TCF 4 tumor suppressor: a molecular target in the prognosis of sporadic colorectal cancer in humans.

BACKGROUND: A huge array of function is played by the Wnt/ß-catenin signaling pathway in development by balancing gene expression through the modulation of cell-specific DNA binding downstream effectors such as T-cell factor/lymphoid enhancer factor (TCF/LEF). The ß-catenin/TCF-4 complex is a central regulatory switch for differentiation and proliferation of intestinal cells (both normal and malignant). Thus, in the present study we evaluated each of 60 cases of sporadic adenocarcinoma, alongside adjoining and normal mucosa specimens of colorectum in humans, for mutation and expression analysis of the gene coding for TCF-4 protein. METHODS: DNA sequencing following PCR amplification and SSCP analysis (single strand conformation polymorphism) was employed to detect TCF-4 gene mutations in the case of exon 1. Quantitative real-time (qRT) PCR, immunohistochemistry (IHC), confocal microscopy and western blot analysis were used to detect TCF-4 gene/protein expression. RESULTS: Sequencing analysis confirmed 5/60 patients with a point mutation in exon 1 of the TCF-4 gene in tumor samples. mRNA expression using qRT-PCR showed approximately 83% decreased TCF-4 mRNA expression in tumor tissue and adjoining mucosa compared to normal mucosa. Similarly, a significant decrease in protein expression using IHC showed decreased TCF-4 protein expression in tumor tissue and adjoining mucosa compared to normal mucosa, which also corresponds to some important clinicopathological factors, including disease metastasis and tumor grade. Mutational alterations and downregulation of TCF-4 mRNA and hence decreased expression of TCF-4 protein in tumors suggest its involvement in the pathogenesis of CRC. CONCLUSIONS: A remarkable decrease in TCF-4 mRNA and protein expression was detected in tumorous and adjoining tissues compared to normal mucosa. Hence the alterations in genomic architecture along with downregulation of TCF-4 mRNA and decreased expression of TCF-4 protein in tumors, which is in accordance with clinical features, suggest its involvement in the pathogenesis of CRC. Thus, deregulation and collaboration of TCF-4 with CRC could be a concrete and distinctive feature in the prognosis of the disease at an early stage of development.

Chemopreventive role of olive oil in colon carcinogenesis by targeting noncoding RNAs and methylation machinery.

Epigenetic therapy induced by dietary components has become a strong interest in the field of cancer prevention. Olive oil, a potent dietary chemopreventive agent, control colon cancer, however, its role in epigenetic therapy remains unclear. Thus, we aimed to investigate the effect of olive oil in a preclinical model of colon cancer by targeting genetic and epigenetic mechanisms. DMH was used to induce colon cancer in rats; while olive oil was given to separate group of rats along with DMH treatment. Tumor burden and incidence in DMH and DMH + olive oil-treated rats was observed by macroscopic examination and histoarchitectural studies. Potent anti-inflammatory, anti-angiogenic and pro-apoptotic activity of olive oil was explored by gene expression and immunohistochemical studies. The effect of olive oil on epigenetic alterations was examined by detecting promoter methylation with MS-HRM and dysregulation of miRNA by TaqMan MicroRNA Assay. We observed that olive oil administration lowered tumor incidence and inhibited the development of tumors in DMH-treated rats. Olive oil markedly decreased the expression of inflammatory and angiogenic markers and restored the expression of pro-apoptotic markers in DMH-treated rats. Furthermore, the inverse relationship between gene expression and DNA methylation, deviant miRNA pattern and miRNA silencing mediated by aberrant DNA methylation was also seen in DMH-treated rats, which was potentially reversible upon olive oil treatment. Our study concludes that olive oil may play a role in the epigenetic therapy by altering NF-κB and apoptotic pathways via targeting noncoding RNAs and methylation machinery that affecting epigenome to prevent colon carcinogenesis.

Toxigenic Clostridium difficile isolates from clinically significant diarrhoea in patients from a tertiary care centre.

