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Methylated
Terms modified by Methylated Selected AbstractsChemInform Abstract: Novel Annulene Dications from Methylated [2.2]Metacyclophane Monoenes and [e]-Ring Benzanellated Dimethyldihydropyrene.CHEMINFORM, Issue 44 2001Kenneth K. Laali Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a "Full Text" option. The original article is trackable via the "References" option. [source] Proteomic analysis of liver cancer cells treated with 5-Aza-2,-deoxycytidine (AZA),DRUG DEVELOPMENT RESEARCH, Issue 1 2009Shujun Bai Abstract 5-Aza-2,-deoxycytidine (AZA) is a potent inhibitor of DNA methylation that exhibits anti-tumor activity in a variety of tumor cells via reactivation of tumor suppressor genes. However, few studies have been done on the biological and clinical significance of AZA in human hepatocellular carcinoma. To identify potential genes that may be aberrantly methylated and confer growth advantage to neoplastic cells and to better understand the molecular mechanism(s) underlying AZA anti-tumor activity, a proteomics approach was used to annotate global gene expression changes of HepG2 cell line pre- and post-treatment with AZA. A total of 56 differentially expressed proteins were identified by 2D gel analysis, 48 of which were up-regulated while the remaining 8 were down regulated. Among the identified proteins, eight of these showed marked changed proteins, including seven up-regulated proteins: glutathione S-transferase P, protein DJ-1, peroxiredoxin-2, UMP-CMP kinase, cytochrome c-type heme lyase, enhancer of rudimentary homolog, profilin-1, and one down-regulated protein, heat-shock protein ,,1. The possible implication of these proteins in hepatocarcinogenesis is discussed. We tested two up-regulated proteins, glutathione S-transferase P and peroxiredoxin-2, using RT-PCR and their expression was consistent with the results obtained in the protein level. Both of these genes were methylated when methylation-specific PCR was used against their promoter regions. Following treatment with AZA, the gene promoter regions were found to be unmethylated, concomitant with overexpression of the proteins compared to HepG2 cells without treatment. These data provide useful information in evaluating the therapeutic potential of AZA for the treatment of HCC. Drug Dev Res 69, 2009. © 2009 Wiley-Liss, Inc. [source] Neuronal and vascular localization of histamine N-methyltransferase in the bovine central nervous systemEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2000Masahiro Nishibori Abstract Histamine N-methyltransferase (HMT) (EC 2.1.1.8) plays a crucial role in the inactivation of the neurotransmitter histamine in the CNS. However, the localization of HMT remains to be determined. In the present study, we investigated immunohistochemical localization of HMT in the bovine CNS using a polyclonal antibody against bovine HMT. The HMT-like immunoreactivity was observed mainly in neurons. Strongly immunoreactive neurons were present in the oculomotor nucleus and ruber nucleus in the midbrain, the facial nucleus in the pons, the dorsal vagal nucleus and hypoglossal nucleus in the medulla oblongata and in the anterior horn as well as intermediolateral zone of the spinal cord. Intermediately immunoreactive neurons were present in the piriform cortex and the inferior olivary nucleus. The grey matter of the forebrain regions was diffusely and faintly stained. In the cerebellum and the striatum, the nerve fibres in the white matter were positive. The tuberomammillary nucleus, where histaminergic neurons are present, were weakly positive. The other immunoreactive structures in the CNS were blood vessels. Almost all of the blood vessel walls, irrespective of whether they were arterial or venous, were variably stained. The glial fibrillary acidic protein- (GFAP-) immunoreactive astrocytes were not stained. These findings indicated that histamine released from histaminergic nerve terminals or varicose fibres is methylated mainly in postsynaptic or extrasynaptic neurons rather than in astrocytes. The localization of HMT in the blood vessel wall may mean that blood-borne histamine and histamine released from mast cells associated with the blood vessels are catabolized in this structure. [source] CpG methylation of the CENP-B box reduces human CENP-B bindingFEBS JOURNAL, Issue 1 2005Yoshinori Tanaka In eukaryotes, CpG methylation is an epigenetic DNA modification that is important for heterochromatin formation. Centromere protein B (CENP-B) specifically binds to the centromeric 17 base-pair CENP-B box DNA, which contains two CpG dinucleotides. In this study, we tested complex formation by the DNA-binding domain of CENP-B with methylated and unmethylated CENP-B box DNAs, and found that CENP-B preferentially binds to the unmethylated CENP-B box DNA. Competition analyses revealed that the affinity of CENP-B for the CENP-B box DNA is reduced nearly to the level of nonspecific DNA binding by CpG methylation. [source] Chromatin structure of the bovine Cyp19 promoter 1.1FEBS JOURNAL, Issue 5 2001DNA hypomethylation correlate with placental expression, DNaseI hypersensitive sites Expression of the Cyp19 gene, encoding aromatase cytochrome P450, is driven by several tissue-specific promoters. The underlying mechanisms of this complex regulation have not yet been elucidated in detail. In the present report we investigate a possible link between chromatin structure and tissue-specific regulation of the bovine Cyp19 gene. We analysed the DNA methylation status and mapped DNaseI hypersensitive sites in the region encompassing the Cyp19 promoter 1.1 (P1.1) which controls Cyp19 expression in the bovine placenta. We show that P1.1 is hypomethylated in placental cotyledons (foetal layer) whereas it is methylated in placental caruncles (maternal layer), testis and corpus luteum. Furthermore, two placenta-specific DNaseI hypersensitive sites, HS1 and HS2, were observed within P1.1. Both DNA hypomethylation and the presence of DNaseI hypersensitive sites correlate with transcriptional activity of P1.1. Sequence analysis of hypersensitive sites revealed potential cis -regulatory elements, an E-box in HS1 and a trophoblast-specific element-like sequence in HS2. It could be demonstrated by electrophoretic