Affect DNA Synthesis Research Articles

DNTP pools determine fork progression and origin usage under replication stress

Intracellular deoxyribonucleoside triphosphate (dNTP) pools must be tightly regulated to preserve genome integrity. Indeed, alterations in dNTP pools are associated with increased mutagenesis, genomic instability and tumourigenesis. However, the mechanisms by which altered or imbalanced dNTP pools affect DNA synthesis remain poorly understood. Here, we show that changes in intracellular dNTP levels affect replication dynamics in budding yeast in different ways. Upregulation of the activity of ribonucleotide reductase (RNR) increases elongation, indicating that dNTP pools are limiting for normal DNA replication. In contrast, inhibition of RNR activity with hydroxyurea (HU) induces a sharp transition to a slow-replication mode within minutes after S-phase entry. Upregulation of RNR activity delays this transition and modulates both fork speed and origin usage under replication stress. Interestingly, we also observed that chromosomal instability (CIN) mutants have increased dNTP pools and show enhanced DNA synthesis in the presence of HU. Since upregulation of RNR promotes fork progression in the presence of DNA lesions, we propose that CIN mutants adapt to chronic replication stress by upregulating dNTP pools.

Platelet‐derived adenosine 5′‐triphosphate suppresses activation of human hepatic stellate cell: In vitro study

Activated hepatic stellate cells (HSC) play a critical role in liver fibrosis. Suppressing abnormal function of HSC or reversion from activated to quiescent form is a hopeful treatment for liver cirrhosis. The interaction between platelets and HSC remains unknown although platelets go through hepatic sinusoids surrounded by HSC. This study aimed at clarifying the hypothesis that platelets control activation of HSC. We used human platelets, platelet extracts, and primary or immortalized human HSC. We examined the effect of platelets on the activation, DNA synthesis, type I collagen production, and fibrosis-relating gene expressions of HSC. We investigated what suppressed activation of HSC within platelets and examined the mechanism of controlling activation in vitro. Platelets and platelet extracts suppressed activation of HSC. Platelets decreased type I collagen production without affecting DNA synthesis. Platelets increased the expression of matrix metallopeptidase 1. As platelet extracts co-cultured with an enzyme of degrading adenosine 5'-triphosphate (ATP) suppressed activation, we detected adenine nucleotides within platelets or on their surfaces and confirmed the degradation of adenine nucleotides by HSC and the production of adenosine. Adenosine and platelets increased the intracellular cyclic adenosine 5'-monophosphate (cAMP), which is important in quiescent HSC. A great amount of adenosine and ATP also suppressed activation of HSC. Activation of human HSC is suppressed by human platelets or platelet-derived ATP via adenosine-cAMP signaling pathway in vitro. Therefore, platelets have the possibility to be used in the treatment of liver cirrhosis.

Abstract B102: Serum compounds involved in the one-carbon metabolism and urothelial cell carcinoma risk: A nested case-control study within EPIC

Abstract Introduction: Folate is, in combination with vitamin B6, vitamin B12 and homocysteine, involved in one-carbon-metabolism affecting DNA synthesis and methylation which may play a role in carcinogenesis. This nested case-control study within the European Prospective Investigation into Cancer and Nutrition (EPIC) is the first prospective study that investigated associations between prediagnostic serum folate, homocysteine, vitamin B6 and vitamin B12 and the risk of urothelial cell carcinomas (UCC). Methods: 847 UCC first incident cases were matched to 847 controls for gender, age at baseline, study center, date of blood collection, time of day of blood collection, and fasting status. Serum concentrations of folate and vitamin B12 were determined with an immuno-analyser. Serum vitamin B6 concentrations were measured by reverse-phase high-performance liquid chromatography (HPLC) and serum homocysteine concentrations by an enzyme cycling assay. Incidence rate ratios (IRR) were estimated with conditional logistic regression with detailed adjustment for smoking status, smoking duration and smoking intensity. All analyses were conducted for total UCC and for two prognostic subgroups of UCC, defined by tumor stage and grade. Ta grade 1 or 2 tumors were defined as non-aggressive (n=377) while all other grade and stage combinations were defined as aggressive (n=395). Results: A reduced risk of UCC was found for higher levels of serum folate (IRR highest vs. lowest quartile 0.73; 95%CI 0.52–1.01, p-trend=0.04), although this was apparent only for current smokers. Serum folate was inversely associated with aggressive UCC risk (IRR highest vs. lowest quartile 0.59; 95%CI 0.37–0.95, p-trend=0.04), but not with the risk of non-aggressive UCC. No associations were observed between serum homocysteine, vitamin B6 and B12 and UCC risk. Conclusion: This large European cohort study shows that high serum concentrations of folate may reduce the risk of UCC, in particular the risk of aggressive UCC. However, residual confounding by smoking can not be excluded. Citation Information: Cancer Prev Res 2011;4(10 Suppl):B102.

