Role In DNA Synthesis Research Articles

Association between folate metabolism-related polymorphisms and colorectal cancer risk.

Folate has essential roles in DNA synthesis, repair and methylation. Folate metabolism-related gene variants may modulate the levels of this vitamin and affect the cancer risk. Thus, whether these polymorphisms play an important role in carcinogenesis, particularly colorectal cancer (CRC) development, has been a subject interest. The present study investigated the association between polymorphisms in the methylenetetrahydrofolate reductase (MTHFR), thymidylate synthase (TS) and the reduced folate carrier 1 (RFC1) genes and CRC risk. Polymorphisms in MTHFR (677C>T and 1298A>C), TS [1494del6 and the TS enhancer region (TSER)] and RFC1 (-43T>C, 80G>A and 696C>T) were characterized using polymerase chain reaction-restriction fragment length polymorphism in 477 CRC cases and 514 controls. Although no polymorphisms were significantly associated with the CRC risk in the overall sample, significant associations between folate metabolism-related polymorphisms and CRC risk were identified in the stratified analyses. The MTHFR 677CT/1298AC and MTHFR 1298AC+CC/TSER 2R3R genotypes in the presence of plasma folate levels ≤4.12 ng/ml were associated with significantly increased CRC risk. In addition, individuals with the MTHFR 677TT/TSER 3R3R or MTHFR 677/TSER 3R3R/TS 1494 0bp6bp+6bp6bp genotypes and diabetes mellitus (DM) were at an increased risk for CRC. Therefore, the data suggest that i) MTHFR polymorphisms combined with low plasma folate levels and ii) polymorphisms in folate metabolism-related genes combined with metabolic syndrome risk factors (hypertension and DM) increase the odds of developing CRC.

Genetic variants in one-carbon metabolism genes and breast cancer risk in European American and African American women.

Folate-mediated one-carbon metabolism plays critical roles in DNA synthesis, repair and DNA methylation. The impact of single nucleotide polymorphisms (SNPs) in folate-metabolizing enzymes has been investigated in risk of breast cancer among European or Asian populations, but not among women of African ancestry. We conducted a comprehensive analysis of SNPs in eleven genes involved in one-carbon metabolism and risk of breast cancer in 1,275 European-American (EA) and 1,299 African-American (AA) women who participated in the Women's Circle of Health Study. Allele frequencies varied significantly between EA and AA populations. A number of these SNPs, specifically in genes including MTR, MTRR, SHMT1, TYMS and SLC19A1, were associated with overall breast cancer risk, as well as risk by estrogen receptor (ER) status, in either EA or AA women. Associations appeared to be modified by dietary folate intake. Although single-SNP associations were not statistically significant after correcting for multiple comparisons, polygenetic score analyses revealed significant associations with breast cancer risk. Per unit increase of the risk score was associated with a modest 19 to 50% increase in risk of breast cancer overall, ER positive or ER negative cancer (all p < 0.0005) in EAs or AAs. In summary, our data suggest that one-carbon metabolizing gene polymorphisms could play a role in breast cancer and that may differ between EA and AA women.

RRM2 (ribonucleotide reductase M2)

Ribonucleotide reductase subunit M2 (RRM2) is located in chromosome-2 p25-p24, converts ribonucleotides to deoxynucleotides which is required for DNA polymerization and repair. It has been shown that RMM2 plays a key role in DNA synthesis, cell growth, and drug resistance of cancer cells. There is accumulating evidence that alteration in the expression level of RRM2 can have a substantial impact on the biological characteristics of cancer cells, including tumor initiation and progression, suggesting its role as a prognostic factor and a possible therapeutic target for cancer therapy. Therefore, this review highlights several recent and clinically relevant aspects of the expression and function of RRM2 in human cancer.

Tyms double (2R) and triple repeat (3R) confers risk for human oral squamous cell carcinoma.

