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The Hippo pathway regulates the development and homeostasis of many tissues and in many species. It controls the activity of two paralogous transcriptional coactivators, YAP and TAZ (YAP/TAZ). Although previous studies have established that aberrant YAP/TAZ activation is detrimental to mammalian brain development, whether and how endogenous levels of YAP/TAZ activity regulate brain development remain unclear. Here, we show that during mammalian cortical development, YAP/TAZ are specifically expressed in apical neural progenitor cells known as radial glial cells (RGCs). The subcellular localization of YAP/TAZ undergoes dynamic changes as corticogenesis proceeds. YAP/TAZ are required for maintaining the proliferative potential and structural organization of RGCs, and their ablation during cortical development reduces the numbers of cortical projection neurons and causes the loss of ependymal cells, resulting in hydrocephaly. Transcriptomic analysis using sorted RGCs reveals gene expression changes in YAP/TAZ-depleted cells that correlate with mutant phenotypes. Thus, our study has uncovered essential functions of YAP/TAZ during mammalian brain development and revealed the transcriptional mechanism of their action.

The tight junction (TJ) and barrier function of colonic epithelium is highly sensitive to ionizing radiation. We evaluated the effect of lysophosphatidic acid (LPA) and its analog, Radioprotein-1, on γ-radiation-induced colonic epithelial barrier dysfunction using Caco-2 and m-IC(C12) cell monolayers in vitro and mice in vivo. Mice were subjected to either total body irradiation (TBI) or partial body irradiation (PBI-BM5). Intestinal barrier function was assessed by analyzing immunofluorescence localization of TJ proteins, mucosal inulin permeability, and plasma lipopolysaccharide (LPS) levels. Oxidative stress was analyzed by measuring protein-thiol oxidation and antioxidant mRNA. In Caco-2 and m-IC(C12) cell monolayers, LPA attenuated radiation-induced redistribution of TJ proteins, which was blocked by a Rho-kinase inhibitor. In mice, TBI and PBI-BM5 disrupted colonic epithelial tight junction and adherens junction, increased mucosal permeability, and elevated plasma LPS; TJ disruption by TBI was more severe in Lpar2(−/−) mice compared to wild type mice. RP1, administered before or after irradiation, alleviated TBI and PBI-BM5-induced TJ disruption, barrier dysfunction, and endotoxemia accompanied by protein thiol oxidation and downregulation of antioxidant gene expression, cofilin activation, and remodeling of the actin cytoskeleton. These data demonstrate that LPAR2 receptor activation prevents and mitigates γ-irradiation-induced colonic mucosal barrier dysfunction and endotoxemia.

The role of reactive oxygen species (ROS) in osmotic stress, dextran sulfate sodium (DSS) and cyclic stretch-induced tight junction (TJ) disruption was investigated in Caco-2 cell monolayers in vitro and restraint stress-induced barrier dysfunction in mouse colon in vivo. Live cell imaging showed that osmotic stress, cyclic stretch and DSS triggered rapid production of ROS in Caco-2 cell monolayers, which was blocked by depletion of intracellular Ca2+ by 1,2-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid. Knockdown of CaV1.3 or TRPV6 channels blocked osmotic stress and DSS-induced ROS production and attenuated TJ disruption and barrier dysfunction. N-Acetyl l-cysteine (NAC) and l-NG-Nitroarginine methyl ester (l-NAME) blocked stress-induced TJ disruption and barrier dysfunction. NAC and l-NAME also blocked stress-induced activation of c-Jun N-terminal kinase (JNK) and c-Src. ROS was colocalized with the mitochondrial marker in stressed cells. Cyclosporin A blocked osmotic stress and DSS-induced ROS production, barrier dysfunction, TJ disruption and JNK activation. Mitochondria-targeted Mito-TEMPO blocked osmotic stress and DSS-induced barrier dysfunction and TJ disruption. Chronic restraint stress in mice resulted in the elevation of intracellular Ca2+, activation of JNK and c-Src, and disruption of TJ in the colonic epithelium. Furthermore, corticosterone administration induced JNK and c-Src activation, TJ disruption and protein thiol oxidation in colonic mucosa. The present study demonstrates that oxidative stress is a common signal in the mechanism of TJ disruption in the intestinal epithelium by different types of cellular stress in vitro and bio behavioral stress in vivo.

The goals of this study were to evaluate the effects of ionizing radiation on apical junctions in colonic epithelium and mucosal barrier function in mice in vivo. Adult mice were subjected to total body irradiation (4 Gy) with or without N-acetyl-l-cysteine (NAC) feeding for 5 days before irradiation. At 2–24 h postirradiation, the integrity of colonic epithelial tight junctions (TJ), adherens junctions (AJ), and the actin cytoskeleton was assessed by immunofluorescence microscopy and immunoblot analysis of detergent-insoluble fractions for TJ and AJ proteins. The barrier function was evaluated by measuring vascular-to-luminal flux of fluorescein isothiocyanate (FITC)-inulin in vivo and luminal-to-mucosal flux in vitro. Oxidative stress was evaluated by measuring protein thiol oxidation. Confocal microscopy showed that radiation caused redistribution of occludin, zona occludens-1, claudin-3, E-cadherin, and β-catenin, as well as the actin cytoskeleton as early as 2 h postirradiation, and this effect was sustained for at least 24 h. Feeding NAC before irradiation blocked radiation-induced disruption of TJ, AJ, and the actin cytoskeleton. Radiation increased mucosal permeability to inulin in colon, which was blocked by NAC feeding. The level of reduced-protein thiols in colon was depleted by radiation with a concomitant increase in the level of oxidized-protein thiol. NAC feeding blocked the radiation-induced protein thiol oxidation. These data demonstrate that radiation rapidly disrupts TJ, AJ, and the actin cytoskeleton by an oxidative stress-dependent mechanism that can be prevented by NAC feeding.