Common fragile sites in colon cancer cell lines: Role of mismatch repair, RAD51 and poly(ADP-ribose) polymerase-1

Common fragile sites (CFS) are specific chromosomal areas prone to form gaps and breaks when cells are exposed to stresses that affect DNA synthesis, such as exposure to aphidicolin (APC), an inhibitor of DNA polymerases. The APC-induced DNA damage is repaired primarily by homologous recombination (HR), and RAD51, one of the key players in HR, participates to CFS stability. Since another DNA repair pathway, the mismatch repair (MMR), is known to control HR, we examined the influence of both the MMR and HR DNA repair pathways on the extent of chromosomal damage and distribution of CFS provoked by APC and/or by RAD51 silencing in MMR-deficient and -proficient colon cancer cell lines (i.e., HCT-15 and HCT-15 transfected with hMSH6, or HCT-116 and HCT-116/3+6, in which a part of a chromosome 3 containing the wild-type hMLH1 allele was inserted). Here, we show that MMR-deficient cells are more sensitive to APC-induced chromosomal damage particularly at the CFS as compared to MMR-proficient cells, indicating an involvement of MMR in the control of CFS stability. The most expressed CFS is FRA16D in 16q23, an area containing the tumour suppressor gene WWOX often mutated in colon cancer. We also show that silencing of RAD51 provokes a higher number of breaks in MMR-proficient cells with respect to their MMR-deficient counterparts, likely as a consequence of the combined inhibitory effects of RAD51 silencing on HR and MMR-mediated suppression of HR. The RAD51 silencing causes a broader distribution of breaks at CFS than that observed with APC. Treatment with APC of RAD51-silenced cells further increases DNA breaks in MMR-proficient cells. The RNAi-mediated silencing of PARP-1 does not cause chromosomal breaks or affect the expression/distribution of CFS induced by APC. Our results indicate that MMR modulates colon cancer sensitivity to chromosomal breaks and CFS induced by APC and RAD51 silencing.

Methylenetetrahydrofolate Reductase (MTHFR) Gene C677T, A1298C and G1793A Polymorphisms: Association with Risk for Clear Cell Renal Cell Carcinoma and Tumour Behaviour in Men

AimsMethylenetetrahydrofolate reductase (MTHFR) plays a crucial role in regulating folate metabolism, which affects DNA synthesis and methylation. This study investigated whether MTHFR C677T, A1298C and G1793A polymorphisms modified clear cell renal cell carcinoma (CCRCC) risk independently as well as in combination with serum total homocysteine (Hcy) and folate levels. Materials and methodsA case–control study of 152 cases (men) and 304 age-matched healthy controls was conducted in one geographical area of Iran. Genotyping of MTHFR gene polymorphisms was carried out using a polymerase chain reaction restriction fragment length polymorphism technique. Serum levels of total Hcy, folate and vitamin B12 were also determined. ResultsThe MTHFR 677T and 1298C allele frequencies were 42.8 and 47.4% in cases, compared with 33.7 and 33.1% in controls. After controlling for confounding factors, a significant increase in CCRCC risk was found among carriers of the 677CT genotype compared with those with the 677CC genotype (odds ratio 2.21, 95% confidence interval 1.31–3.76), with a significant trend (P=0.014). Statistically significant odds ratios were also found in patients homozygous for MTHFR C677T, who have a 1.58-fold higher risk of developing CCRCC (95% confidence interval=1.21–2.44; P=0.024). Compared with the MTHFR 677CC genotype, the odds ratio (95% confidence interval) for the MTHFR 677TT genotype was 6.18 (95% confidence interval=4.75–8.34) for stage IV cancer and 4.68 (95% confidence interval=2.72–6.54) for grade 3 CCRCC (both P=0.0001). After adjustment for selected variants, the MTHFR 1298AC genotype showed a significantly increased risk of CCRCC compared with the wild-type (odds ratio=3.71, 95% confidence interval=2.22–5.33; P=0.001), and the 1298C allele carrier showed a positive association with the risk of CCRCC compared with the wild-type (odds ratio=3.9, 95% confidence interval=2.55–6.02; P=0.001). Furthermore, subjects carrying at least one copy of the variant allele showed a 4.4 times increased risk of developing CCRCC than their control counterparts (odds ratio=4.40, 95% confidence interval=2.41–6.72; P=0.0001). There was not a significant interaction between MTHFR polymorphisms and serum levels of total Hcy and folate in increasing the risk of CCRCC. ConclusionsOur results provide evidence that the MTHFR polymorphisms might contribute to increased CCRCC risk in men.

