Methylation-regulated expression of HLA class I antigens in melanoma.
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Antimetabolites, Antineoplastic↗
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Results for “methylation class”
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A simple method for locating methylated bases in DNA, as applied to detect asymmetric methylation by M.FokIA.
Class-IIS restriction enzymes, which cut the DNA outside their recognition sequence, could be used for locating the bases methylated by a DNA-modification methylase. This is possible because methylation of the class-IIS cut sites does not interfere with the cleavage. The method consists of (i) selection of a nucleotide sequence with appropriate overlap between the methylase recognition site and the class-IIS enzyme cut site, (ii) methylation using S-adenosylmethionine as [3H]methyl donor, (iii) cleavage of the methylated sequence with the class-IIS enzyme, (iv) separation of the cleavage products and identification of the 3H-labelled fragment. Using this simple and straightforward method, we have shown that M.FokIA is an adenine methylase and methylates asymmetrically one strand of the FokI recognition site, resulting in the (Formula: see text) sequence. In addition, it was observed that another class-IIS restriction enzyme, SfaNI, is completely inhibited by methylation of its recognition site, (Formula: see text), by M.FokIA.
Base Sequence↗
4-Methyl-3-(arylsulfonyl)furoxans: a new class of potent inhibitors of platelet aggregation.
A series of 4-methyl-3-(arylthio)furoxans were synthesized by oxidation of 1-(arylthio)-2-methylglyoxymes with dinitrogen tetroxide. Reduction with trimethyl phosphite of the furoxan derivatives afforded the corresponding furazans, while oxidation with an equimolar amount of 30% hydrogen peroxide in acetic acid or with an excess of 81% hydrogen peroxide in trifluoroacetic acid afforded the corresponding arylsulfinyl and arylsulfonyl analogues, respectively. All the furoxan and furazan derivatives showed activity as inhibitors of platelet aggregation. 4-Methyl-3-(arylsulfonyl)furoxans were the most potent derivatives of the series. 4-Methyl-3-(phenylsulfonyl)furoxan (10a), one of the most active derivatives, inhibits the AA-induced increase of cytosolic free Ca2+ and production of malondialdehyde. A primary action of the compound on cyclooxygenase is excluded, as a stable epoxymethano analogue of prostaglandin H2 does not reverse the inhibitory effect of 10a. This compound produces a significant increase in cGMP which is likely to cause inhibition at an early stage of the platelet activation pathway.
Arachidonic Acid↗
4-Methyl-7-(tetradecanoyl)-2H-1-benzopyran-2-one: a novel DNA topoisomerase II inhibitor with adulticidal and embryostatic activity against sub-periodic Brugia malayi.
A compound of the coumarin class, 4-methyl-7-(tetradecanoyl)-2H-1-benzopyran-2-one, was evaluated for antifilarial activity against the human filarial parasite, Brugia malayi (sub-periodic strain) in Mastomys coucha. The test compound brought about a 24.4% reduction in circulating microfilaremia on day 8 after initiation of treatment when administered by the peritoneal route at a dose of 50 mg/kg for 5 consecutive days. The compound also caused a 62.0% mortality in adult parasites. Apart from killing adult filariids, it also brought about sterilization of 81.8% of the surviving female B. malayi. An oral dose of 200 mg/kg for 5 consecutive days was less effective (35.5% adulticidal efficacy and 65.8% sterilization). In vitro, the compound killed adult B. malayi at 100 microM concentration and inhibited DNA topoisomerase II activity in the filarial parasite. Studies are in progress using the compound in combination with standard antifilarials as well as other active agents.
Aedes↗
Epigenetic patterns in the progression of esophageal adenocarcinoma.