mobility shift assays that both sequence motifs are specific targets for placenta-derived nuclear factors. In conclusion, observed tissue-specific differences of the chromatin structure which correlate with tissue-specific promoter activity suggest that chromatin might be an important regulator of aromatase expression in cattle. [source] PCNA clamp facilitates action of DNA cytosine methyltransferase 1 on hemimethylated DNAGENES TO CELLS, Issue 10 2002Tetsuo Iida Background: Proliferating cell nuclear antigen (PCNA) is a ring-shaped protein known as a processivity factor of DNA polymerase ,. In addition to this role, PCNA interacts with a number of other proteins to increase their local concentration at replicated DNA sites. DNA cytosine methyltransferase 1 (Dnmt1), which preserves epigenetic signals by completing the methylation of hemimethylated DNA after DNA replication, has been indicated as one of these PCNA binding proteins by a previous work. However, the molecular mechanisms and functional significance of their association have not yet been studied. Results: Dnmt1 can be readily isolated from nuclear extracts by PCNA affinity chromatography. Studies of the interactions between the two proteins demonstrate that the N-terminal region of Dnmt1, which contains a typical PCNA binding motif, has core PCNA binding activity, and that the remaining portion of the protein exerts a negative influence on the interaction of Dnmt1 with PCNA. The affinity of Dnmt1 for DNA is much higher for DNA bound by PCNA than for free DNA. Furthermore, DNA methylation assays with hemimethylated DNA as a substrate revealed that PCNA clamp-bound DNA is methylated more efficiently by Dnmt1 than is free DNA. Conclusion: These results provide the first biochemical evidence that physical interactions between PCNA and Dnmt1 facilitate the methylation of newly neplicated DNA, on which PCNA remains associated as a functional clamp. [source] Competition between the replication initiator DnaA and the sequestration factor SeqA for binding to the hemimethylated chromosomal origin of E. coli in vitroGENES TO CELLS, Issue 11 2000Aziz Taghbalout Following replication initiation, the replication origin (oriC) in Escherichia coli enters a hemimethylated state at Dam methylation sites which are recognized by the SeqA protein. SeqA binds preferentially to hemimethylated GATC sequences of DNA in vitro. SeqA is essential for the synchronous initiation of chromosome replication from oriC copies in vivo. We show that: (i) purified SeqA binds AT-rich and 13-mers regions and two DnaA boxes, R1 and M, of hemimethylated oriC. (ii) SeqA inhibits the in vitro replication of a hemimethylated oriC plasmid more efficiently than the fully methylated, (iii) SeqA inhibits competitive binding of DnaA protein to the regions of the hemimethylated oriC plasmid, explaining the mechanism of its inhibitory effect. The inhibition of DnaA binding by SeqA also occurs efficiently on a small hemimethylated oriC fragment containing both R1 and M DnaA boxes, but not the 13-mer region. SeqA binds strongly the long region from the AT-rich region to the M DnaA box of the hemimethylated oriC DNA and releases DnaA molecules from the long region. [source] Aberrant methylation impairs low density lipoprotein receptor-related protein 1B tumor suppressor function in gastric cancerGENES, CHROMOSOMES AND CANCER, Issue 5 2010Yen-Jung Lu DNA methylation plays a significant role in tumor progression. In this study, we used CpG microarray and differential methylation hybridization approaches to identify low density lipoprotein receptor-related protein 1B (LRP1B) as a novel epigenetic target in gastric cancer. LRP1B was hypermethylated in four gastric cancer cell lines, and low LRP1B mRNA expression was associated with high methylation levels in gastric cancer cell lines. Addition of a DNA methylation inhibitor (5-Aza-dC) restored the mRNA expression of LRP1B in these cell lines, indicating that DNA methylation is involved in regulating LRP1B expression. In 45 out of 74 (61%) clinical samples, LRP1B was highly methylated; LRP1B mRNA expression was significantly lower in 15 out of 19 (79%, P < 0.001) gastric tumor tissues than in corresponding adjacent normal tissues. In addition, ectopic expression of mLRP1B4 in gastric cancer cell lines suppressed cell growth, colony formation and tumor formation in nude mice. These results collectively indicate that LRP1B is a functional tumor suppressor gene in gastric cancer and that is regulated by DNA methylation. © 2010 Wiley-Liss,Inc. [source] The MLH1 ,93 G>A promoter polymorphism and genetic and epigenetic alterations in colon cancerGENES, CHROMOSOMES AND CANCER, Issue 10 2008Wade S. Samowitz The MLH1 ,93 G>A promoter polymorphism has been reported to be associated with an increased risk of microsatellite unstable colorectal cancer. Other than microsatellite instability, however, the genetic and most epigenetic changes of tumors associated with this polymorphism have not been studied. We evaluated associations between the ,93 G>A polymorphism and CpG island methylator phenotype (CIMP), BRAF V600E mutations, and MLH1 methylation in tumors from a sample of 1,211 individuals with colon cancer and 1,968 controls from Utah, Northern California, and Minnesota. The ,93 G>A polymorphism was determined by the five prime nuclease assay. CIMP was determined previously by methylation-specific PCR of CpG islands in MLH1, methylated in tumors (MINT)1, MINT2, MINT31, and CDKN2A (p16). The BRAF V600E mutation was determined by sequencing exon 15. The MLH1 ,93 G>A promoter polymorphism was associated with CIMP (odds ratio (OR) 3.44, 95% confidence interval (CI) 1.85, 6.42), MLH1 methylation (OR 4.16, 95%CI 2.20, 7.86), BRAF mutations (OR 4.26, 95%CI 1.83, 9.91), and older age at diagnosis (OR 3.65, 95%CI 2.08, 6.39) in microsatellite unstable tumors. These associations were not observed in stable tumors. Increased age at diagnosis and tumor characteristics of microsatellite unstable tumors associated with MLH1 ,93 G>A suggests the polymorphism is acting at a relatively late stage of colorectal carcinogenesis to drive CIMP+ tumors down the microsatellite instability pathway. © 2008 Wiley-Liss, Inc. [source] TCL1 is activated by chromosomal rearrangement or by