Appl1 is dispensable for Akt signaling in vivo and mouse T‐cell development

Appl1 (Adaptor protein containing pleckstrin homology [PH], phosphotyrosine binding [PTB], and Leucine zipper motifs) is an adaptor that participates in cell signaling by interacting with various signaling molecules including Akt, PI3-kinase (PI3K), Rab5, adiponectin receptor, and TrkA. By using RNA knockdown technology, Appl1 has been implicated in zebrafish development and murine glucose metabolism. To investigate the unambiguous role of Appl1 in vivo, we generated a knockout mouse in which exon1 of the Appl1 gene was disrupted using gene trap methodology. Homozygous Appl1 knockout mice with ubiquitous loss of Appl1 protein expression were viable, grossly normal, and born at expected Mendelian ratios. Moreover, activation of Akt and the downstream effecter Gsk3β was unaffected in vivo. We next performed glucose and insulin tolerance tests and found that glucose metabolism is normal in Appl1-null mice. We also tested the effect of Appl1 loss on Akt signaling in T cells, because we discovered that Appl1 strongly interacts with the p110β subunit of PI3K in T lymphocytes. However, such interaction was found to be dispensable for Akt signaling in thymic T cells and T-cell development. Moreover, Appl1 loss did not affect DNA synthesis in cultured thymocytes, although loss of Appl1 was associated with a slight increase in ConA-stimulated splenic T-cell viability/proliferation. Collectively, our findings indicate that Appl1 is dispensable for Akt signaling in vivo and T-cell differentiation.

688 Epigenetic dysregulation of hsa-miR-9 in hepatocellular carcinoma

Common fragile sites (CFS) are specific chromosomal areas prone to form gaps and breaks when cells are exposed to stresses that affect DNA synthesis, such as exposure to aphidicolin (APC), an inhibitor of DNA polymerases. The APC-induced DNA damage is repaired primarily by homologous recombination (HR), and RAD51, one of the key players in HR, participates to CFS stability. Since another DNA repair pathway, the mismatch repair (MMR), is known to control HR, we examined the influence of both the MMR and HR DNA repair pathways on the extent of chromosomal damage and distribution of CFS provoked by APC and/or by RAD51 silencing in MMR-deficient and -proficient colon cancer cell lines (i.e., HCT-15 and HCT-15 transfected with hMSH6, or HCT-116 and HCT-116/3+6, in which a part of a chromosome 3 containing the wild-type hMLH1 allele was inserted). Here, we show that MMR-deficient cells are more sensitive to APC-induced chromosomal damage particularly at the CFS as compared to MMR-proficient cells, indicating an involvement of MMR in the control of CFS stability. The most expressed CFS is FRA16D in 16q23, an area containing the tumour suppressor gene WWOX often mutated in colon cancer. We also show that silencing of RAD51 provokes a higher number of breaks in MMR-proficient cells with respect to their MMR-deficient counterparts, likely as a consequence of the combined inhibitory effects of RAD51 silencing on HR and MMR-mediated suppression of HR. The RAD51 silencing causes a broader distribution of breaks at CFS than that observed with APC. Treatment with APC of RAD51-silenced cells further increases DNA breaks in MMR-proficient cells. The RNAi-mediated silencing of PARP-1 does not cause chromosomal breaks or affect the expression/distribution of CFS induced by APC. Our results indicate that MMR modulates colon cancer sensitivity to chromosomal breaks and CFS induced by APC and RAD51 silencing.

