Metabolism Methylation Research Articles

5-Methyltetrahydrofolate Is Bound in Intersubunit Areas of Rat Liver Folate-binding Protein Glycine N-Methyltransferase

Glycine N-methyltransferase (GNMT) is a key regulatory enzyme in methyl group metabolism. It is abundant in the liver, where it uses excess S-adenosylmethionine (AdoMet) to methylate glycine to N-methylglycine (sarcosine) and produces S-adenosylhomocysteine (AdoHcy), thereby controlling the methylating potential of the cell. GNMT also links utilization of preformed methyl groups, in the form of methionine, to their de novo synthesis, because it is inhibited by a specific form of folate, 5-methyltetrahydrofolate. Although the structure of the enzyme has been elucidated by x-ray crystallography of the apoenzyme and in the presence of the substrate, the location of the folate inhibitor in the tetrameric structure has not been identified. We report here for the first time the crystal structure of rat GNMT complexed with 5-methyltetrahydrofolate. In the GNMT-folate complex, two folate binding sites were located in the intersubunit areas of the tetramer. Each folate binding site is formed primarily by two 1-7 N-terminal regions of one pair of subunits and two 205-218 regions of the other pair of subunits. Both the pteridine and p-aminobenzoyl rings are located in the hydrophobic cavities formed by Tyr5, Leu207, and Met215 residues of all subunits. Binding experiments in solution also confirm that one GNMT tetramer binds two folate molecules. For the enzymatic reaction to take place, the N-terminal fragments of GNMT must have a significant degree of conformational freedom to provide access to the active sites. The presence of the folate in this position provides a mechanism for its inhibition.

Amino acids and the regulation of methyl balance in humans

To outline recent advances in our understanding of the metabolic basis for the maintenance of cellular S-adenosylmethionine levels and, thus, for facilitating the many crucial methylation reactions in the body. Amino acids are intimately involved in these processes. The application of stable-isotope methodology has permitted accurate estimation of the total transmethylation flux in humans. Chemical balance studies have identified the quantitatively major transmethylation reactions. New evidence points to a key role for deranged S-adenosylmethionine metabolism in the pathogenesis of liver disease. Mutations in key enzymes point to the importance of methyl metabolism in closure of the neural tube, synthesis of creatine and metabolic clearance of methionine. Dietary interventions designed to affect S-adenosylmethionine availability to pregnant mice have been shown to modulate the epigenetic DNA methylation of specific genes. These findings are of relevance to the pathogenesis of neural tube defects as well as the interaction between a genetic polymorphism and nutritional status. They also address the issue of methyl group availability and epigenetic regulation. Finally, they are also relevant to the etiology of cirrhosis and steatohepatitis.

Prevention of aneuploidy by S-adenosyl-methionine in human cells treated with sodium arsenite

Alterations in methyl group's metabolism affect availability of S-adenosyl- l-methionine (SAM); these modifications can be originated by enzyme polymorphisms, nutritional deficiencies, and exposure to chemical agents. There are several types of chemicals that interfere with methyl groups, among them is arsenic. It deserves special attention because it modifies a number of cell functions that influence the development of diseases such as cancer. Since part of arsenic's toxicity is influenced by changes on SAM availability, in a previous study we investigated whether exogenous addition of SAM to cells treated with sodium arsenite (NaAsO 2) has an effect on its genotoxicity. Results demonstrated that SAM reduces the frequency of cells presenting micronuclei (MN) and tubulin-cytoskeleton defects after treatment with NaAsO 2. MN are fragments of the cell nucleus that may contain whole chromosomes or chromosome fragments depending on whether they derive either from the aneugenic or from the clastogenic action of chemicals. Therefore one question generated by these results was whether SAM reduced only the frequency MN resulting from aneugenic damage. To answer this question, in the present work we used an all-centromere DNA probe to distinguish the type of MN reduced by SAM after treatment with NaAsO 2 and vinblastine. In addition, the capacity of SAM to reduce clastogenicity was also evaluated. Results show that SAM decreases the frequency of cells with MN containing whole chromosomes in cultures treated either with NaAsO 2 or with vinblastine; however, induction of double-strand breaks by NaAsO 2 was not prevented by SAM.