BACKGROUND & OBJECTIVES: Clostridium difficile is the primary cause of hospital-acquired colitis in patients receiving antibiotics. The pathogenicity of the organism is mainly due to the production of toxins. This study was conducted to investigate the presence of toxigenic C. difficile in the faecal samples of hospitalized patients suspected to have C. difficile infection (CDI) and corroborating the findings with their clinical and demographic data. METHODS: Diarrhoeic samples obtained from 1110 hospitalized patients were cultured for C. difficile and the isolates confirmed by phenotypic and molecular methods. Toxigenicity of the isolates was determined using enzyme-linked immunosorbent assay for toxins A and B. Details of patients included in the study were noted and analyzed. RESULTS: Of the 1110 patients (mean age 39±19.6 yr), 63.9 per cent were males and 36.1 per cent were females. The major antibiotics received by the patients were nitazoxanide (23.9%), penicillins/penicillin combinations (19.0%), quinolones including fluoroquinolones (13.1%), carbapenems (11.5%), glycopeptides (11.0%) and cephalosporins (8.4%). The clinical symptoms predominantly present were watery diarrhoea (56.4%), fever (40.0%) and abdominal pain (35.3%). The underlying diseases were gastrointestinal disorders (52.6%), followed by cancers (13.2%), surgical conditions (8.3%), and hepatic disorders (8.0%). Of the 174 C. difficile isolates, 54.6 per cent were toxigenic. Toxigenic C. difficile was present in all patients with surgical conditions, 65.2 per cent with cancers and 57.1 per cent with gastrointestinal disorders. INTERPRETATION & CONCLUSIONS: C. difficile was found to be an important cause of gastrointestinal infections in hospitalized patients with underlying diseases and on antibiotics. Clinical conditions of the patients correlating with toxigenic culture can be an important tool for establishing CDI diagnosis.

Antioxidant Supplementation: A Linchpin in Radiation-Induced Enteritis.

Radiation enteritis is one of the most feared complications of abdominal and pelvic regions. Thus, radiation to abdominal or pelvic malignancies unavoidably injures the intestine. Because of rapid cell turnover, the intestine is highly sensitive to radiation injury, which is the limiting factor in the permissible dosage of irradiation. Bowel injuries such as fistulas, strictures, and chronic malabsorption are potentially life-threatening complications and have an impact on patient quality of life. The incidence of radiation enteritis is increasing because of the current trend of combined chemotherapy and radiation. The consequences of radiation damage to the intestine may result in considerable morbidity and even mortality. The observed effects of ionizing radiation are mediated mainly by oxygen-free radicals that are generated by its action on water and are involved in several steps of signal transduction cascade, leading to apoptosis. The oxyradicals also induce DNA strand breaks and protein oxidation. An important line of defense against free radical damage is the presence of antioxidants. Therefore, administration of antioxidants may ameliorate the radiation-induced damage to the intestine.

Toxinotyping and Sequencing of Clostridium difficile Isolates from Patients in a Tertiary Care Hospital of Northern India.

BACKGROUND: Clostridium difficile is an important cause of infectious colitis among hospitalized patients across the globe. The pathogenic potential of C. difficile in producing significant morbidity and mortality is mainly due to production of toxins A and B. The outbreaks of C. difficile infection (CDI) are due to changes in the genetic sequences of the organism. There is hardly any molecular study reported on the prevalent types of C. difficile strains in India. Toxinotyping and sequencing of locally circulating C. difficile isolates from patients presenting to our tertiary care center of North India were done. MATERIALS AND METHODS: C. difficile strains (n = 174) isolated from 1,110 fecal samples from patients with suspected CDI were subjected to toxinotyping and partial sequencing of tcdA and tcdB genes. Comparison of nucleotide sequences with reference C. difficile 630 strain using BLAST was made and translated into corresponding amino acid sequences by ExPASy. RESULTS AND DISCUSSION: Of 174 C. difficile isolates, 121 were toxigenic, belonging to toxinotype 0 (n = 76) and VIII (n = 45). Partial sequencing of toxin genes using bioinformatics approaches revealed changes in toxin A sequences of five (50%) C. difficile isolates, but the translated nucleotide sequences showed substitution in only three of them. No variation was seen in the toxin B nucleotide sequences. Interstrain variations were found in the clinical C. difficile isolates in our region. CONCLUSION: PCR amplified toxigenic genes followed by sequencing can help to identify genetic changes and pathogenicity of varied collection of C. difficile isolates.