The oral cancer is responsible for approximately 3 % of cases of cancer in Brazil. Epidemiological studies have associated low folate intake with an increased risk of epithelial cancers, including oral cancer. Folic acid has a key role in DNA synthesis, repair, methylation and this is the basis of explanations for a putative role for folic acid in cancer prevention. The role of folic acid in carcinogenesis may be modulated by polymorphism C677T in MTHFR and tandem repeats 2R/3R in the promoter site of TYMS gene that are related to decreased enzymatic activity and quantity and availability of the enzyme, respectively. These events cause a decrease in the synthesis, repair and DNA methylation, which can lead to a disruption in the expression of tumor suppressor genes as TP53. The objective of this study was investigate the distribution of polymorphisms C677T and tandem repeats 2R/3R associated with the development of oral squamous cell carcinoma (OSCC). 53 paraffin-embedded samples from patients who underwent surgery but are no longer at the institution and 43 samples collected by method of oral exfoliation by cytobrush were selected. 132 healthy subjects were selected by specialists at the dental clinics of the Faculdade de Odontologia de Pernambuco-FOP. The MTHFR genotyping was performed by PCR-RFLP, and the TYMS genotyping was performed by conventional PCR. Fisher's Exact test at significant level of 5 %. Odds ratios (ORs) and 95 % confidence intervals (CIs) were used to measure the strength of association between genotype frequency and OSCC development. The results were statistically significant for the tandem repeats of the TYMS gene (p = 0.015). The TYMS 2R3R genotype was significantly associated with the development of OSCC (OR = 3.582; 95 % CI 1.240-10.348; p = 0.0262) and also the genotype 3R3R (OR = 3.553; 95 % CI 1.293-9.760; p = 0.0345). When analyzed together, the TYMS 2R3R + 3R3R genotypes also showed association (OR = 3.518; 95 % CI 11.188-10.348; p = 0.0177). No differences for the MTHFR C677T polymorphisms distribution were found between the oral cancer patients and controls subjects in our study (p = 0.499). Therefore, these data suggest that determination of TYMS tandem repeats could provide information on the comprehension of the risk factors and prevention of the OSCC.

Enzyme regulation. IRBIT is a novel regulator of ribonucleotide reductase in higher eukaryotes.

Ribonucleotide reductase (RNR) supplies the balanced pools of deoxynucleotide triphosphates (dNTPs) necessary for DNA replication and maintenance of genomic integrity. RNR is subject to allosteric regulatory mechanisms in all eukaryotes, as well as to control by small protein inhibitors Sml1p and Spd1p in budding and fission yeast, respectively. Here, we show that the metazoan protein IRBIT forms a deoxyadenosine triphosphate (dATP)-dependent complex with RNR, which stabilizes dATP in the activity site of RNR and thus inhibits the enzyme. Formation of the RNR-IRBIT complex is regulated through phosphorylation of IRBIT, and ablation of IRBIT expression in HeLa cells causes imbalanced dNTP pools and altered cell cycle progression. We demonstrate a mechanism for RNR regulation in higher eukaryotes that acts by enhancing allosteric RNR inhibition by dATP.

Regulating DNA building blocks

DNA Biochemistry The bulding blocks of DNA, deoxynucleotide triphosphates, are synthesized by the enzyme ribonucleotide reductase (RNR). Its central role in DNA synthesis and its aberrant expression in tumors have made it an important anticancer target, but nucleoside analog inhibitors of RNR can have adverse side effects. Arnaoutov and Dasso identify IRBIT (IP3-receptor-binding protein released with inositol 1,4,5-trisphosphate) as an RNR-binding protein. IRBIT regulates the activity of RNR by stabilizing the inactive form bound to deoxyadenosine triphosphate in the low-affinity A site. In turn, phosophorylation of IRBIT regulates its RNR-binding activity. IRBIT provides a new target for RNR inhibition that might avoid some of the side effects of current drugs. Science , this issue p. [1512][1] [1]: /lookup/doi/10.1126/science.1251550

Quartz crystal microbalance with dissipation and microscale thermophoresis as tools for investigation of protein complex formation between thymidylate synthesis cycle enzymes