Genetic polymorphisms in methylenetetrahydrofolate reductase and clinical response to chemotherapy in non-small cell lung cancer

Methylenetetrahydrofolate reductase (MTHFR) plays an important role in metabolism of folate and DNA methylation. In vitro, many studies have demonstrated that abnormal methylation of some genes may affect the sensitivity of tumor cells to cytotoxic drugs and agents interfering with DNA synthesis. The aim of this study is to investigate the relationship between genetic polymorphisms of MTHFR C677T or A1298C and the response to platinum-based chemotherapy in non-small cell lung cancer (NSCLC). A total of 97 patients with NSCLC were analyzed. MTHFR genotypes were detected in all the patients by PCR-RFLP method. All the patients were treated with platinum-based chemotherapy. (1) Out of all the cases, the frequencies of MTHFR C677T C/C, C/T and T/T genotypes were 34.0%, 50.5% and 15.5%, respectively, while the frequencies of MTHFR A1298C A/A, A/C and C/C genotypes were 64.6%, 29.2% and 6.3%, respectively. The overall response rate (complete and partial response) to platinum-based chemotherapy was 39.2%. (2) No significant difference in response rate to chemotherapy was observed according to the MTHFR C677T or A1298C genotypes. However, MTHFR C677T and A1298C polymorphisms showed a synergic effect on chemotherapeutic efficacy, the response rate of patients with MTHFR C677T T allele and A1298C A/A genotype (51.1%) was significantly higher than those with MTHFR C677T C/T and A1298C C allele (12.5%)(P=0.007, OR=7.30, 95% CI: 1.34-52.47). The results suggest that the synergic effect between MTHFR C677T and A1298C polymorphisms is associated with clinical response to platinum-based chemotherapy. Detection of MTHFR genotypes may indicate the sensitivity of NSCLC patients to platinum-based chemotherapy.

Methyl group metabolism gene polymorphisms as modifier of breast cancer risk in Italian BRCA1/2 carriers

BRCA1 and 2 are major cancer susceptibility genes but their penetrance is highly variable. The folate metabolism plays an important role in DNA methylation and its alterated metabolism is associated with cancer risk. The role of allele variants 677T and 1298C (MTHFR gene) and 2756G (MS gene) has been investigated as potentially modifying factors of BRCA gene penetrance, evaluated as age at first diagnosis of cancer, in 484 BRCA1/BRCA2 carriers and in 108 sporadic breast cancer cases as a control group. The genotype analysis has been performed by means of PCR/RFLP's. The analysis of association between a particular genotype and disease risk was performed using Cox Regression with time to breast or ovarian cancer onset as the end-point. The presence of 677T allele confers an increased risk of breast cancer in BRCA1 carriers (P = 0.007) and the presence of 1298C allele confers an increased risk of breast cancer in sporadic cases (P = 0.015).

Hepatic phosphatidylethanolamine N-methyltransferase expression is increased in diabetic rats.

Phosphatidylcholine is an essential phospholipid that is synthesized by 2 different pathways, the CDP-choline pathway and the methylation of phosphatidylethanolamine by phosphatidylethanolamine N-methyltransferase (PEMT). Recent studies have suggested that PEMT is an important consumer of methyl groups from S-adenosylmethionine (SAM) and is a major determinant of homocysteine pools. Diabetes and all-trans-retinoic acid (ATRA) have been shown to alter the activities of several enzymes involved in methyl group metabolism. Thus, we investigated how diabetes and ATRA, individually and together, affect SAM-dependent phospholipid methylation. Rats received a single injection of streptozotocin (60 mg/kg body wt) or vehicle followed by administration of ATRA (30 mumol/kg body wt) or vehicle for 5 d. The hepatic activity of PEMT increased 50% in both diabetic rat groups, whereas administration of ATRA was without effect. In diabetic rats, plasma total homocysteine decreased 30-35% in all treatment groups as compared with the control group. Thus, alterations in the activity of PEMT were not directly correlated to changes in homocysteine concentrations. Moreover, treatment of diabetic rats with insulin prevented the increase in PEMT activity and abundance. Because these observations support an increased need for SAM-dependent phosphatidylcholine synthesis, this may also indicate an increased choline requirement in diabetes.