hypomethylationGENES, CHROMOSOMES AND CANCER, Issue 4 2001Martin R. Yuille TCL1 is an oncogene activated by recurrent reciprocal translocations at chromosome segment 14q32.1 in the most common of the mature T-cell malignancies, T-cell prolymphocytic leukemia. It acts to transport Akt1 to the nucleus and enhance Akt1's serine-threonine kinase activity. TCL1 is also expressed in the B-cell malignancy, Burkitt's lymphoma (BL). However, 14q32.1 breakpoints have not been detected in BL, and we therefore investigated in more detail how expression was activated. No evidence for rearrangement near TCL1 was found in BL. Instead, a NotI site adjacent to the TATA box in the TCL1 promoter was found to be unmethylated. By contrast, tumor cell lines not expressing TCL1 were fully methylated at this NotI site, while normal somatic cells were hemimethylated. We also found that TCL1 was expressed in B-cell chronic lymphocytic leukemia (CLL) and the related disorder splenic lymphoma with villous lymphocytes (unlike in normal mature B-cells), and that the NotI site was unmethylated on both alleles. This correlation of repression and methylation was tested in vitro. When cells with both alleles methylated at the NotI site were demethylated, TCL1 expression was induced. These data provide evidence that in mature B-cell malignancies there is an alternative mechanism of TCL1 activation that apparently involves loss of methylation of one promoter allele. We discuss the significance of this for CLL tumorigenesis and for genomewide hypomethylation in CLL. © 2001 Wiley-Liss, Inc. [source] DNA methylation variation in cloned miceGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 2 2001Jun Ohgane Abstract Summary: Mammalian cloning has been accomplished in several mammalian species by nuclear transfer. However, the production rate of cloned animals is quite low, and many cloned offspring die or show abnormal symptoms. A possible cause of the low success rate of cloning and abnormal symptoms in many cloned animals is the incomplete reestablishment of DNA methylation after nuclear transfer. We first analyzed tissue-specific methylation patterns in the placenta, skin, and kidney of normal B6D2F1 mice. There were seven spots/CpG islands (0.5% of the total CpG islands detected) methylated differently in the three different tissues examined. In the placenta and skin of two cloned fetuses, a total of four CpG islands were aberrantly methylated or unmethylated. Interestingly, three of these four loci corresponded to the tissue-specific loci in the normal control fetuses. The extent of aberrant methylation of genomic DNA varied between the cloned animals. In cloned animals, aberrant methylation occurred mainly at tissue-specific methylated loci. Individual cloned animals have different methylation aberrations. In other words, cloned animals are by no means perfect copies of the original animals as far as the methylation status of genomic DNA is concerned. genesis 30:45,50, 2001. © 2001 Wiley-Liss, Inc. [source] Chemoselectivity of the Reactions of Diazomethanes with 5-Benzylidene-3-phenylrhodanineHELVETICA CHIMICA ACTA, Issue 9 2009Martin Abstract The reactions of 5-benzylidene-3-phenylrhodanine (2; rhodanine=2-thioxo-1,3-thiazolidin-4-one) with diazomethane (7a) and phenyldiazomethane (7b) occurred chemoselectively at the exocyclic CC bond to give the spirocyclopropane derivatives 9 and, in the case of 7a, also the C -methylated products 8 (Scheme,1). In contrast, diphenyldiazomethane (7c) reacted exclusively with the CS group leading to the 2-(diphenylmethylidene)-1,3-thiazolidine 11via [2+3] cycloaddition and a ,two-fold extrusion reaction'. Treatment of 8 or 9b with an excess of 7a in refluxing CH2Cl2 and in THF at room temperature in the presence of [Rh2(OAc)4], respectively, led to the 1,3-thiazolidine-2,4-diones 15 and 20, respectively, i.e., the products of the hydrolysis of the intermediate thiocarbonyl ylide. On the other hand, the reactions with 7b and 7c in boiling toluene yielded the corresponding 2-methylidene derivatives 16, 21a, and 21b. Finally, the reaction of 11 with 7a occurred exclusively at the electron-poor CC bond, which is conjugated with the CO group. In addition to the spirocyclopropane 23, the C -methylated 22 was formed as a minor product. The structures of the products (Z)- 8, 9a, 9b, 11, and 23 were established by X-ray crystallography. [source] Benzo[a]heptalenes from Heptaleno[1,2- c]furans.HELVETICA CHIMICA ACTA, Issue 4 2007Abstract It is shown in this ,Part 2' that heptaleno[1,2- c]furans 1 react thermally in a Diels,Alder -type [4+2] cycloaddition at the furan ring with vinylene carbonate (VC), phenylsulfonylallene (PSA), , -(acetyloxy)acrylonitrile (AAN), and (1Z)-1,2-bis(phenylsulfonyl)ethene (ZSE) to yield the corresponding 1,4-epoxybenzo[d]heptalenes (cf. Schemes,1, 5, 6, and 8). The thermal reaction of 1a and 1b with VC at 130° and 150°, respectively, leads mainly to the 2,3- endo -cyclocarbonates 2,3- endo - 2a and - 2b and in minor amounts to the 2,3- exo -cyclocarbonates 2,3- exo - 2a and - 2b. In some cases, the (P*)- and (M*)-configured epimers were isolated and characterized (Scheme,1). Base-catalyzed cleavage of 2,3- endo - 2 gave the corresponding 2,3-diols 3, which were further transformed via reductive cleavage of their dimesylates 4 into the benzo[a]heptalenes 5a and 5b, respectively (Scheme,2). In another reaction sequence, the 2,3-diols 3 were converted into their cyclic carbonothioates 6, which on treatment with (EtO)3P gave the deoxygenated 1,4-dihydro-1,4-epoxybenzo[d]heptalenes 7. These were rearranged by acid catalysis into the benzo[a]heptalen-4-ols 8a and 8b, respectively (Scheme,2). Cyclocarbonate 2,3- endo - 2b reacted with lithium diisopropylamide (LDA) at ,70° under regioselective ring opening to the 3-hydroxy-substituted benzo[d]heptalen-2-yl carbamate 2,3- endo - 9b (Scheme,3). The latter was O -methylated to 2,3- endo -(P*)- 10b. The further way, to get finally the benzo[a]heptalene 13b with MeO groups in 1,2,3-position, could not be realized due to the fact that we found no way to cleave the carbamate group of 2,3- endo -(P*)- 10b without touching its 1,4-epoxy bridge (Scheme,3). The reaction of 1a with PSA in toluene at 120° was successful, in a way that we found regioisomeric as well as epimeric