Thymidylate synthase (TS) and dihydrofolate reductase (DHFR) play essential role in DNA synthesis, repair and cell division by catalyzing two subsequent reactions in thymidylate biosynthesis cycle. The lack of either enzyme leads to thymineless death of the cell, therefore inhibition of the enzyme activity is a common and successful tool in cancer chemotherapy and treatment of other diseases. However, the detailed mechanism of thymidylate synthesis cycle, especially the interactions between cycle enzymes and its role remain unknown.In this paper we are the first to show that human TS and DHFR enzymes form a strong complex which might be essential for DNA synthesis. Using two unique biosensor techniques, both highly sensitive to biomolecular interactions, namely quartz crystal microbalance with dissipation monitoring (QCM-D) and microscale thermophoresis (MST) we have been able to determine DHFR–TS binding kinetic parameters such as the Kd value being below 10µM (both methods), kon=0.46×104M−1s−1 and koff=0.024s−1 (QCM-D). We also calculated Gibbs free energy as in the order of −30kJ/mol and DHFR/TS molar ratio pointing to binding of 6 DHFR monomers per 1 TS dimer (both methods). Moreover, our data from MST analysis have pointed to positive binding cooperativity in TS–DHFR complex formation. The results obtained with both methods are comparable and complementary.

Do Folate, Vitamins B&lt;sub&gt;6&lt;/sub&gt; and B&lt;sub&gt;12&lt;/sub&gt; Play a Role in the Pathogenesis of Migraine? The Role of Pharmacoepigenomics

Migraine is a neurovascular disease that has classically been attributed to multifactorial aetiologies, with genetic components and environmental interactions considered the main influence. Genes such as flavoenzyme 5, 10- methylenetetrahydrofolate reductase (MTHFR), especially the C677T variant, have been associated with elevated plasma homocysteine levels. This elevation in homocysteine results in an array of metabolic disorders and increased risk of complex diseases, including migraine. Catalysation of homocysteine requires the presence of vitamins B6, B12 and folate. Deficiencies in these cofactor vitamins result in hypomethylation, which triggers migraine. Because migraine predominantly affects females, it is hypothesised that fluctuating oestrogen levels, which are governed by oestrogen receptor 1 polymorphisms, are important. Another important factor is homocysteine, the production of which is dependent upon MTHFR and B vitamins. Gene expression is modulated through epigenetic mechanisms, which involve methionine. Additionally, folate plays a major role in DNA synthesis. We propose that vitamin B intake, coupled with MTHFR and oestrogen receptor 1 polymorphisms, causes differential DNA methylation and gene expression that may contribute to the occurrence of migraine.

Knockdown of TNFAIP1 inhibits growth and induces apoptosis in osteosarcoma cells through inhibition of the nuclear factor-κB pathway.

Tumor necrosis factor-α-inducible protein-1 (TNFAIP1) plays a role in DNA synthesis, DNA repair, cell apoptosis and human diseases including cancer, and may be involved in tumor progression and metastases. However, little is known concerning the function of TNFAIP1 in human osteosarcoma (OS). The aim of the present study was to investigate the function and underlying mechanisms of TNFAIP1 in human OS. The expression of TNFAIP1 was examined by immunohistochemical assay using a tissue microarray procedure. A loss-of-function experiment was performed to explore the effects of lentiviral-mediated TNFAIP1 siRNA (siTNFAIP1) on cell proliferation, invasive potential and apoptosis by MTT and Transwell assays and flow cytometric analysis in OS (MG-63 and U-2 OS) cells. The results showed that the expression of TNFAIP1 protein was significantly increased in OS tissues compared with that in adjacent non-cancerous tissues (ANCTs) (73.3 vs. 48.9%, P=0.018), and was correlated with the distant metastasis of the patients with OS (P=0.029). Knockdown of TNFAIP1 suppressed cell proliferation and invasion, and induced cell apoptosis in the OS cells together with the downregulation of p65 nuclear factor-κB (NF-κB), proliferating cell nuclear antigen (PCNA) and matrix metalloproteinase-2 (MMP-2) and upregulation of caspase-3. Collectively, our findings indicate that high expression of TNFAIP1 is associated with distant metastasis of OS, and knockdown of TNFAIP1 inhibits the growth and invasion, and induces apoptosis in OS cells through inhibition of the NF-κB pathway, suggesting that TNFAIP1 may act as a potential therapeutic target for the treatment of cancer.

