Serine Hydroxymethyltransferase 1 Research Articles

MiR-218-5p Suppresses the Killing Effect of Natural Killer Cell to Lung Adenocarcinoma by Targeting SHMT1.

PurposeLung adenocarcinoma (LA) is one of the major types of lung cancer. MicroRNAs (miRNAs) play an essential role in regulating responses of natural killer (NK) cells to cancer malignancy. However, the mechanism of miR-218-5p involved in the killing effect of NK cells to LA cells remains poorly understood.Materials and MethodsThe expression of miR-218-5p was examined by quantitative real-time polymerase chain reaction (qRT-PCR). Serine hydroxymethyl transferase 1 (SHMT1) level was detected by qRT-PCR or western blots. Cytokines production of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) were detected by ELISA. The killing effect of NK cells to LA cells was investigated using lactate dehydrogenase cytotoxicity assay kit. The interaction of miR-218-5p and SHMT1 was probed by luciferase activity assay. Xenograft model was established to investigate the killing effect of NK cells in vivo.ResultsmiR-218-5p was enhanced and SHMT1 was inhibited in NK cells of LA patients, whereas stimulation of interleukin-2 (IL-2) reversed their abundances. Addition of miR-218-5p reduced IL-2-induced cytokines expression and cytotoxicity in NK-92 against LA cells. Moreover, SHMT1 was negatively regulated by miR-218-5p and attenuated miR-218-5p-mediated effect on cytotoxicity, IFN-γ and TNF-α secretion in IL-2-activated NK cells. In addition, miR-218-5p exhaustion inhibited tumor growth by promoting killing effect of NK cells.ConclusionmiR-218-5p suppresses the killing effect of NK cells to LA cells by targeting SHMT1, providing a potential target for LA treatment by ameliorating NK cells function.

Serine hydroxymethyltransferase 1 promoter hypermethylation increases the risk of essential hypertension.

Serine hydroxymethyltransferase 1 (SHMT1) is an enzyme involved in folic acid metabolism and is known to contribute to the development of hypertension. We evaluated the relationship between SHMT1 promoter methylation and essential hypertension (EH). Quantitative methylation-specific polymerase chain reaction was used to measure the SHMT1 promoter methylation level in 241 EH patients and 288 age- and gender-matched healthy individuals. The diagnostic value of SHMT1 promoter hypermethylation was analyzed using a receiver operating characteristic (ROC) curve. The Gene Expression Omnibus (GEO) database and dual-luciferase reporter assay were used to validate our findings. Compared with the control group, significant differences in SHMT1 promoter methylation were found in both EH and hyperhomocysteinemia groups (P<0.001 and P=0.029, respectively). The area under the curve of the diagnosis of SHMT1 promoter hypermethylation for EH was 0.808, with a sensitivity and specificity of 73.9% and 77.8%, respectively. The risk of SHMT1 promoter hypermethylation was significantly higher in the >65-year group than in the ≤65-year group (odds ratio=3.925; 95% confidence interval=2.141-7.196). In addition, GEO database analysis showed that 5-aza-deoxycytidine increased gene expression in several carotid endothelial cell lines. A dual-luciferase reporter assay revealed that the target sequence in the SHMT1 promoter upregulated gene expression. Our findings indicate that SHMT1 promoter hypermethylation increases the risk of EH and may be a promising biomarker for EH.

Role of Cytosolic Serine Hydroxymethyl Transferase 1 (SHMT1) in Phosphate-Induced Vascular Smooth Muscle Cell Calcification

