Characteristic promoter hypermethylation signatures in male germ cell tumors.
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BACKGROUND: Human male germ cell tumors (GCTs) arise from undifferentiated primordial germ cells (PGCs), a stage in which extensive methylation reprogramming occurs. GCTs exhibit pluripotentiality and are highly sensitive to cisplatin therapy. The molecular basis of germ cell (GC) transformation, differentiation, and exquisite treatment response is poorly understood. RESULTS: To assess the role and mechanism of promoter hypermethylation, we analyzed CpG islands of 21 gene promoters by methylation-specific PCR in seminomatous (SGCT) and nonseminomatous (NSGCT) GCTs. We found 60% of the NSGCTs demonstrating methylation in one or more gene promoters whereas SGCTs showed a near-absence of methylation, therefore identifying distinct methylation patterns in the two major histologies of GCT. DNA repair genes MGMT, RASSF1A, and BRCA1, and a transcriptional repressor gene HIC1, were frequently methylated in the NSGCTs. The promoter hypermethylation was associated with gene silencing in most methylated genes, and reactivation of gene expression occurred upon treatment with 5-Aza-2' deoxycytidine in GCT cell lines. CONCLUSIONS: Our results, therefore, suggest a potential role for epigenetic modification of critical tumor suppressor genes in pathways relevant to GC transformation, differentiation, and treatment response. (+info)
Class II transactivator (CIITA) promoter methylation does not correlate with silencing of CIITA transcription in trophoblasts.
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Trophoblast cells are unique because they do not express major histocompatibility complex (MHC) class II antigens, either constitutively or after exposure to interferon-gamma (IFN-gamma). The absence of MHC class II antigens on trophoblasts is thought to play a critical role in preventing rejection of the fetus by the maternal immune system. The inability of trophoblasts to express MHC class II genes is primarily due to lack of the class II transactivator (CIITA), a transacting factor that is required for constitutive and IFN-gamma-inducible MHC class II transcription. We, therefore, investigated the silencing of CIITA expression in trophoblasts. In transient transfection assays, transcription from the IFN-gamma-responsive CIITA type IV promoter was upregulated by IFN-gamma in trophoblasts, which suggests that CIITA is silenced by an epigenetic mechanism in these cells. Polymerase chain reaction analysis demonstrated that the CIITA type IV promoter is methylated in both the human choriocarcinoma cell lines JEG-3 and Jar and in 2fTGH fibrosarcoma cells, which are IFN-gamma inducible for CIITA. Conversely, methylation of the CIITA type IV promoter was not observed in human primary cytotrophoblasts isolated from term placentae or in mouse or rat trophoblast cell lines. Simultaneous treatment with IFN-gamma and the histone deacetylase inhibitor trichostatin A weakly activated CIITA transcription in mouse trophoblasts. Stable hybrids between human choriocarcinoma and fibrosarcoma cells and between mouse trophoblasts and fibroblasts expressed CIITA following treatment with IFN-gamma. These results suggest that silencing of CIITA transcription is recessive in trophoblasts and involves an epigenetic mechanism other than promoter methylation. The fact that CIITA is expressed in the stable hybrids implies that trophoblasts may be missing a factor that regulates chromatin structure at the CIITA promoter. (+info)
Disruption of imprinted gene methylation and expression in cloned preimplantation stage mouse embryos.
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Cloning by somatic cell nuclear transfer requires that epigenetic information possessed by the donor nucleus be reprogrammed to an embryonic state. Little is known, however, about this remodeling process, including when it occurs, its efficiency, and how well epigenetic markings characteristic of normal development are maintained. Examining the fate of epigenetic information associated with imprinted genes during clonal development offers one means of addressing these questions. We examined transcript abundance, allele specificity of imprinted gene expression, and parental allele-specific DNA methylation in cloned mouse blastocysts. Striking disruptions were seen in total transcript abundance and allele specificity of expression for five imprinted genes. Only 4% of clones recapitulated a blastocyst mode of expression for all five genes. Cloned embryos also exhibited extensive loss of allele-specific DNA methylation at the imprinting control regions of the H19 and Snprn genes. Thus, epigenetic errors arise very early in clonal development in the majority of embryos, indicating that reprogramming is inefficient and that some epigenetic information may be lost. (+info)
Epigenetic characteristics and development of embryos cloned from donor cells treated by trichostatin A or 5-aza-2'-deoxycytidine.
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Development to blastocyst following nuclear transfer is dependent on the donor cell's ability to reprogram its genome to that of a zygote. This reprogramming step is inefficient and may be dependent on a number of factors, including chromatin organization. Trichostatin A (TSA; 0-5 microM), a histone deacetylase inhibitor, was used to increase histone acetylation and 5-aza-2'-deoxycytidine (5-aza-dC; 0-5 microM), a DNA methyl-transferase inhibitor, was used to decrease methylation of chromatin in donor cells in an attempt to improve their reprogrammability. Adult fibroblast cells treated with 1.25 or 5 microM TSA had elevated histone H3 acetylation compared to untreated controls. Cells treated with 0.3 microM 5-aza-dC had decreased methylation compared to untreated controls. Both drugs at 0.08 microM caused morphological changes of the donor cells. Development to blastocysts by embryos cloned from donor cells after 0.08 or 0.3 microM 5-aza-dC treatments was lower than in embryos cloned from untreated control cells (9.7% and 4.2%, respectively, vs. 25.1%), whereas 0.08 microM TSA treatment of donor cells increased blastocyst development compared to controls (35.1% vs. 25.1%). These results indicate that partial erasure of preexisting epigenetic marks of donor cells improves subsequent in vitro development of cloned embryos. (+info)
Significant reduction of WT1 gene expression, possibly due to epigenetic alteration in Wilms' tumor.
