Gtr2p localized to chromatin and could activate transcription. Gtr1p and Gtr2p were found to be involved in chromatin silencing in the vicinity of telomeres. Gtr1p and Gtr2p were required to repress nitrogen catabolite-repressed genes, which are repressed by the TOR signaling pathway. We propose that Gtr1p and Gtr2p are involved in epigenetic control of gene expression in the TOR signaling pathway.
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Posted in research articles at 11:30 am by admin
Chen YL, Law PY, Loh HH
Biochem Biophys Res Commun (Apr 2008)
The G protein-coupled delta opioid receptor gene (dor) has been associated with neuronal survival, differentiation, and neuroprotection. Our previous study identified PI3K/Akt/NF-kappaB signaling is a main downstream signaling pathway in nerve growth factor (NGF)-induced temporal expression of the dor gene in the PC12 cell model. It is still unknown how NGF/PI3K signaling regulates the expression of the dor gene in the nucleus. In the current study, we investigated how PI3K signaling affected epigenetic modifications of histone H3 Lys(9) (H3K9) in the 5′-UTR region of the rat dor gene locus. NGF treatment resulted in the global reversal of H3K9 trimethylation in cells. Moreover, the locus-specific reversal of H3K9 trimethylation and acetylation of H3K9 were dependent upon NGF/PI3K signaling and temporally well correlated with NGF-induced gene expression. These results indicate the importance of epigenetic modifications of H3K9, particularly the reversal of trimethylated H3K9, in the regulation of NGF/PI3K-dependent genes during neuronal differentiation.
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Posted in research articles at 11:30 am by admin
Chan EM, Yates F, Boyer LF, Schlaeger TM, Daley GQ
Cloning Stem Cells (2008)
Human embryonic stem cells (hESCs) can be cultured abundantly and indefinitely, but are subject to accumulations of chromosomal aberrations. To preserve their genetic integrity, hESCs are commonly maintained as cell aggregates or clumps during passaging. However, clump passaging hinders large-scale culture and complicates the isolation of single cell clones. To facilitate the isolation of genetically modified clones of hESCs while preserving their genetic integrity, we employed trypsin single-cell passaging for brief periods before returning to clump passaging for long-term maintenance. We observed that accommodation to trypsin passage as single cells is an adaptive process where over three to four passages considerably increases the plating efficiency. However, trypsin passage was associated with abnormalities of chromosomes 12 and 17. Nevertheless, the high plating efficiency of trypsin passaged hESCs is a reversible phenotype, regardless of chromosomal abnormalities, suggesting that epigenetic events are responsible for the switch in phenotype.
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Posted in research articles at 11:30 am by admin
Li X, Kato Y, Tsuji Y, Tsunoda Y
Cloning Stem Cells (2008)
Trichostatin A (TSA) is the most potent histone deacetylase (HDAC) inhibitor known. We previously reported that treatment of mouse somatic cell nuclear-transferred (SCNT) oocytes with TSA significantly increased the blastocyst rate, blastocyst cell number, and full-term development. How TSA enhances the epigenetic remodeling ability of somatic nuclei and the expression of development-related genes, however, is not known. In the present study, we compared the expression patterns of nine genes involved in chromatin structure and DNA methylation, and seven development-related genes in blastocysts developed from SCNT oocytes treated with and without TSA, and in blastocysts developed in vivo and in vitro using real-time reverse transcription-polymerase chain reaction. In vivo-recovered blastocysts and blastocysts developed from TSA-treated SCNT oocytes exhibited similar expression patterns for Hdac1, 2, and 3, CBP, PCAF, and Dnmt3b genes compared with in vitro-developed blastocysts and blastocysts developed from SCNT oocytes without TSA treatment. There were significantly lower expression levels of Hdac1 and Hdac2 transcripts in TSA-treated and in vivo-recovered blastocysts than in TSA-untreated and in vitro-developed blastocysts. The finding that TSA treatment of SCNT oocytes significantly upregulated Sox2 and cMyc transcripts in blastocysts indicated that both transcripts are TSA-responsive genes. Thus, TSA treatment of mouse SCNT oocytes decreased the expression of chromatin structure- and DNA methylation-related genes, and increased the expression of Sox2 and cMyc genes in blastocysts. Such modifications might be a reason for the high developmental potential of mouse SCNT oocytes treated with TSA.