Background/Aims: Hyperphosphatemia promotes medial vascular calcification, at least partly, by induction of osteo-/chondrogenic transdifferentiation of vascular smooth muscle cells (VSMCs). The complex signaling pathways regulating this process are still incompletely understood. The present study investigated the role of cytosolic serine hydroxymethyl transferase 1 (SHMT1) in phosphate-induced vascular calcification. Methods: Endogenous expression of SHMT1 was suppressed by silencing in primary human aortic smooth muscle cells (HAoSMCs) followed by treatment without and with phosphate or antioxidants. Results: In HAoSMCs, SHMT1 mRNA expression was up-regulated by phosphate. Silencing of SHMT1 alone was sufficient to induce osteo-/chondrogenic transdifferentiation of HAoSMCs, as shown by increased tissue-nonspecific alkaline phosphatase (ALPL) activity and osteogenic markers MSX2, CBFA1 and ALPL mRNA expression. Furthermore, phosphate-induced ALPL mRNA expression and activity as well as calcification were augmented in SHMT1 silenced HAoSMCs as compared to negative control siRNA transfected HAoSMCs. Silencing of SHMT1 decreased total antioxidant capacity and up-regulated NADH/NADPH oxidase system components NOX4 and CYBA mRNA expression in HAoSMCs, effects paralleled by increased mRNA expression of matrix metalloproteinase MMP2 as well as BAX/BCL2 ratio. More importantly, additional treatment with antioxidants TEMPOL or TIRON blunted the increased osteogenic markers mRNA expression in SHMT1 silenced HAoSMCs. Conclusion: Silencing of SHMT1 promotes osteo-/chondrogenic signaling in VSMCs, at least in part, by inducing cellular oxidative stress. It thus aggravates phosphate-induced calcification of VSMCs. The present findings support a regulatory role of SHMT1 in vascular calcification during conditions of hyperphosphatemia such as chronic kidney disease.

Neuro-fuzzy model of homocysteine metabolism.

In view of well-documented association of hyperhomocysteinaemia with a wide spectrum of diseases and higher incidence of vitamin deficiencies in Indians, we proposed a mathematical model to forecast the role of demographic and genetic variables in influencing homocysteinemetabolism and investigated the influence of life style modulations in controlling homocysteine levels. Total plasma homocysteine levels were measured in fasting samples using reverse phase HPLC. Multiple linear regression (MLR) and neuro-fuzzy models were developed. The MLR model explained 64% variability in homocysteine, while the neurofuzzy model showed higher accuracy in predicting homocysteine with a mean absolute error of 0.00002 μmol/L. Methylene tetrahydrofolate reductase (MTHFR) C677T, 5-methyltetrahydrofolate homocysteine methyltransferase (MTR) A2756G and 5- methyltetrahydrofolate homocysteine methyltransferase reductase (MTRR) A66G were shown to be positively associatiated with homocysteine, while nonvegetarian diet, serine hydroxymethyltransferase 1 (SHMT1) C1420T and TYMS 5'-UTR 28 bp tandem repeat exhibited negative association with homocysteine. The protective role of SHMT1 C1420T was attributed to more H-bonding interactions in the mutant modelled compared to the wild type, as shown through in silico analysis. To conclude, polymorphisms in genes regulating remethylation of homocysteine strongly influence homocysteine levels. The restoration of one-carbon homeostasis by SHMT1 C1420T or increased flux of folate towards remethylation due to TYMS 5'-UTR 28 bp tandem repeat or nonvegetarian diet can lower homocysteine levels.

Serine hydroxymethyl transferase 1 stimulates pro-oncogenic cytokine expression through sialic acid to promote ovarian cancer tumor growth and progression

High-grade serous (HGS) ovarian cancer accounts for 90% of all ovarian cancer-related deaths. However, factors that drive HGS ovarian cancer tumor growth have not been fully elucidated. In particular, comprehensive analysis of the metabolic requirements of ovarian cancer tumor growth has not been performed. By analyzing The Cancer Genome Atlas mRNA expression data for HGS ovarian cancer patient samples, we observed that six enzymes of the folic acid metabolic pathway were overexpressed in HGS ovarian cancer samples compared with normal ovary samples. Systematic knockdown of all six genes using short hairpin RNAs (shRNAs) and follow-up functional studies demonstrated that serine hydroxymethyl transferase 1 (SHMT1) was necessary for ovarian cancer tumor growth and cell migration in culture and tumor formation in mice. SHMT1 promoter analysis identified transcription factor Wilms tumor 1 (WT1) binding sites, and WT1 knockdown resulted in reduced SHMT1 transcription in ovarian cancer cells. Unbiased large-scale metabolomic analysis and transcriptome-wide mRNA expression profiling identified reduced levels of several metabolites of the amino sugar and nucleotide sugar metabolic pathways, including sialic acid N-acetylneuraminic acid (Neu5Ac), and downregulation of pro-oncogenic cytokines interleukin-6 and 8 (IL-6 and IL-8) as unexpected outcomes of SHMT1 loss. Overexpression of either IL-6 or IL-8 partially rescued SHMT1 loss-induced tumor growth inhibition and migration. Supplementation of culture medium with Neu5Ac stimulated expression of IL-6 and IL-8 and rescued the tumor growth and migratory phenotypes of ovarian cancer cells expressing SHMT1 shRNAs. In agreement with the ovarian tumor-promoting role of Neu5Ac, treatment with Neu5Ac-targeting glycomimetic P-3Fax-Neu5Ac blocked ovarian cancer growth and migration. Collectively, these results demonstrate that SHMT1 controls the expression of pro-oncogenic inflammatory cytokines by regulating sialic acid Neu5Ac to promote ovarian cancer tumor growth and migration. Thus, targeting of SHMT1 and Neu5Ac represents a precision therapy opportunity for effective HGS ovarian cancer treatment.

