The role of epigenetics in resistance to cisplatin chemotherapy in lung cancer

Non-small cell lung cancer (NSCLC) is the most common cause of cancer related death in the world. Cisplatin and carboplatin are the most commonly used cytotoxic chemotherapeutic agents to treat the disease. These agents, usually combined with drugs such as gemcitabine or pemetrexed, induce objective tumor responses in only 20-30% of patients. Aberrant epigenetic regulation of gene expression is a frequent event in NSCLC. In this article we review the emerging evidence that epigenetics and the cellular machinery involved with this type of regulation may be key elements in the development of cisplatin resistance in NSCLC. © 2011 by the authors; licensee MDPI, Basel, Switzerland.

Role of the apolipoprotein E and catechol-O-methyltransferase genes in prospective and retrospective memory traits

Human memory is a complex neurocognitive process. By combining psychological and molecular genetics expertise, we examined the APOE ε4 allele, a known risk factor for Alzheimer's disease, and the COMT Val 158 polymorphism, previously implicated in schizophrenia, for association with lowered memory functioning in healthy adults. To assess memory type we used a range of memory tests of both retrospective and prospective memory. Genotypes were determined using RFLP analysis and compared with mean memory scores using univariate ANOVAs. Despite a modest sample size (n=197), our study found a significant effect of the APOE ε4 polymorphism in prospective memory. Supporting our hypothesis, a significant difference was demonstrated between genotype groups for means of the Comprehensive Assessment of Prospective Memory total score (p=0.036; ε4 alleles=1.99; all other alleles=1.86). In addition, we demonstrate a significant interactive effect between the APOE ε4 and COMT polymorphisms in semantic memory. This is the first study to investigate both APOE and COMT genotypes in relation to memory in non-pathological adults and provides important information regarding the effect of genetic determinants on human memory.

Isolation and expression analysis of multiple isoforms of putative farnesoic acid O-methyltransferase in several crustacean species

Farnesoic acid O-methyltransferase (FaMeT) is the enzyme responsible for the conversion of farnesoic acid (FA) to methyl farnesoate (MF) in the final step of MF synthesis. Multiple isoforms of putative FaMeT were isolated from six crustacean species belonging to the families Portunidae, Penaeidae, Scyllaridae and Parastacidae. The portunid crabs Portunus pelagicus and Scylla serrata code for three forms: short, intermediate and long. Two isoforms (short and long) were isolated from the penaeid prawns Penaeus monodon and Fenneropenaeus merguiensis. Two isoforms were also identified in the scyllarid Thenus orientalis and parastacid Cherax quadricarinatus. Putative FaMeT sequences were also amplified from the genomic DNA of P. pelagicus and compared to the putative FaMeT transcripts expressed. Each putative FaMeT cDNA isoform was represented in the genomic DNA, indicative of a multi-gene family. Various tissues from P. pelagicus were individually screened for putative FaMeT expression using PCR and fragment analysis. Each tissue type expressed all three isoforms of putative FaMeT irrespective of sex or moult stage. Protein domain analysis revealed the presence of a deduced casein kinase II phosphorylation site present only in the long isoform of putative FaMeT.

Separate and interacting effects within the catechol-O-methyltransferase (COMT) are associated with schizophrenia

Several lines of evidence have implicated the catechol-O-methyltransferase (COMT) gene as a candidate for schizophrenia (SZ) susceptibility, not only because it encodes a key dopamine catabolic enzyme but also because it maps to the velocardiofacial syndrome region of chromosome 22q11 which has long been associated with SZ predisposition. The interest in COMT as a candidate SZ risk factor has led to numerous case-control and family-based studies, with the majority placing emphasis on examining a functional Val/Met polymorphism within this enzyme. Unfortunately, these studies have continually produced conflicting results. To assess the genetic contribution of other COMT variants to SZ susceptibility, we investigated three single-nucleotide polymorphisms (SNPs) (rs737865, rs4633, rs165599) in addition to the Val/Met variant (rs4680) in a highly selected sample of Australian Caucasian families containing 107 patients with SZ. The Val/Met and rs4633 variants showed nominally significant associations with SZ (P<0.05), although neither of the individual SNPs remained significant after adjusting for multiple testing (most significant P=0.1174). However, haplotype analyses showed strong evidence of an association; the most significant being the three-marker haplotype rs737865-rs4680-rs165599 (global P=0.0022), which spans more than 26 kb. Importantly, conditional analyses indicated the presence of two separate and interacting effects within this haplotype, irrespective of gender. In addition, our results indicate the Val/Met polymorphism is not disease-causing and is simply in strong linkage disequilibrium with a causative effect, which interacts with another as yet unidentified variant approximately 20 kb away. These results may help explain the inconsistent results reported on the Val/Met polymorphism and have important implications for future investigations into the role of COMT in SZ susceptibility.