CHKA and PCYT1Agene polymorphisms, choline intake and spina bifida risk in a California population

BackgroundNeural tube defects (NTDs) are among the most common of all human congenital defects. Over the last two decades, accumulating evidence has made it clear that periconceptional intake of folic acid can significantly reduce the risk of NTD affected pregnancies. This beneficial effect may be related to the ability of folates to donate methyl groups for critical physiological reactions. Choline is an essential nutrient and it is also a methyl donor critical for the maintenance of cell membrane integrity and methyl metabolism. Perturbations in choline metabolism in vitro have been shown to induce NTDs in mouse embryos.MethodsThis study investigated whether single nucleotide polymorphisms (SNPs) in human choline kinase A (CHKA) gene and CTP:phosphocholine cytidylytransferase (PCYT1A) gene were risk factors for spina bifida. Fluorescence-based allelic discrimination analysis was performed for the two CHKA intronic SNPs hCV1562388 (rs7928739) and hCV1562393, and PCYT1A SNP rs939883 and rs3772109. The study population consisted of 103 infants with spina bifida and 338 non-malformed control infants who were born in selected California counties in the period 1989–1991.ResultsThe CHKA SNP hCV1562388 genotypes with at least one C allele were associated with a reduced risk of spina bifida (odds ratio = 0.60, 95%CI = 0.38–0.94). The PCYT1A SNP rs939883 genotype AA was associated with a twofold increased risk of spina bifida (odds ratio = 1.89, 95% CI = 0.97–3.67). These gene-only effects were not substantially modified by analytic consideration to maternal periconceptional choline intake.ConclusionOur analyses showed genotype effects of CHKA and PCYT1A genes on spina bifida risk, but did not show evidence of gene-nutrient interactions. The underlying mechanisms are yet to be resolved.

Genetic Polymorphisms of Methylenetetrahydrofolate Reductase Genes and Diffuse Large B-Cell Lymphoma Risk in Middle Eastern Population.

Diffuse large B-Cell Lymphoma (DLBCL) is one of the most common non-Hodgkin lymphoma types and increased incidence has also been reported during the past 30 years. Methylenetetrahydrofolate (MTHFR) balances the pool of folate coenzymes in one carbon metabolism of DNA synthesis and methylation, both are implicated in carcinogenesis of many types of cancer including lymphoma. Two common variants in the MTHFR gene (C677T and A1298C) have been associated with reduced enzyme activity, thereby making MTHFR polymorphisms a potential candidate caner predisposing factor. These genetic differences are highly race specific and have never been screened in the Saudi DLBCL patients. We conducted hospital-based case control study in the Saudi DLBCL patients.To evaluate the MTHFR C677T and A1298C functional polymorphisms in the MTHFR gene and their association with Saudi DLBCL risk. A hospital based case control study was conducted on a Saudi population- which is known for their genetic homogeneity and high consanguinity- consisting of 187 histologically confirmed DLBCL cases and 513 Saudi controls without a history of cancer. A PCR-RFLP method was used for MTHFR polymorphism genotyping.Data showed that Saudi individuals carrying MTHFR 1298 CC genotype (p<0.001) and genotypes carrying MTHFR 1298C allele (p= 0.012) had 4.23 and 1.73-fold higher risk of developing DLBCL, respectively. Additionally, combined genotype CCCC (MTHFR 677CC+ MTHFR 1298CC) among intermediate MTHFR activity group was associated with 3.489 fold and CTCC (MTHFR 677 CT + 1298CC) among low MTHFR activity group was related to 9.515 fold higher risk, compared with full MTHFR enzyme activity.Our findings suggest that polymorphisms of MTHFR enzyme genes support for the important role of folate metabolism in lymphomagenesis and may be associated with the individual susceptibility to develop DLBCL in Saudi Arabian population.Table 1Distribution of MTHFR polymorphisms in healthy population and lymphoma patients.PolymorphismGenotypeControl groupLymphoma patientspMTHFR C677TCC372 (72.8%)109 (68.1%)CT126 (24.7%)45 (28.1%)0.3461.219TT13 (2.5%)6 (3.8%)0.4041.575CT+TT139 (27.2%)51 (31.9%)0.2691.252MTHFR A1298CAA239 (46.8%)38 (33.6%)AC220 (43.1%)40 (35.4%)0.6251.144CC52 (10.2%)35 (31%)<0.0014.233AC+CC272 (53.2%)75 (66.4%)0.0121.734-Methylenetetrahydrofolate reductase (MTHFR)Table 2Distribution of combined C677T and A1298C MTHFR genotypes in case and control group.GenotypeControlCasepORFull Activity groupCCAA157 (30.8%)22 (27.8%)Intermediate Activity groupCCAC169 (33.2%)28 (35.4%)0.6491.182CCCC45 (8.8%)22 (27.8%)<0.0013.489CTAA69 (13.6%)13 (16.5%)0.4391.345TOTAL283630.1041.589Low Activity GroupCTAC50 (9.8%)5 (6.3%)0.6340.714CTCC6 (1.2%)8 (10.1%)<0.0019.515TTAA12 (2.4%)1 (1.3%)10.595TTAC02 (2.5%)0.017NDTTCC1 (0.2%)---TOTAL69160.1891.655Intermediate + Low352 (69.1%)790.0731.602ND=Not detected