Cobalamin E defect, a rare inborn error of vitamin B12 metabolism: Value of early diagnosis and treatment

Cobalamin and its metabolites play a crucial role in DNA synthesis and cellular energy metabolism. Disorders of cobalamin metabolism are rare, autosomal recessive, conditions that present with neurological dysfunction of varying severity. We report a child with cobalamin E defect presenting in early infancy with vertical nystagmus, developmental delay, deceleration in head growth, status epilepticus and leukoencephalopathy, with only mild haematological abnormalities. Resolution of seizures and subsequent improvement in development and head growth was observed following early treatment with parenteral hydroxocobalamin, betaine, folate and methionine, emphasising the importance of early diagnosis and treatment in these conditions.

Trichinella pseudospiralis vs. T. spiralis thymidylate synthase gene structure and T. pseudospiralis thymidylate synthase retrogene sequence

BackgroundThymidylate synthase is a housekeeping gene, designated ancient due to its role in DNA synthesis and ubiquitous phyletic distribution. The genomic sequences were characterized coding for thymidylate synthase in two species of the genus Trichinella, an encapsulating T. spiralis and a non-encapsulating T. pseudospiralis.MethodsBased on the sequence of parasitic nematode Trichinella spiralis thymidylate synthase cDNA, PCR techniques were employed.ResultsEach of the respective gene structures encompassed 6 exons and 5 introns located in conserved sites. Comparison with the corresponding gene structures of other eukaryotic species revealed lack of common introns that would be shared among selected fungi, nematodes, mammals and plants. The two deduced amino acid sequences were 96% identical. In addition to the thymidylate synthase gene, the intron-less retrocopy, i.e. a processed pseudogene, with sequence identical to the T. spiralis gene coding region, was found to be present within the T. pseudospiralis genome. This pseudogene, instead of the gene, was confirmed by RT-PCR to be expressed in the parasite muscle larvae.ConclusionsIntron load, as well as distribution of exon and intron phases in thymidylate synthase genes from various sources, point against the theory of gene assembly by the primordial exon shuffling and support the theory of evolutionary late intron insertion into spliceosomal genes. Thymidylate synthase pseudogene expressed in T. pseudospiralis muscle larvae is designated a retrogene.

Plasma homocysteine levels and genetic polymorphisms in folate metablism are associated with breast cancer risk in chinese women

BackgroundFolate plays a pivotal role in DNA synthesis, repair, methylation and homocysteine (Hcy) metabolism. Therefore, alterations in the folate-mediated one-carbon metabolism may lead to abnormal methylation proliferation, increases of tumor/neoplasia and vein thrombosis/cardiovascular risk. The serine hydroxymethyhransferase (SHMT), methionine synthase (MS), methionine synthase reductase (MTRR) and cystathionine beta synthase (CBS) regulate key reactions in the folate and Hcy metabolism. Therefore, we investigated whether the genetic variants of the SHMT, MS, MTRR and CBS gene can affect plasma Hcy levels and are associated with breast cancer risk.MethodsGenotyping was performed by PCR-RFLP method. Plasma Hcy levels were measured by the fluorescence polarization immunoassay on samples of 96 cases and 85 controls.Results(a) The SHMT 1420 T, MS 2756G, MTRR 66G allele frequency distribution showed significant difference between case and controls (p < 0.01 ~ 0.05). (b) The concentration of plasma Hcy levels of SHMT 1420TT was significantly lower than that of the wild type, while the plasma Hcy levels of MS 2756GG, CBS 699TT/1080TT significantly higher than that of the wild type both in case and controls. The plasma Hcy levels of MTRR 66GG was significantly higher than that of wild type in cases. The plasma Hcy levels of the same genotype in cases were significantly higher than those of controls except SHMT 1420CC, MS 2756AA, MTRR 66GG; (c) Multivariate Logistic regression analysis showed that SHMT C1420T (OR = 0.527, 95% CI = 0.55 ~ 1.24), MS A2756G (OR = 2.32, 95% CI = 0.29 ~ 0.82), MTRR A66G (OR = 1.84, 95% CI = 0.25 ~ 1.66) polymorphism is significantly associated with breast cancer risk. And elevated plasma Hcy levels were significantly linked to increased risk of breast cancer (adjusted OR = 4.45, 95% CI = 1.89-6.24 for the highest tertile as compared with the lowest tertile).ConclusionsThe current study results seem to suggest a possibility that SHMT C1420T mutation may be negatively correlated with breast cancer susceptibility; while MS A2756G and MTRR A66G mutation may be positively associated with breast cancer risk. SHMT C1420T, MS A2756G, MTRR A66G, CBS C1080T, CBS C699T locus mutation may be factors affecting plasma levels of Hcy. The plasma Hcy levels could be metabolic risk factor for breast cancer risk to a certain extent.