cycloadducts (Scheme,5). Unfortunately, the attempts to rearrange the products under strong-base catalysis as it had been shown successfully with other furan,PSA adducts were unsuccessful (Scheme,4). The thermal cycloaddition reaction of 1a and 1b with AAN yielded again regioisomeric and epimeric adducts, which could easily be transformed into the corresponding 2- and 3-oxo products (Scheme,6). Only the latter ones could be rearranged with Ac2O/H2SO4 into the corresponding benzo[a]heptalene-3,4-diol diacetates 20a and 20b, respectively, or with trimethylsilyl trifluoromethanesulfonate (TfOSiMe3/Et3N), followed by treatment with NH4Cl/H2O, into the corresponding benzo[a]heptalen-3,4-diols 21a and 21b (Scheme,7). The thermal cycloaddition reaction of 1 with ZSE in toluene gave the cycloadducts 2,3- exo - 22a and - 22b as well as 2- exo,3- endo - 22c in high yields (Scheme,8). All three adducts eliminated, by treatment with base, benzenesulfinic acid and yielded the corresponding 3-(phenylsulfonyl)-1,4-epoxybenzo[d]heptalenes 25. The latter turned out to be excellent Michael acceptors for H2O2 in basic media (Scheme,9). The Michael adducts lost H2O on treatment with Ac2O in pyridine and gave the 3-(phenylsulfonyl)benzo[d]heptalen-2-ones 28a and 3- exo - 28b, respectively. Rearrangement of these compounds in the presence of Ac2O/AcONa lead to the formation of the corresponding 3-(phenylsulfonyl)benzo[a]heptalene-1,2-diol diacetates 30a and 30b, which on treatment with MeONa/MeI gave the corresponding MeO-substituted compounds 31a and 31b. The reductive elimination of the PhSO2 group led finally to the 1,2-dimethoxybenzo[a]heptalenes 32a and 32b. Deprotonation experiments of 32a with t -BuLi/N,N,N,,N,-tetramethylethane-1,2-diamine (tmeda) and quenching with D2O showed that the most acid CH bond is HC(3) (Scheme,9). Some of the new structures were established by X-ray crystal-diffraction analyses (cf. Figs.,1, 3, 4, and 5). Moreover, nine of the new benzo[a]heptalenes were resolved on an anal. Chiralcel OD-H column, and their CD spectra were measured (cf. Figs.,8 and 9). As a result, the 1,2-dimethoxybenzo[a]heptalenes 32a and 32b showed unexpectedly new Cotton -effect bands just below 300,nm, which were assigned to chiral exciton coupling between the heptalene and benzo part of the structurally highly twisted compounds. The PhSO2 -substituted benzo[a]heptalenes 30b and 31b showed, in addition, a further pair of Cotton -effect bands in the range of 275,245,nm, due to chiral exciton coupling of the benzo[a]heptalene chromophore and the phenylsulfonyl chromophore (cf. Fig.,10). [source] Epigenetic and genetic alterations of PTEN in hepatocellular carcinomaHEPATOLOGY RESEARCH, Issue 5 2007Li Wang Aim:, To investigate the roles of epigenetic and genetic alterations of the phosphatase and tensin homologue on chromosome 10 gene (PTEN) in carcinogenesis and the development of hepatocellular carcinomas (HCC). Methods:, A total of 56 cases of HCC tissues and six liver cell lines were studied for the expression of PTEN by immunohistochemistry and Western blot analysis. The PTEN gene mutations in exon5 and exon8 were detected by a combination of single-strand conformation polymorphism (SSCP) analysis and DNA sequencing. Methylation-specific PCR (MSP) was used to identify PTEN promoter methylation. Results:, Of the 56 cases of HCC, 24 (42.9%) expressed the PTEN protein. All surrounding liver tissues of the hepatoma (32 cases) were positive for PTEN. Of the six cell lines, three liver cancer cell lines showed a low expression of PTEN. Five mutations of 56 HCC samples were detected. All of them were located at intron4. No mutation was found in exon5 and exon8. After MSP analysis, we found nine cases of PTEN promoter methylation in 56 specimens (16.1%). However, no CpG island of PTEN was found to be methylated in all six liver cell lines. Conclusion:, The level of PTEN protein was altered in part of the HCC. The downregulation of PTEN expression may not be mainly associated with the PTEN mutations, but partly due to PTEN promoter methylation and other epigenetic regulation. [source] Rapid detection of methylation change at H19 in human imprinting disorders using methylation-sensitive high-resolution melting,HUMAN MUTATION, Issue 10 2008Tomasz K. Wojdacz Abstract Beckwith Wiedemann syndrome (BWS) and Russell Silver syndrome (RS) are growth disorders with opposing epimutations affecting the H19/IGF2 imprinting center at 11p15.5. Overgrowth and tumor risk in BWS is caused by aberrant expression of the paternally expressed, imprinted IGF2 gene, occurring as a consequence of mosaic hypermethylation within the imprinting center, or to mosaic paternal uniparental disomy (UPD). RS is characterized by severe intrauterine growth retardation (IUGR). A subset of RS cases were recently shown to have mosaic hypomethylation within the H19/IGF2 imprinting center, predicted to silence paternally expressed IGF2 in early development. Molecular diagnosis for BWS and RS involves methylation analysis of the H19 locus, enabling discrimination of allelic methylation patterns. In this study, methylation-sensitive high-resolution melting analysis (MS-HRM) was used to analyze methylation within the intergenic region of the H19 locus. A total of 36 samples comprising normal control (11), BWS (19), and RS (six) DNA were analyzed in a blinded study and scored as hypermethylated, normal, or hypomethylated. Results were compared with those derived by methylation-sensitive Southern blotting using the restriction enzymes Rsa I and Hpa II. A total of 100% concordance was obtained for the Southern blotting and MS-HRM scores. A total of three samples with paternal duplication affecting the H19/IGF2 region were scored as equivocal by both methods; however, 33 out of 36 (92%) the samples were unambiguously scored as being hypermethylated, hypomethylated, or normally methylated using MS-HRM. We conclude that MS-HRM is a rapid, cost-effective, and sensitive method for screening mosaic methylation changes at the H19 locus in BWS and RS. Hum Mutat 0,1,6, 2008. © 2008 Wiley-Liss, Inc. [source] Epigenetic detection of human chromosome 14 uniparental disomy,HUMAN MUTATION, Issue 1 2003S.K. Murphy Abstract The recent demonstration of genomic imprinting of DLK1 and MEG3 on human chromosome 