Methionine and Vitamin B 6 Intake and Risk of Pancreatic Cancer: A Prospective Study of Swedish Women and Men

It has been hypothesized that dietary factors involved in methyl group metabolism, such as methionine, folate, and vitamin B(6), may modify cancer risk. We have previously reported an inverse association between folate intake and pancreatic cancer risk in a prospective population-based cohort of Swedish women and men. In the present study, we used data from this prospective study to examine whether methionine and vitamin B(6) intakes were associated with the incidence of exocrine pancreatic cancer. Our study population comprised 81,922 Swedish women and men, aged 45-83 years, who were free from cancer and completed a self-administered food-frequency questionnaire in 1997. We used Cox proportional hazards models to estimate rate ratios with 95% confidence intervals (CI), adjusted for age, sex, education, smoking, body mass index, diabetes, and intakes of total energy and dietary folate. During a mean follow-up of 7.2 years, through June 2005, 147 incident cases of pancreatic cancer were diagnosed. Methionine intake was significantly inversely associated with risk of pancreatic cancer, whereas no significant association was observed for dietary or total vitamin B(6) intake. The multivariate rate ratios comparing the highest with the lowest quartile of methionine intake were 0.44 (95% CI, 0.26-0.73; P for trend = .0005) in women and men combined, 0.59 (95% CI, 0.28-1.21; P for trend = .07) in women, and 0.32 (95% CI, 0.15-0.65; P for trend = .002) in men. These findings suggest that higher methionine intake may reduce the risk of pancreatic cancer.

Genome-wide Hypomethylation in Human Glioblastomas Associated with Specific Copy Number Alteration, Methylenetetrahydrofolate Reductase Allele Status, and Increased Proliferation

Genome-wide reduction in 5-methylcytosine is an epigenetic hallmark of human tumorigenesis. Experimentally induced hypomethylation in mice promotes genomic instability and is sufficient to initiate tumorigenesis. Here, we report that global hypomethylation is common in primary human glioblastomas [glioblastoma multiforme (GBM)] and can affect up to an estimated 10 million CpG dinucleotides per haploid tumor genome. Demethylation involves satellite 2 (Sat2) pericentromeric DNA at chromosomes 1 and 16, the subtelomeric repeat sequence D4Z4 at chromosomes 4q and 10q, and interspersed Alu elements. Severe hypomethylation of Sat2 sequences is associated with copy number alterations of the adjacent euchromatin, suggesting that hypomethylation may be one factor predisposing to specific genetic alterations commonly occurring in GBMs. An additional apparent consequence of global hypomethylation is reactivation of the cancer-testis antigen MAGEA1 via promoter demethylation, but only in GBMs and GBM cell lines exhibiting a 5-methylcytosine content below a threshold of approximately 50%. Primary GBMs with significant hypomethylation tended to be heterozygous or homozygous for the low-functioning Val allele of the rate-limiting methyl group metabolism gene methylenetetrahydrofolate reductase (MTHFR), or had a deletion encompassing this gene at 1p36. Tumors with severe genomic hypomethylation also had an elevated proliferation index and deletion of the MTHFR gene. These data suggest a model whereby either excessive cell proliferation in the context of inadequate methyl donor production from MTHFR deficiency promotes genomic hypomethylation and further genomic instability, or that MTHFR deficiency-associated demethylation leads to increased proliferative activity in GBM.