Positive association between MTHFR C677T polymorphism and oral cancer risk: a meta-analysis

Methylenetetrahydrofolate reductase (MTHFR) is a central enzyme involved in regulating the metabolic function of folate, which plays a pivotal role in DNA synthesis, repair, and methylation. The role of MTHFR C677T polymorphism in oral cancer risk has been reported with conflicting evidence. We conducted this study to appropriately estimate the effect size. We searched eligible studies in medicine-specific databases (PubMed, EMBASE, and Web of Knowledge) using (polymorphism) OR (polymorphisms) AND (methylenetetrahydrofolate reductase) OR (MTHFR) AND (oral cancer). A total of seven studies were summarized. This meta-analysis of the combined data showed a marginal association of MTHFR C677T polymorphism with oral cancer risk [odds ratio (OR) = 0.86, 95% confidence interval (CI) = 0.73-1.00 for CT vs. CC]. We also found decreased oral cancer risk in Asian population and hospital-based studies. Moreover, heavy drinkers were found to have a significantly higher risk of developing such cancer as compared to the non-heavy drinkers. These results suggest that MTHFR C677T polymorphism may play a role in oral cancer carcinogenesis in Asian population and heavy drinkers.

Vitamin B12 Gene Polymorphisms and Chronic Diseases

Vitamin B12 or cobalamin is an essential nutrient with important roles in DNA synthesis, repair and methylation. It is also required in the one carbon metabolism pathway to reduce plasma homocysteine concentrations. Several epidemiological studies have indicated that genes and metabolites of the B vitamin-mediated one-carbon metabolic pathway are associated with chronic diseases. This short review describes polymorphisms in the MTHFR, FUT2 and TCN2 genes which have been implicated in cardiovascular diseases and neural tube defects amongst others.

The Dual Effect of Folic Acid Supplementation in Preventing Colorectal Carcinomas

Folic acid, a water-soluble B vitamin contained in vegetables, is essential for maintaining DNA stability through donating one-carbon-unit for cellular metabolism. So it plays an important role in DNA synthesis and replication. Early research suggested that folic acid could reduce the risk of colorectal cancer, but recent studies have questioned about this. Folic acid plays dual roles in the chemoprevention of colorectal cancer. Both the hopes for rapid cancer prevention and the fears about rapidly increased cancer risk from folic acid supplementation were not confirmed by folic acid supplementation. Folate deficiency may induce both gene-specific DNA hypermethylation and global DNA hypomethylation by its DNA-damaging effect. Meanwhile, the effect of folic acid in the development of colorectal cancer has a time-and dose-dependent pattern. Therefore, folate concentrations determined in duodenum or sigmoid colon epithelia are recommended so that a rational regimen could be made individually. We discussed the relationships between the effects of folic acid supplementation and colorectal cancer chemoprevention in the present review. The pros and cons of folic acid supplementation in tumor chemoprevention are still vague and need more efforts in the future.

MTHFR C677T and A1298C polymorphisms and risk of nonsyndromic orofacial clefts in a south Indian population

ObjectivesSeveral lines of evidence suggest that the decrease in folate in periconceptional period or maternal use of folate antagonists has been associated with a higher risk of orofacial clefts (OFCs). MTHFR is a critical enzyme in folate metabolism that catalyzes the irreversible conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, thus playing a vital role in DNA synthesis and DNA methylation. The aim of our study was to determine whether there is any association between the susceptibility to Nonsyndromic cleft lip with or without cleft palate (NSCL/P) amongst the variations of MTHFR genotypes in South Indian population. MethodsOur sample comprised 123 cases with NSCL/P and 141 controls without clefts or family history of clefting. The most common polymorphisms C677T (rs1801133) and A1298C (rs1801131) on the MTHFR gene were screened for the genotypes using PCR-RFLP. ResultsBoth C677T and A1298C are polymorphic with minor allele frequencies of 0.131 and 0.429, respectively, for controls. Genotype data in control and cleft groups are following the Hardy Weinberg Equilibrium. There were no significant differences in genotypes of both polymorphisms between controls and NSCL/P. The pairwise LD values (D′ and r2) between C677T and A1298C are 1.0 and 0.096 respectively indicating no significant LD between these two SNPs. Haplotype phenotype analysis did not show the evidence for association. Gene–gene interaction showed the distribution of the observed combinations of the two MTHFR polymorphisms was not different between NSCL/P and controls (p=0.887). ConclusionsOur results do not support the hypothesis, that variants in the MTHFR gene confer a risk for NSCL/P in the South Indian population.