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03.03.08
Posted in research articles at 10:37 am by admin
Morris MJ
Clin Exp Pharmacol Physiol (Apr 2008)
1. Obesity is an important risk factor for hypertension and its incidence is increasing around the world. 2. The mechanisms underlying obesity-related hypertension include sympathetic activation, altered vascular responses, hormonal changes, enhanced inflammatory markers and structural changes. 3. This review summarizes recent evidence of the underlying impact of obesity on blood pressure. A number of candidate mechanisms include increased sympathetic activity, activation of the renin-angiotensin system, altered vasoconstrictor or dilator responses and the attendant systemic inflammatory state. 4. While adult lifestyle factors undoubtedly contribute to the incidence of obesity and its attendant hypertension, evidence suggests that the programming of obesity may occur following over-nutrition during development. A growing body of evidence links maternal obesity, offspring obesity and hypertension. 5. Finally, epigenetic modification of genes relevant to hypertension may contribute to the development of hypertension following a suboptimal intrauterine environment. To date the cardiovascular effects of early nutritional changes have been largely investigated following maternal under-nutrition or protein restriction; further work is necessary to determine the impact of maternal obesity.
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Posted in research articles at 10:37 am by admin
Anway MD, Skinner MK
Prostate (Apr 2008)
PURPOSE: The ability of an endocrine disruptor exposure during gonadal sex determination to promote a transgenerational prostate disease phenotype was investigated in the current study. METHODS: Exposure of an F0 gestating female rat to the endocrine disruptor vinclozolin during F1 embryo gonadal sex determination promoted a transgenerational adult onset prostate disease phenotype. The prostate disease phenotype and physiological parameters were determined for males from F1 to F4 generations and the prostate transcriptome was assessed in the F3 generation. RESULTS: Although the prostate in prepubertal animals develops normally, abnormalities involving epithelial cell atrophy, glandular dysgenesis, prostatitis, and hyperplasia of the ventral prostate develop in older animals. The ventral prostate phenotype was transmitted for four generations (F1-F4). Analysis of the ventral prostate transcriptome demonstrated 954 genes had significantly altered expression between control and vinclozolin F3 generation animals. Analysis of isolated ventral prostate epithelial cells identified 259 genes with significantly altered expression between control and vinclozolin F3 generation animals. Characterization of regulated genes demonstrated several cellular pathways were influenced, including calcium and WNT. A number of genes identified have been shown to be associated with prostate disease and cancer, including beta-microseminoprotein (Msp) and tumor necrosis factor receptor superfamily 6 (Fadd). CONCLUSIONS: The ability of an endocrine disruptor to promote transgenerational prostate abnormalities appears to involve an epigenetic transgenerational alteration in the prostate transcriptome and male germ-line. Potential epigenetic transgenerational alteration of prostate gene expression by environmental compounds may be important to consider in the etiology of adult onset prostate disease. Prostate 68: 517-529, 2008. (c) 2008 Wiley-Liss, Inc.
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Posted in research articles at 10:37 am by admin
Alkelai A, Baum A, Carless M, Crowley J, Dasbanerjee T, Dempster E, Docherty S, Hare E, Galsworthy MJ, Grover D, Glubb D, Karlsson R, Mill J, Sen S, Quinones MP, Vallender EJ, Verma R, Vijayan NN, Villafuerte S, Voineskos AN, Volk H, Yu L, Zimmermann P, Delisi LE
Am J Med Genet B Neuropsychiatr Genet (Mar 2008)
The World Congress of Psychiatric Genetics (WCPG) has become an annual event since the early 1990’s sponsored by the International Society of Psychiatric Genetics (ISPG). Each year the latest published and unpublished findings are aired for discussion by representatives of the majority of research programs on this topic world-wide. The 2007 congress was held in New York City and attracted over 1000 researchers. The topics emphasized included results from whole genome association studies, the significance of copy number variation and the important contributions of epigenetic events to psychiatric disorders. There were over 20 oral sessions devoted to these and other topics of interest. Young investigator recipients of travel awards served as rapporteurs to summarize sessions and these summaries follow.
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Posted in research articles at 10:37 am by admin
Magnani L, Lee K, Fodor WL, Machaty Z, Cabot RA
Mol Reprod Dev (May 2008)
Somatic cell nuclear transfer (SCNT) still retains important limitations. Impaired epigenetic reprogramming is considered responsible for altered gene expression and developmental failure in SCNT-derived embryos. After nuclear transfer the donor cell nucleus undergoes extensive changes in gene expression that involve epigenetic modifications and chromatin remodeling. We hypothesized that SNF2-type ATP-dependent chromatin factors contribute to epigenetic reprogramming and the relative amount of these factors in the donor cell affects developmental potential of the reconstructed embryos. In order to test this hypothesis, we assessed the relative amount of SNF2-type ATPases (Brahma, Brg1, SNF2H, SNF2L, CHD3, and CHD5) in three different donor cells as well as in porcine metaphase II oocytes. We performed SCNT with fetal fibroblast cells, olfactory bulb (OB) progenitor cells, and porcine skin originating sphere stem cells (PSOS). We found that OB-NT embryos and PSOS-NT embryos resulted in a higher morulae/blastocysts ratio as compared to fibroblast-NT embryos (23.53%, 16.98%, and 11.63%, respectively; P < 0.05). Fibroblast cells contained a significantly higher amount of SNF2L and CHD3 transcripts while Brg1 and SNF2H were the most expressed transcripts in all the cell lines analyzed. Metaphase II oocyte expression profile appeared to be unique compared to the cell lines analyzed. This work supports our hypothesis that an array of chromatin-remodeling proteins on donor cells may influence the chromatin structure, effect epigenetic reprogramming, and developmental potential. Mol. Reprod. Dev. 75: 766-776, 2008. (c) 2008 Wiley-Liss, Inc.