Esophageal adenocarcinoma (EAC) arises after normal squamous mucosa undergoes metaplasia to specialized columnar epithelium (intestinal metaplasia or Barrett's esophagus), which can then ultimately progress to dysplasia and subsequent malignancy. Epigenetic studies of this model have thus far been limited to the DNA methylation analysis of a few genes. In this study, we analyzed a panel of 20 genes using a quantitative, high-throughput methylation assay, METHYLIGHT: We used this broader approach to gain insight into concordant methylation behavior between genes and to generate epigenomic fingerprints for the different histological stages of EAC. Our study included a total of 104 tissue specimens from 51 patients with different stages of Barrett's esophagus and/or associated adenocarcinoma. We screened 84 of these samples with the full panel of 20 genes and found distinct classes of methylation patterns in the different types of tissue. The most informative genes were those with an intermediate frequency of significant hypermethylation [ranging from 15% (CDKN2A) to 60% (MGMT) of the samples]. This group could be further subdivided into three classes, according to the absence (CDKN2A, ESR1, and MYOD1) or presence (CALCA, MGMT, and TIMP3) of methylation in normal esophageal mucosa and stomach, or the infrequent methylation of normal esophageal mucosa accompanied by methylation in all normal stomach samples (APC). The other genes were less informative, because the frequency of hypermethylation was below 5% (ARF, CDH1, CDKN2B, GSTP1, MLH1, PTGS2, and THBS1), completely absent (CTNNB1, RB1, TGFBR2, and TYMS1), or ubiquitous (HIC1 and MTHFR), regardless of tissue type. Each class undergoes unique epigenetic changes at different steps of disease progression of EAC, suggesting a step-wise loss of multiple protective barriers against CpG island hypermethylation. The aberrant hypermethylation occurs at many different loci in the same tissues, suggestive of an overall deregulation of methylation control in EAC tumorigenesis. However, we did not find evidence for a distinct group of tumors with a CpG island methylator phenotype. Finally, we found that normal and metaplastic tissues from patients with evidence of associated dysplasia or cancer had a significantly higher incidence of hypermethylation than similar tissues from patients with no further progression of their disease. The fact that the samples from these two groups of patients were histologically indistinguishable, yet molecularly distinct, suggests that the occurrence of such hypermethylation may provide a clinical tool to identify patients with premalignant Barrett's who are at risk for further progression.
Adenocarcinoma↗
N-methyl antitumour agents. A distinct class of anticancer drugs?
This article reviews the structure-activity characteristics, mode of action, pharmacokinetics and clinical utility of a group of chemically dissimilar antitumour agents which have as a common structural feature the N-methyl moiety. The importance of this feature is shown by the fact that molecules without a substituent on the nitrogen or compounds with N-alkyl groups other than methyl are usually inactive in experimental systems. This observation is supported by structure-activity studies with N-alkyl derivatives of s-triazines, triazenes, formamides, hydrazines and nitrosoureas. Representatives of these structural types which have found clinical application are, respectively, hexamethylmelamine, dacarbazine, N-methylformamide, procarbazine and streptozotocin. Mode of action studies have shown that dacarbazine, procarbazine and streptozotocin can give rise to species capable of methylating nucleic acid. This may be the lesion which produces antitumour activity. The mechanism of action of N-methylmelamines and N-methylformamide remains unclear. There is good evidence that, with the exception of N-methylnitrosoureas, host metabolism is prerequisite for activity with these agents. Although not pronounced, the clinical activity of N-methyl antitumour agents is useful, particularly as activity is not associated with severe haematological toxicity. Furthermore, responses may be observed in patients resistant to bifunctional alkylating agents. It is concluded that the drugs reviewed herein show a degree of coincidence in terms of their biological properties which may warrant a common classification. The term N-methyl antitumour agent is proposed.
Alkylating Agents↗
Cajal body-specific small nuclear RNAs: a novel class of 2'-O-methylation and pseudouridylation guide RNAs.
Cajal (coiled) bodies are conserved subnuclear organelles that are present in the nucleoplasm of both animal and plant cells. Although Cajal bodies were first described nearly 100 years ago, their function has remained largely speculative. Here, we describe a novel class of human small nuclear RNAs that localize specifically to Cajal bodies. The small Cajal body-specific RNAs (scaRNAs) are predicted or have already been demonstrated to function as guide RNAs in site-specific synthesis of 2'-O-ribose-methylated nucleotides and pseudouridines in the RNA polymerase II-transcribed U1, U2, U4 and U5 spliceosomal small nuclear RNAs (snRNAs). Our results provide strong support for the idea that the Cajal body, this mysterious nuclear organelle, provides the cellular locale for post-transcriptional modification of spliceosomal snRNAs.
Animals↗
Synthesis of L-thiocitrulline, L-homothiocitrulline, and S-methyl-L-thiocitrulline: a new class of potent nitric oxide synthase inhibitors.
Nitric oxide synthase catalyzes the NADPH- and O2-dependent conversion of L-arginine to L-citrulline and nitric oxide. L-Thiocitrulline, L-homothiocitrulline, and S-methyl-L-thiocitrulline, novel citrulline analogs, have been synthesized and are shown to be potent inhibitors of both the constitutive brain and the inducible smooth muscle isoforms of nitric oxide synthase. Although many N omega-monosubstituted arginine derivatives inhibit nitric oxide synthase, inhibitory citrulline derivatives have not previously been reported. S-Methyl-L-thiocitrulline is significantly more potent than N omega-methyl-L-arginine, the prototypic nitric oxide synthase inhibitor.