Background Disruption of epithelial tight junctions (TJ), gut barrier dysfunction and endotoxemia play crucial role in the pathogenesis of alcoholic tissue injury. Occludin, a transmembrane protein of TJ, is depleted in colon by alcohol. However, it is unknown whether occludin depletion influences alcoholic gut and liver injury. Methods Wild type (WT) and occludin deficient (Ocln −/− ) mice were fed 1–6% ethanol in Lieber–DeCarli diet. Gut permeability was measured by vascular-to-luminal flux of FITC-inulin. Junctional integrity was analyzed by confocal microscopy. Liver injury was assessed by plasma transaminase, histopathology and triglyceride analyses. The effect of occludin depletion on acetaldehyde-induced TJ disruption was confirmed in Caco-2 cell monolayers. Results Ethanol feeding significantly reduced body weight gain in Ocln −/− mice. Ethanol increased inulin permeability in colon of both WT and Ocln −/− mice, but the effect was 4-fold higher in Ocln −/− mice. The gross morphology of colonic mucosa was unaltered, but ethanol disrupted the actin cytoskeleton, induced redistribution of occludin, ZO-1, E-cadherin and β-catenin from the junctions and elevated TLR4, which was more severe in Ocln −/− mice. Occludin knockdown significantly enhanced acetaldehyde-induced TJ disruption and barrier dysfunction in Caco-2 cell monolayers. Ethanol significantly increased liver weight and plasma transaminase activity in Ocln −/− mice, but not in WT mice. Histological analysis indicated more severe lesions and fat deposition in the liver of ethanol-fed Ocln −/− mice. Ethanol-induced elevation of liver triglyceride was also higher in Ocln −/− mice. Conclusion This study indicates that occludin deficiency increases susceptibility to ethanol-induced colonic mucosal barrier dysfunction and liver damage in mice.

Background Acetaldehyde, the toxic ethanol (EtOH) metabolite, disrupts intestinal epithelial barrier function. Aldehyde dehydrogenase (ALDH) detoxifies acetaldehyde into acetate. Subpopulations of Asians and Native Americans show polymorphism with loss-of-function mutations in ALDH2. We evaluated the effect of ALDH2 deficiency on EtOH-induced disruption of intestinal epithelial tight junctions and adherens junctions, gut barrier dysfunction, and liver injury. Methods Wild-type and ALDH2-deficient mice were fed EtOH (1 to 6%) in Lieber–DeCarli diet for 4 weeks. Gut permeability in vivo was measured by plasma-to-luminal flux of FITC-inulin, tight junction and adherens junction integrity was analyzed by confocal microscopy, and liver injury was assessed by the analysis of plasma transaminase activity, histopathology, and liver triglyceride. Results EtOH feeding elevated colonic mucosal acetaldehyde, which was significantly greater in ALDH2-deficient mice. ALDH2−/− mice showed a drastic reduction in the EtOH diet intake. Therefore, this study was continued only in wild-type and ALDH2+/− mice. EtOH feeding elevated mucosal inulin permeability in distal colon, but not in proximal colon, ileum, or jejunum of wild-type mice. In ALDH2+/− mice, EtOH-induced inulin permeability in distal colon was not only higher than that in wild-type mice, but inulin permeability was also elevated in the proximal colon, ileum, and jejunum. Greater inulin permeability in distal colon of ALDH2+/− mice was associated with a more severe redistribution of tight junction and adherens junction proteins from the intercellular junctions. In ALDH2+/− mice, but not in wild-type mice, EtOH feeding caused a loss of junctional distribution of tight junction and adherens junction proteins in the ileum. Histopathology, plasma transaminases, and liver triglyceride analyses showed that EtOH-induced liver damage was significantly greater in ALDH2+/− mice compared to wild-type mice. Conclusions These data demonstrate that ALDH2 deficiency enhances EtOH-induced disruption of intestinal epithelial tight junctions, barrier dysfunction, and liver damage.

Disruption of intestinal epithelial tight junctions is an important event in the pathogenesis of ulcerative colitis. Dextran sodium sulfate (DSS) induces colitis in mice with symptoms similar to ulcerative colitis. However, the mechanism of DSS-induced colitis is unknown. We investigated the mechanism of DSS-induced disruption of intestinal epithelial tight junctions and barrier dysfunction in Caco-2 cell monolayers in vitro and mouse colon in vivo. DSS treatment resulted in disruption of tight junctions, adherens junctions and actin cytoskeleton leading to barrier dysfunction in Caco-2 cell monolayers. DSS induced a rapid activation of c-Jun N-terminal kinase (JNK), and the inhibition or knockdown of JNK2 attenuated DSS-induced tight junction disruption and barrier dysfunction. In mice, DSS administration for 4 days caused redistribution of tight junction and adherens junction proteins from the epithelial junctions, which was blocked by JNK inhibitor. In Caco-2 cell monolayers, DSS increased intracellular Ca2+ concentration, and depletion of intracellular Ca2+ by 1,2-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid tetrakis(acetoxymethyl ester) (BAPTA/AM) or thapsigargin attenuated DSS-induced JNK activation, tight junction disruption and barrier dysfunction. Knockdown of apoptosis signal-regulated kinase 1 (Ask1) or MKK7 blocked DSS-induced tight junction disruption and barrier dysfunction. DSS activated c-Src by a Ca2+ and JNK-dependent mechanism. Inhibition of Src kinase activity or knockdown of c-Src blocked DSS-induced tight junction disruption and barrier dysfunction. DSS increased tyrosine phosphorylation of occludin, zonula occludens-1 (ZO-1), E-cadherin and β-catenin. SP600125 abrogated DSS-induced tyrosine phosphorylation of junctional proteins. Recombinant JNK2 induced threonine phosphorylation and auto-phosphorylation of c-Src. The present study demonstrates that Ca2+/Ask1/MKK7/JNK2/cSrc signalling cascade mediates DSS-induced tight junction disruption and barrier dysfunction.