Characterization of an N6 adenine methyltransferase from Helicobacter pylori strain 26695 which methylates adjacent adenines on the same strand

Genomic sequences of Helicobacter pylori strains 26695, J99, HPAGI and G27 have revealed an abundance of restriction and modification genes. hp0050, which encodes an N6 adenine DNA methyltransferase, was cloned, overexpressed and purified to near homogeneity. It recognizes the sequence 5'-GRRG-3' (where R is A or G) and, most intriguingly, methylates both adenines when R is A (5'-GAAG-3'). Kinetic analysis suggests a nonprocessive (repeated-hit) mechanism of methylation in which HP0050 methyltransferase methylates one adenine at a time in the sequence 5'-GAAG-3'. This is the first report of an N6 adenine DNA methyltransferase that methylates two adjacent residues on the same strand. Interestingly, HP0050 homologs from two clinical strains of H. pylori (PG227 and 128) methylate only 5'-GAGG-3' compared with 5'-GRRG-3' in strain 26695. HP0050 methyltransferase is highly conserved as it is present in more than 90% of H. pylori strains. Inactivation of hp0050 in strain PG227 resulted in poor growth, suggesting its role in the biology of H. pylori. Collectively, these findings provide impetus for exploring the role(s) of this conserved DNA methyltransferase in the cellular processes of H. pylori.

Mutations in hpyAVIBM, C5 cytosine DNA methyltransferase from Helicobacter pylori result in relaxed specificity

The genome of Helicobacter pylori is rich in restrictionmodification (RM) systems. Approximately 4% of the genome codes for components of RM systems. hpyAVIBM, which codes for a phase-variable C5 cytosine methyltransferase (MTase) from H. pylori, lacks a cognate restriction enzyme. Over-expression of M.HpyAVIB in Escherichia coli enhances the rate of mutations. However, when the catalytically inactive F9N or C82W mutants of M.HpyAVIB were expressed in E. coli, mutations were not observed. The M.HpyAVIB gene itself was mutated to give rise to different variants of the MTase. M.HpyAVIB variants were purified and differences in kinetic properties and specificity were observed. Intriguingly, purified MTase variants showed relaxed substrate specificity. Homologues of hpyAVIBM homologues amplified and sequenced from different clinical isolates showed similar variations in sequence. Thus, hpyAVIBM presents an interesting example of allelic variations in H. pylori where changes in the nucleotide sequence result in proteins with new properties.

Epigenetic remodeling of meiotic crossover frequency in Arabidopsis thaliana DNA methyltransferase mutants.

