Review. Meiotic drive and sex determination: molecular and cytological mechanisms of sex ratio adjustment in birds.

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Rutkowska J, Badyaev AV
Philos Trans R Soc Lond B Biol Sci (May 2008)

Differences in relative fitness of male and female offspring across ecological and social environments should favour the evolution of sex-determining mechanisms that enable adjustment of brood sex ratio to the context of breeding. Despite the expectation that genetic sex determination should not produce consistent bias in primary sex ratios, extensive and adaptive modifications of offspring sex ratio in relation to social and physiological conditions during reproduction are often documented. Such discordance emphasizes the need for empirical investigation of the proximate mechanisms for modifying primary sex ratios, and suggests epigenetic effects on sex-determining mechanisms as the most likely candidates. Birds, in particular, are thought to have an unusually direct opportunity to modify offspring sex ratio because avian females are heterogametic and because the sex-determining division in avian meiosis occurs prior to ovulation and fertilization. However, despite evidence of strong epigenetic effects on sex determination in pre-ovulatory avian oocytes, the mechanisms behind such effects remain elusive. Our review of molecular and cytological mechanisms of avian meiosis uncovers a multitude of potential targets for selection on biased segregation of sex chromosomes, which may reflect the diversity of mechanisms and levels on which such selection operates in birds. Our findings indicate that pronounced differences between sex chromosomes in size, shape, size of protein bodies, alignment at the meiotic plate, microtubule attachment and epigenetic markings should commonly produce biased segregation of sex chromosomes as the default state, with secondary evolution of compensatory mechanisms necessary to maintain unbiased meiosis. We suggest that it is the epigenetic effects that modify such compensatory mechanisms that enable context-dependent and precise adjustment of primary sex ratio in birds. Furthermore, we highlight the features of avian meiosis that can be influenced by maternal hormones in response to environmental stimuli and may account for the precise and adaptive patterns of offspring sex ratio adjustment observed in some species.

Helicobacter pylori regulates p21(WAF1) by histone H4 acetylation.

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Xia G, Schneider-Stock R, Diestel A, Habold C, Krueger S, Roessner A, Naumann M, Lendeckel U
Biochem Biophys Res Commun (May 2008)

Helicobacter pylori are bacteria that colonize the stomach persistently, which confers risk of serious diseases, including peptic ulceration and gastric neoplasia. Aberrant expression of cell cycle control proteins has been demonstrated in H. pylori infected gastric epithelial cells, suggesting that perturbation of the cell cycle plays a role in the pathogenesis of various H. pylori associated diseases. In this study, we investigate the modulation of the cell cycle control protein p21(WAF1) by H. pylori in the gastric carcinoma cell line NCI-N87 and in primary gastric cells derived from healthy tissue. We observed an up-regulation of p21(WAF1) in both NCI-N87 and primary cells. Chromatin immunoprecipitation analysis revealed that the increased expression of p21(WAF1) induced by H. pylori is associated with the release of HDAC-1 from the p21(WAF1) promoter and hyper-acetylation of histone H4. The elucidation of the epigenetic regulation of p21(WAF1) by H. pylori may help to dissect the pathogenetic mechanisms underlying the development and progression of H. pylori associated diseases.

Promoter methylation correlates with reduced Smad4 expression in advanced prostate cancer.

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Aitchison AA, Veerakumarasivam A, Vias M, Kumar R, Hamdy FC, Neal DE, Mills IG
Prostate (May 2008)

BACKGROUND: Transforming growth factor-beta (TGF-beta) is a potent growth inhibitor in a wide range of cell types. A transducer of TGF-beta signaling known as Mothers against decapentaplegic homologue 4 (Smad4) is a known tumor suppressor found on chromosome 18q21.1 and is typically inactivated by deletion or mutation in pancreatic and colorectal cancers. The purpose of the article is to investigate Smad4 expression, gene copy number and methylation status in advanced cases of prostate cancer. METHODS: We have employed Methylation Specific PCR (MSP) to identify methylation sites within the Smad4 promoter and combined this with quantitative real-time PCR to look for correlates between methylation status and Smad4 expression and to examine androgen receptor (AR) expression. Bacterial artificial chromosome-comparative genomic hybridization (BAC-CGH) has been used to look for genomic amplifications and deletions which may also contribute to expression changes. RESULTS: We fail to find evidence of genomic deletions or amplifications affecting the Smad4 locus on chromosome 18 but show a correlation between promoter methylation and the loss of Smad4 expression in the same material. We confirm that the AR locus on the X chromosome is amplified in 30% of the advanced clinical samples and that this correlates with increased transcript levels as previously reported by other groups. CONCLUSION: This indicates that epigenetic changes affect the expression of the Smad4 protein in prostate cancer and points to methylation of the promoter as a novel marker of and contributor to the disease warranting further study. Prostate 68: 661-674, 2008. (c) 2008 Wiley-Liss, Inc.