Abstract PR10: Serine biosynthesis from glucose regulates folate availability

Abstract It has been established that de novo serine biosynthesis is important for some cancers. We have previously shown that phosphoglycerate dehydrogenase (PHGDH), the enzyme responsible for catalyzing the first step in glycolytic serine biosynthesis, is amplified in certain cancers, including breast cancer and melanoma. Knockdown of PHGDH results in decreased proliferation of PHGDH-amplified cells, demonstrating a dependence on PHDGH in these cells; however, it has not been possible to rescue this defect with exogenous serine, suggesting that glycolytic serine biosynthesis has an alternate function in cells. Here we have investigated four mutually exclusive hypotheses each consistent with the inability of serine to rescue decreased glycolytic serine biosynthesis: 1: There exists an alternative function of PHGDH; 2: Pathway activity is important for TCA cycle anapleurosis; 3: Metabolic channeling of de novo serine; and 4: A unique product of this pathway other than serine is critical for proliferation. Using a combination of genetic and biochemical approaches, we show that flux through the serine biosynthesis pathway is required for cytosolic serine-glycine conversion via serine hydroxymethyltransferase 1 (SHMT1). In many cells flux through SHMT1 occurs in the glycine-to-serine direction, including PHDGH-dependent cell lines, and loss of PHGDH activity results in wasting of folates from the one-carbon pool to make serine. This reduces availability of one-carbon units in the folate pool for nucleotide biosynthesis, resulting in S-phase arrest and eventual cell death. These data link serine biosynthesis from glucose with the regulation of one-carbon folate metabolism, and provide an explanation for why cancer cells are dependent on de novo serine production even when serine is abundant. Citation Format: Caroline A. Lewis, Brian P. Fiske, Michael E. Pacold, Aaron M. Hosios, Katherine R. Mattaini, David M. Sabatini, Matthew G. Vander Heiden. Serine biosynthesis from glucose regulates folate availability. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr PR10.

SHMT1 C1420T polymorphism contributes to the risk of non-Hodgkin lymphoma: evidence from 7309 patients.

BackgroundSerine hydroxymethyltransferase 1 (SHMT1) is a key enzyme in the folate metabolic pathway that plays an important role in biosynthesis by providing one carbon unit. SHMT1 C1420T may lead to the abnormal biosynthesis involved in DNA synthesis and methylation, and it may eventually increase cancer susceptibility. Many epidemiologic studies have explored the association between C1420T polymorphism and the risk of non-Hodgkin lymphoma (NHL), but the results have been contradictory. Therefore, we performed this meta-analysis to evaluate the relationship.MethodsThe meta-analyses were conducted to evaluate the effect of SHMT1 C1420T polymorphism on NHL risk. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to measure the strength of the association.ResultsEight studies encompassing 3232 cases and 4077 controls were included. A statistically significant association was found between SHMT1 C1420T polymorphism and NHL risk under the allelic comparison (T vs. C: OR = 1.09, 95% CI 1.01–1.17); a borderline association was found between SHMT1 C1420T polymorphism and NHL risk under the homozygote model (TT vs. CC: OR = 1.18, 95% CI 1.00–1.39) and the dominant model (CT+TT vs. CC: OR = 1.10, 95% CI 1.00–1.21).ConclusionSHMT1 C1420T polymorphism may be associated with NHL risk, which needs to be validated in large, prospective studies.

MiR-198 targets SHMT1 to inhibit cell proliferation and enhance cell apoptosis in lung adenocarcinoma.