The Effects of Arsenic Trioxide on DNA Synthesis and Genotoxicity in Human Colon Cancer Cells

Colon cancer is the third leading cause of cancer-related deaths worldwide. Recent studies in our laboratory have demonstrated that arsenic trioxide is cytotoxic in human colon cancer (HT-29), lung (A549) and breast (MCF-7) carcinoma cells. The purpose of the present study is to investigate the effects of arsenic trioxide on DNA synthesis and the possible genotoxic effects on human colon cancer cells. HT-29 cells were cultured according to standard protocol, followed by exposure to various doses (0, 2, 4, 6, 8, 10, and 12 μg/mL) of arsenic trioxide for 24 h. The proliferative response (DNA synthesis) to arsenic trioxide was assessed by [3H]thymidine incorporation. The genotoxic effects of arsenic-induced DNA damage in a human colon cancer cell line was evaluated by the alkaline single cell gel electrophoresis. Results indicated that arsenic trioxide affected DNA synthesis in HT-29 cells in a biphasic manner; showing a slight but not significant increase in cell proliferation at lower levels of exposure (2, 4 and 6 μg/mL) followed by a significant inhibition of cell proliferation at higher doses (i.e., 8 and 10 μg/mL). The study also confirmed that arsenic trioxide exposure caused genotoxicity as revealed by the significant increase in DNA damage, comet tail-lengths, and tail moment when compared to non-exposed cells. Results of the [3H]thymidine incorporation assay and comet assay revealed that exposure to arsenic trioxide affected DNA synthesis and exhibited genotoxic effects in human colon cancer cells.

A Novel Fibroblast Growth Factor-1 (FGF1) Mutant that Acts as an FGF Antagonist

BackgroundCrosstalk between integrins and FGF receptors has been implicated in FGF signaling, but the specifics of the crosstalk are unclear. We recently discovered that 1) FGF1 directly binds to integrin αvβ3, 2) the integrin-binding site and FGF receptor (FGFR) binding site are distinct, and 3) the integrin-binding-defective FGF1 mutant (R50E) is defective in inducing FGF signaling although R50E still binds to FGFR and heparin and induces transient ERK1/2 activation.Principal FindingsWe tested if excess R50E affect DNA synthesis and cell survival induced by WT FGF1 in BaF3 mouse pro-B cells expressing human FGFR1. R50E suppressed DNA synthesis and cell proliferation induced by WT FGF1. We tested if WT FGF1 and R50E generate integrin-FGF1-FGFR ternary complex. WT FGF1 induced ternary complex formation (integrin-FGF-FGFR1) and recruitment of SHP-2 to the complex in NIH 3T3 cells and human umbilical endothelial cells, but R50E was defective in these functions. It has been reported that sustained ERK1/2 activation is integrin-dependent and crucial to cell cycle entry upon FGF stimulation. We thus determined the time-course of ERK1/2 activation induced by WT FGF1 and R50E. We found that WT FGF1 induced sustained activation of ERK1/2, but R50E was defective in this function.Conclusions/SignificanceOur results suggest that 1) R50E is a dominant-negative mutant, 2) Ternary complex formation is involved in FGF signaling, 3) The defect of R50E to bind to integrin may be directly related to the antagonistic action of R50E. Taken together, these results suggest that R50E has potential as a therapeutic in cancer.