Folate status modulates the induction of hepatic glycineN-methyltransferase and homocysteine metabolism in diabetic rats

A diabetic state induces the activity and abundance of glycine N-methyltransferase (GNMT), a key protein in the regulation of folate, methyl group, and homocysteine metabolism. Because the folate-dependent one-carbon pool is a source of methyl groups and 5-methyltetrahydrofolate allosterically inhibits GNMT, the aim of this study was to determine whether folate status has an impact on the interaction between diabetes and methyl group metabolism. Rats were fed a diet containing deficient (0 ppm), adequate (2 ppm), or supplemental (8 ppm) folate for 30 days, after which diabetes was initiated in one-half of the rats by streptozotocin treatment. The activities of GNMT, phosphatidylethanolamine N-methyltransferase (PEMT), and betaine-homocysteine S-methyltransferase (BHMT) were increased about twofold in diabetic rat liver; folate deficiency resulted in the greatest elevation in GNMT activity. The abundance of GNMT protein and mRNA, as well as BHMT mRNA, was also elevated in diabetic rats. The marked hyperhomocysteinemia in folate-deficient rats was attenuated by streptozotocin, likely due in part to increased BHMT expression. These results indicate that a diabetic state profoundly modulates methyl group, choline, and homocysteine metabolism, and folate status may play a role in the extent of these alterations. Moreover, the upregulation of BHMT and PEMT may indicate an increased choline requirement in the diabetic rat.

P4-054: Effects of folate, B12 and B6 deprivation on methylation metabolism and amyloid processing in human neuroblastoma and glioblastoma cells

P4-054: Effects of folate, B12 and B6 deprivation on methylation metabolism and amyloid processing in human neuroblastoma and glioblastoma cells

Choline metabolism in high-producing dairy cows: Metabolic and nutritional basis

Choline, the beta-hydroxyethyltrimethylammonium ion, is a strong base containing a trimethylated quaternary nitrogen. Choline occurs widely in biological materials as the compound itself, as acetylcholine and as various phospholipids. In feed ingredients and crude unprocessed fat sources, most choline is present as phosphatidylcholine (lecithin). Although soybean, soybean meal, rapeseed meal, fish meal and dried yeast are relatively rich sources of choline, the bioavailability of choline in these feedstuffs is only moderate. In dairy ruminants, choline is extensively degraded in the rumen. For this reason, dietary choline contributes insignificantly to the choline body pool, and methyl group metabolism is generally conservative with a relatively low rate of methyl catabolism and an elevated rate of de novo synthesis of methyl groups via the tetrahydrofolate system. Gluconeogenic precursors are the primary sources of the new methyl groups. Depending on energy balance and physiological state, such precursors can be deficient in ruminants. In situations of glucose imbalance, for example at the onset of lactation in the dairy cow, choline may become a limiting nutrient for milk production, owing to the extra demand for methyl groups. Furthermore, choline is a lipotropic substance and it may optimize the balance between fat retained and fat metabolized by the liver, thereby improving lipid metabolism in general. The data reviewed in this paper are consistent with these assumptions and support the fact that choline supplementation for dairy cows, in a form protected from ruminal degradation, may improve methyl group metabolism and the status of other nutrients, including vitamin E. Key words: Choline, methyl groups, methionine, dairy cows, rumen-protected choline