The apical junctional complex (AJC), which includes tight junctions (TJs) and adherens junctions (AJs), determines the epithelial polarity, cell-cell adhesion and permeability barrier. An intriguing characteristic of a TJ is the dynamic nature of its multiprotein complex. Occludin is the most mobile TJ protein, but its significance in TJ dynamics is poorly understood. On the basis of phosphorylation sites, we distinguished a sequence in the C-terminal domain of occludin as a regulatory motif (ORM). Deletion of ORM and expression of a deletion mutant of occludin in renal and intestinal epithelia reduced the mobility of occludin at the TJs. ORM deletion attenuated Ca(2+) depletion, osmotic stress and hydrogen peroxide-induced disruption of TJs, AJs and the cytoskeleton. The double point mutations T403A/T404A, but not T403D/T404D, in occludin mimicked the effects of ORM deletion on occludin mobility and AJC disruption by Ca(2+) depletion. Both Y398A/Y402A and Y398D/Y402D double point mutations partially blocked AJC disruption. Expression of a deletion mutant of occludin attenuated collective cell migration in the renal and intestinal epithelia. Overall, this study reveals the role of ORM and its phosphorylation in occludin mobility, AJC dynamics and epithelial cell migration.

The intestinal epithelium is subjected to various types of mechanical stress. In this study, we investigated the impact of cyclic stretch on tight junction and adherens junction integrity in Caco-2 cell monolayers. Stretch for 2 h resulted in a dramatic modulation of tight junction protein distribution from a linear organization into wavy structure. Continuation of cyclic stretch for 6 h led to redistribution of tight junction proteins from the intercellular junctions into the intracellular compartment. Disruption of tight junctions was associated with redistribution of adherens junction proteins, E-cadherin and β-catenin, and dissociation of the actin cytoskeleton at the actomyosin belt. Stretch activates JNK2, c-Src, and myosin light-chain kinase (MLCK). Inhibition of JNK, Src kinase or MLCK activity and knockdown of JNK2 or c-Src attenuated stretch-induced disruption of tight junctions, adherens junctions, and actin cytoskeleton. Paracellular permeability measured by a novel method demonstrated that cyclic stretch increases paracellular permeability by a JNK, Src kinase, and MLCK-dependent mechanism. Stretch increased tyrosine phosphorylation of occludin, ZO-1, E-cadherin, and β-catenin. Inhibition of JNK or Src kinase attenuated stretch-induced occludin phosphorylation. Immunofluorescence localization indicated that phospho-MLC colocalizes with the vesicle-like actin structure at the actomyosin belt in stretched cells. On the other hand, phospho-c-Src colocalizes with the actin at the apical region of cells. This study demonstrates that cyclic stretch disrupts tight junctions and adherens junctions by a JNK2, c-Src, and MLCK-dependent mechanism.

Ethanol is metabolized into acetaldehyde in most tissues. In this study, we investigated the synergistic effect of ethanol and acetaldehyde on the tight junction integrity in Caco-2 cell monolayers. Expression of alcohol dehydrogenase sensitized Caco-2 cells to ethanol-induced tight junction disruption and barrier dysfunction, whereas aldehyde dehydrogenase attenuated acetaldehyde-induced tight junction disruption. Ethanol up to 150 mM did not affect tight junction integrity or barrier function, but it dose-dependently increased acetaldehyde-mediated tight junction disruption and barrier dysfunction. Src kinase and MLCK inhibitors blocked this synergistic effect of ethanol and acetaldehyde on tight junction. Ethanol and acetaldehyde caused a rapid and synergistic elevation of intracellular calcium. Calcium depletion by BAPTA or Ca2+-free medium blocked ethanol and acetaldehyde-induced barrier dysfunction and tight junction disruption. Diltiazem and selective knockdown of TRPV6 or CaV1.3 channels, by shRNA blocked ethanol and acetaldehyde-induced tight junction disruption and barrier dysfunction. Ethanol and acetaldehyde induced a rapid and synergistic increase in reactive oxygen species by a calcium-dependent mechanism. N-acetyl-L-cysteine and cyclosporine A, blocked ethanol and acetaldehyde-induced barrier dysfunction and tight junction disruption. These results demonstrate that ethanol and acetaldehyde synergistically disrupt tight junctions by a mechanism involving calcium, oxidative stress, Src kinase and MLCK.

Psoriasis is a chronic inflammatory skin disease with a clear genetic contribution, characterized by keratinocyte proliferation and immune cell infiltration. Various closely interacting cell types, including innate immune cells, T cells, and keratinocytes, are known to contribute to inflammation. Innate immune cells most likely initiate the inflammatory process by secretion of IL-23. IL-23 mediates expansion of T helper 17 (Th17) cells, whose effector functions, including IL-17A, activate keratinocytes. Keratinocyte activation in turn results in cell proliferation and chemokine expression, the latter of which fuels the inflammatory process through further immune cell recruitment. One question that remains largely unanswered is how genetic susceptibility contributes to this process and, specifically, which cell type causes disease due to psoriasis-specific genetic alterations. Here we describe a mouse model based on the human psoriasis susceptibility locus TNIP1, also referred to as ABIN1, whose gene product is a negative regulator of various inflammatory signaling pathways, including the Toll-like receptor pathway in innate immune cells. We find that Tnip1-deficient mice recapitulate major features of psoriasis on pathological, genomic, and therapeutic levels. Different genetic approaches, including tissue-specific gene deletion and the use of various inflammatory triggers, reveal that Tnip1 controls not only immune cells, but also keratinocyte biology. Loss of Tnip1 in keratinocytes leads to deregulation of IL-17-induced gene expression and exaggerated chemokine production in vitro and overt psoriasis-like inflammation in vivo. Together, the data establish Tnip1 as a critical regulator of IL-17 biology and reveal a causal role of keratinocytes in the pathogenesis of psoriasis.