MTR polymorphic variant A2756G and retinoblastoma risk in Brazilian children

Polymorphisms in the genes of folate and methionine metabolism enzymes have been associated with some forms of cancer by affecting DNA synthesis, repair, and methylation. A case-control study of 72 retinoblastoma cases and 98 cancer-free children controls was performed to investigate whether the polymorphisms of the methylenetetrahydrofolate reductase (MTHFR C677T and A1298C), methionine synthase (MTR A2756G), carrier of reduced folate 1 (RFC-1 A80G) and thymidylate synthase (TYMS 2R > 3R) altered the risk for retinoblastoma. MTR A2756G AG plus GG genotype frequencies were higher in patients than in controls (45% vs. 26%, P = 0.03). Individual carriers of the variant allele G had a 2.02 (95% CI: 1.05-3.92)-fold increased risk for retinoblastoma. In contrast, no association was observed with respect to MTHFR C677T and A1298C, RFC A80G, and TYMS polymorphisms. This study presents evidence for an association between the MTR A2756G polymorphism and retinoblastoma susceptibility in a Northeast population from Brazil.

Lenalidomide induces cell death in an MDS-derived cell line with deletion of chromosome 5q by inhibition of cytokinesis

Myelodysplastic syndromes (MDS) are a group of hematopoietic stem cell disorders characterized by refractory cytopenias and susceptibility to leukemic transformation. On a subset of MDS patients with deletion of the long arm of chromosome 5 (del(5q)), lenalidomide exerts hematological and cytogenetic effects, but the underlying pharmacological mechanisms are not fully understood. In this study, we have investigated the in vitro effects of lenalidomide on an MDS-derived cell line, MDS-L, which carries del(5q) and complex chromosome abnormalities. We found that the growth of MDS-L cells was specifically suppressed mainly by apoptosis, and in addition, multinucleated cells were frequently formed and finally died out in the presence of lenalidomide. Time-lapse microscopic observation and the DNA ploidy analysis revealed that lenalidomide does not affect DNA synthesis but inhibits cytokinesis of MDS-L cells. The gene expression profile showed decreased expression of M phase-related genes such as non-muscle myosin heavy-chain 10, polo-like kinase 1, aurora kinase B, citron kinase and kinesin family member 20A(KIF20A). Interestingly, KIF20A is located at 5q31. These data contribute to the understanding of action mechanisms of lenalidomide on MDS with del(5q) and complex abnormalities.

Cell cycle defects in polyhomeotic mutants are caused by abrogation of the DNA damage checkpoint

Polycomb group (PcG) genes are required for heritable silencing of target genes. Many PcG mutants have chromatin bridges and other mitotic defects in early embryos. These phenotypes can arise from defects in S phase or mitosis, so the phenotype does not show when PcG proteins act in cell cycle regulation. We analyzed the cell cycle role of the proximal subunit of Polyhomeotic (PhP) in Drosophila. Time-lapse imaging reveals that chromatin bridges formed during mitosis are able to resolve but sometimes result in chromosome breakage. Chromosome bridging is also observed in canonical cell cycles occurring in larval brains and is therefore not unique to the rapid embryonic cycles. PhP colocalizes with chromatin in S phase but not in mitosis in early embryos, indicating a direct role in DNA synthesis. Time lapse imaging of php mutants reveals an acceleration of S phase, showing that php regulates S phase length. Like php mutations, mutations in DNA damage checkpoints result in S phase acceleration. Consistent with this model, mutations in ph do not affect DNA synthesis rates, but exhibit impaired ability to block cell cycle progression following exposure to gamma-rays. Our data show that the mitotic defects of php are caused by defects in the DNA damage response that occurs after DNA replication in S phase, and we propose that PhP has a direct role in DNA damage repair.

Vitamins B2 and B6 and Genetic Polymorphisms Related to One-Carbon Metabolism as Risk Factors for Gastric Adenocarcinoma in the European Prospective Investigation into Cancer and Nutrition

