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At least 55 records · Page 3Linked to original sources
PubMed2004

Hypermethylation of the GATA genes in lung cancer.

PURPOSE: In lung cancer, DNA hypermethylation is known to be a common event. EXPERIMENTAL DESIGN: Gene expression and methylation status of GATA-4, GATA-5, and GATA-6 were analyzed with cell lines and primary human lung cancers. Methylation profiles of primary lung cancers were analyzed and correlated with clinical as well as histopathological data. RESULTS: Complete methylation was detected by methylation-specific PCR for both GATA-4 and GATA-5 in four cell lines (H358, DMS-53, A549, and H1299), all of which had no expression by reverse transcription-PCR analysis. Demethylation with 5-aza-2'deoxycytidine restored expression in each case. GATA-6 was ubiquitously expressed in all of the six cell lines. GATA-4 bisulfite sequencing revealed complete methylation of the GATA-4 promoter in H358 cells, correlating well with its lack of expression at the mRNA level. Only a few CpG sites showed methylation by bisulfite sequencing within the GATA-4 promoter in a cell line that expressed the gene. In 63 cases of primary lung cancers, GATA-4 and GATA-5 promoter methylation was detected in (42 of 63) 67% and (26 of 63) 41%, respectively. GATA-6 remained unmethylated both in cell lines and primary tumors. Six autopsy specimens of normal lung tissue showed no aberrant promoter hypermethylation for the GATA genes. Correlation of concomitant GATA-4 and GATA-5 methylation with clinicopathological parameters only found a statistically significant increase in methylation frequency with increasing patient age (P < 0.001). CONCLUSIONS: These epigenetic changes in the GATA genes in lung cancer are tumor-specific, relate to the loss of GATA gene expression, and occur increasingly in the elderly.

Adenocarcinoma↗
PubMed2004

Loss of estrogen receptor-alpha expression is associated with hypermethylation near its ATG start codon in gastric cancer cell lines.

The proportion of gastric cancers positive for estrogen receptor (ER)-alpha expression is reported to be between 0-67%, depending upon the study. The role of ER-alpha in gastric carcinogenesis is unclear. The ER-alpha gene is located at chromosome 6q25.1, and the long arm of chromosome 6 has been known as a site with frequent loss of heterozygosity (LOH) in gastric cancer. ER expression is linked to suppression of cell proliferation in vitro. Epigenetic inactivation might explain the loss of ER-alpha gene expression in gastric cancer. Given there is no information available regarding the methylation status of the ER-alpha gene promoter region in gastric cancer, we investigated such methylation in 13 gastric cancer cell lines. Western blot analysis, reverse transcription-polymerase chain reaction (PCR), methylation-specific PCR (MS-PCR) and bisulfite sequencing analyses were used. ER-alpha protein was not detected in any cell line, although ER-alpha mRNA was detected in 1 of 13 gastric cancer cell lines. MS-PCR and bisulfite sequencing showed all 13 gastric cancer cell lines had methylated CpG regions in their ER-alpha gene promoters. In conclusion, inactivation of ER-alpha gene expression in gastric cancer cell lines appears associated with CpG island methylation near the TGA initiation codon of the ER-alpha gene.

Base Sequence↗
PubMed2004

Isolation and characterization of a novel pituitary tumor apoptosis gene.

To determine mechanisms for pituitary neoplasia we used methylation-sensitive arbitrarily primed-PCR to isolate novel genes that are differentially methylated relative to normal pituitary. We report the isolation of a novel differentially methylated chromosome 22 CpG island-associated gene (C22orf3). Sodium bisulfite sequencing of pooled tumor cohorts, used in the isolation of this gene, showed that only a proportion of the adenomas within the pools were methylated; however, expression analysis by quantitative RT-PCR of individual adenoma irrespective of subtype showed the majority (30 of 38; 79%) failed to express this gene relative to normal pituitary. Sodium bisulfite sequencing of individual adenomas showed that 6 of 30 (20%) that failed to express pituitary tumor apoptosis gene (PTAG) were methylated; however, genetic change as determined by loss of heterozygosity and sequence analysis was not apparent in the remaining tumors that failed to express this gene. In those cases where the CpG island of these genes was methylated it was invariably associated with loss of transcript expression. Enforced expression of C22orf3 in AtT20 cells had no measurable effects on cell proliferation or viability; however, in response to bromocriptine challenge (10-40 microm) cells expressing this gene showed a significantly augmented apoptotic response as determined by both acridine orange staining and TUNEL labeling. The apoptotic response to bromocriptine challenge was inhibited in coincubation experiments with the general caspase inhibitor z-VAD-fmk. In addition, in time course experiments, direct measurement of active caspases by fluorochrome-labeled inhibition of caspases, showed an augmented increase (approximately 2.4 fold) in active caspases in response to bromocriptine challenge in cells expressing C22orf3 relative to those harboring an empty vector control. The pituitary tumor derivation and its role in apoptosis of this gene led us to assign the acronym PTAG to this gene and its protein product. The ability of cells, showing reduced expression of PTAG, to evade or show a blunted apoptotic response may underlie oncogenic transformation in both the pituitary and other tumor types.