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02.29.08
Posted in research articles at 10:27 pm by admin
Nowak-Imialek M, Wrenzycki C, Herrmann D, Lucas-Hahn A, Lagutina I, Lemme E, Lazzari G, Galli C, Niemann H
Mol Reprod Dev (May 2008)
Histone modification genes in bovine embryos: The mRNA expression pattern of histone-related genes was determined in bovine oocytes and embryos. We compared immature and in vitro-matured oocytes, either before or after enucleation and activation, in vitro produced embryos (zygotes, 8-16 cell stages, blastocysts), embryos cloned with female or male donor cells; parthenogenetic embryos, and in vivo-derived blastocysts to detect deviations from the normal expression pattern. A sensitive semi-quantitative endpoint RT-PCR assay was used to reveal differences in histone deacetylation [histone deacetylase 2 (HDAC2)]; histone acetylation [histone acetyltransferase 1 (HAT1)]; histone methylation [histone methyltransferases (SUV39H1, G9A)]; heterochromatin formation [heterochromatin protein 1 (HP1)]; and chromatin-mediated transcription regulation [zygote arrest 1 (ZAR1)]. With the exception of ZAR1, these mRNAs were present throughout preimplantation development. The relative abundance of mRNAs for histone methyltransferases (SUV39H1 and G9A) and for heterochromatin-associated protein (HP1) differed significantly before and after activation of the bovine embryonic genome. The similarity of HAT1 gene expression in 8-16 cell embryos and blastocysts suggests that histone acetylation is primarily affected by in vitro culture only prior to embryonic genome activation. HDAC2 gene mRNA expression was not affected by in vitro culture and/or cloning before and after activation of the embryonic genome. The donor cell line affected mRNA expression patterns of genes involved in reprogramming cloned embryos suggesting epigenetic dysregulation. Results show that both in vitro production and somatic cloning alter the mRNA expression of histone modifying genes in bovine embryos. Mol. Reprod. Dev. 75: 731-743, 2008. (c) 2007 Wiley-Liss, Inc.
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Posted in research articles at 10:27 pm by admin
Magnani L, Lee K, Fodor WL, Machaty Z, Cabot RA
Mol Reprod Dev (May 2008)
Somatic cell nuclear transfer (SCNT) still retains important limitations. Impaired epigenetic reprogramming is considered responsible for altered gene expression and developmental failure in SCNT-derived embryos. After nuclear transfer the donor cell nucleus undergoes extensive changes in gene expression that involve epigenetic modifications and chromatin remodeling. We hypothesized that SNF2-type ATP-dependent chromatin factors contribute to epigenetic reprogramming and the relative amount of these factors in the donor cell affects developmental potential of the reconstructed embryos. In order to test this hypothesis, we assessed the relative amount of SNF2-type ATPases (Brahma, Brg1, SNF2H, SNF2L, CHD3, and CHD5) in three different donor cells as well as in porcine metaphase II oocytes. We performed SCNT with fetal fibroblast cells, olfactory bulb (OB) progenitor cells, and porcine skin originating sphere stem cells (PSOS). We found that OB-NT embryos and PSOS-NT embryos resulted in a higher morulae/blastocysts ratio as compared to fibroblast-NT embryos (23.53%, 16.98%, and 11.63%, respectively; P < 0.05). Fibroblast cells contained a significantly higher amount of SNF2L and CHD3 transcripts while Brg1 and SNF2H were the most expressed transcripts in all the cell lines analyzed. Metaphase II oocyte expression profile appeared to be unique compared to the cell lines analyzed. This work supports our hypothesis that an array of chromatin-remodeling proteins on donor cells may influence the chromatin structure, effect epigenetic reprogramming, and developmental potential. Mol. Reprod. Dev. 75: 766-776, 2008. (c) 2008 Wiley-Liss, Inc.
