Epigenetics is alive and growing.
The conference 'Epigenetics of Cancer,' organized by the American Association for Cancer Research, was held 17-21 October 2001 in Palm Desert, CA.
Chromatin↗
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Results for “DNA Modification Methylases”
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Human embryonic stem cell methyl cycle enzyme expression: modelling epigenetic programming in assisted reproduction?
To investigate a possible mechanism for inducing epigenetic defects in the preimplantation embryo, a human embryonic stem cell model was developed, and gene expression of the key methyl cycle enzymes, MAT2A, MAT2B, GNMT, SAHH, CBS, CGL, MTR, MTRR, BHMT, BHMT2, mSHMT, cSHMT and MTHFR was demonstrated, while MAT1 was barely detectable. Several potential acceptors of cycle-generated methyl groups, the DNA methyltransferases (DNMT1, DNMT3A, DNMT3B and DNMT3L), glycine methyltransferase and the polyamine biosynthetic enzymes, SAM decarboxylase and ornithine decarboxylase, were also expressed. Expression of folate receptor alpha suggests a propensity for folate metabolism. Methotrexate-induced depletion of folate resulted in elevated intracellular homocysteine concentration after 7 days in culture and a concomitant increase in cysteine and glutathione, indicating clearance of homocysteine through the transulphuration pathway. These studies indicate that altered methyl group metabolism provides a potential mechanism for inducing epigenetic changes in the preimplantation embryo.
Cell Proliferation↗
Exceptionally high protection of photocarcinogenesis by topical application of (--)-epigallocatechin-3-gallate in hydrophilic cream in SKH-1 hairless mouse model: relationship to inhibition of UVB-induced global DNA hypomethylation.
(--)-Epigallocatechin-3-gallate (EGCG) has been shown to have potent antiphotocarcinogenic activity, but it was required to develop a cream-based formulation for topical application. For topical application, we tested hydrophilic cream as a vehicle for EGCG. Treatment with EGCG ( approximately 1 mg/cm(2) skin area) in hydrophilic cream resulted in exceptionally high protection against photocarcinogenesis when determined in terms of tumor incidence, tumor multiplicity, and tumor size in a SKH-1 hairless mouse model. EGCG also inhibited malignant transformation of ultraviolet B (UVB)-induced papillomas to carcinomas. In order to determine the mechanism of prevention of photocarcinogenesis, we determined the effect of EGCG on global DNA methylation pattern using monoclonal antibodies against 5-methyl cytosine and DNA methyltransferase in the long-term UV-irradiated skin because altered DNA methylation silencing is recognized as a molecular hallmark of human cancer. We found that treatment with EGCG resulted in significant inhibition of UVB-induced global DNA hypomethylation pattern. Long-term application of EGCG did not show any apparent sign of toxicity in mice when determined in terms of skin appearance, lean mass, total bone mineral content, and total bone mineral density but showed reduction in fat mass when analyzed using dual-energy X-ray absorptiometry. These data suggest that hydrophilic cream could be a suitable vehicle for topical application of EGCG, and that EGCG is a promising candidate for future cancer therapies based on its influence on the epigenetic pathway.
5-Methylcytosine↗
Methylation in fertilised and parthenogenetic preimplantation mouse embryos.
DNA methylation is one of the proposed biochemical mechanisms involved in cell differentiation and in genomic imprinting, and DNA methyltransferase (DMT) is a key enzyme in the embryo since mutation of its gene is lethal early in development. In order to verify that non-viability of uniparental embryos was not due to a defect in the regulation of DMT activity, we compared the metabolism of methylation in parthenogenetic embryos (maternal genome) and in fertilised embryos (maternal and paternal genomes). As regards total methylation, estimated by a measure of S-adenosyl methionine (SAM) and S-adenosyl homocysteine (SAH) formation, no significant difference was found between the two kinds of embryos during preimplantation development. Mean values were 4.5 +/- 0.6 fmol (SAM+SAH)/h per 2-cell embryo and 0.40 +/- 0.05 fmol SAH/h per 2-cell embryo, i.e. a SAH/(SAM+SAH) ratio of 9%; there was no detectable SAH formation in blastocysts. The same observation can be made for DMT activity, with mean values of: 7.8 fmol/h per oocyte, 8.5 fmol/h per 2-cell embryo, 6.1 fmol/h per 4-cell embryo, 4.1 fmol/h per morula, and no detectable activity in blastocysts. Total methylation as well as DNA methylation is characterised by a progressive drop in activity during preimplantation development.
Animals↗
Synthesis and in vitro evaluation of biotinylated RG108: a high affinity compound for studying binding interactions with human DNA methyltransferases.