Adenoma↗
PubMed2005

Hypermethylation of the prostacyclin synthase (PTGIS) promoter is a frequent event in colorectal cancer and associated with aneuploidy.

Inactivation of specific tumor suppressor genes by transcriptional silencing associated with hypermethylation of the promoter is a common event in cancer. We have applied the amplification of intermethylated sites (AIMS) technique to a 100 human colorectal cancers and seven cell lines to identify recurrent alterations that may unveil silenced tumor suppressor genes. Bisulfite sequencing was used to confirm differential DNA methylation results. Gene expression analysis was performed by real-time RT-PCR. An AIMS band recurrently displayed in tumors but not in normal tissues was isolated and identified as part of the CpG island of the prostacyclin synthase (PTGIS) gene promoter. PTGIS promoter was hypermethylated in 43 out of 100 colorectal cancers and in all cell lines. Bisulfite sequencing and clonal analysis confirmed the results obtained by AIMS and demonstrated biallelic hypermethylation of PTGIS promoter. Hypermethylation of the PTGIS promoter was associated with diminished gene expression, that was restored after treatment with demethylating and histone deacetylases inhibitor agents. PTGIS hypermethylation was associated with aneuploidy and p53 mutations. In the adjusted model, PTGIS methylation, but not p53 mutation, maintained the association with aneuploidy. We conclude that epigenetic inactivation of the PTGIS gene is a recurrent alteration in colorectal carcinogenesis.

Adenoma↗
PubMed2004

Mapping chromatin structure in vivo using DNA methyltransferases.

Cytosine-5 DNA methyltransferases (C5 DMTases) are effective reagents for analyzing chromatin and footprinting DNA-bound factors in vivo. Cytosine methylation in accessible regions is assayed positively by the PCR-based technique of bisulfite sequencing. In this article, we outline two complementary uses for the DNA methyltransferase CviPI (M.CviPI, GC specificity) in probing chromatin organization. First, we describe the use of the naturally occurring, free enzyme as a diffusible probe to map changes in nucleosome structure and to footprint factor interactions at cis-regulatory sequences. In a second application, termed targeted gene methylation (TAGM), the DMTase is targeted via in-frame fusion to a DNA-binding factor. The rapid accumulation of DNA methylation enables highly sensitive detection of factor binding. Both strategies can be applied with any C5 DMTase, such as M.SssI, which also possesses a short-recognition specificity (CG). A description of methods for constructing C5 DMTase-expressing strains of Saccharomyces cerevisiae and analyzing chromatin regions is provided. We also include comprehensive protocols for the isolation and bisulfite treatment of genomic DNA as well as the subsequent bisulfite sequencing steps. Data demonstrating the efficacy of both DMTase probing techniques, theoretical considerations, and experimental analyses are presented at GAL1 and PHO5.

Acid Phosphatase↗
PubMed1998

Selective variegated methylation of the p15 CpG island in acute myeloid leukemia.

Both p15 and p16 are tumor suppressor genes that have 5' CpG islands; aberrant cytosine methylation of these islands has been associated with silencing of their expression. Deoxycytidine kinase (dCK) converts prodrugs to their cytotoxic form, has a 5' CpG island and is a candidate gene for inactivation by hypermethylation. In our study, we used sodium bisulfite sequencing to generate high resolution maps of 5-methylcytosine in the CpG islands associated with p15, p16 and dCK in normal human bone marrow (BM), peripheral blood lymphocytes (PBL) and cytosine arabinoside (ara-C)-resistant acute myeloid leukemia (AML) patients, and established human hematopoietic tumor cell lines. In normal cells the p15, p16 and dCK CpG islands were largely unmethylated. The p16 and dCK CpG islands were also unmethylated in the 8 AML specimens. In contrast, the p15 CpG island was aberrantly methylated in 6 of the 8 AML specimens. Furthermore, bisulfite sequencing revealed that the p15 CpG island is heterogeneously methylated in AML, with large intra-individual and inter-individual variability.