English

BACKGROUND: Vitamin B12 (Cobalamin) plays an important role in DNA synthesis and neurologic function. Deficiency can lead to a wide spectrum of hematologic and neuropsychiatric disorders that can often be reversed by early diagnosis and prompt treatment. MATERIAL AND METHODS: Cross sectional descriptive study carried out in Medicine department, SMIMER, Surat during the period from June 2009 to December 2010. 30 patients whose Serum Vitamin B12 level <250 pg/ml were included. RESULT: The most common complaint was fatigue present in 29(96.7%) followed by anorexia, present in 19(63.3%) patients. Anemia was observed in 25(83.3%) patients. Out of this 25 patients with anemia 13(52%) patients had leucopenia, and 15(60%) patients had thrombocytopenia (platelet count<150000/cumm), while 10 (40%) patient had normal platelet count. 13(52%) out of 25 anemic patients had MCV of 80-100 fl, while 11(44%) had MCV more than 100 fl. 15 patients had neurological features, myeloneuropathy (4 patients) and neurocognitive disorder with neuropathy(4 patients) were the commonest feature. CONCLUSION: In patients with Vitamin B12 deficiency anorexia and fatigue are most common symptoms even in absence of anaemia. Anaemia can be associated with normal or high MCV. Pancytopenia is a common feature. The most common neurological presentations were neuropathy and neuropsychiatric involvement.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Pif1 helicase and Polδ promote recombination-coupled DNA synthesis via bubble migration

During DNA repair by homologous recombination (HR), DNA synthesis copies information from a template DNA molecule. Multiple DNA polymerases have been implicated in repair-specific DNA synthesis1–3, but it has remained unclear whether a DNA helicase is involved in this reaction. A good candidate is Pif1, an evolutionarily conserved helicase in S. cerevisiae important for break-induced replication (BIR)4 as well as HR-dependent telomere maintenance in the absence of telomerase5 found in 10–15% of all cancers6. Pif1 plays a role in DNA synthesis across hard-to-replicate sites7, 8 and in lagging strand synthesis with Polδ9–11. Here we provide evidence that Pif1 stimulates DNA synthesis during BIR and crossover recombination. The initial steps of BIR occur normally in Pif1-deficient cells, but Polδ recruitment and DNA synthesis are decreased, resulting in premature resolution of DNA intermediates into half crossovers. Purified Pif1 protein strongly stimulates Polδ-mediated DNA synthesis from a D-loop made by the Rad51 recombinase. Importantly, Pif1 liberates the newly synthesized strand to prevent the accumulation of topological constraint and to facilitate extensive DNA synthesis via the establishment of a migrating D-loop structure. Our results uncover a novel function of Pif1 and provide insights into the mechanism of HR.

Vitamin B12 Metabolism during Pregnancy and in Embryonic Mouse Models

Vitamin B12 (cobalamin, Cbl) is required for cellular metabolism. It is an essential coenzyme in mammals for two reactions: the conversion of homocysteine to methionine by the enzyme methionine synthase and the conversion of methylmalonyl-CoA to succinyl-CoA by the enzyme methylmalonyl-CoA mutase. Symptoms of Cbl deficiency are hematological, neurological and cognitive, including megaloblastic anaemia, tingling and numbness of the extremities, gait abnormalities, visual disturbances, memory loss and dementia. During pregnancy Cbl is essential, presumably because of its role in DNA synthesis and methionine synthesis; however, there are conflicting studies regarding an association between early pregnancy loss and Cbl deficiency. We here review the literature about the requirement for Cbl during pregnancy, and summarized what is known of the expression pattern and function of genes required for Cbl metabolism in embryonic mouse models.