14q32 indicates that these genes might contribute to the discordant phenotypes associated with uniparental disomy (UPD) of chromosome 14. Regulation of imprinted expression of DLK1 and MEG3 involves a differentially methylated region (DMR) that encompasses the MEG3 promoter. We exploited the normal differential methylation of the DLK1/MEG3 region to develop a rapid diagnostic PCR assay based upon an individual's epigenetic profile. We used methylation-specific multiplex PCR in a retrospective analysis to amplify divergent lengths of the methylated and unmethylated MEG3 DMR in a single reaction and accurately identified normal, maternal UPD14, and paternal UPD14 in bisulfite converted DNA samples. This approach, which is based solely on differential epigenetic profiles, may be generally applicable for rapidly and economically screening for other imprinting defects associated with uniparental disomy, determining loss of heterozygosity of imprinted tumor suppressor genes, and identifying gene-specific hypermethylation events associated with neoplastic progression. Hum Mutat 22:92,97, 2003. © 2003 Wiley-Liss, Inc. [source] Epigenetic regulation and downstream targets of the Rhox5 homeobox geneINTERNATIONAL JOURNAL OF ANDROLOGY, Issue 5 2008S. Shanker Summary The discovery of the Rhox homeobox gene cluster on the X chromosome opens up new vistas in the regulation of reproductive processes in mammals. In mice, this cluster comprises more than 30 genes that are selectively expressed in reproductive tissues. A subset of Rhox genes are androgen and AR regulated in postnatal and adult Sertoli cells, making them candidates to mediate androgen-dependent steps during spermatogenesis. The best characterized of these androgen/AR-regulated genes is Rhox5 (Pem), the founding member of the Rhox gene cluster. Targeted deletion of Rhox5 in mice causes male subfertility marked by increased germ-cell apoptosis and decreased sperm count and motility. Microarray analyses identified a wide variety of genes regulated by Rhox5 in Sertoli cells. One of them is the tumour suppressor UNC5C, a pro-apoptotic molecule previously only known to be involved in brain development. Targeted deletion of Unc5c causes decreased germ-cell apoptosis in postnatal and adult testes, indicating that it also has a role in spermatogenesis and supporting a model in which Rhox5 promotes germ-cell survival by downregulating Unc5c. Rhox5 has two independently regulated promoters that have distinct expression patterns. The unique tissue-specific and developmentally regulated transcription pattern of these two promoters appear to be controlled by DNA methylation. Both promoters are methylated in tissues in which they are not expressed, suggesting that DNA methylation serves to repress Rhox5 expression in inappropriate cell types and tissues. In summary, the Rhox gene cluster is an epigenetically regulated set of genes encoding a large number of transcription factors that are strong candidates to regulate gametogenesis and other aspects of reproduction. [source] Methylation of cystatin M promoter is associated with unfavorable prognosis in operable breast cancerINTERNATIONAL JOURNAL OF CANCER, Issue 12 2009Magdalini Kioulafa Abstract The methylation status of cystatin M (CST6) gene in breast tumors was investigated and its prognostic significance as a novel breast cancer biomarker was evaluated. Using methylation-specific PCR (MSP), CST6 promoter methylation was examined in 134 formalin fixed paraffin-embedded tissues (FFPEs): 10 pairs of breast tumors and their surrounding normal tissues, 10 breast fibroadenomas, 11 normal breast tissues and 93 breast tumors. Methylation of CST6 promoter was observed in 2/21 (9.5%) noncancerous breast tissues, 1/10 (10%) benign breast tumors (fibroadenomas) and 52 (55.9%) operable breast cancer tumor samples. CST6 was rarely methylated in the normal tissue surrounding the tumor (10%). During the follow-up period, 24 (25.8%) patients relapsed and 19 (20.4%) died. CST6 methylation was detected in 19 (79.2%) of patients who relapsed and in 15 (78.9%) of patients who died. Disease-free-interval (DFI) and overall survival (OS) were significantly associated with CST6 promoter methylation (p = 0.004 and p = 0.001 respectively). Multivariate analysis revealed that CST6 methylation is an independent prognostic factor for DFI (HR = 3.484; 95% CI: 1.155,10.511; p = 0.027). and OS (HR = 9.190; 95% CI: 1.989,42.454; p = 0.004). CST6 promoter methylation status in tumor cells seems to provide important prognostic information in operable breast cancer and merits to be further evaluated and validated in a larger cohort of patients. © 2009 UICC [source] Distinct CpG island methylation profiles and BRAF mutation status in serrated and adenomatous colorectal polypsINTERNATIONAL JOURNAL OF CANCER, Issue 11 2008Yong Ho Kim Abstract A subset of colorectal cancers with CpG island methylator phenotype-high (CIMP-H) is frequently associated with MSI and BRAF V600E mutation. Since limited data are available on different histological types of colorectal polyps, we compared the pattern and the frequency of promoter methylation, CIMP-H, MSI, KRAS and BRAF V600E mutations and the relationship among these molecular parameters and the clinicopathologic characteristics in 110 serrated polyps (48 hyperplastic polyps, 32 sessile serrated adenomas and 30 serrated adenomas) and 32 tubular adenomas using 7 commonly used tumor-associated gene loci. No significant difference in the frequency of overall methylation frequency (86% vs. 100%) and CIMP-H (39% vs. 28%) between serrated polyps and tubular adenomas was observed, but proximally located serrated polyps showed more frequent methylation at 5 of 7 loci examined, and were more likely to be CIMP-H (62% vs. 22%). MGMT methylation was more common in tubular adenomas while MLH1 and HIC1 were more frequently methylated in serrated polyps. BRAF mutation was frequently present in all types of serrated polyps (80%), but was absent in tubular adenomas and was not associated with CIMP or MSI status. These results show comparable frequencies of promoter methylation of tumor-associated genes and CIMP-H, but distinct differences in gene-specific or colonic site-specific methylation profiles occur in serrated polyps and tubular adenomas. BRAF