Base Sequence↗
PubMed2006

Aberrant methylation of the eyes absent 4 gene in ulcerative colitis-associated dysplasia.

BACKGROUND & AIMS: This study explored the eyes absent 4 (EYA4) gene promoter methylation in noncolitic colorectal tissues and assessed its discrimination for neoplasia in chronic ulcerative colitis (CUC). METHODS: The methylation status of noncolitic specimens was confirmed by direct bisulfite sequencing. Methylation-specific polymerase chain reaction (MSP) primers were designed to evaluate colorectal tissues, including 50 noncolitic patients comprising 24 normal epithelia, 14 polyps, and 12 cancers. The assay was tested on tissues from 67 CUC patients including 31 surveillance neoplasia-positive patients and nonneoplastic controls including 22 CUC surveillance-negative and 14 CUC short-disease duration. Remote colonic tissue was included from each of 27 of the 31 CUC neoplasia cases. The expression of EYA4 was quantified in cell lines by use of reverse-transcription polymerase chain reaction. RESULTS: Within noncolitic tissues, bisulfite sequencing showed EYA4 promoter hypermethylation in 80% (8 of 10) of colorectal cancers but in none (0 of 9) of the normal tissues. MSP was positive in 81% (21 of 26) of cancers and polyps and in only 4% (1 of 14) of normal mucosa. In CUC, MSP was positive in 81% (25 of 31) of neoplastic cases but in none (0 of 36) of the nonneoplastic controls. RNA expression was decreased in methylated compared with unmethylated cell lines (P < .001). Treatment with 5-Aza-2'-deoxycytidine (DAC)/Trichostatin (TSA) increased the overall messenger RNA expression (P = .005). CONCLUSIONS: The EYA4 gene promoter is hypermethylated commonly in sporadic and colitic neoplasia and may be associated with gene silencing. EYA4 methylation represents a candidate marker for CUC surveillance.

Azacitidine↗
PubMed2004

Aberrant promoter hypermethylation of the death-associated protein kinase gene is early and frequent in murine lung tumors induced by cigarette smoke and tobacco carcinogens.

Loss of expression of the death-associated protein (DAP)-kinase gene by aberrant promoter methylation may play an important role in cancer development and progression. The purpose of this investigation was to determine the commonality for inactivation of the DAP-kinase gene in adenocarcinomas induced in mice by chronic exposure to mainstream cigarette smoke, the tobacco carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and vinyl carbamate, and the occupational carcinogen methylene chloride. The timing for inactivation was also determined in alveolar hyperplasias that arise in lung cancer induced in the A/J mouse by NNK. The DAP-kinase gene was not expressed in three of five NNK-induced lung tumor-derived cell lines or in a spontaneously arising lung tumor-derived cell line. Treatment with 5-aza-2'-deoxycytidine restored expression; dense methylation throughout the DAP-kinase CpG island detected by bisulfite sequencing supported methylation as the inactivating event in these cell lines. Methylation-specific PCR detected inactivation of the DAP-kinase gene in 43% of tumors associated with cigarette smoke, a frequency similar to those reported in human non-small cell lung cancer. In addition, DAP-kinase methylation was detected in 52%, 60%, and 50% of tumors associated with NNK, vinyl carbamate, and methylene chloride, respectively. Methylation was observed at similar prevalence in both NNK-induced hyperplasias and adenocarcinomas (46% versus 52%), suggesting that inactivation of this gene is one pathway for tumor development in the mouse lung. Bisulfite sequencing of both premalignant and malignant lesions revealed dense methylation, substantiating that this gene is functionally inactivated at the earliest histological stages of adenocarcinoma development. This study is the first to use a murine model of cigarette smoke-induced lung cancer and demonstrate commonality for inactivation by promoter hypermethylation of a gene implicated in the development of this disease in humans.

Animals↗
PubMed2003

Carcinogen exposure differentially modulates RAR-beta promoter hypermethylation, an early and frequent event in mouse lung carcinogenesis.