Doxycycline Promotes Carcinogenesis & Metastasis via Chronic Inflammatory Pathway: An In Vivo Approach.

BACKGROUND: Doxycycline (DOX) exhibits anti-inflammatory, anti-tumor, and pro-apoptotic activity and is being tested in clinical trials as a chemotherapeutic agent for several cancers, including colon cancer. MATERIALS & METHODS: In the current study, the chemotherapeutic activity of doxycycline was tested in a rat model of colon carcinogenesis, induced by colon specific cancer promoter, 1,2, dimethylhydrazine (DMH) as well as study the effect of DOX-alone on a separate group of rats. RESULTS: Doxycycline administration in DMH-treated rats (DMH-DOX) unexpectedly increased tumor multiplicity, stimulated progression of colonic tumor growth from adenomas to carcinomas and revealed metastasis in small intestine as determined by macroscopic and histopathological analysis. DOX-alone treatment showed markedly enhanced chronic inflammation and reactive hyperplasia, which was dependent upon the dose of doxycycline administered. Moreover, immunohistochemical analysis revealed evidence of inflammation and anti-apoptotic action of DOX by deregulation of various biomarkers. CONCLUSION: These results suggest that doxycycline caused chronic inflammation in colon, small intestine injury, enhanced the efficacy of DMH in tumor progression and provided a mechanistic link between doxycycline-induced chronic inflammation and tumorigenesis. Ongoing studies thus may need to focus on the molecular mechanisms of doxycycline action, which lead to its inflammatory and tumorigenic effects.

Frequent activation of the ß-catenin gene in sporadic colorectal carcinomas: A mutational & expression analysis.

ß-catenin (CTNNB1), an oncogene/onco-protein and an adhesion molecule is a key effector in colorectal cancer (CRC). Its activation, and subsequent up-regulation of Wnt-signaling, is an important event in the development of certain human cancers including CRC. Mutations in the ß-catenin gene in the region of serine-threonine glycogen kinase (GSK)-3ß phosphorylation target sites have been identified in colorectal cancer in humans. In the current study, we investigated 60 sporadic colorectal adenocarcinomas along with adjoining and normal mucosa cases in humans for ß-catenin mutations. Thirteen of sixty colorectal tumors from humans had point mutations with a frequency of 21.66% at codons 24, 26, 27, 32, 34, 35, 41, 42,43, 46, 49, 54, 55, or 67 sites which are mutated in colorectal cancer and some of these sites in other cancers. Thus, there appears to be a key involvement of ß-catenin activation in human colorectal carcinogenesis. mRNA expression analysis using q-Real Time PCR showed 21.5-fold up-regulation of ß-catenin mRNA in tumor tissue compared to normal and adjoining mucosa. Protein expression analysis using immunohistochemistry, confocal microscopy, and Western blot confirmed aberrant accumulation of ß-catenin protein along the nucleus and cytoplasm following mutation. The observed mutations and up-regulation of mRNA in tumors, and the increased expression of ß-catenin protein in CRC suggest that these alterations are early and prognostic events in sporadic colorectal carcinogenesis in humans. © 2015 Wiley Periodicals, Inc.

Prevalence and molecular types of Clostridium difficile isolates from faecal specimens of patients in a tertiary care centre.