Small-molecule inhibitors of DNA methyltransferases such as RG108 represent promising candidates for cancer drug development. We report the synthesis and in vitro analysis of a biotinylated RG108 conjugate, 2-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-3-(5-[3-[5-(2-oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)pentanoylamino]propoxy]-1H-indol-3-yl)propionic acid (bio-RG108), for the evaluation of interactions with DNA methyltransferase enzymes. The structural design of the chemically modified inhibitor was aided by molecular modeling, which suggested the possibility for extensive chemical modifications at the 5-position of the tryptophan moiety in RG108. The inhibitory activity of the corresponding derivative was confirmed in a cell-free biochemical assay, where bio-RG108 showed an undiminished inhibition of DNA methyltransferase activity (IC50 = 40 nM). Bio-RG108 therefore represents a suitable bioconjugate for the elucidation of inhibitory mechanisms and for the affinity purification of RG108-associated proteins.
Biotin↗
EcoRI DNA methyltransferase-DNA interactions.
We present a novel strategy with synthetic hemimethylated DNA substrates containing uracil for thymine and inosine for guanosine replacements and EcoRI DNA methyltransferase to characterize the importance of major groove hydrophobic groups to the sequence-specific modification of DNA. The bacterial Mtase uses S-adenosyl-L-methionine to methylate the double-stranded DNA site 5'GAATTC3' at the N6 position of the central adenosine of each strand. Uracil substitution in either strand at the outer thymine (5'GAATUC3') causes 2.2- and 1.7-fold improvements in specificity (kcat/KmDNA). The fact that the specificity constant for the substrate containing uracil in both strands is identical to the value expected for noninteracting substitutions suggests that no significant methyltransferase-DNA interactions are altered beyond the site of either substitution. Similar analysis of the internal thymine (5'GAAUTC3') also shows these methyl groups to make a negative contribution to specificity, although the observed nonadditivity with the doubly modified substrate clearly shows methyltransferase-DNA interactions beyond the site of substitution to be affected in this case. To further probe the effect of analogue incorporation on methyltransferase-DNA interactions beyond the site of substitution, the relatively "silent" and additive uracil changes (5'GAATUC3') were combined with inosine for guanosine substitutions (e.g., 5'IAATTC3') known to have significant negative effects on specificity. In contrast to the additivity observed with the outer thymines, these studies show significant changes in methyltransferase-DNA interactions caused by the removal of the thymine methyls. Our results implicate a complex and flexible methyltransferase-DNA interface in which subtle structural changes in the substrate are transmitted over the entire canonical site.(ABSTRACT TRUNCATED AT 250 WORDS)
Base Sequence↗
Identification of borinic esters as inhibitors of bacterial cell growth and bacterial methyltransferases, CcrM and MenH.
As bacteria continue to develop resistance toward current antibiotics, we find ourselves in a continual battle to identify new antibacterial agents and targets. We report herein a class of boron-containing compounds termed borinic esters that have broad spectrum antibacterial activity with minimum inhibitory concentrations (MIC) in the low microgram/mL range. These compounds were identified by screening for inhibitors against Caulobacter crescentus CcrM, an essential DNA methyltransferase from gram negative alpha-proteobacteria. In addition, we demonstrate that borinic esters inhibit menaquinone methyltransferase in gram positive bacteria using a new biochemical assay for MenH from Bacillus subtilis. Our data demonstrate the potential for further development of borinic esters as antibacterial agents as well as leads to explore more specific inhibitors against two essential bacterial enzymes.
Anti-Bacterial Agents↗
Psammaplins from the sponge Pseudoceratina purpurea: inhibition of both histone deacetylase and DNA methyltransferase.
Four novel bisulfide bromotyrosine derivatives, psammaplins E (9), F (10), G (11), and H (12), and two new bromotyrosine derivatives, psammaplins I (13) and J (14), were isolated from the sponge Pseudoceratina purpurea, along with known psammaplins A (4), B (6), C (7), and D (8) and bisaprasin (5). The structures of psammaplins E (9) and F (10), which each contain an oxalyl group rarely found in marine organisms, were determined by spectroscopic analysis. Compounds 4, 5, and 10 are potent histone deacetylase inhibitors and also show mild cytotoxicity. Furthermore, compounds 4, 5, and 11 are potent DNA methyltransferase inhibitors. The biogenetic pathway previously proposed for the psammaplins class is also revisited.
Animals↗
Peyssonenynes a and B, novel enediyne oxylipins with DNA methyl transferase inhibitory activity from the red marine alga peyssonneliacaulifera.
Two novel omega3 fatty acids, obtained as monoacyl glycerol derivatives, were isolated as DNA methyl transferase inhibitors following bioassay-guided fractionation of the Fijian red marine alga Peyssonnelia caulifera. Both active metabolites, peyssonenynes A (1) and B (2), possess an unusual enediyne motif, whereas an inactive co-metabolite, peyssopyrone (3), contains an unusual gamma-pyrone functionality. The molecular structures of all three compounds were determined by NMR spectroscopy in combination with UV, IR, and MS data analysis. The instability of the enediyne monoacyl glycerol derivatives prevented their complete stereochemical assignments.