S -adenosylmethionine stabilizes cystathionine β-synthase and modulates redox capacity

The transsulfuration pathway converts homocysteine to cysteine and represents the metabolic link between antioxidant and methylation metabolism. The first and committing step in this pathway is catalyzed by cystathionine beta-synthase (CBS), which is subject to complex regulation, including allosteric activation by the methyl donor, S-adenosylmethionine (AdoMet). In this study, we demonstrate that methionine restriction leads to a >10-fold decrease in CBS protein levels, and pulse proteolysis studies reveal that binding of AdoMet stabilizes the protein against degradation by approximately 12 kcal/mol. These observations predict that under pathological conditions where AdoMet levels are diminished, CBS, and therefore glutathione levels, will be reduced. Indeed, we demonstrate this to be the case in a mouse model for spontaneous steatohepatitis in which the gene for the MAT1A isoenzyme encoding AdoMet synthetase has been disrupted, and in human hepatocellular carcinoma, where MAT1A is silenced. Furthermore, diminished CBS levels are associated with reduced cell viability in hepatoma cells challenged with tert-butyl hydroperoxide. This study uncovers a mechanism by which CBS is allosterically activated by AdoMet under normal conditions but is destabilized under pathological conditions, for redirecting the metabolic flux toward methionine conservation. A mechanistic basis for the coordinate changes in redox and methylation metabolism that are a hallmark of several complex diseases is explained by these observations.

Perinatal Choline Influences Brain Structure and Function

Choline is derived not only from the diet, but also from de novo synthesis. It is important for methyl-group metabolism, the formation of membranes, kidney function, and neurotransmission. When deprived of dietary choline, most adult men and postmenopausal women develop signs of organ dysfunction (fatty liver or muscle damage) and have a decreased capacity to convert homocysteine to methionine. Choline is critical during fetal development, when it influences stem cell proliferation and apoptosis, thereby altering brain structure and function (memory is permanently enhanced in rodents exposed to choline during the latter part of gestation).

Hepatic and renal metabolism of methyl groups and homocysteine are altered by diabetes and glucocorticoid treatment in rats

Homocysteine (Hcys) is produced from methionine via transmethylation reactions. The supply and utilization of methyl groups for transmethylation reactions are proposed to be regulated by an abundant hepatic protein, glycine N-methyltransferase (GNMT). Our previous short-term (5–7 d) studies show that GNMT activity and abundance are increased in streptozotocin-induced diabetic rats. However, a concurrent increase in remethylation by betaine-homocysteine methyltransferase may contribute to a decrease in plasma Hcys levels. Elevated plasma Hcys is an independent risk factor for the development of cardiovascular disease (CVD). Elevated plasma Hcys levels are frequently observed in diabetic patients, particularly those with diminished renal function, and the diabetic condition itself is a risk factor for CVD. Thus, the aim of the current study was to investigate temporal changes in hepatic and renal methyl group and Hcys metabolism past the acute diabetic stage. Rat treatments included injection of streptozotocin (60 mg/kg BW) or dexamethasone (DEX, 1 mg/kg BW) to induce a diabetic/gluconeogenic state. Renal GNMT activity and abundance were elevated by DEX. By two weeks, plasma Hcys levels were significantly decreased and hepatic GNMT activity and abundance were markedly increased in the diabetic rat. These results indicate that renal, like hepatic, methyl group metabolism was significantly altered. Future research studies should focus on a more thorough evaluation of hepatic and renal methyl group and Hcys metabolism to understand the temporal progression from hypohomocysteinemia to hyperhomocysteinemia during diabetes. (Support: Am. Diab. Assoc.)