The retinoic acid receptor beta (RAR-beta) gene encodes one of the primary receptors for retinoic acid, an important signaling molecule in lung growth, differentiation and carcinogenesis. RAR-beta has been shown to be down-regulated by methylation in human lung cancer. We have used previously lung tumors induced in mice to evaluate the timing and effect of specific carcinogen exposures on targeting genes altered in human lung cancer. These studies were extended to characterize the role of methylation of the RAR-beta gene in murine lung cancers. After treatment with the demethylating agent 5-aza-2'-deoxycytidine (DAC), RAR-beta was re-expressed in silenced cell lines or expressed at a higher rate than without DAC, supporting methylation as the inactivating mechanism. Bisulfite sequencing detected dense methylation in the area of the CpG island that contained the 5' untranslated region and the first translated exon in non-expressing cell lines, compared with minimal and heterogeneous methylation in normal mouse lung. Methylation-specific PCR revealed that this gene is targeted differentially by carcinogen exposures with the detection of methylated alleles in virtually all primary tumors associated with cigarette smoke or 4-methylnitrosamino-1-(3-pyridyl)-butanone (NNK) in contrast to half of tumors induced by methylene chloride or vinyl carbamate. RAR-beta methylation was also detected in 54% of preneoplastic hyperplasias induced by treatment with NNK. Bisulfite sequencing of both premalignant and malignant lesions detected dense methylation in the same area observed in cell lines, substantiating that this gene is functionally inactivated at the earliest histologic stage of adenocarcinoma development. These studies demonstrate that aberrant methylation of RAR-beta is an early and common alteration in murine lung tumors induced by several environmentally relevant exposures.

Adenocarcinoma↗
PubMed2004

Epigenetic CRBP downregulation appears to be an evolutionarily conserved (human and mouse) and oncogene-specific phenomenon in breast cancer.

BACKGROUND: The cellular retinol binding protein I gene (CRBP) is downregulated in a subset of human breast cancers and in MMTV-Myc induced mouse mammary tumors. Functional studies suggest that CRBP downregulation contributes to breast tumor progression. What is the mechanism underlying CRBP downregulation in cancer? Here we investigated the hypothesis that CRBP is epigenetically silenced through DNA hypermethylation in human and mouse breast cancer. RESULTS: Bisulfite sequencing of CRBP in a panel of 6 human breast cancer cell lines demonstrated that, as a rule, CRBP hypermethylation is closely and inversely related to CRBP expression and identified one exception to this rule. Treatment with 5-azacytidine, a DNA methyltransferase inhibitor, led to CRBP reexpression, supporting the hypothesis that CRBP hypermethylation is a proximal cause of CRBP silencing. In some cells CRBP reexpression was potentiated by co-treatment with retinoic acid, an inducer of CRBP, and trichostatin A, a histone deacetylase inhibitor. Southern blot analysis of a small panel of human breast cancer specimens identified one case characterized by extensive CRBP hypermethylation, in association with undetectable CRBP mRNA and protein. Bisulfite sequencing of CRBP in MMTV-Myc and MMTV-Neu/NT mammary tumor cell lines extended the rule of CRBP hypermethylation and silencing (both seen in MMTV-Myc but not MMTV-Neu/NT cells) from human to mouse breast cancer and suggested that CRBP hypermethylation is an oncogene-specific event. CONCLUSION: CRBP hypermethylation appears to be an evolutionarily conserved and principal mechanism of CRBP silencing in breast cancer. Based on the analysis of transgenic mouse mammary tumor cells, we hypothesize that CRBP silencing in human breast cancer may be associated with a specific oncogenic signature.

Animals↗
PubMed2026

Integrative analysis of transcriptome and DNA methylome dynamics during caudal fin regeneration in silver pomfret (Pampus argenteus).

Caudal fin regeneration in teleost fish is a complex, multi-stage process involving coordinated molecular and cellular changes. While the role of epigenetic regulation particularly DNA methylation has been studied in model freshwater species such as zebrafish, its contribution to regeneration in marine teleosts remains largely unexplored. In this study, we integrated transcriptomic and DNA methylomic data to characterize the temporal dynamics of gene expression and methylation during caudal fin regeneration in the silver pomfret (Pampus argenteus). Using RNA-sequencing and reduced representation bisulfite sequencing (RRBS) at three biologically critical time points 1, 3, and 7&#xa0;days post-amputation (dpa), we characterized the spatiotemporal molecular landscape of caudal fin regeneration. These time points capture the key transitional phases of wound healing and inflammation (1 dpa), blastema formation and progenitor proliferation (3 dpa), and regenerative outgrowth with tissue remodeling (7 dpa), enabling robust detection of the major molecular programs underlying epimorphic regeneration. Concurrently, CG-methylome analysis identified thousands of dynamically changing differentially methylated regions (DMRs). A strong global inverse correlation was observed between promoter methylation and gene expression. Integrative analysis pinpointed key regeneration genes (fgf20a, msxb, sox9b) whose expression was associated with dynamic methylation changes in their promoters or gene bodies. We conclude that DNA methylation is a dynamic and key regulatory layer that acts in concert with transcriptional reprogramming to coordinate tissue regeneration, providing new insights into the epigenetic mechanisms underlying complex regenerative processes in teleosts.