Purpose Carcinogens causes DNA damage, including oxidative lesions that are removed efficiently by the base excision repair (BER) pathway. Variations in BER genes may reduce DNA repair capacity, leading to development of urological cancers. Methods This study included 195 prostate cancer (PCa) and 212 bladder cancer (BC) patients and 250 controls who had been frequency matched by age, sex, and ethnicity. We genotyped XRCC1 Exon 6 (C > T), 9 (G > A), 10 (G > A), OGG1 Exon 7 (C > G) and APE1 Exon 5 (T > G) genes polymorphism using PCR-RFLP and ARMS. Results GA of XRCC1 Exon 9 demonstrated increased risk with PCa as well as in BC (p = 0.001; p = 0.006). Similarly variant containing genotype revealed association with PCa (p = 0.031). Haplotype of XRCC1 also associated with significant risk for PCa and BC. The APE1 GG genotype showed a decreased risk of BC (OR = 0.25; p = 0.017). Variant genotype GG of OGG1 demonstrated significant risk with BC (p = 0.028). Conclusions Our observations suggested increased risk for PCa and BC in case of GA genotype for XRCC1, and variant GG in case of OGG1. However APE1 GG genotype conferred a protective association with BC susceptibility. Larger studies and the more SNPs in the same pathway are needed to verify these findings.

MicroRNAs (miRNA) are a class of small noncoding RNA molecules that have been implicated in a wide variety of cellular functions through post-transcriptional regulations on target genes. Common genetic variants (single-nucleotide polymorphisms, SNPs) in pre-miRNA genes may alter their expression and/or maturation effecting thousands of target mRNAs, resulting in varied functional consequences. Three common SNPs (hsa-mir-146a GC rs2910164, hsa-mir-196a2 CT rs11614913, and hsa-mir-499 TC rs3746444) in pre-miRNAs were investigated to evaluate their association with urinary bladder cancer risk. The hospital-based case-control study comprised of 212 histologically confirmed patients with urinary bladder cancer and 250 healthy controls who were unrelated, of similar ethnicity, and age and gender matched. Genotyping was done using polymerase chain reaction-restriction fragment length polymorphism methodology. Our results showed that the heterozygous genotype of rs11614913 was higher in cases than controls but the results were marginally significant (p = 0.055; odds ratio, 1.44). Smoking had no impact in modulating the effect of any of the three miRNA SNPs studied. No association was observed with either the tumor stage or grade in patients with bladder cancer. Even though there was no association between the individuals carrying the variant genotypes of the three miRNA studied and bladder cancer risk, marginal significance of heterozygousity in rs11614913 suggested further characterization of miRNA SNPs in a large cohort of varied ethnicity. This could further provide new prospects for understanding the underlying mechanisms between miRNAs and disease etiology.

The DNA double strand break repair gene XRCC4, an important caretaker of genome stability and XRCC3 are suggested to play an imperative role in the development of carcinogenesis. However, no evidence has been provided showing that these genes are associated with risk of urinary bladder cancer (UBC). The study was designed to examine the polymorphisms associated with two genes namely XRCC4 G1394T (rs6869366), intron 3 (rs28360317), intron 7 rs1805377 and rs2836007 and XRCC3 (rs861539 and rs1799796), respectively and inves- tigate their role as susceptible markers for UBC risk in North Indian cohort. In this hospital-based case-control study histologically confirmed 211 UBC patients and 244 age and gender matched controls of similar ethnicity were genotyped by means of PCR-RFLP. Significant different distributions in the frequency of the XRCC4 intron 3 genotype, but not the XRCC4 G1394T or intron 7 genotypes, between the UBC and control groups were observed. XRCC4 intron 7 Del/Del conferred enhanced risk (OR 1.94; P 0.017) in UBC. Inter- estingly, XRCC -1394 G(T variant genotype GG was associated with reduced risk (OR 0.27; P 0.020). However, none of the four polymorphisms in XRCC4 were associated with tobacco smoking and risk of recurrence in patients treated with BCG immunotherapy. Similarly, none of the XRCC3 polymorphisms were associated with UBC suscep- tibility. Our results suggested that the XRCC4 intron 3 rs6869366 genotype and intron 7 rs28360317 may be asso- ciated with UBC risk and may be a novel useful marker for primary prevention and anticancer intervention.

Matrix metalloproteinases (MMPs) contribute to tumor invasion and microenvironment, hence are associated with bladder cancer risk. We therefore, tested whether polymorphisms in MMP genes modify the risk of bladder cancer (BC) and whether smoke exposure modifies this risk.Genotyping was performed in 200 BC patients and 200 controls by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).MMP1-1607 2G/2G andMMP7-181 GG genotype were associated with increased risk of BC (pMMP1-519-1607 (p< 0.001; OR, 2.62; 95% CI1.68–4.09). The 2G allele carrier (1G/2G + 2G/2G) ofMMP1-1607 showed a protective effect and high recurrence free survival in Bacillus Calmette-Guerin (BCG) treated non muscle invasive BC (NMIBC) patients (log rank p, 0.030). Our datasuggested that MMP11607 2G and MMP7181 G allele were associated with high risk of BC, which was quite evident amongst smokers too. BCG treated NMIBC patients reflected protective effect for 2G allele carrier (1G/2G+2G/2G) ofMMP1-1607. This study provided new support for the association of MMP1-1607 and MMP7-181 in bladder cancer development, the tumorigenic effect of which was observed to be more enhanced in case of tobacco exposure.