Amino Acid Oxidoreductases↗
ING2 PHD domain links histone H3 lysine 4 methylation to active gene repression.
Dynamic regulation of diverse nuclear processes is intimately linked to covalent modifications of chromatin. Much attention has focused on methylation at lysine 4 of histone H3 (H3K4), owing to its association with euchromatic genomic regions. H3K4 can be mono-, di- or tri-methylated. Trimethylated H3K4 (H3K4me3) is preferentially detected at active genes, and is proposed to promote gene expression through recognition by transcription-activating effector molecules. Here we identify a novel class of methylated H3K4 effector domains--the PHD domains of the ING (for inhibitor of growth) family of tumour suppressor proteins. The ING PHD domains are specific and highly robust binding modules for H3K4me3 and H3K4me2. ING2, a native subunit of a repressive mSin3a-HDAC1 histone deacetylase complex, binds with high affinity to the trimethylated species. In response to DNA damage, recognition of H3K4me3 by the ING2 PHD domain stabilizes the mSin3a-HDAC1 complex at the promoters of proliferation genes. This pathway constitutes a new mechanism by which H3K4me3 functions in active gene repression. Furthermore, ING2 modulates cellular responses to genotoxic insults, and these functions are critically dependent on ING2 interaction with H3K4me3. Together, our findings establish a pivotal role for trimethylation of H3K4 in gene repression and, potentially, tumour suppressor mechanisms.
Amino Acid Motifs↗
Dynamic regulation of estrogen receptor-beta expression by DNA methylation during prostate cancer development and metastasis.
Estrogen receptor (ER)-beta is thought to exert anti-proliferative effects in the normal prostate but supports prostate cancer (PCa) cell survival. We previously reported that the receptor's expression declined as PCa developed in the gland but reappeared in lymph node and bone metastases. To investigate whether hypermethylation was the underlying mechanism for these phenomena, we first identified two CpG islands (CGIs) encompassing 41 CpG dinucleotides, located separately in the untranslated exon 0N and the promoter region of ER-beta. Using immunostained, laser capture-microdissected samples from 56 clinical specimens, we demonstrated an inverse relationship exists between the extent of ER-beta CGI methylation and receptor expression in normal, hyperplastic, premalignant, and malignant foci of the prostate and in lymph node and bone metastases. Treatment of PCa cell lines (LNCaP and DU145), that express little ER-beta mRNA, with a demethylating agent increased levels of receptor expression thus corroborating our in vivo findings that methylation is involved in ER-beta silencing. Methylation centers in the promoter region and exon 0N were identified by hierarchical cluster analysis of bisulfite sequencing data obtained from 710 alleles. Methylation at these centers was insignificant in normal epithelium, reached 80 to 90% in grade 4/5 PCa, but declined to less than 20% in bone metastases. In addition, progressive methylation spreading from the exonic CGI to the promoter CGI, which correlated with loss of ER-beta expression, was detected in microdissected samples and in cell cultures. Using a new class of methylated oligonucleotides that mediate sequence-specific methylation in cellulo, we demonstrated that methylation of the promoter CGI, but not the exonic CGIs, led to transcriptional inactivation of ER-beta. Our results present the first evidence that epigenetic regulation of ER-beta is a reversible and tumor stage-specific process and that gene silencing via methylated oligonucleotides may have therapeutic potential in the treatment of advanced PCa.
Base Sequence↗
State of the translational science: summary of Baltimore workshop on gene re-expression as a therapeutic target in cancer January 2003.
A workshop was held in Baltimore, Maryland in January 2003 to discuss translational aspects of cancer therapies targeted at impacting aberrant gene transcription due to epigenetic changes. The mission of the meeting was the development of strategies for scientifically sound, clinically feasible applications targeting epigenetics in cancer therapy. Sessions included preclinical discussions of DNA methylation, the histone code, chromatin remodeling, and transcriptional control. Data on the histone deacetylase and DNA methyltransferase inhibitors under preclinical and clinical investigation were presented and discussed. The optimal correlative laboratory studies for monitoring clinical trials with these agents remain controversial. DNA methyltransferase and histone deacetylase inhibitors will be combined with each other to maximally re-express genes silenced through promoter methylation. Other classes of agents that may be rationally combined with these classes of drugs include retinoids, steroid hormones, and cytotoxic drugs.