Animals↗
PubMed2006

Bisulfite-modified target DNA array for aberrant methylation analysis.

Aberrant DNA methylation of CpG islands is among the earliest and most frequent alterations in cancer. It is of great importance to develop simple and high-throughput methods of methylation analysis for earlier cancer diagnosis or the detection of recurrence. In this study, bisulfite-modified target DNA arrays were prepared on positively charged nylon membrane with two different procedures: fixing PCR products and fixing genomic DNA. First, a bisulfite PCR product array was prepared through fixing PCR products amplified in bisulfite sequencing primers from the bisulfite-modified genomic DNA of different clinical samples on membrane. Furthermore, bisulfite-modified genomic DNA of the different samples was directly fixed on membrane to fabricate bisulfite genomic DNA arrays. The two kinds of arrays were hybridized by probes labeled with digoxigenin, and the hybridization signals were obtained through chemiluminescent detection. The methylation statuses of the IGFBP7 gene for breast tumor and normal tissue samples and for normal human blood cell samples were detected successfully by the two procedures. It was shown that the methods are reliable and sensitive and that they have high potential in screening molecular methylation markers from a large number of clinical samples.

Base Sequence↗
PubMed2005

Active demethylation of individual genes in intracytoplasmic sperm injection rabbit embryos.

Intracytoplasmic sperm injection (ICSI), as an assisted reproduction technique, has been widely used in animal and human. However, its possible effect on epigenetic changes has not been well studied. To investigate whether ICSI can induce aberrant DNA methylation changes in rabbit preimplantation embryos, we examined the methylation status of the SP-A promoter region and the satellite sequence Rsat IIE by bisulfite-sequencing technology. The SP-A promoter region was extensively demethylated before the first round of DNA replication commences, and the unmethylated status was maintained until morula when dynamic remethylation occurred. A similar but more moderate demethylation process was observed in satellite sequence Rsat IIE. These results are in contrast with the previous reports of no active demethylation in normal rabbit embryos, suggesting that the active demethylation we observed may be induced by ICSI.

Animals↗
PubMed2025

Expression regulation network in papillae of sea cucumbers: Whole-transcriptome and DNA methylation datasets.

To elucidate the expression regulation network of papilla size of sea cucumbers (Apostichopus japonicus), the whole-transcriptome and DNA methylome datasets of different sizes of papillae in sea cucumbers were generated. Average clean bases of whole-transcriptome (16.35&#x2009;G) and DNA methylome (28.92&#x2009;G) were obtained using RNA sequencing and whole-genome bisulfite sequencing techniques. A total of 3,188 ceRNA networks were also identified including 3,081 long non-coding RNAs (lncRNA)/microRNAs (miRNA)/mRNA networks and 107 circular RNA (circRNA)/miRNA/mRNA networks. Methylome data indicate that there were 3,307 and 3,776 differentially methylated regions (DMRs) with high-level methylation as well as 3,125 and 3,016 DMRs with low-level methylation in big papillae compared to small papillae. The identified DMRs were mainly distributed in introns, promotors, or exons. The whole-transcriptome and DNA methylome datasets generated from this study not only established a robust theoretical foundation (especially from the epigenetic aspect) for elucidating expression regulation network determining papilla size in sea cucumbers but also can be a valuable resource of biomarker mining for papilla appearance-based selective breeding in sea cucumbers.

DNA Methylation↗
PubMed2001

Typical demethylation events in cloned pig embryos. Clues on species-specific differences in epigenetic reprogramming of a cloned donor genome.

We investigated whether a genomic demethylation process occurs in pig preimplantation embryos produced by either normal fertilization or somatic cell nuclear transfer. The methylation status of the centromeric satellite and the PRE-1 short interspersed element (SINE) sequences was characterized using bisulfite-sequencing technology. Typical demethylation processes were identified in these repetitive sequences of the cloned donor genome during cleavage, the patterns of which were similar to the ones detected in fertilized counterparts. These findings are different from previous observations with cloned bovine embryos where various repeated regions of the donor genome exhibited aberrant methylation patterns. Our results indicate that species-specific differences exist in modifying the epigenetic status of cloned donor genomes.

Animals↗