Clostridium difficile infection (CDI) leads to considerable morbidity and mortality among hospitalized patients. Faecal specimens from 1110 hospitalized patients suspected for CDI were cultured for isolation of C. difficile and characterization of virulence genes. PCR was carried out for toxigenic genes tcdA, tcdB, cdtA and cdtB and PCR-RFLP for fliC and slpA genes. Of 174 (15.7%) C. difficile isolates, 121 (69.5%) were toxigenic, amongst which 68 (56.2%) also had both tcdA and tcdB genes. The remaining 53 (43.8%) of the isolates also had at least one of the toxin genes. Binary toxin genes (cdtA and cdtB) with only one of the two components were present in 16 (9.2%) of the 174 isolates. The other virulence genes - fliC and slpA - were present in 100% of the isolates. The most frequent PCR-RFLP type of fliC gene was type I (n = 101), followed by type VII (n = 49) and type III (n = 24). The slpA gene presented with three combinations of patterns. Characterization of virulence genes in C. difficile isolates is of extreme importance for epidemiological surveillance and control of outbreaks owing to the capacity of this bacterium to adapt to new environmental circumstances, leading to the emergence of new epidemic strains.

Lactase genetic polymorphisms and coeliac disease in children: a cohort study.

BACKGROUND: Lactase activity declines during childhood in the majority of human populations leading to adult-type hypolactasia (AtH). C/T-13910 and G/A-22018 single nucleotide polymorphisms (SNPs) have been suggested to be associated with AtH in different human populations. Coeliac disease (CD) is an autoimmune condition characterized by damage to intestinal cells leading to ultimate deterioration. AIM: This study investigated the association between coeliac disease (CD) and SNPs leading to AtH in children from North India. SUBJECTS AND METHODS: Intestinal biopsies and saliva samples were obtained from 52 children with CD diagnosis and 102 control subjects. Biopsies were assayed for disaccharidase activities and samples were genotyped for given SNPs. RESULTS: Prevalence of C/C and G/G genotypes of AtH was almost equal in the CD and control group. The CD group had low lactase activity compared to the control group, irrespective of genotype at C/T -13910 and G/A -22018 SNPs (p < 0.05). For the control group, lactase activity was high in children with C/T + G/A genotypes compared to C/C + G/G (p < 0.05). CONCLUSION: There appears to be no significant correlation between C/T -13910 or G/A -22018 SNPs of AtH and CD. Children with C/C or G/G genotype of AtH may not be at greater risk of CD.

Promoter methylation and immunohistochemical expression of hMLH1 and hMSH2 in sporadic colorectal cancer: a study from India.

To determine the etiological factors of human colorectal cancer (CRC) we assessed the frequency and prognostic significance of hMLH1 and hMSH2 genes in conjunction with hMLH1 and hMSH2 protein expression in 30 Indian CRC patients. The protein expression and promoter methylation of hMLH1 and hMSH2; Mismatch Repair genes (MMR) were analyzed by immunohistochemistry and methylation-specific PCR (MSP), respectively. A loss of hMLH1 expression was recognized in 4(13.3%) and loss of hMSH2 expression was recognized in 2(6.6%) of 30 CRC cases whereas 50% tumors showed reduced expression of hMLH1 and 33.3% showed reduced expression of hMSH2 protein. One tumor showed a loss of both hMLH1 and hMSH2 expression. Normal nuclear staining pattern of hMLH1 and hMSH2 was observed in almost all the adjoining and normal mucosa. Promoter hypermethylation of the hMLH1 gene was detected in 15 of 30 CRC cases (50%) and of hMSH2 gene was only in 3 of 30 CRC cases (10%). No promoter methylation of hMLH1 and hMSH2 genes was observed in adjoining and normal mucosa. Combination of methylation of hMLH1 and hMSH2 gene was observed in two tumors (6.6%). A significant correlation between histological grade of the tumor, methylation and expression of hMLH1 gene (p < 0.05) was observed. Normal expression of hMLH1 and hMSH2 was seen in all of the unmethylated tumors (100%). Nuclear staining and promoter methylation of hMLH1 and hMSH2 did not significantly influence survival. hMLH1 methylation was common and was significantly correlated with loss of hMLH1 protein expression. In contrast, hMSH2 methylation was infrequent. These findings suggest that the inactivation of MMR gene expression probably via hypermethylation may lead to inactivation of their functions which finally leads to tumor aggressiveness and the immunostaining of hMLH1 protein can be used as a prognostic factor for determining the grade of the tumor.