MiR-198 is involved in tumorigenesis, migration, invasion, and metastasis of various malignant cancers. However, the exact expression levels of miR-198 and the molecular mechanism underlying its role in lung adenocarcinoma require further exploration. In this study, quantitative real-time PCR was applied to study miR-198 and serine hydroxymethyltransferase 1 (SHMT1) expression in 47 paired lung adenocarcinoma tissues and adjacent nontumor lung tissues. Clinicopathological characters were analyzed. Pearson's correlation analysis was used to detect the relationship between miR-198 and SHMT1 expression. The function of miR-198 was explored by measuring cell proliferation, cell apoptosis, and the cell-cycle in vitro and in vivo. The target gene of miR-198 was certified using dual luciferase report assay. We found that in lung adenocarcinoma, miR-198 was significantly downregulated and SHMT1 was inversely upregulated. A strong negative correlation was noticed between miR-198 and SHMT1 expression. Further analysis revealed that miR-198 expression was associated with TNM stage and lymph node metastasis. Upregulated miR-198 could inhibit cell proliferation, enhance cell apoptosis, and lead to cell-cycle arrest in lung adenocarcinoma, which showed a more effective alteration than SHMT1 siRNA. Moreover, we identified SHMT1 as a target gene of miR-198. In conclusion, miR-198 suppressed proliferation of lung adenocarcinoma cells both in vitro and in vivo by directly targeting SHMT1. miR-198 may be a potential therapeutic target for lung adenocarcinoma in the near future.

Maternal dietary uridine causes, and deoxyuridine prevents, neural tube closure defects in a mouse model of folate-responsive neural tube defects

Folic acid prevents neural tube closure defects (NTDs), but the causal metabolic pathways have not been established. Serine hydroxymethyltransferase 1 (SHMT1) is an essential scaffold protein in folate-dependent de novo thymidylate synthesis in the nucleus. SHMT1-deficient mice provide a model to investigate folic acid-responsive NTDs wherein disruption of de novo thymidylate synthesis impairs neural tube closure. We examined the effects of maternal supplementation with the pyrimidine nucleosides uridine, thymidine, or deoxyuridine with and without folate deficiency on NTD incidence in the Shmt1 mouse model. Shmt1(+/+) and Shmt1(-/-) female mice fed folate-replete or folate-deficient diets and supplemented with uridine, thymidine, or deoxyuridine were bred, and litters (n = 10-23 per group) were examined for the presence of NTDs. Biomarkers of impaired folate status and metabolism were measured, including plasma nucleosides, hepatic uracil content, maternal plasma folate concentrations, and incorporation of nucleoside precursors into DNA. Shmt1(+/-) and Shmt1(-/-) embryos from dams fed the folate-deficient diet were susceptible to NTDs. No NTDs were observed in litters from dams fed the folate-deficient diet supplemented with deoxyuridine. Surprisingly, uridine supplementation increased NTD incidence, independent of embryo genotype and dietary folic acid. These dietary nucleosides did not affect maternal hepatic uracil accumulation in DNA but did affect plasma folate concentrations. Maternal deoxyuridine supplementation prevented NTDs in dams fed the folate-deficient diet, whereas maternal uridine supplementation increased NTD incidence, independent of folate and embryo genotype. These findings provide new insights into the metabolic impairments and mechanisms of folate-responsive NTDs resulting from decreased Shmt1 expression.

Azoxymethane-Induced Colon Carcinogenesis in Mice Occurs Independently of De Novo Thymidylate Synthesis Capacity1–3