Acetylation↗
Cytotoxic and DNA-damaging effects of 1,2-bis(sulfonyl)hydrazines on human cells of the Mer+ and Mer- phenotype.
A series of 1,2-bis(sulfonyl)hydrazines with the capacity to function as alkylating agents have been evaluated for their toxicity towards Mer- HT29 and Mer- BE cells, and for their ability to produce DNA damage expressed as single-strand breaks and DNA interstrand cross-links. Compounds of this class with methylating potential showed a marked difference in their capacity to inhibit the growth of Mer- and Mer+ cells, being considerably more toxic to BE Mer- cells. Dose-dependent DNA single-strand breaks were induced by these agents, with the quantity of breaks produced in Mer- and Mer+ cells being essentially the same. Maintenance of these lesions did not appear to explain the differential in toxicity to BE and HT29 cells. A chloroethylating compound of this class was also more toxic to Mer- BE cells than to Mer+ HT29 cells, but the differential toxicity was considerably less than that of the methylating agents of the series. The chloroethylating agent did not produce measurable single-strand breaks of the DNA of treated cells, but caused more DNA interstrand cross-links in Mer- cells than in Mer+ cells. Thus, DNA interstrand cross-links may be at least in part responsible for the cell kill produced by this agent. The findings suggest that methylating and chloroethylating derivatives of the 1,2-bis(sulfonyl)hydrazine family have different biochemical determinants of their cytodestructive actions.
Alkylating Agents↗
Methylation status and transcriptional expression of the MHC class I loci in human trophoblast cells from term placenta.
Of the various molecular regulatory mechanisms that may be used by human trophoblast cells to down-regulate expression of HLA class I genes, we chose to investigate the methylation of DNA, generally associated with inhibition of transcription. We analyzed the methylation status of different HLA class I loci in villous and extravillous cytotrophoblast cells and in vitro-differentiated syncytiotrophoblast, purified from human term placenta, as well as in the human trophoblast-derived JAR and JEG-3 cell lines. We then compared methylation status and transcriptional activity. An inverse relationship was established between JAR and JEG-3: HLA-A, -B, and -G are methylated and repressed in JAR, whereas in JEG-3, HLA-A is methylated and repressed but HLA-B and -G are partially methylated and transcribed. HLA-E is unmethylated and transcribed in both cell lines. Apart from HLA-E, which is always unmethylated and transcribed, no such relationship exists for the other class I loci in trophoblast cells. Whereas nonclassical HLA-G and classical HLA-A and -B class I genes are undermethylated in both cytotrophoblast and syncytiotrophoblast, they are clearly transcribed in the former but minimally transcribed in the latter subpopulation. Thus, the down-regulation of class I gene expression in the in vitro-differentiated syncytiotrophoblast is unlikely to be caused by DNA methylation. Furthermore, there is no detectable expression of any class I molecule at the cell surface of either trophoblast cell subpopulation, suggesting a negative control on translation and/or on the secretory pathway to the plasma membrane.
Base Sequence↗
Guanine nucleotides stimulate carboxyl methylation of kidney cytosolic proteins.
We studied the effect of guanine nucleotides on the carboxyl methylation catalyzed by class II protein carboxylmethyltransferases (PCMT). Addition of guanosine 5'-O-(gamma-thio)triphosphate (GTP gamma S) promoted a time- and concentration-dependent enhancement of protein methylation in the cytosolic fraction isolated from kidney cortex. GTP gamma S affected the kinetics of the methylation reaction, as reflected by alterations of both apparent Km and Vmax of the methyltransferase. This effect was specific for guanine nucleotides and was completely abolished by addition of S-adenosyl-L-homocysteine, a well-known inhibitor of methyltransferase-catalyzed reactions. No GTP gamma S stimulation of methylation was found in cytosolic extracts from any of the other tissues studied, including brain, testis, spleen, and liver, nor in brush-border membranes isolated from the kidney cortex. The methylated proteins were highly sensitive to moderately alkaline conditions, suggesting that the methyl esters were formed on L-isoaspartyl residues and thus methylated by a class II PCMT. These results suggest that class-II-associated protein methylation activity from the soluble fraction of the kidney can be regulated by guanine nucleotides.
Animals↗
Separation of methylated free bile acids from their taurine and methyl glycine conjugates by thin-layer chromatography.