Effect of antioxidant supplementation on digestive enzymes in radiation induced intestinal damage in rats.

PURPOSE: Intestinal mucosa, a rapidly proliferating tissue, is highly sensitive to radiation and undergoes apoptosis as a consequence of over generation of oxidative free radicals and the lack of the antioxidants. Thus the present study was designed to investigate the intestinal damage induced by radiation and to study if supplementation of the diet with antioxidant vitamins could ameliorate the intestinal damage and its digestive activity, as determined by the expression of various border enzymes. MATERIALS AND METHODS: Swiss Albino rats (150-200 g body weight) were divided into six groups. Group I: Control untreated; Group II: Irradiated; Group III: Irradiated + vitamin A; Group IV: Irradiated + vitamin C; Group V: Irradiated + vitamin E; and Group VI: Irradiated + lycopene. Animals were exposed to whole body γ-radiation from (60)Co at the rate of 8 Gy for 15 min/rat. Intestinal morphology and changes in various digestive enzymes together with, DNA damage was studied in six groups and each group consisted of 18 animals. RESULTS: The gastrointestinal toxicity resulted in malabsorption, diarrhoea, weight loss, loss of appetite, abdominal haemorrhage and hair loss. The activities of sucrase and alkaline phosphatase were elevated and those of lactase, leucine aminopeptidase (LAP) and gamma-glutamyl transpeptidase or tranferase (γ-GTP) were markedly reduced. Antioxidant vitamin A, C or E supplementations prevented changes in brush border enzyme activities as compared to lycopene administration in rat intestine by radiation exposure. Intestinal histology showed that the vitamin supplementation to irradiated rats minimized the intestinal damage in rats. CONCLUSION: These findings suggest that the epithelial lining of the intestine is highly sensitive to radiation exposure and supplementation of antioxidant vitamins is helpful in minimizing the intestinal damage and supplementation by vitamin E was most potent in ameliorating the intestinal aberrations.

Alterations in K-ras, APC and p53-multiple genetic pathway in colorectal cancer among Indians.

The incidence of colorectal cancer (CRC) is increasing rapidly in Asian countries during the past few decades, but no comprehensive analysis has been done to find out the exact cause of this disease. In this study, we investigated the frequencies of mutations and expression pattern of K-ras, APC (adenomatosis polyposis coli) and p53 in tumor, adjoining and distant normal mucosa and to correlate these alterations with patients clinicopathological parameters as well as with the survival. Polymerase chain reaction (PCR)-restriction digestion was used to detect mutations in K-ras and PCR-SSCP (Single Strand Conformation Polymorphism) followed by DNA sequencing was used to detect mutations in APC and p53 genes. Immunohistochemistry was used to detect the expression pattern of K-ras, APC and p53 proteins. The frequencies of mutations of K-ras, APC and p53 in 30 tumor tissues samples were 26.7 %, 46.7 % and 20 %, respectively. Only 3.3 % of tumors contained mutations in all the three genes. The most common combination of mutation was APC and p53 whereas mutation in both p53 and K-ras were extremely rare. There was no association between the mutations and expression pattern of K-ras, APC and p53 (p>0.05). In Indians, the frequency of alterations of K-ras and APC is similar as in Westerns, whereas the frequency of p53 mutation is slightly lower. The lack of multiple mutations in tumor specimens suggests that these genetic alterations might have independent influences on CRC development and there could be multiple alternative genetic pathways to CRC in our present study cohort.

Correlation of G/A -22018 single-nucleotide polymorphism with lactase activity and its usefulness in improving the diagnosis of adult-type hypolactasia among North Indian children.