Perturbations in the cell cycle and apoptotic genes have been implicated in human malignancies. Cell cycle (MDM2 and Cyclin D1) and apoptotic (Fas) genes that are differentially expressed in urinary bladder cancer (UBC) were investigated with the susceptibility to UBC in northern India. A total of 212 UBC patients and age- and sex-matched 250 controls were investigated for MDM2 G309T, Cyclin D1 G870A, and Fas A670G polymorphisms by polymerase chain reaction and restriction fragment length polymorphism. MDM2 309GG was at reduced risk for developing UBC when compared with TT genotype (p = 0.027; odds ratio, 0.55). GG genotype was also associated with reduced risk in nonmuscle invasive bladder cancer (NMIBC) and muscle invasive BC (p = 0.006 and p = 0.049). Cyclin D1 AA genotype was associated with high risk in NMIBC (intermediate risk) stage (p = 0.015; odds ratio, 4.55). MDM2-Cyclin D1 interaction revealed overall protective effect. The GG genotype of MDM2 also showed a protective effect and high recurrence-free survival in Bacillus Calmette-Guerin-treated NMIBC patients (log rank p = 0.008). MDM2 GG genotype had protective effect and MDM2T309G polymorphism had profound influence in Bacillus Calmette-Guerin-treated UBC patients in the North Indian population.

The objective of the study was to investigate the association of Xeroderma Pigmentoum group C (XPC) Lys939Gln (A > C) and poly (AT) insertion deletion (PAT -/+) gene polymorphism with the risk of cervical cancer. We enrolled 220 cervical cancer patients and 237 healthy individuals. Polymorphism for XPC gene (Lys939Gln, PAT -/+) was genotyped by polymerase chain reaction and restriction fragment length polymorphism method. XPC Lys939Gln CC genotype was associated with elevated risk of cervical cancer (OR = 2.15, p = 0.036). CC genotype of Lys939Gln (OR = 3.68; p = 0.02) and +/+ genotype of PAT (OR = 7.99; p = 0.01) were observed to have statistically significant elevated risk in stage IV of cervical cancer. Among tobacco users, carriers of the +/+ genotype of PAT showed a borderline significance (OR = 3.72, p = 0.032). Haplotypes A+ and C- (A > C of Lys939Gln, --> + of PAT) were significantly associated with higher susceptibility to cervical cancer (OR = 2.01, p = 0.005 and OR = 1.76, p = 0.002, respectively). Polymorphisms and haplotypes in XPC appear to influence cervical cancer risk and may modify risk attributable to tobacco usage.

Dysregulation of apoptosis plays a crucial role in carcinogenesis. Our aim was to investigate the association of Caspase 9 and Caspase 8 gene polymorphism with bladder cancer (BC) susceptibility. We undertook a case–control study of 212 (BC) cases and 250 controls to investigate the association between Caspase 9-1263A > G, Caspase 9-293del, and Caspase 8-6 N ins/del polymorphism and BC susceptibility by polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) method, and further to study the influence on recurrence in patients after Bacillus Calmette–Guerin (BCG) immunotherapy. Overall, no statistically significant association was observed in Caspase 9-293del and Caspase 8. Nevertheless, Caspase 9-1263GG genotype was at reduced risk of BC [p = 0.010; odds ratio (OR) = 0.487]. Caspase 9-1263AG genotype was also observed to be significantly associated with reduced risk with high-risk non-muscle-invasive bladder cancer (NMIBC) (TaG2-3, T1G1-3) and invasive tumors (T2 +) of BC (P = 0.042, OR = 0.39 and P = 0.013, OR = 0.028 respectively). Caspase 9-293del, heterozygous (–/+) genotype, too, demonstrated protective effect in high-risk NMIBC (P = 0.017; OR = 0.205). Haplotype analysis revealed variant genotypes Caspase 9AG + GG/Caspase 8 DI + II to be at reduced risk of BC (= 0P.014, OR = 0.47). The GG genotype of Caspase 9-1263 was associated with reduced risk for recurrence in BCG-treated patients [hazard ratio (HR) = 0.217, P = 0.005], thus showing increased recurrence-free survival (log-rank P = 0.024). Polymorphism in Caspase 9-1263 was observed to play a protective role in susceptibility to BC risk. Caspase 9 gene variants were also associated with reduced risk of NMBIC stages. The variant G allele at Caspase 9-1263 may be responsible for increased recurrence-free survival in BCG-treated patients.

Objectives To explore the association between xerodema pigmentosum group D ( XPD ) Asp 312 Asn and Lys 751 Gln and apurinic apyrimidic endonuclease 1 ( APE1) Asp 148 Glu gene polymorphism and risk of bladder cancer (BC) susceptibility. Methods This hospital-based case-control study included 206 patients with newly diagnosed bladder transitional cell carcinoma and 250 cancer-free controls who had been frequency matched by age, sex, and ethnicity. Polymorphisms in XPD Asp 312 Asn and Lys 751 Gln and APE1 Asp 148 Glu gene using polymerase chain reaction-restriction fragment length polymorphism and amplification refractory mutation system were genotyped. Results The XPD Asp 312 Asn AA genotype was associated with an elevated risk of BC (odds ratio [OR] 3.30, P = .001.) The AA genotype was significantly associated with nonmuscle-invasive BC (OR 4.62, corrected P = .003). Both the heterozygous GA and the homozygous AA was associated with a greater risk of low-grade (grade 1) BC (OR 2.51, corrected P = .006 and OR 5.21, corrected P = .003, respectively). The APE1 GG genotype showed a decreased risk of BC (OR 0.27, P = .027.) Haplotype AC (codon 312A-codon 751C) of XPD demonstrated an association with a greater susceptibility to BC (OR 2.16, correct P = .0008). Conclusions Reduced DNA repair capacity due to XPD Asp 312 Asn AA genotype might be a risk factor for BC. The AA genotype predisposed to a greater risk at the initial stage and grade of BC. The APE1 148GG genotype conferred a protective association with BC susceptibility.