B vitamins and polymorphisms in genes coding for enzymes involved in one-carbon metabolism may affect DNA synthesis and methylation and thereby be implicated in carcinogenesis. Previous data on vitamins B2 and B6 and genetic polymorphisms other than those involving MTHFR as risk factors for gastric cancer (GC) are sparse and inconsistent. In this case-control study nested within the European Prospective Investigation into Cancer and Nutrition cohort, cases (n = 235) and controls (n = 601) were matched for study center, age, sex, and time of blood sampling. B2 and B6 species were measured in plasma, and the sum of riboflavin and flavin mononucleotide was used as the main exposure variable for vitamin B2 status, whereas the sum of pyridoxal 5'-phosphate, pyridoxal, and 4-pyridoxic acid was used to define vitamin B6 status. In addition, we determined eight polymorphisms related to one-carbon metabolism. Relative risks for GC risk were calculated with conditional logistic regression, adjusted for Helicobacter pylori infection status and smoking status. Adjusted relative risks per quartile (95% confidence interval, P(trend)) were 0.85 (0.72-1.01, 0.06) for vitamin B2 and 0.78 (0.65-0.93, <0.01) for vitamin B6. Both relations were stronger in individuals with severe chronic atrophic gastritis. The polymorphisms were not associated with GC risk and did not modify the observed vitamin-cancer associations. In summary, results from this large European cohort study showed an inverse association between vitamin B2 and GC risk, which is borderline significant, and a significant inverse association between vitamin B6 and GC risk.

Altered Folate Metabolism Maybeo the Develop Linked tment of Acute Lymphoblastic Leukemia in African American Children.

Abstract 4118 BackgroundAcute lymphoblastic leukaemia (ALL) is the most common form of childhood cancer in the United States. The incidence of ALL is approximately 2-3-fold higher in Caucasian compared to African American (AA) children, suggesting potential differences in genetic susceptibility and/or exogenous exposures. Multiple epidemiologic studies have examined both genetic and environmental factors linked to the development of childhood ALL, primarily in Caucasian populations. Hence, identifying factors associated with racial differences in incidence of leukemia may provide new insights into the role of endogenous versus exogenous factors in the development of leukemia. A number of studies have reported relationships between folate metabolism and the risk of developing ALL including: i)maternal folate supplementation during pregnancy (reduced risk of ALL in offspring); and ii)polymorphisms of genes encoding enzymes involved in folate metabolism, including 5,10-methylenetetrahydrofolate reductase (MTHFR) (increased and decreased risks). To date, no studies have been performed specifically examining the role of folate metabolism in AA children. The objective of this study was to identify factors associated with folate metabolism which may be linked to the development of ALL in AA children compared to healthy controls. Patients and MethodsAA children with B-precursor (BP) ALL were enrolled from the Hematology/Oncology Division of Children's Hospital of Michigan, while healthy AA children were enrolled as controls. Patients' racial backgrounds were based on parental reporting. The frequencies of polymorphisms in the MTHFR [677C>T, 1298A>C], thymidylate synthase [TS 2R3R], cystathionine-β-synthase [CBS 844ins(68)], and reduced folate carrier [RFC 80G>A] genes were determined by genotyping between AA childhood BP-ALL [n=26; 14 males] and healthy AA children [n=87; 47 males]. The distributions of genotypes between cases and controls were compared using Fisher's exact test. ResultsThe genotype distributions of the polymorphisms of the folate pathway genes are summarized in Table 1. The frequencies of the MTHFR gene variants 677 CT/TT were 2-fold higher in the ALL cohort than that in the healthy control cohort. MTHFR 677 CT/TT was significantly associated with a risk of developing ALL in the AA patients. There were no significant differences in the distributions of the TS, CBS, or RFC polymorphisms between the groups. High birth weight has been associated with an increased risk of developing ALL, though we found no significant difference in birth weights between ALL and control groups. ConclusionOur study is the first to demonstrate that there is a higher frequency of the variant MTHFR C677T polymorphism (associated with reduced enzyme activity and altered distribution of folate forms) in AA children with ALL compared to healthy controls. Low MTHFR enzyme activity leads to imbalances in the thymidylate and de novo purine biosynthetic pathways, ultimately affecting DNA synthesis and repair and likely increasing the risk of leukemia. Thus, the role of altered folate metabolism may contribute to the development of ALL in AA children similar to Caucasian children, although additional studies are still required to identify factors linked to the higher incidence of ALL in Caucasian children and/or low incidence in AA children.Table 1Distribution of Genotypes in AA Children with BP-ALL Patients and Healthy ControlsMTHFR VariantGenotypeBP-ALL cases (N=26)Control cases (N=87)pC677TCC15 (57.69%)71 (81.61%)CT9 (34.62%)14 (16.09%)0.044TT2 (7.69%)2 (2.30%)0.160CT+TT11 (42.31%)16 (18.39%)0.018A1298CAA18 (69. 23%)67 (77.01%)AC8 (30.77%)19 (21.84%)0.434CC0 (0%)1 (1.15%)1AC+CC8 (30.77%)20 (22.99%)0.444 Disclosures:No relevant conflicts of interest to declare.