Meiosis is a specialized eukaryotic cell division that generates haploid gametes required for sexual reproduction. During meiosis, homologous chromosomes pair and undergo reciprocal genetic exchange, termed crossover (CO). Meiotic CO frequency varies along the physical length of chromosomes and is determined by hierarchical mechanisms, including epigenetic organization, for example methylation of the DNA and histones. Here we investigate the role of DNA methylation in determining patterns of CO frequency along Arabidopsis thaliana chromosomes. In A. thaliana the pericentromeric regions are repetitive, densely DNA methylated, and suppressed for both RNA polymerase-II transcription and CO frequency. DNA hypomethylated methyltransferase1 (met1) mutants show transcriptional reactivation of repetitive sequences in the pericentromeres, which we demonstrate is coupled to extensive remodeling of CO frequency. We observe elevated centromere-proximal COs in met1, coincident with pericentromeric decreases and distal increases. Importantly, total numbers of CO events are similar between wild type and met1, suggesting a role for interference and homeostasis in CO remodeling. To understand recombination distributions at a finer scale we generated CO frequency maps close to the telomere of chromosome 3 in wild type and demonstrate an elevated recombination topology in met1. Using a pollen-typing strategy we have identified an intergenic nucleosome-free CO hotspot 3a, and we demonstrate that it undergoes increased recombination activity in met1. We hypothesize that modulation of 3a activity is caused by CO remodeling driven by elevated centromeric COs. These data demonstrate how regional epigenetic organization can pattern recombination frequency along eukaryotic chromosomes.

Variants in the catechol-o-methyltransferase (COMT) gene are associated with schizophrenia in Irish high-density families

The enzyme catechol-o-methyltransferase (COMT) transfers a methyl group from adenosylmethionine to catecholamines including the neurotransmitters dopamine, epinephrine and norepinephrine. This methylation results in the degradation of catecholamines. The involvement of the COMT gene in the metabolic pathway of these neurotransmitters has made it an attractive candidate gene for many psychiatric disorders. In this article, we reported our study of association of COMT with schizophrenia in Irish families with a high density of schizophrenia. Three single nucleotide polymorphisms (SNPs) were genotyped for the 274 such families and within-family transmission disequilibrium tests were performed. SNP rs4680, which is the functional Val/Met polymorphism, showed modest association with the disease by the TRANSMIT, FBAT and PDT programs, while the other two SNPs were negative. These SNPs showed lower level of LDs with each other in the Irish subjects than in Ashkenazi Jews. Haplotype analysis indicated that a haplotype, haplotype A-G-A for SNPs rs737865-rs4680-rs165599, was preferentially transmitted to the affected subjects. This was different from the reported G-G-G haplotype found in Ashkenazi Jews, but both haplotypes shared the Val allele. We concluded that COMT gene is associated with schizophrenia and carries a small but significant risk to the susceptibility in the Irish subjects.

Catechol-O-methyltransferase and the clinical features of psychosis

A functional polymorphism (Val-158-Met) at the Catechol-O-methyltransferase (COMT) locus has been identified as a potential etiological factor in schizophrenia. Yet the association has not been convincingly replicated across independent samples. We hypothesized that phenotypic heterogeneity might be diluting the COMT effect. To clarify the putative association, we performed an exploratory analysis to test for association between COMT and five psychosis symptom scales. These were derived through factor analysis of the Operational Criteria Checklist for Psychiatric Illness. Our sample was the Irish Study of High Density Schizophrenia Families, a large collection consisting of 268 multiplex families. This sample has previously shown a small but significant effect of the COMT Val allele in conferring risk for schizophrenia. We tested for preferential transmission of COMT alleles from parent to affected offspring (n = 749) for each of the five factor-derived scales (negative symptoms, delusions, hallucinations, mania, and depression). Significant overtransmission of the Val allele was found for mania (P <0.05) and depression (P = 0.01) scales. Examination of odds ratios (ORs) revealed a heterogeneous effect of COMT, whereby it had no effect on Negative Symptoms, but largest impact on Depression (OR = 1.4). These results suggest a modest affective vulnerability conferred by this allele in psychosis, but will require replication.

The impact of catechol-O-methyltransferase genotype on the acute responsiveness of vascular reactivity to a green tea extract