In this study we characterized the effects of radiation injury on the expression and function of the autotaxin (ATX)–LPA2 GPCR axis. In IEC-6 crypt cells and jejunum enteroids quantitative RT-PCR showed a time- and dose-dependent upregulation of lpa2 in response to γ-irradiation that was abolished by mutation of the NF-κB site in the lpa2 promoter or by inhibition of ATM/ATR kinases with CGK-733, suggesting that lpa2 is a DNA damage response gene upregulated by ATM via NF-κB. The resolution kinetics of the DNA damage marker γ-H2AX in LPA-treated IEC-6 cells exposed to γ-irradiation was accelerated compared to vehicle, whereas pharmacological inhibition of LPA2 delayed the resolution of γ-H2AX. In LPA2-reconstituted MEF cells lacking LPA1&3 the levels of γ-H2AX decreased rapidly, whereas in Vector MEF were high and remained sustained. Inhibition of ERK1&2 or PI3K/AKT signaling axis by pertussis toxin or the C311A/C314A/L351A mutation in the C-terminus of LPA2 abrogated the effect of LPA on DNA repair. LPA2 transcripts in Lin−Sca-1+c-Kit+ enriched for bone marrow stem cells were 27- and 5-fold higher than in common myeloid or lymphoid progenitors, respectively. Furthermore, after irradiation higher residual γ-H2AX levels were detected in the bone marrow or jejunum of irradiated LPA2-KO mice compared to WT mice. We found that γ-irradiation increases plasma ATX activity and LPA level that is in part due to the previously established radiation-induced upregulation of TNFα. These findings identify ATX and LPA2 as radiation-regulated genes that appear to play a physiological role in DNA repair.

The objective of the study was to investigate the association of Xeroderma Pigmentoum group C (XPC) Lys939Gln (AC) and poly (AT) insertion deletion (PAT -/+) gene polymorphism with the risk of cervical cancer. We enrolled 220 cervical cancer patients and 237 healthy individuals. Polymorphism for XPC gene (Lys939Gln, PAT -/+) was genotyped by polymerase chain reaction and restriction fragment length polymorphism method. XPC Lys939Gln CC genotype was associated with elevated risk of cervical cancer (OR = 2.15, p = 0.036). CC genotype of Lys939Gln (OR = 3.68; p = 0.02) and +/+ genotype of PAT (OR = 7.99; p = 0.01) were observed to have statistically significant elevated risk in stage IV of cervical cancer. Among tobacco users, carriers of the +/+ genotype of PAT showed a borderline significance (OR = 3.72, p = 0.032). Haplotypes A+ and C- (AC of Lys939Gln, --+ of PAT) were significantly associated with higher susceptibility to cervical cancer (OR = 2.01, p = 0.005 and OR = 1.76, p = 0.002, respectively). Polymorphisms and haplotypes in XPC appear to influence cervical cancer risk and may modify risk attributable to tobacco usage.

Recent evidence indicates the involvement of microRNAs (miRNAs), in cell growth control, differentiation, and apoptosis, thus playing a role in tumorigenesis. Single-nucleotide polymorphisms (SNPs) located at miRNA-binding sites (miRNA-binding SNPs) are likely to affect the expression of the miRNA target and may contribute to the susceptibility of humans to common diseases. We genotyped SNPs hsa-mir196a2 (rs11614913), hsa-mir146a (rs2910164), and hsa-mir499 (rs3746444) in a case–control study including 159 prostate cancer patients and 230 matched controls. Patients with heterozygous genotype in hsa-mir196a2 and hsa-mir499, showed significant risk for developing prostate cancer (P = 0.01; OR = 1.70 and P ≤ 0.001; OR = 2.27, respectively). Similarly, the variant allele carrier was also associated with prostate cancer, (P = 0.01; OR = 1.66 and P ≤ 0.001; OR = 1.97, respectively) whereas, hsa-mir146a revealed no association in prostate cancer. None of the miRNA polymorphisms were associated with Gleason grade and bone metastasis. This is the first study on Indian population substantially presenting that individual as well as combined genotypes of miRNA-related variants may be used to predict the risk of prostate cancer and may be useful for identifying patients at high risk.

Interindividual variation in prostate cancer (PCa) susceptibility may be modulated in part through genetic polymorphisms in the DNA repair genes, especially the genes involved in the base excision repair and nucleotide excision repair pathway. Two of the common single-nucleotide polymorphisms X-ray repair cross-complementing group 1 (XRCC1) and Xeroderma pigmentosum group D (XPD) genes in PCa, which is one of the most common neoplasias in men all over the world, have been studied. In a case-control study of 171 PCa patients and 200 age-matched healthy controls, of similar ethnicity, genotyping was done to determine XPD exon 10 (G23592A), exon 23 (A35931C), and XRCC1 exon 6 (C26304T), exon 9 (G27466A), exon 10 (G23591A) gene polymorphisms by amplification refractory mutation-specific and polymerase chain reaction-restriction fragment length polymorphism methods, respectively. We observed that XPD exon 10 variant AA genotype was associated with increased risk for PCa (adjusted odds ratio [OR] 2.63, 95% confidence interval [95% CI] 1.40-4.92, p = 0.003), and XRCC1 exon 9 GA genotype was also statistically associated with PCa (adjusted OR 2.61, 95% CI 1.53-4.43, p0.001). However, XPD exon 23 (AC) and XRCC1 exon 6 (CT) and exon 10 (GA) did not have significantly increased risk for PCa. The haplotype analysis of XPD exon 10 and exon 23 G-C (OR 3.44, 95% CI 2.15-5.51, p0.0001) and A-A (OR 4.96, 95% CI 3.08-7.98, p0.0001) was associated with a significant increase in PCa risk. Similarly, the combined analysis of XRCC1 exon 6, exon 9, and exon 10 C-G-A (OR 2.93, 95% CI 1.91-4.50, p0.001) and C-A-G (OR 2.48, 95% CI 1.62-3.80, p0.001) demonstrated statistically significant risk in PCa. XRCC1 exon 9 GA genotype showed protective association with high grade of PCa. Our results suggest a positive association of XRCC1 exon 9 and XPD exon 10 genotypes that may play an important role in the pathophysiology and may modulate the risk of PCa.