Epigenetic-based treatments emphasize the biologic differences of core-binding factor acute myeloid leukemias.

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Serrano E, Carnicer MJ, Lasa A, Orantes V, Pena J, Brunet S, Aventn A, Sierra J, Nomdedu JF
Leuk Res (Jun 2008)

Acute myeloid leukemia (AML) is a heterogeneous group of disorders characterized by an abnormal proliferation of the myeloid precursors and a maturation block. The most common chromosomal lesions in AML are the t(8;21) and inv(16). To better understand the leukemogenic mechanism of these fusion proteins, we performed gene expression studies in samples from (8;21), AML1 mutated and inv(16) patients, as well as from the Kasumi-1 cell line and a U937 cell line expressing the AML1-ETO fusion gene. To assess the influence of associated epigenetic lesions, we performed gene expression studies in Kasumi-1 cells and cells extracted from an Inv(16) patient, both treated with demethylating and HDAC inhibitor agents. Shared deregulated genes in the different types of core-binding factor leukemias were identified. We found a tight link between Inv(16) and mutant AML1 samples. Furthermore, some of the genes deregulated by the leukemogenic process reverted to their normal expression with demethylating and HDAC inhibitor treatment, highlighting the role of chromatin remodeling processes in AML.

The current state of preclinical prostate cancer animal models.

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Pienta KJ, Abate-Shen C, Agus DB, Attar RM, Chung LW, Greenberg NM, Hahn WC, Isaacs JT, Navone NM, Peehl DM, Simons JW, Solit DB, Soule HR, Vandyke TA, Weber MJ, Wu L, Vessella RL
Prostate (May 2008)

Prostate cancer continues to be a major cause of morbidity and mortality in men around the world. The field of prostate cancer research continues to be hindered by the lack of relevant preclinical models to study tumorigenesis and to further development of effective prevention and therapeutic strategies. The Prostate Cancer Foundation held a Prostate Cancer Models Working Group (PCMWG) Summit on August 6th and 7th, 2007 to address these issues. The PCMWG reviewed the state of prostate cancer preclinical models and identified the current limitations of cell line, xenograft and genetically engineered mouse models that have hampered the transition of scientific findings from these models to human clinical trials. In addition the PCMWG identified administrative issues that inhibit the exchange of models and impede greater interactions between academic centers and these centers with industry. The PCMWG identified potential solutions for discovery bottlenecks that include: (1) insufficient number of models with insufficient molecular and biologic diversity to reflect human cancer, (2) a lack of understanding of the molecular events that define tumorigenesis, (3) a lack of tools for studying tumor-host interactions, (4) difficulty in accessing model systems across institutions, and (5) addressing why preclinical studies appear not to be predictive of human clinical trials. It should be possible to apply the knowledge gained molecular and epigenetic studies to develop new cell lines and models that mimic progressive and fatal prostate cancer and ultimately improve interventions. Prostate 68: 629-639, 2008. (c) 2008 Wiley-Liss, Inc.

Molecular and cytogenetic evidence for an allotetraploid origin of Trifolium dubium (Leguminosae).