DNA Modification Methylases↗
DNA methyl transferase inhibiting halogenated monoterpenes from the Madagascar red marine alga Portieria hornemannii.
Three new halogenated monoterpenes, 2, 3, and 4, along with the known compounds halomon (1) and two analogues, 5 and 6, were isolated from the Madagascar red marine alga Portieria hornemannii. The structures of all three new compounds were determined by NMR spectroscopy in combination with mass spectrometric data analysis. Two of these monoterpenes (1 and 2) were low micromolar inhibitors of DNA methyl transferase-1.
DNA Modification Methylases↗
DNA methyltransferase-moderated click chemistry.
[reaction: see text]. Biological methylation plays a vital role in regulatory mechanisms of gene transcription. Methylation of both promoter sequences within the genome, as well as protein substrates, has a profound impact upon gene transcription. Yet, few tools exist by which to identify sites of biological methylation in complex biological mixtures. We have generated a novel adenosine-derived N-mustard that serves as an efficient synthetic cofactor and allows for subsequent "click" chemistry involving the modified nucleic acid substrate.
Adenosine↗
Targeting DNA methyltransferase in cancer.
DNA methyltransferase is an enzyme responsible for generating and maintaining DNA methylation patterns. DNA methylation patterns control different genome functions, thus they are an important component of the epigenetic information. It has been recently postulated that DNA methyltransferase plays an important role in oncogenesis and that it is a candidate target for anticancer therapy. This commentary discusses the possible mechanisms through which DNA methyltransferase participates in oncogenesis and the rationale for targeting it in cancer.
Animals↗
Expression of MAGE and BAGE genes in Japanese breast cancers.
BACKGROUND: The MAGE and BAGE genes code for distinct antigens, which are recognized on melanoma cells as well as on other various tumor cells by autologous cytolytic T lymphocytes. These antigens may thus constitute useful targets for specific immunotherapy, since no expression of MAGE or BAGE genes has been recognized in normal tissue except for the testis. PATIENTS AND METHODS: We studied the MAGE-1, MAGE-3, and BAGE gene expression observed in 49 Japanese breast cancers. Gene expression was evaluated by reverse transcription polymerase chain reaction. RESULTS: Out of 49 tumor tissue specimens of primary breast cancers, the expression of MAGE-1, -3 and BAGE was recognized in 15 (31%), 12 (24%), and 4 (8%) tumors, respectively. The expression of MAGE and BAGE genes is not recognized in normal breast tissue. The expression of the MAGE-3 gene was frequently recognized in tumors with lymphatic and/or vascular vessel permeations. Either MAGE-1 or -3 gene expressions were induced in 1 of 3 MAGE-1 negative breast cell lines or 1 of 3 MAGE-3 negative breast cell lines by the treatment with 5-aza-2'-deoxycytidine. CONCLUSIONS: These findings suggest that: 1) the identification of such antigens coded by MAGE or BAGE genes may thus offer the possibility of using specific immunotherapy, and 2) the use of a demethylating agent may increase the number of patients who might be candidates for MAGE specific immunotherapy.
Antigens, Neoplasm↗
Putative DNA-(amino)methyltransferases in eucaryotes.
By computer analysis of the known data bases, we have established that the open reading frames (ORF) coding for proteins that possess high degree of homology with procaryotic DNA-(amino)methyltransferases are present in the genomes of Leishmania major, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Arabidopsis thaliana, Drosophila melanogaster, Caenorhabditis elegans, and Homo sapiens. Conservative motifs typical for bacterial DNA-(amino)methyltransferases are detected in the amino acid sequences of these putative proteins. The ORF of all putative eucaryotic DNA-(amino)methyltransferases found are encoded in nuclear DNA. In mitochondrial genomes including a few fully sequenced higher plant mtDNA, nucleotide sequences significantly homologous to genes of procaryotic DNA-(amino)methyltransferases are not found. Thus, ORF homologous to bacterial adenine DNA-methyltransferases are present in nuclei of protozoa, yeasts, insects, nematodes, vertebrates, higher plants, and other eucaryotes. A special search for corresponding proteins and, in particular, adenine DNA-methyltransferases in these organisms and a study of their functions are quite promising.
Adenine↗
A novel histone deacetylase inhibitor, scriptaid, enhances expression of functional estrogen receptor alpha (ER) in ER negative human breast cancer cells in combination with 5-aza 2'-deoxycytidine.