Adult-type hypolactasia (AtH or lactase non-persistence) is the physiological decline in lactase activity that manifests in majority of the world's population after weaning. Recently, various single-nucleotide polymorphisms (SNPs) upstream of lactase gene (LCT) have been suggested to be associated with AtH or the lactase persistent trait in different human populations. C/T -13910 SNP was found be completely associated with AtH in Finnish population, and G/A -22018 SNP was found to be strongly, but not completely, associated with AtH. The aim of this study was to correlate G/A -22018 SNP with intestinal lactase activity in North Indian children. These children were also genotyped for C/T -13910 SNP. We also examined the differences in milk consumption and milk-related clinical symptoms in children with different genotypes of G/A -22018 and C/T -13910 SNPs. Intestinal biopsies were obtained from 231 children aged 2-16 years undergoing routine endoscopy for various abdominal complaints. The biopsies were assayed for lactase, sucrase, and maltase activities and genotyped for G/A -22018 and C/T -13910 SNPs using restriction fragment length polymorphism and DNA sequencing analysis. There was a significant correlation between lactase activity and different genotypes of G/A -22018 SNP. Children with G/G -22018 genotype had low lactase activity. With a reference value of <10 U/g protein (lactase activity) to be indicative of AtH, the sensitivity and specificity of genetic test based on G/A -22018 SNP was 94.4 and 94.1 %, respectively. Furthermore, the consumption of milk was lower in children with G/G -22018 genotype. Flatulence was the only symptom significantly more frequent among the children with G/G -22018 genotype compared to those with G/A and A/A -22018 genotypes. However, most of the children with G/G -22018 genotype seem to tolerate small amounts of milk without any significant difference in gastrointestinal symptoms from those with G/A and A/A -22018 genotypes.

Extracellular matrix remodeling in Takayasu's arteritis: role of matrix metalloproteinases and adventitial inflammation.

Takayasu's arteritis (TA) is an inflammatory fibrosing arteritis affecting predominately the aorta and its main branches. Pathogenesis of this disease remains enigmatic. Despite the numerous studies, the role of adventitia in vascular lesion formation in the setting of TA has been ignored. Virtually nothing is known about the mechanism regulating inflammation in the adventitia in the setting of TA. The present study included subjects with Takayasu's arteritis and normal healthy control subjects. Isolated T cells from peripheral blood mononuclear cells (PBMCs) using nylon wool and HUT-78 (human cutaneous T lymphoma cell line) were stimulated with PHA for 24 h. Stimulated cell were fixed with paraformaldehyde and fractionated into membrane, cytosolic and nuclear fractions. These cellular fractions were co-cultured with human fibrosarcoma cell line (HT-1080) and transcriptional expression of matrix metalloproteinases (MMP-1, 3, 9 and TIMP-1) was determined using semiquantitative RT-PCR. Stimulation of MMPs-TIMP synthesis by HT-1080 cells was mimicked by a membranous fraction derived from activated T-cell isolated from TA subjects and activated HUT-78 cells, whereas cytosolic and nuclear fractions were ineffective. In conclusion, for the first time we provide evidence for the presence of a cell surface-specific antigenic moiety on T-cells of TA subjects, which is responsible for activation of fibroblasts (cells predominantly present in adventitia) to enhance MMP production and, therefore, may lead to extracellular matrix degradation.

Effect of C/T -13910 cis-acting regulatory variant on expression and activity of lactase in Indian children and its implication for early genetic screening of adult-type hypolactasia.