Background Cyclooxygenase-2 (COX-2) is an enzyme involved in the synthesis of prostaglandins and thromboxanes, which are regulators of processes that are relevant to cancer development. It is involved in carcinogenesis, immune response suppression, apoptosis inhibition, angiogenesis, and tumor cell invasion and metastasis. The gene for COX-2, designated as prostaglandin-endoperoxide synthase 2 (PTGS-2), carries polymorphisms, such as -765G>C, 1195G>A in the promoter region, and 8473T>C in the 3'-untranslated region (UTR), which have been associated with susceptibility to malignant disease. Methods We undertook a case-control study of 212 urothelial bladder cancer (UBC) cases and 250 controls to investigate the association between COX-2 polymorphism and bladder cancer susceptibility, using the polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) method and also investigated gene–environment interactions. Results Cox-2 765G>C, a variant(C) allele carrier, was at an increased risk of UBC (odds ratio [OR] = 1.90; P = .004); however, –1195G>A; –1290A>G; and 3'UTR 8473T>C polymorphisms of COX-2 gene were not significantly associated with UBC. 765G>C also was associated with the invasive stage of a bladder tumor (OR = 2.73; P = .033). High risk for UBC also was observed with respect to COX-2 haplotypes C-765T8473A-1195A-1290 (OR = 3.47; P = .014). In case-only analysis, COX-2 765 variant allele carrier genotypes also showed an increased risk among former and current smokers (OR = 3.06; P = .041 and OR = 4.39; P = .032, respectively). Conclusion COX-2 -765G>C polymorphism confers UBC susceptibility particularly in smokers and in patients with invasive tumors. 765C allele carrier genotypes also are influenced with a high risk of recurrence in Bacillus Calmette-Guerin-treated patients. Collectively, these findings confirm that the COX-2 -765G>C polymorphism is a risk factor for the development of bladder cancer and can provide a plausible mechanistic explanation. Further validation in large population-based studies is needed.

Background The etiology of cervical cancer is associated with excessive–inflammation-mediated tumorigenesis. Interleukin-6 ( IL-6), a multifunctional cytokine, regulates inflammation and various physiological processes. We therefore aimed to evaluate the association of the IL-6 –174G>C polymorphism with predisposition to cervical cancer. Materials and methods The present case-control study comprised 160 histopathologically confirmed cases of cervical cancer and 200 healthy controls. Polymorphism for the IL-6 gene was genotyped by amplification refractory mutation system (ARMS) polymerase chain reaction (PCR). Results We observed a significant association of the IL-6 –174CC genotype with risk of cervical cancer (OR=3.16; p=0.014). An increased risk of developing stage I tumors was found in individuals with a heterozygous (GC) genotype (OR=3.63, p=0.003). In a case-only analysis, the risk was further increased in patients consuming tobacco products (OR=3.14; p=0.033). Conclusion The CC genotype in the IL-6 promoter region may confer a high risk of cervical cancer, which is further modulated in patients who are tobacco users.

Objective Polymorphisms in DNA repair genes may be associated with altered DNA repair capacity, thereby influencing an individual's susceptibility to smoking-related cancers such as bladder cancer. Therefore, we sought to examine the correlation between single nucleotide polymorphisms in DNA repair genes and bladder cancer. Methodology We undertook a case-control study of 212 urothelial bladder cancer (UBC) cases and 250 controls to investigate the association between OGG1 (C1245G rs1052133), XRCC3 (C18067T, rs861539) and XRCC7 (G6721T, rs7003908) polymorphisms and bladder cancer susceptibility by PCR–RFLP and the ARMS method. We also investigated gene-environment interactions. Results The OGG1 GG genotype was associated with an elevated risk of urothelial bladder cancer (UBC) (OR, 2.10; p, 0.028). XRCC7 + 6721 GG was also associated with increased susceptibility to UBC (OR, 4.45; p, 0.001). In a recessive model, the OGG1 GG genotype showed an increased risk of TaG2,3 + T1G1–3 tumors. Additionally, the OGG1 GG genotype in non-smokers represented a 2.46-fold greater risk (OR, 2.46; p, 0.035) in bladder cancer patients. Subsequent analysis demonstrated more pronounced association of XRCC7 with smokers (OR, 4.39; p, 0.001). XRCC7 also showed increased association with TaG2,3 + T1G1–3 tumors and muscle invasive tumors (OR, 3.16; p, 0.001 and OR, 4.24; p, 0.001, respectively). Multiple Cox regression analysis in non-muscle invasive bladder tumor (NMIBT) patients demonstrated an association of the OGG1 GG polymorphism with a high risk of recurrence in patients on cystoscopic surveillance (HR, 4.04; p, 0.013). Subsequently, shorter recurrence-free survival (log rank p, 0.024; CC/GG, 42/24) was observed. Conclusion Our data suggest association of a variant (GG) genotype of OGG1 with increased UBC susceptibility and a high risk of tumor recurrence in NMIBT patients on cystoscopic surveillance. XRCC7 G allele carriers (TG + GG) are also at an elevated risk for susceptibility to UBC as evidenced by a high odds ratio throughout the analysis.

Identifying risk factors for human cancers should consider combinations of genetic variations and environmental exposures. Several polymorphisms in DNA repair genes have impact on repair and cancer susceptibility. We focused on X-ray repair cross-complementing group 1 (XRCC1), Xeroderma pigmentosum D (XPD) and apurinic/apyrimidinic endonuclease (APE1) as these are most extensively studied in cancer. Present study was conducted to determine distribution of XRCC1 C26304T, G27466A, G23591A, APE1 T2197G and XPD A35931C gene polymorphisms in North Indian population and compare with different populations globally. PCR-based analysis was conducted in 209 normal healthy individuals of similar ethnicity. Allelic frequencies in wild type of XRCC1 C26304T were 91.1% C(Arg); G27466A 62.9% G(Arg); G23591A 60.3% G(Arg); APE1 T2197G 75.1% T(Asp) and XPD A35931C 71.8% A(Lys). The variant allele frequency were 8.9% T(Trp) in XRCC1 C26304T; 37.1% A(His) in G27466A; 39.7% A(Gln) in G23591A; 24.9% G(Glu) in APE1 and 28.2% C(Gln) in XPD respectively. We further compared frequency distribution for these genes with various published studies in different ethnicity. Our results suggest that frequency in these DNA repair genes exhibit distinctive pattern in India that could be attributed to ethnicity variation. This could assist in high-risk screening of humans exposed to environmental carcinogens and cancer predisposition in different ethnic groups.