Folate metabolism affects DNA synthesis, methylation, mutation rates, genomic stability, and gene expression, which are altered in colon cancer. Serine hydroxymethyltransferase 1 (SHMT1) regulates thymidylate (dTMP) biosynthesis and uracil accumulation in DNA, and as such affects genome stability. Previously, we showed that decreased SHMT1 expression inShmt1 knockout mice (Shmt1−/+) or its impaired nuclear localization, as occurs in mice over-expressing anShmt1 transgene (Shmt1tg+), results in elevated uracil incorporation into DNA, which could affect colon cancer risk. We used these 2 models to determine the effect of altered SHMT1 expression and localization, and its interaction with folate insufficiency, on azoxymethane (AOM)-induced colon cancer in mice.Shmt1−/+ andShmt1tg+ mice were weaned to a control or folate-and-choline-deficient (FCD) diet and fed the diet for 28 or 32 wk, respectively. At 6 wk of age, mice were injected weekly for 6 wk with 10 mg/kg AOM (w/v in saline). Colon uracil concentrations in nuclear DNA were elevated 2–7 fold inShmt1−/+ andShmt1tg+ mice. However, colon tumor incidence and numbers were not dependent on SHMT1 expression inShmt1−/+ orShmt1−/− mice. The FCD diet reduced tumor load independent ofShmt1 genotype. In contrast,Shmt1tg+ mice exhibited a 30% reduction in tumor incidence, a 50% reduction in tumor number, and a 60% reduction in tumor load compared with wild-type mice independent of dietary folate intake. Our data indicate that uracil accumulation in DNA does not predict tumor number in AOM-mediated carcinogenesis. Furthermore, enrichment of SHMT1 in the cytoplasm, as observed inShmt1tg+ mice, protects against AOM-mediated carcinogenesis independent of its role in nuclear de novo dTMP biosynthesis.

Expression of serine and glycine-related enzymes in phyllodes tumor.

Expression patterns of proteins involved in serine and glycine metabolism, and correlations of these patterns with clinicopathologic factors in phyllodes tumor were investigated. Tissue microarrays were prepared from 203 phyllodes tumors (PT) and stained with antibodies specific for glycine decarboxylase (GLDC), phosphoserine aminotransferase 1 (PSAT1), phosphoserine phosphatase (PSPH), phosphoglycerate dehydrogenase (PHGDH), and serine hydroxymethyltransferase 1 (SHMT1). These immunohistochemical results and clinicopathologic parameters were analyzed for correlation. Numbers of benign, borderline, and malignant tumors were 155, 32, and 16, respectively. Stromal expression of PHGDH, PSAT1, PSPH, SHMT1, and GLDC increased with increasing tumor grade, and epithelial expression of SHMT1 also increased with increasing tumor grade (p<0.001, and p=0.005, respectively). On univariate analysis, positive stainings for stromal PHGDH (p<0.001), stromal PSAT1 (p<0.001), stromal PSPH (p=0.003), epithelial SHMT1 (p=0.001), stromal SHMT1 (p=0.022), and stromal GLDC (p<0.001) were each associated with shorter disease-free survival. Stromal GLDC was associated with shorter overall survival (p<0.001). In conclusion, expression of proteins related to serine and glycine metabolism increased with increasing histologic grade in stromal components of phyllodes tumor. glycine, tumor grade, metabolism, phyllodes tumor, serine.

Disruption of Shmt1 Impairs Hippocampal Neurogenesis and Mnemonic Function in Mice1–3

Impaired folate-mediated one-carbon metabolism (OCM) has emerged as a risk factor for several diseases associated with age-related cognitive decline, but the underlying mechanisms remain unknown and thus hinder the identification of subpopulations most vulnerable to OCM disruption. Here we investigated the role of serine hydroxymethyltransferase 1 (SHMT1), a folate-dependent enzyme regulating de novo thymidylate biosynthesis, in influencing neuronal and cognitive function in the adult mouse. We observed Shmt1 expression in the hippocampus, including the granule cell layer of the dentate gyrus (DG), and examined hippocampal neurogenesis and hippocampal-dependent fear conditioning in mice deficient for Shmt1. We used a 3 × 3 design in which adult male Shmt1+/+, Shmt1+/−, and Shmt1−/− mice were fed folic acid control (2 mg/kg), folic acid–deficient (0 mg/kg), or folic acid–supplemented (8 mg/kg) diets from weaning through the duration of the study. Proliferation within the DG was elevated by 70% in Shmt1+/− mice, yet the number of newborn mature neurons was reduced by 98% compared with that in Shmt1+/+ mice. Concomitant with these alterations, Shmt1+/− mice showed a 45% reduction in mnemonic recall during trace fear conditioning. Dietary folate manipulations alone did not influence neural outcomes. Together, these data identify SHMT1 as one of the first enzymes within the OCM pathway to regulate neuronal and cognitive profiles and implicate impaired thymidylate biosynthesis in the etiology of folate-related neuropathogenesis.