Are We Hitting the Right Combination for Hormonally Sensitive Breast Cancer?

Are We Hitting the Right Combination for Hormonally Sensitive Breast Cancer?

L‐leucine increases [3H]‐thymidine incorporation in chicken hepatocytes: Involvement of the PKC, PI3K/Akt, ERK1/2, and mTOR signaling pathways

This study examined how L-leucine affected DNA synthesis and cell cycle regulatory protein expression in cultured primary chicken hepatocytes. L-Leucine promoted DNA synthesis in a dose- and time-dependent manner, with concomitant increases in cyclin D1 and cyclin E expression. Phospholipase C (PLC) and protein kinase C (PKC) mediated the L-leucine-induced increases in [3H]-thymidine incorporation and cyclin D1/CDK4 and cyclin E/CDK2 expression, as U73122 (a PLC inhibitor) or bisindolylmaleimide I (a PKC blocker) inhibited these effects. L-Leucine also increased PKC phosphorylation and intracellular Ca2+ levels. L-Leucine-mediated increases in [3H]-thymidine incorporation and cyclin/CDK expression were sensitive to LY 294002 (PI3K inhibitor), Akt inhibitor, PD 98059 (MEK inhibitor). It was also observed that L-leucine-induced increases of cyclin/CDK expression were inhibited by PI3K siRNA and ERK siRNA; L-leucine increased extracellular signal-regulated kinases 1/2 (ERK1/2) and Akt phosphorylation levels. Bisindolylmaleimide I attenuated L-leucine-induced phosphorylation of ERK1/2 but did not influence Akt phosphorylation, and PI3K siRNA and LY 294002 inhibited L-leucine-induced ERK1/2 phosphorylation, suggesting some cross-talk between the PKC and ERK1/2 or PI3K/Akt and ERK1/2 pathways. L-Leucine also increased the levels of phosphorylated molecular target of rapamycin (mTOR) and two of its targets, ribosomal protein S6 kinase (p70S6K), and 4E binding protein 1 (4E-BP1); furthermore, rapamycin (an mTOR inhibitor) blocked all of the mitogenic effects of L-leucine. In addition, Akt inhibitor blocked L-leucine-induced mTOR phosphorylation. In conclusion, L-leucine stimulated DNA synthesis and promoted cell cycle progression in primary cultured chicken hepatocytes through PKC, ERK1/2, PI3K/Akt, and mTOR.

Polymorphisms of methylenetetrahydrofolate reductase and methionine synthase genes and bladder cancer risk: a case–control study with meta-analysis

Folate deficiency due to the methylenetetrahydrofolate reductase (MTHFR) and methionine synthase (MS) variants leads to carcinogenesis by affecting DNA synthesis, repair, and methylation. We hypothesized that the MTHFR C677T, A1298C, and MS A2756G polymorphisms are associated with risk of bladder cancer. In a case-control study of 239 bladder cancer cases and 250 cancer-free controls, we found that the MTHFR 677TT genotype was statistically significantly associated with an increased risk of bladder cancer compared with the 677CC genotype (OR = 2.06, 95% CI = 1.16-3.64). Furthermore, the TA haplotype was associated with a significantly increased bladder cancer risk (OR = 1.38, 95% CI = 1.05-1.81) than was the most common haplotype, CA (e.g., CA denotes MTHFR 677C -1298A). We also found that the combined genotypes with 4-6 variant (risk) alleles (i.e., MTHFR 677T, 1298A, and MS 2756G alleles) were associated with an increased risk of bladder cancer (OR = 1.62, 95% CI = 1.03-2.53) compared with those with 0-3 variants, and this increased risk was more pronounced among subgroup of older people (OR = 1.71, 95% CI = 1.03-2.83). A meta-analysis of seven studies did not show a significant risk of bladder cancer in the MTHFR polymorphisms. The MTHFR polymorphisms and their haplotypes appear to jointly contribute to risk of bladder cancer.