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Posted in research articles at 10:27 pm by admin
Muretto P, Ruzzo A, Pizzagalli F, Graziano F, Maltese P, Zingaretti C, Berselli E, Donnarumma N, Magnani M
Ann Oncol (Mar 2008)
BACKGROUND: We investigated whether an endogastric capsule (EC) may be a valuable tool for collecting DNA from exfoliated cells from the gastric mucosa and for carrying out an analysis of promoter methylation status of the E-cadherin (CDH1) gene in poorly differentiated, diffuse gastric cancer (DGC). MATERIAL AND METHODS: Consecutive patients with a confirmed diagnosis of poorly differentiated DGC underwent collection of gastric juice by EC. Subjects without cancer and premalignant lesions were also accrued as controls. The samples of gastric juice were processed for DNA isolation and amplification. Then they were used for analysis of CDH1 promoter hypermethylation. RESULTS: The procedure successfully allowed the analysis of CDH1 promoter hypermethylation in 20 patients and 14 controls. This pilot study showed feasibility of the procedure and a significantly different CDH1 promoter hypermethylation status between DGC patients and controls was detected. CONCLUSIONS: The EC may represent an innovative and noninvasive tool for the analysis of a specific epigenetic change in DGC patients. Our findings deserve additional studies as this method may represent a cost-effective tool for early detection of sporadic as well as hereditary DGC in CDH1 germline mutations carriers.
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Posted in research articles at 10:27 pm by admin
Anway MD, Skinner MK
Prostate (Apr 2008)
PURPOSE: The ability of an endocrine disruptor exposure during gonadal sex determination to promote a transgenerational prostate disease phenotype was investigated in the current study. METHODS: Exposure of an F0 gestating female rat to the endocrine disruptor vinclozolin during F1 embryo gonadal sex determination promoted a transgenerational adult onset prostate disease phenotype. The prostate disease phenotype and physiological parameters were determined for males from F1 to F4 generations and the prostate transcriptome was assessed in the F3 generation. RESULTS: Although the prostate in prepubertal animals develops normally, abnormalities involving epithelial cell atrophy, glandular dysgenesis, prostatitis, and hyperplasia of the ventral prostate develop in older animals. The ventral prostate phenotype was transmitted for four generations (F1-F4). Analysis of the ventral prostate transcriptome demonstrated 954 genes had significantly altered expression between control and vinclozolin F3 generation animals. Analysis of isolated ventral prostate epithelial cells identified 259 genes with significantly altered expression between control and vinclozolin F3 generation animals. Characterization of regulated genes demonstrated several cellular pathways were influenced, including calcium and WNT. A number of genes identified have been shown to be associated with prostate disease and cancer, including beta-microseminoprotein (Msp) and tumor necrosis factor receptor superfamily 6 (Fadd). CONCLUSIONS: The ability of an endocrine disruptor to promote transgenerational prostate abnormalities appears to involve an epigenetic transgenerational alteration in the prostate transcriptome and male germ-line. Potential epigenetic transgenerational alteration of prostate gene expression by environmental compounds may be important to consider in the etiology of adult onset prostate disease. Prostate 68: 517-529, 2008. (c) 2008 Wiley-Liss, Inc.
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Posted in research articles at 10:27 pm by admin
Han DW, Im YB, Do JT, Gupta MK, Uhm SJ, Kim JH, Schler HR, Lee HT
Mol Reprod Dev (May 2008)
This study was designed to identify the putative differentially methylated regions (DMRs) of the porcine imprinted genes insulin-like growth factor 2 and H19 (IGF2-H19), and to assess the genomic imprinting status of IGF2-H19 by identifying the methylation patterns of these regions in germ cells, and in tissues from porcine fetuses, an adult pig, as well as cloned offspring produced by somatic cell nuclear transfer (SCNT). Porcine IGF2-H19 DMRs exhibit a normal monoallelic methylation pattern (i.e., either the paternally- or the maternally derived allele is methylated) similar to the pattern observed for the same genes in the human and mice genomes. Examination of the methylation patterns of the IGF2-H19 DMRs revealed that the zinc finger protein binding sites CTCF1 and 2 did not exhibit differential methylation in both control and cloned offspring. In contrast, the CTCF3 and DMR2 loci of the IGF2 gene showed abnormal methylation in cloned offspring, but a normal differential or moderate methylation pattern in tissues from control offspring and an adult pig. Our data thus suggest that regulation of genomic imprinting at the porcine IGF2-H19 loci is conserved among species, and that the abnormal methylation pattern in the regulatory elements of imprinted genes may lead to an alteration in the coordinated expression of genes required for successful reprogramming, which, in consequence, may contribute to the low efficiency of porcine genome reprogramming induced by nuclear transfer. Mol. Reprod. Dev. 75: 777-784, 2008. (c) 2008 Wiley-Liss, Inc.
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