The beneficial effects of green tea catechins, such as the proposed improvement in endothelial function, may be influenced by phase II metabolism during and after absorption. The methylation enzyme, catechol-O-methyltransferase (COMT), has a missense mutation rs4680 (G to A), proposed to result in a 40 % reduction in enzyme activity. In the present pilot study, twenty subjects (ten of each homozygous COMT genotype) were recruited. Green tea extract capsules (836 mg green tea catechins) were given in a fasted state, and a high-carbohydrate breakfast was given after 60 min. Blood samples and vascular function measurements were taken at regular intervals. The change in digital volume pulse stiffness index (SI) from baseline was shown to be different between genotype groups at 120 and 240 min, with a lower SI in the GG individuals (P ≤ 0·044). The change in blood pressure from baseline also differed between genotype groups, with a greater increase in systolic (P = 0·023) and diastolic (P = 0·034) blood pressure at 120 min in the GG group. The AA group was shown to have a greater increase in insulin concentrations at 120 min (P = 0·019) and 180 min (P = 0·008) compared with baseline, despite similar glucose profiles. No genotypic differences were found in vascular reactivity measured using laser Doppler iontophoresis, total nitrite, lipids, plasma total antioxidant capacity or inflammatory markers after ingestion of the green tea extract. In conclusion, SI and insulin response to the glucose load differed between the COMT genotype groups, and this may be suggestive of a green tea extract and genotype interaction.

A preliminary investigation of the impact of catechol-O-methyltransferase genotype on the absorption and metabolism of green tea catechins

Purpose Green tea is thought to possess many beneficial effects on human health. However, the extent of green tea polyphenol biotransformation may affect its proposed therapeutic effects. Catechol-O-methyltransferase (COMT), the enzyme responsible for polyphenolic methylation, has a common polymorphism in the genetic code at position 158 reported to result in a 40% reduction in enzyme activity in in vitro studies. The current preliminary study was designed to investigate the impact of COMT genotype on green tea catechin absorption and metabolism in humans. Methods Twenty participants (10 of each homozygous COMT genotype) were recruited, and plasma concentration profiles were produced for epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG), epicatechin (EC) and 4′-O-methyl EGCG after 1.1 g of Sunphenon decaffeinated green tea extract (836 mg green tea catechins), with a meal given after 60 min. Results For the entire group, EGCG, EGC, EC, ECG and 4′-O-methyl EGCG reached maximum concentrations of 1.09, 0.41, 0.33, 0.16 and 0.08 μM at 81.5, 98.5, 99.0, 85.5 and 96.5 min, respectively. Bimodal curves were observed for the non-gallated green tea catechins EGC and EC as opposed to single-peaked curves for the gallated green tea catechins EGCG and ECG. No significant parametric differences between COMT genotype groups were found. Conclusions In conclusion, the COMT Val(158/108)Met does not appear to have a dramatic influence on EGCG absorption and elimination. However, further pharmacokinetic research is needed to substantiate these findings.

The impact of the catechol-O-methyltransferase genotype on vascular function and blood pressure after acute green tea ingestion

SCOPE: Evidence for the benefits of green tea catechins on vascular function is inconsistent, with genotype potentially contributing to the heterogeneity in response. Here, the impact of the catechol-O-methyltransferase (COMT) genotype on vascular function and blood pressure (BP) after green tea extract ingestion are reported. METHODS AND RESULTS: Fifty subjects (n = 25 of the proposed low-activity [AA] and of the high-activity [GG] COMT rs4680 genotype), completed a randomized, double-blind, crossover study. Peripheral arterial tonometry, digital volume pulse (DVP), and BP were assessed at baseline and 90 min after 1.06 g of green tea extract or placebo. A 5.5 h and subsequent 18.5 h urine collection was performed to assess green tea catechin excretion. A genotype × treatment interaction was observed for DVP reflection index (p = 0.014), with green tea extract in the AA COMT group attenuating the increase observed with placebo. A tendency for a greater increase in diastolic BP was evident at 90 min after the green tea extract compared to placebo (p = 0.07). A genotypic effect was observed for urinary methylated epigallocatechin during the first 5.5 h, with the GG COMT group demonstrating a greater concentration (p = 0.049). CONCLUSION: Differences in small vessel tone according to COMT genotype were evident after acute green tea extract.