Protective Effects of Partially Hydrolyzed Guar Gum Against Intestinal Epithelial Barrier Dysfunction Atsushi Majima, Yuji Naito, Osamu Handa, Yuriko Onozawa, Yukiko Uehara, Hideki Horie, Mayuko Morita, Yasuki Higashimura, Katsura Mizushima, Tetsuya Okayama, Kazuhiro Katada, Kazuhiro Kamada, Kazuhiko Uchiyama, Ishikawa Takeshi, Tomohisa Takagi, Yoshito Itoh, Zenta Yasukawa, Makoto Tokunaga, Tsutomu Okubo

Background & objectives: Genetic variation in the DNA repair genes might be associated with altered DNA repair capacities (DRC). Reduced DRC due to inherited polymorphisms may increase the susceptibility to cancers. Base excision and nucleotide excision are the two major repair pathways. We investigated the association between two base excision repair (BER) genes (APE1 exon 5, OGG1 exon 7) and two nucleotide excision repair (NER) genes (XPC PAT, XPC exon 15) with risk of prostate cancer (PCa). Methods: The study was designed with 192 histopathologically confirmed PCa patients and 224 age matched healthy controls of similar ethnicity. Genotypes were determined by amplification refractory mutation specific (ARMS) and PCR-restriction fragment length polymorphism (RFLP) methods. Results: Overall, a significant association in NER gene, XPC PAT Ins/Ins (I/I) genotype with PCa risk was observed (Adjusted OR- 2.55, 95%CI-1.22-5.33, P=0.012). XPC exon 15 variant CC genotypes presented statistically significant risk of PCa (Adjusted OR- 2.15, 95% CI-1.09-4.23, P=0.026). However, no association was observed for polymorphism with BER genes. Diplotype analysis of XPC PAT and exon 15 revealed that the frequency of the D-C and I-A diplotype was statistically significant in PCa. The variant genotypes of NER genes were also associated with high Gleason grade. Interpretation & conclusions: The results indicated that there was a significant modifying effect on the association between genotype XPC PAT and exon 15 polymorphism and PCa risk which was further confirmed by diplotype analysis of XPC PAT and exon 15 in north Indian population.

XRCC1 protein plays crucial role in base excision repair (BER)by acting as a scaffold for other BER enzymes. Variants in XRCC1 gene might alter protein structure/function or create alternatively spliced protein influencing BER efficiency and affect individual susceptibility/recurrence to urinary bladder cancer (BC). We tested whether polymorphisms in XRCC1 gene were associated with BC risk and further to substantiate risk of recurrence after Bacillus Calmette-Guerin (BCG) immunotherapy. Genotyping for three polymorphic sites of XRCC1 gene at codon Arg194Trp (PvuII), Arg280His (RsaI) and Arg399Gln (MspI) in 140 BC cases and 190 controls by PCR-RFLP method was done. We observed significant association in heterozygous genotype (GA) of codon 280 and 399 with BC risk (OR = 1.96, p = 0.021 and OR = 1.81, p = 0.021, respectively), however no association was seen for variant AA genotype. A trend of increased risk with high stage and grade in patients with codon 194 variant genotypes (CT + TT) was observed. Haplotype analysis showed that individuals with haplotype 194C-280G-399A were at3-fold higher risk for BC (OR = 3.48, p = 0.01). The A/A genotype of codon 399 was associated with high risk for recurrence in BCG treated patients (HR = 5.05, p = 0.01) thus, showing reduced recurrence free survival (AA/GG = 12/60 months; log rank p = 0.004). The study suggested no association of variant genotypes with the susceptibility to BC. Haplotype analysis however, revealed that XRCC1 399 A allele may have a major role as patients with haplotype 194C-280G-399A carrying variant allele of 399 were at higher risk.

Background Alcohol consumption is one of the major risk factors for colorectal cancer. However, the mechanism involved in this effect of alcohol is unknown. Methods We evaluated the effect of chronic ethanol feeding on azoxymethane and dextran sulfate sodium (AOM/DSS)-induced carcinogenesis in mouse colon. Inflammation in colonic mucosa was assessed at a precancerous stage by evaluating mucosal infiltration of neutrophils and macrophages, and analysis of cytokine and chemokine gene expression. Results Chronic ethanol feeding significantly increased the number and size of polyps in colon of AOM/DSS treated mice. Confocal microscopic and immunoblot analyses showed a significant elevation of phospho-Smad, VEGF and HIF1α in the colonic mucosa. RT-PCR analysis at a precancerous stage indicated that ethanol significantly increases the expression of cytokines IL-1α, IL-6 and TNFα, and the chemokines CCL5/RANTES, CXCL9/MIG and CXCL10/IP-10 in the colonic mucosa of AOM/DSS treated mice. Confocal microscopy showed that ethanol feeding induces a dramatic elevation of myeloperoxidase, Gr1 and CD68-positive cells in the colonic mucosa of AOM/DSS-treated mice. Ethanol feeding enhanced AOM/DSS-induced suppression of tight junction protein expression and elevated cell proliferation marker, Ki-67 in the colonic epithelium. Conclusion This study demonstrates that chronic ethanol feeding promotes colonic tumorigenesis potentially by enhancing inflammation and elevation of proinflammatory cytokines and chemokines.