mutation occurs independently of CIMP and MSI in all types of serrated polyps and may serve as a marker of serrated pathway of colorectal carcinogenesis. © 2008 Wiley-Liss, Inc. [source] Identification of novel DNA methylation markers in cervical cancer,INTERNATIONAL JOURNAL OF CANCER, Issue 1 2008Hung-Cheng Lai Abstract Testing for DNA methylation has potential in cancer screening. Most previous studies of DNA methylation in cervical cancer used a candidate gene approach. The aim our study was to identify novel genes that are methylated in cervical cancers and to test their potential in clinical applications. We did a differential methylation hybridization using a CpG island (CGI) microarray containing 8640 CGI tags to uncover methylated genes in squamous cell carcinomas (SCC) of the uterine cervix. Pooled DNA from cancer tissues and normal cervical swabs were used for comparison. Methylation-specific polymerase chain reaction, bisulfite sequencing and reverse transcription polymerase chain reaction were used to confirm the methylation status in cell lines, normal cervices (n = 45), low-grade lesions (n = 45), high-grade lesions (HSIL; n = 58) and invasive squamous cell carcinomas (SCC; n = 22 from swabs and n = 109 from tissues). Human papillomavirus (HPV) was detected using reverse line blots. We reported 6 genes (SOX1, PAX1, LMX1A, NKX6-1, WT1 and ONECUT1) more frequently methylated in SCC tissues (81.5, 94.4, 89.9, 80.4, 77.8 and 20.4%, respectively) than in their normal controls (2.2, 0, 6.7, 11.9, 11.1 and 0%, respectively; p < 0.0001). Parallel testing of HPV and PAX1 methylation in cervical swabs confers an improved sensitivity than HPV testing alone (80% vs. 66%) without compromising specificity (63% vs. 64%) for HSIL/SCC. Testing PAX1 methylation marker alone, the specificity for HSIL/SCC is 99%. The analysis of these novel DNA methylations may be a promising approach for the screening of cervical cancers. © 2008 Wiley-Liss, Inc. [source] DNA demethylation of vascular endothelial growth factor-C is associated with gene expression and its possible involvement of lymphangiogenesis in gastric cancerINTERNATIONAL JOURNAL OF CANCER, Issue 8 2007Shunji Matsumura Abstract Previous studies have indicated that lymphangiogenesis in solid tumors is associated with lymphatic metastasis. Overexpression of Vascular endothelial growth factor (VEGF)-C plays a major role in lymphangiogenesis in cancers. In the present study, DNA methylation and expression of the VEGF-C gene was investigated in gastric cancer (GC). Four GC cell lines (MKN-45, MKN-74, HSC-39 and HSC-43) showed no expression of VEGF-C, and the VEGF-C gene was found to be methylated in these cells. In contrast, 7 GC cell lines (MKN-1, MKN-7, MKN-28, TMK-1, KATO-III, SH101-P4 and HSC-44PE) expressed VEGF-C, and the VEGF-C gene was found to be unmethylated in these cell lines. In addition, expression of VEGF-C mRNA was retrieved by treatment with a demethylating agent, Aza-2,-deoxycytidine. In GC tissue samples, bisulfite DNA sequencing analysis revealed that VEGF-C was not methylated in 9 (29.0%) of 31 GC samples, whereas demethylation was not observed in corresponding non-neoplastic mucosa samples. Overexpression of VEGF-C mRNA was observed in 16 (51.6%) of 31 GC samples by quantitative reverse transcription-polymerase chain reaction. Of the 9 GC cases with VEGF-C demethylation, 8 (88.9%) overexpressed VEGF-C. In contrast, of the 22 GC cases without VEGF-C demethylation, 8 (36.4%) overexpressed VEGF-C (p = 0.0155). Furthermore, lymphatic vessel density determined by immunostaining of podoplanin in GC tissues was associated with overexpression of VEGF-C (p < 0.0001). These results suggest that demethylation and activation of the VEGF-C gene is likely involved in lymphangiogenesis in GC. © 2007 Wiley-Liss, Inc. [source] The expression of metastasis suppressor MIM/MTSS1 is regulated by DNA methylationINTERNATIONAL JOURNAL OF CANCER, Issue 10 2006Jochen Utikal Abstract MIM/MTSS1 was initially described as a gene missing in invasive bladder cancer cell lines. Functional analysis revealed that MIM is an actin binding protein involved in the regulation of actin cytoskeleton dynamics. MIM was shown to be sonic hedgehog (Shh) signaling dependent and synergizes with the effects of Gli transcription factors. Overexpression of MIM in cell lines leads to the inhibition of cell proliferation. In this study, we showed that the inhibition of cell growth by MIM is anchorage independent. We identified and cloned the promoter region of MIM and located the main promoter activity to 276 bp of 5, flanking sequence sited within a CpG island. Analysis of DNA methylation using bisulphite sequencing revealed that MIM promoter is methylated in its 5, region in cells and tissue samples with reduced endogenous MIM expression. Using luciferase reporter assay, we demonstrated that nonmethylated MIM promoter has a similar activity in cell lines with different endogenous MIM expression. Inhibition of DNA methylation by 5-Aza-2,-deoxycytidine led to upregulation of MIM expression in a low expressing cell line. In conclusion, we clearly demonstrate here that the expression of metastasis suppressor MIM is regulated by DNA methylation of a CpG island within its promoter region. © 2006 Wiley-Liss, Inc. [source] Regional DNA hypermethylation and DNA methyltransferase (DNMT) 1 protein overexpression in both renal tumors and corresponding nontumorous renal tissuesINTERNATIONAL JOURNAL OF CANCER, Issue 2 2006Eri Arai Abstract To evaluate the significance of altered DNA methylation during renal tumorigenesis, tumorous tissues (T) and corresponding nontumorous renal tissues (N) from 94 patients with renal tumors, and normal renal tissues (C) from 16 patients without renal tumors were investigated. DNA methylation status on CpG islands of the p16, human MutL homologue 1 (hMLH1), von-Hippel Lindau (VHL) and thrombospondin-1 (THBS-1) genes and the methylated in tumor (MINT) -1, -2, -12, -25 and -31 clones and DNA methyltransferase (DNMT) 1 expression were examined by bisulfite modification and immunohistochemistry, respectively. The average number of methylated CpG islands was significantly higher in N than in C, and was even higher in T. The average number of methylated CpG islands in N was significantly