Inhibition of DNA methyltransferase stimulates the expression of signal transducer and activator of transcription 1, 2, and 3 genes in colon tumor cells

Inhibitors of DNA methyltransferase, typified by 5-aza-2′-deoxycytidine (5-Aza-CdR), induce the expression of genes transcriptionally down-regulated by de novo methylation in tumor cells. We utilized gene expression microarrays to examine the effects of 5-Aza-CdR treatment in HT29 colon adenocarcinoma cells. This analysis revealed the induction of a set of genes that implicated IFN signaling in the HT29 cellular response to 5-Aza-CdR. Subsequent investigations revealed that the induction of this gene set correlates with the induction of signal transducer and activator of transcription (STAT) 1, 2, and 3 genes and their activation by endogenous IFN-α. These observations implicate the induction of the IFN-response pathway as a major cellular response to 5-Aza-CdR and suggests that the expression of STATs 1, 2, and 3 can be regulated by DNA methylation. Consistent with STAT’s limiting cell responsiveness to IFN, we found that 5-Aza-CdR treatment sensitized HT29 cells to growth inhibition by exogenous IFN-α2a, indicating that 5-Aza-CdR should be investigated as a potentiator of IFN responsiveness in certain IFN-resistant tumors.

The DNMT3B DNA methyltransferase gene is mutated in the ICF immunodeficiency syndrome

DNA methylation is an important regulator of genetic information in species ranging from bacteria to humans. DNA methylation appears to be critical for mammalian development because mice nullizygous for a targeted disruption of the DNMT1 DNA methyltransferase die at an early embryonic stage. No DNA methyltransferase mutations have been reported in humans until now. We describe here the first example of naturally occurring mutations in a mammalian DNA methyltransferase gene. These mutations occur in patients with a rare autosomal recessive disorder, which is termed the ICF syndrome, for immunodeficiency, centromeric instability, and facial anomalies. Centromeric instability of chromosomes 1, 9, and 16 is associated with abnormal hypomethylation of CpG sites in their pericentromeric satellite regions. We are able to complement this hypomethylation defect by somatic cell fusion to Chinese hamster ovary cells, suggesting that the ICF gene is conserved in the hamster and promotes de novo methylation. ICF has been localized to a 9-centimorgan region of chromosome 20 by homozygosity mapping. By searching for homologies to known DNA methyltransferases, we identified a genomic sequence in the ICF region that contains the homologue of the mouse Dnmt3b methyltransferase gene. Using the human sequence to screen ICF kindreds, we discovered mutations in four patients from three families. Mutations include two missense substitutions and a 3-aa insertion resulting from the creation of a novel 3′ splice acceptor. None of the mutations were found in over 200 normal chromosomes. We conclude that mutations in the DNMT3B are responsible for the ICF syndrome.

The Saccharomyces cerevisiae Prenylcysteine Carboxyl Methyltransferase Ste14p Is in the Endoplasmic Reticulum Membrane

Eukaryotic proteins containing a C-terminal CAAX motif undergo a series of posttranslational CAAX-processing events that include isoprenylation, C-terminal proteolytic cleavage, and carboxyl methylation. We demonstrated previously that the STE14 gene product of Saccharomyces cerevisiae mediates the carboxyl methylation step of CAAX processing in yeast. In this study, we have investigated the subcellular localization of Ste14p, a predicted membrane-spanning protein, using a polyclonal antibody generated against the C terminus of Ste14p and an in vitro methyltransferase assay. We demonstrate by immunofluorescence and subcellular fractionation that Ste14p and its associated activity are localized to the endoplasmic reticulum (ER) membrane of yeast. In addition, other studies from our laboratory have shown that the CAAX proteases are also ER membrane proteins. Together these results indicate that the intracellular site of CAAX protein processing is the ER membrane, presumably on its cytosolic face. Interestingly, the insertion of a hemagglutinin epitope tag at the N terminus, at the C terminus, or at an internal site disrupts the ER localization of Ste14p and results in its mislocalization, apparently to the Golgi. We have also expressed the Ste14p homologue from Schizosaccharomyces pombe, mam4p, in S. cerevisiae and have shown that mam4p complements a Δste14 mutant. This finding, plus additional recent examples of cross-species complementation, indicates that the CAAX methyltransferase family consists of functional homologues.