Epigenetic mechanisms, such as DNA methylation and histone deacetylation, may play a role in loss of estrogen receptor alpha (ER) expression in ER negative human breast cancer cells. Our previous studies showed that pharmacologic inhibition of these mechanisms using the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (AZA), and the histone deacetylase (HDAC) inhibitor, Trichostatin A (TSA), resulted in expression of functional ER mRNA and protein. Therefore, we sought to characterize the effects of a recently described HDAC inhibitor, Scriptaid, on cell growth and ER expression and function in ER negative human breast cancer cell lines. Scriptaid treatment of three ER negative cell lines, MDA-MB-231, MDA-MB-435 and Hs578t, resulted in significant growth inhibition and increased acetylation of H3 and H4 histone tails. Quantitative Real Time PCR showed 2000-20,000-fold increase of ER mRNA transcript in all three cell lines after 48 h of Scriptaid treatment. Further, dose dependent re-expression of an estrogen responsive gene, the progesterone receptor (PR), indicated that induced ER is functional. As seen with TSA and AZA, Scriptaid and AZA co-treatment was more effective in inducing ER than Scriptaid or AZA alone. In vivo analysis using a xenograft mouse model bearing MDA-MB-231 tumors showed decreased tumor growth following Scriptaid or TSA treatment. Our results indicate that the novel HDAC inhibitor, Scriptaid, inhibits tumor growth in vitro and in vivo and, in conjunction with AZA, acts to re-express functional ER. These data suggest that Scriptaid or related HDAC inhibitors are candidates for further study in breast cancer.
Acetylation↗
Role for DNA methylation in genomic imprinting.
The paternal and maternal genomes are not equivalent and both are required for mammalian development. The difference between the parental genomes is believed to be due to gamete-specific differential modification, a process known as genomic imprinting. The study of transgene methylation has shown that methylation patterns can be inherited in a parent-of-origin-specific manner, suggesting that DNA methylation may play a role in genomic imprinting. The functional significance of DNA methylation in genomic imprinting was strengthened by the recent finding that CpG islands (or sites) in three imprinted genes, H19, insulin-like growth factor 2 (Igf-2), and Igf-2 receptor (Igf-2r), are differentially methylated depending on their parental origin. We have examined the expression of these three imprinted genes in mutant mice that are deficient in DNA methyltransferase activity. We report here that expression of all three genes was affected in mutant embryos: the normally silent paternal allele of the H19 gene was activated, whereas the normally active paternal allele of the Igf-2 gene and the active maternal allele of the Igf-2r gene were repressed. Our results demonstrate that a normal level of DNA methylation is required for controlling differential expression of the paternal and maternal alleles of imprinted genes.
Alleles↗
Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer.
Densely methylated DNA associates with transcriptionally repressive chromatin characterized by the presence of underacetylated histones. Recently, these two epigenetic processes have been dynamically linked. The methyl-CpG-binding protein MeCP2 appears to reside in a complex with histone deacetylase activity. MeCP2 can mediate formation of transcriptionally repressive chromatin on methylated promoter templates in vitro, and this process can be reversed by trichostatin A (TSA), a specific inhibitor of histone deacetylase. Little is known, however, about the relative roles of methylation and histone deacetylase activity in the stable inhibition of transcription on densely methylated endogenous promoters, such as those for silenced alleles of imprinted genes, genes on the female inactive X chromosome and tumour-suppressor genes inactivated in cancer cells. We show here that the hypermethylated genes MLH1, TIMP3 (TIMP3), CDKN2B (INK4B, p15) and CDKN2A (INK4, p16) cannot be transcriptionally reactivated with TSA alone in tumour cells in which we have shown that TSA alone can upregulate the expression of non-methylated genes. Following minimal demethylation and slight gene reactivation in the presence of low dose 5-aza-2'deoxycytidine (5Aza-dC), however, TSA treatment results in robust re-expression of each gene. TSA does not contribute to demethylation of the genes, and none of the treatments alter the chromatin structure associated with the hypermethylated promoters. Thus, although DNA methylation and histone deacetylation appear to act as synergistic layers for the silencing of genes in cancer, dense CpG island methylation is dominant for the stable maintenance of a silent state at these loci.
Adaptor Proteins, Signal Transducing↗
Direct transfer of extended groups from synthetic cofactors by DNA methyltransferases.
S-Adenosyl-L-methionine (AdoMet) is the major methyl donor for biological methylation reactions catalyzed by methyltransferases. We report the first chemical synthesis of AdoMet analogs with extended carbon chains replacing the methyl group and their evaluation as cofactors for all three classes of DNA methyltransferases. Extended groups containing a double or triple bond in the beta position to the sulfonium center were transferred onto DNA in a catalytic and sequence-specific manner, demonstrating a high utility of such synthetic cofactors for targeted functionalization of biopolymers.
Binding Sites↗