correlated with a higher histological grade of corresponding conventional renal cell carcinomas (RCCs). The average number of methylated CpG islands in RCCs was significantly correlated with macroscopic configuration with extranodular or multinodular growth, higher histological grade, infiltrating growth pattern and vascular involvement. The recurrence-free survival rate of patients with RCCs showing accumulation of DNA methylation was significantly lower than that of patients not showing this feature. The incidence of nuclear immunoreactivity for DNMT1 tended to be higher in proximal tubules from N than in those from C, and was significantly higher in RCCs. From the viewpoint of altered DNA methylation, N is at the precancerous stage, and N showing accumulation of DNA methylation may generate more malignant RCCs. Regional DNA hypermethylation may be associated with renal tumorigenesis from a precancerous condition to malignant progression and become a predictor of patient prognosis. © 2006 Wiley-Liss, Inc. [source] Comparison of DNA hypermethylation patterns in different types of uterine cancer: Cervical squamous cell carcinoma, cervical adenocarcinoma and endometrial adenocarcinomaINTERNATIONAL JOURNAL OF CANCER, Issue 9 2006Sokbom Kang Abstract The incidence of cervical adenocarcinoma (CA) is rising, whereas the incidence of cervical squamous cell carcinoma (CSCC) continues to decrease. However, it is still unclear whether different molecular characteristics underlie these 2 types of cervical carcinoma. To better understand the epigenetic characteristics of cervical carcinoma, we investigated the DNA promoter hypermethylation profiles in CA and CSCC. In addition, we investigated whether DNA hypermethylation patterns might be used for the molecular diagnosis of CA and endometrial adenocarcinoma (EA). Using the bisulfite-modification technique and methylation-specific PCR, we examined the aberrant promoter hypermethylation patterns of 9 tumor suppressor genes (APC, DAPK, CDH1, HLTF, hMLH1, p16, RASSF1A, THBS1 and TIMP3) in 62 CSCCs, 30 CAs and 21 EAs. After Bonferroni correction adjustment (statistically significant at p < 0.0055), we found that the aberrant hypermethylations of CDH1 and DAPK were more frequent in CSCCs than in CAs (80.6% vs. 43.3%, p = 0.001; 77.4% vs. 46.7%, p = 0.005), whereas HLTF and TIMP3 were more frequently methylated in CAs (3.2% vs. 43.3%, p < 0.001; 8.1% vs. 53.3%, p = 0.001). The hypermethylations of RASSF1A and APC were more frequent in CAs than in CSCCs, but this was not significant (9.7% vs. 33.3%, p = 0.008; and 14.5% vs. 40.0%, respectively, p = 0.009). In addition, RASSF1A hypermethylation was significantly more frequent in EAs than in CAs (81.0% vs. 33.3%, p = 0.001). In conclusion, the existence of these unique methylation patterns in these cancers suggests that their tumorigenesis may involve different epigenetic mechanisms. © 2005 Wiley-Liss, Inc. [source] Methylation profile in tumor and sputum samples of lung cancer patients detected by spiral computed tomography: A nested case,control studyINTERNATIONAL JOURNAL OF CANCER, Issue 5 2006Rosalia Cirincione Abstract We evaluated the aberrant promoter methylation profile of a panel of 3 genes in DNA from tumor and sputum samples, in view of a complementary approach to spiral computed tomography (CT) for early diagnosis of lung cancer. The aberrant promoter methylation of RAR,2, p16INK4A and RASSF1A genes was evaluated by methylation-specific PCR in tumor samples of 29 CT-detected lung cancer patients, of which 18 had tumor-sputum pairs available for the analysis, and in the sputum samples from 112 cancer-free heavy smokers enrolled in a spiral CT trial. In tumor samples from 29 spiral CT-detected patients, promoter hypermethylation was identified in 19/29 (65.5%) cases for RAR,2, 12/29 (41.4%) for p16INK4A and 15/29 (51.7%) for RASSF1A. Twenty-three of twenty-nine (79.3%) samples of the tumors exhibited methylation in at least 1 gene. In the sputum samples of 18 patients, methylation was detected in 8/18 (44.4%) for RAR,2 and 1/18 (5%) for both RASSF1A and p16INK4A. At least 1 gene was methylated in 9/18 (50%) sputum samples. Promoter hypermethylation in sputum from 112 cancer-free smokers was observed in 58/112 (51.7%) for RAR,2 and 20/112 (17.8%) for p16, whereas methylation of the RASSF1A gene was found in only 1/112 (0.9%) sputum sample. Our study indicates that a high frequency of hypermethylation for RAR,2, p16INK4A and RASSF1A promoters is present in spiral CT-detected tumors, whereas promoter hypermethylation of this panel of genes in uninduced sputum has a limited diagnostic value in early lung cancer detection. © 2005 Wiley-Liss, Inc. [source] DNA methylation patterns in adenomas from FAP, multiple adenoma and sporadic colorectal carcinoma patientsINTERNATIONAL JOURNAL OF CANCER, Issue 4 2006Coral V.A. Wynter Abstract Colorectal adenomas have traditionally been regarded as homogeneous. The aim of our study was to identify molecular features that may differentiate sporadic adenomas from familial adenomas such as Familial Adenomatous Polyposis (FAP) and Multiple Adenoma patients. DNA methylation was tested at Methylated IN Tumor (MINT) loci (1,2,12,31) and the CpG promoter region of genes MLH1, HPP1, MGMT, p14ARF and p16INK4a in FAP-associated adenomas (33) from 5 patients with a known APC mutation (Group 1, FAP), adenomas (29) from 4 Multiple Adenoma patients (Group 2 Multiple), adenomas (14) from 3 patients with sporadic colorectal cancers showing high microsatellite instability (Group 3, MSI-H) and adenomas (16) from 7 patients, with sporadic colorectal cancers showing microsatellite stable or low level instability (Group 4, MSS/MSI-L). Aberrant Crypt Foci (ACFs), Hyperplastic Polyps (HPs) and cancers were also examined for methylation status as well as K- ras mutation. Multiple Adenoma patients were examined for germline polymorphisms in the base excision repair gene, MYH. The familial syndrome, FAP -associated adenomas showed a significantly low frequency of MINT methylation (15.5%,) compared to sporadic MSS/MSI-L-associated adenomas (35.5%). Group 3 (MSI-H) adenomas were different in that many showed serration and a high level of methylation (57.1%). Group 2, Multiple Adenoma cases, resembled sporadic MSS/MSI-L-associated