UBIQUITIN-SPECIFIC PROTEASE16 Modulates Salt Tolerance in Arabidopsis by Regulating Na+/H+ Antiport Activity and Serine Hydroxymethyltransferase Stability

Protein ubiquitination is a reversible process catalyzed by ubiquitin ligases and ubiquitin-specific proteases (UBPs). We report the identification and characterization of UBP16 in Arabidopsis thaliana. UBP16 is a functional ubiquitin-specific protease and its enzyme activity is required for salt tolerance. Plants lacking UBP16 were hypersensitive to salt stress and accumulated more sodium and less potassium. UBP16 positively regulated plasma membrane Na(+)/H(+) antiport activity. Through yeast two-hybrid screening, we identified a putative target of UBP16, SERINE HYDROXYMETHYLTRANSFERASE1 (SHM1), which has previously been reported to be involved in photorespiration and salt tolerance in Arabidopsis. We found that SHM1 is degraded in a 26S proteasome-dependent process, and UBP16 stabilizes SHM1 by removing the conjugated ubiquitin. Ser hydroxymethyltransferase activity is lower in the ubp16 mutant than in the wild type but higher than in the shm1 mutant. During salt stress, UBP16 and SHM1 function in preventing cell death and reducing reactive oxygen species accumulation, activities that are correlated with increasing Na(+)/H(+) antiport activity. Overexpression of SHM1 in the ubp16 mutant partially rescues its salt-sensitive phenotype. Taken together, our results suggest that UBP16 is involved in salt tolerance in Arabidopsis by modulating sodium transport activity and repressing cell death at least partially through modulating SMH1stability and activity.

Competition between Sumoylation and Ubiquitination of Serine Hydroxymethyltransferase 1 Determines Its Nuclear Localization and Its Accumulation in the Nucleus

Serine hydroxymethyltransferase 1 (SHMT1) expression limits rates of de novo dTMP synthesis in the nucleus. Here we report that SHMT1 is ubiquitinated at the small ubiquitin-like modifier (SUMO) consensus motif and that ubiquitination at that site is required for SHMT1 degradation. SHMT1 protein levels are cell cycle-regulated, and Ub-SHMT1 levels are lowest at S phase when SHMT1 undergoes SUMO modification and nuclear transport. Mutation of the SUMO consensus motif increases SHMT1 stability. SHMT1 interacts with components of the proteasome in both the nucleus and cytoplasm, indicating that degradation occurs in both compartments. Ubc13-mediated ubiquitination is required for SHMT1 nuclear export and increases stability of SHMT1 within the nucleus, whereas Ubc9-mediated modification with Sumo2/3 is involved in nuclear degradation. These data demonstrate that SUMO and ubiquitin modification of SHMT1 occurs on the same lysine residue and determine the localization and accumulation of SHMT1 in the nucleus.

Impact of SHMT1 polymorphism on the clinical outcome of patients with metastatic colorectal cancer treated with first-line FOLFIRI+bevacizumab

The impact of thymidylate synthase (TYMS), methylenetetrahydrofolate reductase (MTHFR), and serine hydroxymethyltransferase 1 (SHMT1) gene polymorphisms and that of dihydropyrimidine dehydrogenase (DPD) enzyme activity, serum total homocysteine level, and estimated serum creatinine clearance on first-line 5-fluorouracil, leucovorin, irinotecan, and bevacizumab (FOLFIRI+bevacizumab) regimen efficacy in metastatic colorectal cancer patients was investigated. DNA was extracted from peripheral blood mononuclear cells. Genotyping was performed for TYMS 5'UTR variable number tandem repeat, TYMS 3'UTR ins/del, MTHFR C677T, and SHMT1 C1420T polymorphisms. The DPD activity of peripheral blood mononuclear cells was also determined. The univariate and multivariate analyses demonstrated that the SHMT1 1420T allele was associated with better response (P=0.025) and longer progression-free survival (PFS) (P=0.00004) and overall survival (OS) (P=0.034). Grade ≥2 hypertension was also an independent prognostic factor of longer progression-free survival and OS. Bevacizumab-related hypertension might be a predictive marker of treatment efficacy (P=0.0002 for OS) in the case of wild (CC) SHMT1 1420 genotypes only.