Modulation of methyl group and homocysteine metabolism by insulin treatment in diabetic rats

Modulation of methyl group and homocysteine metabolism by insulin treatment in diabetic rats

Diabetes upregulates phospholipid methylation

Diabetes and all-trans-retinoic acid (ATRA) have been shown to disrupt methyl group metabolism, including a decrease in homocysteine concentrations due to elevated expression of the folate-independent enzyme betaine-homocysteine S-methyltransferase (BHMT) and stimulation of the folate-dependent enzyme methionine synthase, respectively. Phosphatidylcholine (PC) is an essential phospholipid that can be synthesized by either the CDP-choline pathway or S-adenosylmethionine (SAM)-dependent methylation of phosphatidylethanolamine by phosphatidylethanolamine N-methyltransferase (PEMT). It has recently been suggested that PEMT is the major consumer of hepatic methyl groups and thus is a key contributor to elevated plasma homocysteine levels, an independent risk factor for cardiovascular disease. We investigated how diabetes and ATRA, both individually and in combination, affect SAM-dependent phospholipid methylation. Rats received a single injection of streptozotocin (STZ, 60 mg/kg body weight) or vehicle followed by administration of ATRA (30 μmol/kg body weight) or vehicle for 5 d. Diabetes increased the hepatic activity of PEMT 50%, whereas ATRA supplementation was without affect. Although PC concentrations were not altered, the proportion of PC (% total phospholipids) was increased in all treatment groups. Plasma homocysteine concentrations were decreased 30-35% in all treatment groups as compared to control values. Thus, diabetes-mediated alterations in PEMT activity were not directly correlated to plasma homocysteine levels or PC concentrations. Taken together, the increase in PEMT as well as BHMT may indicate an increased requirement for choline in the diabetic state. (Support: Am. Diabetes Assoc.)

DNA hypermethylation in the normal colonic mucosa of patients with colorectal cancer

The CpG-island methylator phenotype (CIMP+) in colorectal cancer (CRC) is characterised by frequent hypermethylation of promoter regions in tumour suppressor genes. Low level methylation of some CpG islands is also seen in the normal colonic mucosa and increases with age; however, it is still unclear what other factors regulate this phenomenon. The first aim of our study was to determine whether the level of promoter methylation is elevated in the normal colonic mucosa of patients with CIMP+ tumours. The second aim was to investigate whether common, functional polymorphisms in genes involved in methyl group metabolism are associated with the level of methylation in this tissue. CpG islands within the ERα, MYOD, P16(INK4A), MLH1, APC, P14(ARF), DAPK and TIMP3 genes were quantitatively evaluated for methylation in normal colonic mucosa from a large series of CRC patients using the MethyLight assay. Genotyping was carried out for polymorphisms in the MTHFR, TS, MS, MTHFD1 and DNMT3b genes. Methylation of ERα and MYOD in normal colonic mucosa increased with age and was higher in female subjects. Methylation of P16(INK4A), MLH1, TIMP3 and DAPK in normal mucosa occurred at a lower level than ERα and MYOD but also increased with age and was significantly higher in patients with CIMP+ tumours. The DNMT3b C46359T polymorphism was associated with significantly less methylation of MYOD and MLH1 and with trends for lower methylation in each of the other CpG islands examined. Our results demonstrate that age, gender and genetic factors can influence the methylation level of CpG islands in gene promoter regions of normal colonic mucosa. Further work is required to determine whether such methylation is associated with the development of CIMP+ CRC.

Methyl-Group Metabolism and the Response of Colorectal Cancer to 5-Fluorouracil

The major mechanism of action of 5-fluorouracil (5FU)-based therapies is thought to be inhibition of thymidylate synthase (TS). This enzyme catalyzes synthesis of the thymidine nucleotide precursor using a methyl group provided by a folate cofactor. In addition to TS activity levels, various elements of methyl-group metabolism could also be predictive for the response of colorectal cancer (CRC) to 5FU. These include the activity of enzymes involved in folate metabolism, the concentrations of intracellular folate intermediates, and surrogate markers of aberrant methyl-group metabolism, such as DNA methylation and microsatellite instability. The factors of age, gender, common genetic variants, and diet have been shown to influence both systemic and tumor methyl-group metabolism. This has important implications for the prediction of toxicity and response to 5FU, respectively. Identification of predictive factors within the methyl-group metabolism pathway should assist in targeting 5FU treatment to the most responsive CRC patient groups. This is particularly important for early-stage disease where conclusive demonstration of a survival benefit from 5FU in the overall CRC group has thus far proven difficult.