Class separation of methylated free bile acids from bile acids conjugated with taurine and methylglycine was accomplished using a solvent system of 2,2,4-trimethylpentane-absolute ethanol 10:1 (v/v). By developing a silica thin-layer plate two times with solvent in a Brinkmann sandwich tank, the difficult resolution between methyl cholate and methyl glycolithocholate was achieved. Evidence is presented that this separation system may be useful as a preparative step in the analysis of bile acids by gas-liquid chromatography or high pressure liquid chromatography.--Bolt, M. J. G. Separation of methylated free bile acids from their taurine and methyl glycine conjugates by thin-layer chromatography.
Bile Acids and Salts↗
A novel class of FokI restriction endonuclease mutants that cleave hemi-methylated substrates.
A genetic screen was used to identify amino acid substitutions that enable the FokI restriction endonuclease to cleave DNA in cells that express the cognate methyltransferase activity. Missense mutations that give rise to this phenotype were isolated at eight different positions (G188K, P196S, T343I, S388N, S395F, E407K, E410K, D421N), clustered in two regions of the polypeptide sequence of FokI. Two of the mutant endonucleases (P196S and D421N) were purified to homogeneity and analyzed in detail. Both mutants cleave FokI target sites (5'-GGATG-3') in a manner similar to the wild-type enzyme. Neither mutant cleaved noncanonical sequences, but both efficiently cleaved DNA substrates containing hemi-methylated FokI sites. This class of mutations has not been observed with other restriction enzymes.
Base Sequence↗
Cyclosporin treatment alters protein phosphorylation in kidney membranes.
Phosphorylation, protein carboxyl methylation, and ADP-ribosylation were assayed in renal basolateral membranes and brush border membranes isolated from rats treated by subcutaneous administration of 5 or 10 mg/(kg.day) of cyclosporin A (CsA) for 10 days to investigate potential alterations in signal transduction in kidney cortex. Protein carboxyl methylation of class II measured in membranes and in cytosolic fraction was not affected by CsA treatment. ADP-ribosylation performed in the presence of pertussis or cholera toxin was also similar in control and treated rats. However, changes in phosphorylation of endogenous substrates were observed in membranes and cytosol isolated from rats treated with 10 mg/(kg.day) of CsA. Phosphorylation was increased for two brush border membrane proteins (56 and 77 kilodaltons (kDa)) by 47 and 24% and for two basolateral membrane proteins (51 and 80 kDa) by 28 and 29%, respectively. In the cytosolic fraction, phosphorylation of two proteins (31 and 65 kDa) was increased by 37% and that of 25- and 43-kDa proteins was reduced by 29%. Protein kinase A, protein kinase C, and tyrosine protein kinase activities were also determined in membranes. Increases in protein kinase C and tyrosine protein kinase activities were observed in basolateral membranes, but not in brush border membranes after cyclosporin A administration. Endogenous substrates for tyrosine kinase were also detected with an antiphosphotyrosine (PY20) monoclonal antibody. Densitometric analysis indicated that the phosphorylation of three proteins of high molecular masses (61, 132, and 183 kDa) was stimulated by CsA in basolateral membranes.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenosine Diphosphate Ribose↗
Diastereoselective addition of amines to vinyl sulfone modified carbohydrates: a highly flexible methodology for the synthesis of new classes of deoxyaminosugars
Methyl 2,3-dideoxy-4,6-O-(phenylmethylene)-3-C-phenylsulfonyl-alpha- D-erythro-hex-2-enopyranoside (5 alpha) and methyl 2,3-dideoxy-4,6-O-(phenylmethylene)-3-C-phenylsulfonyl- beta-D-erythro-hex-2-enopyranoside (5 beta) have been subjected to Michael addition reaction with various amines to develop a new methodology for the synthesis of new classes of aminosugars. Compound 5 alpha reacted with primary amines to generate gluco- derivatives, but secondary amines produced both gluco- (major) and manno- (minor) isomers. Compound 5 beta, on the other hand, produced only gluco- isomers with both primary and secondary amines. The stereochemical course of addition of some of the amines to 5 alpha and 5 beta are significantly different from that of the addition of amines to 3-nitroenopyranoses. The present route to the syntheses of various aminosugars with gluco- configurations from 5 alpha and 5 beta constitutes a novel method for the introduction of N-monoalkylated and N,N-dialkylated amines to the C-2 carbon of pyranoses in equatorial configurations.
Journal Article↗