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Ansari HA, Ellison NW, Williams WM
Chromosoma (Apr 2008)

Suckling clover, Trifolium dubium Sibth., is a European grassland legume that has spread to many parts of the world. The present work shows that it is an allotetraploid (2n = 4x = 30) combining the genomes of T. campestre Schreb. (2n = 2x = 14) and T. micranthum Viv. (2n = 2x = 16), two diploid species of similar geographic distribution. T. dubium has two nuclear ITS sequences that closely match those of T. campestre and T. micranthum. Genomic in situ hybridisation using genomic DNA of T. campestre and T. micranthum as probes has differentiated the ancestral sets of chromosomes in T. dubium cells. Comparative fluorescence in situ hybridisation analyses of 5S and 18S-26S rDNA loci were also consistent with an allotetraploid structure of the T. dubium genome. A marked preponderance of ITS repeats from T. campestre over those from T. micranthum indicated that concerted evolution has resulted in partial homogenisation of these sequences by depletion of the T. micranthum-derived 18S-26S rDNA repeats. In parallel with this, the epigenetic phenomenon of nucleolar dominance has been observed in T. dubium such that the chromatin associated with the 18S-26S rDNA loci derived from T. campestre is decondensed (transcriptionally active), whilst that from T. micranthum remains highly condensed throughout the cell cycle. T. dubium, therefore, appears to have arisen by way of hybridisation between forms of the diploid species T. campestre and T. micranthum accompanied by chromosome doubling. The observed genomic changes in rDNA resulting from interspecific hybridisation provide evidence for the process of genome diploidisation in T. dubium.

A quantitative high-throughput screen identifies potential epigenetic modulators of gene expression.

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Johnson RL, Huang W, Jadhav A, Austin CP, Inglese J, Martinez ED
Anal Biochem (Apr 2008)

Epigenetic regulation of gene expression is essential in embryonic development and contributes to cancer pathology. We used a cell-based imaging assay that measures derepression of a silenced green fluorescent protein (GFP) reporter to identify novel classes of compounds involved in epigenetic regulation. This locus derepression (LDR) assay was screened against a 69,137-member chemical library using quantitative high-throughput screening (qHTS), a titration-response method that assays compounds at multiple concentrations. From structure-activity relationships of the 411 actives recovered from the qHTS, 6 distinct chemical series were chosen for further study. A total of 48 qHTS actives and analogs were counterscreened using the parental line of the LDR cells, which lack the GFP reporter. Three series-8-hydroxy quinoline, quinoline-8-thiol, and 1,3,5-thiadiazinane-2-thione-were not fluorescent and reconfirmed activity in the LDR cells. The three active series did not inhibit histone deacetylase activity in nuclear extracts or reactivate the expression of the densely methylated p16 gene in cancer cells. However, one series induced expression of the methylated CDH13 gene and inhibited the viability of several lung cancer lines at submicromolar concentrations. These results suggest that the identified small molecules act on epigenetic or transcriptional components and validate our approach of using a cell-based imaging assay in conjunction with qHTS.

Proliferative inflammatory atrophy: a background lesion of prostate cancer?

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Woenckhaus J, Fenic I
Andrologia (Apr 2008)

Proliferative inflammatory atrophy (PIA) belongs to the atrophic lesions that frequently occur in the prostate. The location of PIA in the periphery of the gland near to prostate carcinoma or even showing direct transition to malignant or pre-malignant epithelia suggested a connection between PIA and prostate cancer. Further findings in PIA, such as imbalance between proliferation and apoptosis and detection of molecular-biological abnormalities specific for oxidative stress or malignancy, supported this hypothesis. Recently, epidemiological studies including large cohorts of patients have been undertaken in order to investigate the causal connection between PIA and prostate carcinoma. The current understanding of PIA allows us to consider it as a benign lesion with certain genetic instability which can degenerate into prostate intraepithelial neoplasia or carcinoma, provided that the balance between anti-carcinogens and carcinogens is perturbed. To evaluate the role of PIA as a precursor lesion of prostate cancer, further examinations of genetic or epigenetic aberrations are required.

Potentiation of reactive oxygen species is a marker for synergistic cytotoxicity of MS-275 and 5-azacytidine in leukemic cells.

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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.

Promoter analysis of epigenetically controlled genes in bladder cancer.

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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

Genetic evidence that Ras-like GTPases, Gtr1p, and Gtr2p, are involved in epigenetic control of gene expression in Saccharomyces cerevisiae.

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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.

NGF/PI3K signaling-mediated epigenetic regulation of delta opioid receptor gene expression.

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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.

Enhanced Plating Efficiency of Trypsin-Adapted Human Embryonic Stem Cells is Reversible and Independent of Trisomy 12/17.

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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.

The effects of trichostatin a on mRNA expression of chromatin structure-, DNA methylation-, and development-related genes in cloned mouse blastocysts.

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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.

Cardiovascular and metabolic effects of obesity.

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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.