SHMT1 1420 and MTHFR 677 variants are associated with rectal but not colon cancer

BackgroundAssociation between rectal or colon cancer risk and serine hydroxymethyltransferase 1 (SHMT1) C1420T or methylenetetrahydrofolate reductase (MTHFR) C677T polymorphisms was assessed. The serum total homocysteine (HCY), marker of folate metabolism was also investigated.MethodsThe SHMT1 and MTHFR genotypes were determined by real-time PCR and PCR-RFLP, respectively in 476 patients with rectal, 479 patients with colon cancer and in 461 and 478, respective controls matched for age and sex. Homocysteine levels were determined by HPLC kit. The association between polymorphisms and cancer risk was evaluated by logistic regression analysis adjusted for age, sex and body mass index. The population stratification bias was also estimated.ResultsThere was no association of genotypes or diplotypes with colon cancer. The rectal cancer risk was significantly lower for SHMT1 TT (OR = 0.57, 95% confidence interval (CI) 0.36-0.89) and higher for MTHFR CT genotypes (OR = 1.4, 95%CI 1.06-1.84). A gene-dosage effect was observed for SHMT1 with progressively decreasing risk with increasing number of T allele (p = 0.014). The stratified analysis according to age and sex revealed that the association is mainly present in the younger (< 60 years) or male subgroup. As expected from genotype analysis, the SHMT1 T allele/MTHFR CC diplotype was associated with reduced rectal cancer risk (OR 0.56, 95%CI 0.42-0.77 vs all other diplotypes together). The above results are unlikely to suffer from population stratification bias. In controls HCY was influenced by SHMT1 polymorphism, while in patients it was affected only by Dukes' stage. In patients with Dukes' stage C or D HCY can be considered as a tumor marker only in case of SHMT1 1420CC genotypes.ConclusionsA protective effect of SHMT1 1420T allele or SHMT1 1420 T allele/MTHFR 677 CC diplotype against rectal but not colon cancer risk was demonstrated. The presence of SHMT1 1420 T allele significantly increases the HCY levels in controls but not in patients. Homocysteine could be considered as a tumor marker in SHMT1 1420 wild-type (CC) CRC patients in Dukes' stage C and D. Further studies need to clarify why SHMT1 and MTHFR polymorphisms are associated only with rectal and not colon cancer risk.

Association of genetic variation in cystathionine-beta-synthase and arsenic metabolism.

Variation in individual susceptibility to arsenic-induced disease may be partially explained by genetic differences in arsenic metabolism. Mounting epidemiological evidence and in vitro studies suggest that methylated arsenic metabolites, particularly monomethylarsonic (MMA3), are more acutely toxic than inorganic arsenic; thus, MMA3 may be the primary toxic arsenic species. To test the role of genetic variation in arsenic metabolism, polymorphisms in genes involved in one-carbon metabolism [methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTR), cystathionine-beta-synthase (CBS), thymidylate synthase (TYMS), dihydrofolate reductase (DHFR), serine hydroxymethyltransferase 1 (SHMT1)] and glutathione biosynthesis [glutathione-S-transferase omega 1 (GSTO1)] were examined in an arsenic-exposed population to determine their influence in urinary arsenic metabolite patterns. In 142 subjects in Cordoba Province, Argentina, variant genotypes for CBS rs234709 and rs4920037 SNPs compared with wild-type homozygotes were associated with 24% and 26% increases, respectively, in the mean proportion of arsenic excreted as monomethylarsonic acid (%MMA). This difference is within the range of differences in %MMA seen between people with arsenic-related disease and those without such disease in other studies. Small inverse associations with CBS rs234709 and rs4920037 variants were also found for the mean levels of the proportion of arsenic excreted as dimethylarsinous acid (%DMA). No other genetic associations were found. These findings are the first to suggest that CBS polymorphisms may influence arsenic metabolism in humans and susceptibility to arsenic-related disease.