adenomas. However the promoter regions of key genes, MGMT, p14ARF and p16INK4a were methylated to a greater extent than MINTs in both sporadic and familial adenomas. Genetic profiling of adenomas supports the concept that adenomas belonging to familial syndromes pursue a different pathway to tumorigenesis than their sporadic counterpar/ts from their earliest formation. © 2005 Wiley-Liss, Inc. [source] Identification of differentially methylated CpG islands in prostate cancerINTERNATIONAL JOURNAL OF CANCER, Issue 5 2004Yasushi Yamada Abstract Epigenetic change such as DNA methylation is one important mechanism for regulating gene expression as genetic change, such as mutation or loss of heterozygosity. Methylation of cancer-related genes has been shown to play an important role in carcinogenesis and tumor progression. Using methylated CpG island amplification (MCA)/representational difference analysis (RDA), we identified four CpG islands in neurotrophin tyrosine kinase receptor type 2 (NTRK2), Protocadherine Flamingo1 and MFPC (Methylated Fragments in Prostate Cancer) 7 and 8. Bisulfite sequencing revealed that 2 regions of NTRK2 as well as MFPC7 and MFPC8 were aberrantly methylated in prostate cancer cell lines, and COBRA showed that 48 (76.24%), 37 (58.7%) and 14 (22.2%) of 63 prostate cancer tissues were methylated, respectively, for these sites. On the other hand, none of 13 benign prostate samples were methylated, except for 1 (7.7%) with NTRK2. For NTRK2, mRNA expression was negative in prostate cancer cell lines (LNCaP and DU145) but was recovered on a methyltransferase inhibitor (5-Aza-CdR) treatment. The role of NTRK2 within NTRK remains unclear. Our results suggest that these 3 hypermethylated DNA fragments also may be markers of prostate cancer detection. © 2004 Wiley-Liss, Inc. [source] Aberrant methylation of multiple genes in the upper aerodigestive tract epithelium of heavy smokersINTERNATIONAL JOURNAL OF CANCER, Issue 4 2003Sabine Zöchbauer-Müller Abstract An important method for silencing tumor suppressor genes in cancers is by aberrant methylation (referred to as methylation) of CpG islands in gene promoter regions. In lung cancer, methylation of the genes retinoic acid receptor ,-2 (RAR,- 2), CDH13 (H-cadherin), p16INK4a (p16), RASSF1A (RAS association domain family I) is frequent. Thus, we investigated methylation of these genes in 4 different types of specimens (oropharyngeal brushes, sputum samples, bronchial brushes and bronchioloalveolar lavage [BAL] samples) of the upper aerodigestive tract epithelium from heavy smokers without evidence of cancer but with morphometric evidence of sputum atypia and compared the frequencies of methylation in the different types of specimens. In addition, we also analyzed sputum samples from 30 never smokers for methylation of these genes. Our major findings are: (i) At least one gene was methylated in one or more specimens from 48% of the smokers. However, methylation was statistically significant less frequently in never smokers compared to smokers. (ii) In general, methylation occurred more frequently in samples from the central airways (sputum, bronchial brushes) compared to the peripheral airways (BAL) and only occasionally in the oropharynx. (iii) RAR,- 2 was the most frequently methylated gene, whereas the frequency of methylation for the other genes was lower. (iv) Data from sputum samples and bronchial brushes were comparable. Our findings suggest that detection of methylation should be investigated as an intermediate marker for lung cancer risk assessment and response to chemopreventive regimens. © 2003 Wiley-Liss, Inc. [source] Ditopic Cyclodextrin-Based Receptors: New Perspectives in Aqueous Organometallic CatalysisADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 9 2010Natacha Six Abstract The mass transfer properties of mono- and ditopic ,-cyclodextrin-based receptors have been evaluated in a biphasic palladium-catalyzed Tsuji,Trost reaction and compared to one of the best mass-transfer promoters, namely the randomly methylated ,-cyclodextrin. While monotopic receptors appeared to be poor mass-transfer promoters of long alkyl chain allyl carbonates or urethanes, cooperative effects have been evidenced with ditopic cyclodextrin-based receptors, opening new perspectives in aqueous organometallic catalysis. [source] DNA methylation and histone modification regulate silencing of OPG during tumor progression,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2009Tung-Ying Lu Abstract The identification of molecules that are down-regulated in malignant phenotype is important for understanding tumor biology and their role in tumor suppression. We compared the expression profile of four normal nasal mucosal (NNM) epithelia and a series of nasopharyngeal cancinoma (NPC) cell lines using cDNA microarray and confirmed the actual expression of the selected genes, and found osteoprotegerin (OPG) to be ubiquitously deficient in NPC cells. We also found OPG to be down-regulated in various cancer cell lines, including oral, cervical, ovarian, lung, breast, pancreas, colon, renal, prostate cancer, and hepatoma. Administration of recombinant OPG (rOPG) brought about a reduction in cancer cell growth through apoptotic mechanism. We generated eleven monoclonal antibodies (MAbs) against OPG to study OPG's expression and biological functions in cancer cells. OPG was detected in the tumor stromal regions, but not in the cancer cell per se in surgical specimens of liver cancer. Quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR) revealed that OPG was down-regulated in NPC tissues compared with normal nasal polyp (NNP) tissues. In addition, we showed OPG silencing to be associated with promoter methylation as well as histone modifications. In OPG-silenced cancer cell lines, the OPG gene promoter CpG dinucleotides were highly methylated. Compared to normal cells, silenced OPG gene in cancer cells were found to have reduced histone 3 lysine 4 tri-methylation (H3K4me3) and increased histone 3 lysine 27 tri-methylation (H3K27me3). Taken together, these results suggest that OPG silencing in carcinoma cancer cells occurs through epigenetic repression. J. Cell. Biochem. 108: 315,325, 2009. © 2009 Wiley-Liss, Inc. [source] | |||||||||||||||||||||||||||||||||||||