Therapeutic touch affects DNA synthesis and mineralization of human osteoblasts in culture

Complementary and alternative medicine (CAM) techniques are commonly used in hospitals and private medical facilities; however, the effectiveness of many of these practices has not been thoroughly studied in a scientific manner. Developed by Dr. Dolores Krieger and Dora Kunz, Therapeutic Touch is one of these CAM practices and is a highly disciplined five-step process by which a practitioner can generate energy through their hands to promote healing. There are numerous clinical studies on the effects of TT but few in vitro studies. Our purpose was to determine if Therapeutic Touch had any effect on osteoblast proliferation, differentiation, and mineralization in vitro. TT was performed twice a week for 10 min each on human osteoblasts (HOBs) and on an osteosarcoma-derived cell line, SaOs-2. No significant differences were found in DNA synthesis, assayed by [(3)H]-thymidine incorporation at 1 or 2 weeks for SaOs-2 or 1 week for HOBs. However, after four TT treatments in 2 weeks, TT significantly (p = 0.03) increased HOB DNA synthesis compared to controls. Immunocytochemistry for Proliferating Cell Nuclear Antigen (PCNA) confirmed these data. At 2 weeks in differentiation medium, TT significantly increased mineralization in HOBs (p = 0.016) and decreased mineralization in SaOs-2 (p = 0.0007), compared to controls. Additionally, Northern blot analysis indicated a TT-induced increase in mRNA expression for Type I collagen, bone sialoprotein, and alkaline phosphatase in HOBs and a decrease of these bone markers in SaOs-2 cells. In conclusion, Therapeutic Touch appears to increase human osteoblast DNA synthesis, differentiation and mineralization, and decrease differentiation and mineralization in a human osteosarcoma-derived cell line.

ATP-dependent chromatin remodeling shapes the DNA replication landscape

The eukaryotic DNA replication machinery must traverse every nucleosome in the genome during S phase. As nucleosomes are generally inhibitory to DNA-dependent processes, chromatin structure must undergo extensive reorganization to facilitate DNA synthesis. However, the identity of chromatin-remodeling factors involved in replication and how they affect DNA synthesis is largely unknown. Here we show that two highly conserved ATP-dependent chromatin-remodeling complexes in Saccharomyces cerevisiae, Isw2 and Ino80, function in parallel to promote replication fork progression. As a result, Isw2 and Ino80 have especially important roles for replication of late-replicating regions during periods of replication stress. Both Isw2 and Ino80 complexes are enriched at sites of replication, suggesting that these complexes act directly to promote fork progression. These findings identify ATP-dependent chromatin-remodeling complexes that promote DNA replication and define a specific stage of replication that requires remodeling for normal function.

Chronic health effects of sulphur mustard exposure with special reference to Iranian veterans

The widespread use of sulphur mustard (SM) as an incapacitating chemical warfare agent in the past century has proved its long-lasting toxic effects. It may also be used as a chemical terrorist agent. Therefore, all health professionals should have sufficient knowledge and be prepared for any such chemical attack. SM exerts direct toxic effects on the eyes, skin, and respiratory tissue, with subsequent systemic action on the nervous, immunological, haematological, digestive, and reproductive systems. SM is an alkylating agent that affects DNA synthesis, and, thus, delayed complications have been seen since the First World War. Cases of malignancies in the target organs, particularly in haematopoietic, respiratory, and digestive systems, have been reported. Important delayed respiratory complications include chronic bronchitis, bronchiectasis, frequent bronchopneumonia, and pulmonary fibrosis, all of which tend to deteriorate with time. Severe dry skin, delayed keratitis, and reduction of natural killer cells with subsequent increased risk of infections and malignancies are also among the most distressing long-term consequences of SM intoxication. However, despite a lot of research over the past decades on Iranian veterans, there are still major gaps in the SM literature. Immunological and neurological dysfunction, as well as the relationship between SM exposure and mutagenicity, carcinogenicity, and teratogenicity are important fields that require further studies, particularly on Iranian veterans with chronic health effects of SM poisoning. There is also a paucity of information on the medical management of acute and delayed toxic effects of SM poisoning-a subject that greatly challenges health care specialists.