Expression of methylation pathway enzymes in bovine oocytes and preimplantation embryos

The methylation pathway, which consists of two metabolic cycles of nutrients, i.e., the methionine and folate cycles, generates S-adenosylmethionine, the methyl donor for the methylation of DNA and histones. Using reverse transcription-polymerase chain reaction, we examined the gene expression patterns of the methylation pathway enzymes during bovine oocyte maturation and preimplantation embryonic development up to the blastocyst stage. Bovine oocytes were demonstrated to have the mRNA of all methylation pathway enzymes examined, namely, methionine adenosyltransferase 1A (MAT1A), MAT2A, MAT2B, S-adenosylhomocysteine hydrolase (AHCY), 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR), betaine-homocysteine methyltransferase (BHMT), serine hydroxymethyltransferase 1 (SHMT1), SHMT2, and 5,10-methylenetetrahydrofolate reductase (MTHFR). All the transcripts were consistently expressed throughout all developmental stages, except for MAT1A, which was not detected from the 8-cell stage onward and BHMT, which was not detected in the 8-cell stage. Immunofluorescence analysis of MAT1A protein revealed the relatively higher expression in oocytes and early cleavage stage embryos up to the 8-cell stage compared with the morula and blastocyst stage. Further, to investigate the effects of methylation pathway disruption during the earliest stages of embryonic development, the effects of exogenous homocysteine on preimplantation development and DNA methylation of bovine embryos were investigated in vitro. As results, high concentrations of homocysteine induced hypermethylation of genomic DNA as well as developmental retardation in bovine embryos. These results provide a new insight into nutrient-sensitive epigenetic regulation and perturbation at the earliest stage of our life.

A UV-responsive Internal Ribosome Entry Site Enhances Serine Hydroxymethyltransferase 1 Expression for DNA Damage Repair

Thymidine nucleotides are required for faithful DNA synthesis and repair, and their de novo biosynthesis is regulated by serine hydroxymethyltransferase 1 (SHMT1). The SHMT1 transcript contains a heavy chain ferritin, heterogeneous nuclear ribonucleoprotein H2, and CUG-binding protein 1-responsive internal ribosome entry site (IRES) that regulates SHMT1 translation. In this study a non-lethal dose of UVC is shown to increase SHMT1 IRES activity and protein levels in four different cell lines. The mechanism for the UV-induced activation of the SHMT1 IRES involves an increase in heavy chain ferritin and heterogeneous nuclear ribonucleoprotein H2 expression and the translocation of CUG-binding protein 1 from the nucleus to the cytoplasm. The UV-induced increase in SHMT1 translation is accompanied by an increase in the small ubiquitin-like modifier-dependent nuclear localization of the de novo thymidylate biosynthesis pathway and a decrease in DNA strand breaks, indicating a role for SHMT1 and nuclear folate metabolism in DNA repair.

Mechanism of the Internal Ribosome Entry Site-mediated Translation of Serine Hydroxymethyltransferase 1

The 5'-untranslated region (UTR) of serine hydroxymethyltransferase 1 (SHMT1) contains an internal ribosome entry site (IRES) that regulates SHMT1 expression, a rate-limiting enzyme in de novo thymidylate biosynthesis. In this study, we show that the SHMT1 IRES is the first example of a cellular IRES that is poly(A) tail-independent. Interactions between the 5'-UTR and 3'-UTR functionally replaced interactions between the poly(A) tail and the poly(A)-binding protein (PABP) to achieve maximal IRES-mediated translational efficiency. Depletion of the SHMT1 IRES-specific trans-acting factor (ITAF) CUG-binding protein 1 (CUGBP1) from in vitro translation extracts or deletion of the CUGBP1 binding site on the 3'-UTR of the SHMT1 transcript decreased the IRES activity of non-polyadenylylated biscistronic mRNAs relative to polyadenylylated biscistronic mRNAs and resulted in a requirement for PABP. We also identified a novel ITAF, heterogeneous nuclear ribonucleoprotein H2 (hnRNP H2), that stimulates SHMT1 IRES activity by binding to the 5'-UTR of the transcript and interacting with CUGBP1. Collectively, these data support a model for the IRES-mediated translation of SHMT1 whereby the circularization of the mRNA typically provided by the eukaryotic initiation factor (eIF) 4G/PABP/poly(A) tail interaction is achieved instead through the hnRNP H2/CUGBP1-mediated interaction of the 5'- and 3'-UTRs of the SHMT1 transcript. This circularization enhances the IRES activity of SHMT1 by facilitating the recruitment and/or recycling of ribosomal subunits, which bind to the transcript in the middle of the 5'-UTR and migrate to the initiation codon via eIF4A-mediated scanning.