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WT1 at 11p13 is a tumor suppressor gene, an aberration of which causes Wilms' tumor (WT). Since WT1 expression is reduced in a certain proportion of WTs and its mutation is found only in 10-20% of WTs, we examined WT1 gene silencing due to epigenetic alteration in a total of 22 WTs. WT1 expression was significantly reduced in half of WTs without any mutation in the WT1 gene itself, suggesting that the reduction of expression was possibly epigenetic. We found promoter hypermethylation in one WT with loss of heterozygosity (LOH) and showed that promoter methylation reduced reporter gene activity by a reporter assay. These data suggested that methylation was an epigenetic mechanism leading to WT1 silencing and that the expression-reduced allele by hypermethylation combined with LOH was consistent with the revised two-hit model. In addition, as the beta-catenin mutation is frequently associated with the WT1 mutation, the association of WT1 silencing with the beta-catenin mutation was also investigated. beta-catenin mutated in only one WT without WT1 silencing, suggesting that the beta-catenin mutation was not associated with the reduction of WT1 expression. (+info)
The long (LINEs) and the short (SINEs) of it: altered methylation as a precursor to toxicity.
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Although once thought of as "junk" DNA, the importance of interspersed elements in the genome has become increasingly appreciated in recent years. In a broad sense these are collectively referred to as transposable elements, which encompass both transposons and retrotransposons. The latter include long interspersed nuclear elements (LINEs) and short interspersed nuclear elements (SINEs). Expression of these elements leads to genetic instability. Therefore, it is important that they remain transcriptionally silenced, and DNA methylation plays a key role in this regard. A framework for understanding the possible interplay between altered DNA methylation, an epigenetic change, and mutational events is presented. A case is made as to how retrotransposable elements, specifically LINEs and SINEs, are likely to emerge as key players in furthering our understanding of mechanisms underlying a variety of toxicities, including carcinogenesis but not limited to this endpoint. (+info)
Epigenetic regulation of proprotein convertase PACE4 gene expression in human ovarian cancer cells.
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Proprotein convertases (PC) are a family of serine endoproteases that play important roles in regulating cell function by converting proproteins to biologically active molecules. Several lines of evidence suggest that overexpression of PCs contributes to tumor formation and progression in various types of cancer. In this study, we examined PC expression in six normal ovarian surface epithelium (OSE) cultures, nine primary ovarian cancer (OC) cultures, and three established OC cell lines (Hey, HeyC2, and OCC-1). Our results show that furin and PC7 expression in OC cells was comparable to that in normal OSE. However, PACE4 expression was greatly reduced in all OC samples studied. PACE4 promoter activity was measured in HeyC2 and OCC-1 cells using transiently transfected luciferase reporter plasmids. Both cell lines supported PACE4 promoter activity, showing that the transcription factors critical for PACE4 expression are present in OC cells. The observation that established OC cell lines have reduced PACE4 expression, but maintained the ability to support PACE4 promoter activity, led to the hypothesis that reduced expression may be due to epigenetic modification of the PACE4 gene, such as DNA methylation and histone deacetylation. Methylation analysis of 79 CpG dinucleotides within the PACE4 promoter and exon I (-196/+340) revealed that the percentage of methylated cytosine nucleotides was 8-9% in normal OSE, but 58-93% in OC cells. Treatment with the demethylating agent 5-aza-2'-deoxycytidine and/or the histone deacetylase inhibitor trichostatin A greatly increased PACE4 expression in OC cells. These data suggest that the reduction of PACE4 expression in OC cells is caused, in part, by DNA hypermethylation and histone deacetylation. (+info)
Molecular biology of prostate cancer.
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In spite of progress in diagnosis and treatment, prostate cancer has become one of the most frequent lethal cancers in males in many Western industrialized countries. Research on the molecular biology of prostate cancer is expected to reveal those aspects of Western lifestyle contributing to its high incidence with the aims of improving prevention, distinguishing slow-growing from aggressive clinically relevant cancers, and providing targets for treatment, particularly of locally advanced and of metastatic disease. Traditionally, prostate cancer research focused on androgens. More recently, tumour suppressors and proto-oncogenes important in other human cancers have been intensely investigated. Current approaches include the search for genes mutated in familial cases, identification of recurrent chromosomal alterations and their associated potential tumour suppressor genes, determination of gene expression profiles characterizing tumour stages and subclasses, and elucidation of the importance of epigenetic alterations. Results from such studies have begun to be translated into the clinic. Further successful transfer of results from molecular biology to the clinic will, however, require integration of the amassed molecular data into a biological framework model of prostate carcinoma. (+info)