BACKGROUND: Absorption of milk sugar (lactose) is regulated by the activity of lactase enzyme in gut wall. Intestinal lactase activity declines during childhood in majority of human populations leading to adult-type hypolactasia (primary lactose malabsorption), limiting the use of fresh milk due to lactose intolerance. Aim of this study was to correlate lactase expression and activity with C/T -13910 variant in Indian children, determine the age of onset of down-regulation of lactase activity and assess the applicability of the C/T -13910 variant as a diagnostic marker for identifying children genetically inclined to develop adult-type hypolactasia. METHODS: Intestinal biopsies were obtained from 176 children aged 1-16 years undergoing routine endoscopy for various abdominal complaints. The biopsies were assayed for lactase, sucrase and maltase activities and genotyped for C/T 13910 variant using PCR-RFLP analysis. The functional effect of the C/T -13910 variant on expression of lactase mRNA and protein in these children was examined using reverse-transcription PCR and western blotting. RESULTS: Among the 176 children investigated in our study, 56.8% (100/176) carried the C/C -13910 genotype, which has been associated with the onset of adult-type hypolactasia, while 40.9% (72/176) carried the C/T -13910 genotype and 2.3% (4/176) the T/T -13910 genotype. There was a significant correlation between lactase activity and C/T -13910 variant (P<0.001). The mean level of lactase activity among children with C/C -13910 genotype was 15.9 U/g protein and with C/T and T/T -13910 genotypes was 30.9 U/g protein. The age of onset of down-regulation of lactase activity in children with C/C -13910 genotype was between 3 and 5 years and keeping 10 U/g protein lactase activity as cut off, adult-type hypolactasia was evident in all the individuals>8 years of age for this genotype. C/C -13910 genotype was associated with low expression of lactase mRNA and protein compared with C/T genotype. Considering lactase activity of 10 U/g protein as gold standard, predictive value of genetic test based on C/T -13910 variant for adult-type hypolactasia was 100% in children>8 years of age. CONCLUSION: C/T -13910 cis-acting regulatory variant located ≈14 kb upstream of lactase gene (LCT) completely correlates with lactase phenotype in Indian children. The genetic testing for the C/T -13910 variant may be helpful in the diagnosis of adult-type hypolactasia in Indian children.

Aberrant promoter methylation of p16 in colorectal adenocarcinoma in North Indian patients.

AIM: To investigate p16 gene methylation and its expression in 30 patients with sporadic colorectal adenocarcinoma in a North Indian population. METHODS: Methylation specific polymerase chain reaction was used to detect p16 gene methylation and immunohistochemistry was used to study the p16 expression in 30 sporadic colorectal tumors as well as adjoining and normal tissue specimens. RESULTS: Aberrant promoter methylation of p16 gene was detected in 12 (40%) tumor specimens, whereas no promoter methylation was observed in adjoining and normal tissue. Immunohistochemistry showed expression of p16 protein in 26 (86.6%) colorectal tumors whereas complete loss of expression was seen in 4 (13.3%) and reduced expression was observed in 12 (40%) tumors. In the adjoining mucosa, expression of p16 was in 11 (36.6%) whereas no clear positivity for p16 protein was seen in normal tissue. There was a significant difference in the expression of p16 protein in tumor tissue and adjoining mucosa (P < 0.001). The methylation of the p16 gene had a significant effect on the expression of p16 protein (P = 0.021). There was a significant association of methylation of p16 gene with the tumor size (P = 0.015) and of the loss/reduced expression of p16 protein with the proximal site of the tumor (P = 0.047). Promoter methylation and expression of p16 had no relation with the survival of the patients (P > 0.05). CONCLUSION: Our study demonstrated that promoter hypermethylation of the p16 gene results in loss/reduced expression of p16 protein and this loss/reduced expression may contribute to tumor enlargement.

Ethanol-induced changes in lipid peroxidation of enterocytes across the crypt-villus axis in rats.

BACKGROUND: Reactive oxygen species (ROS) have been implicated in the turnover of epithelial cells in the rat intestine. The metabolism of ethanol generates ROS, which are implicated in cellular injury, but the levels of lipid peroxidation in intestine in chronic alcoholism are unknown. AIM: To investigate the effects of ethanol ingestion on lipid peroxidation, and anti- and pro-oxidant enzyme systems in enterocytes across the crypt-villus axis in intestine. METHODS: Wistar rats (90-100 g) were administered 1 mL of 30% ethanol daily for 39 days. Intestinal epithelial cells were isolated in fractions. Malondialdehyde levels, and activities of glutathione-S-transferase (GST), glutathione reductase (GR), superoxide dismutase (SOD) and catalase were determined in various cell fractions. Incorporation of H3-thymidine into DNA of enterocytes was also determined. RESULTS: Lipid peroxidation was elevated by two- to three-folds in both villus and crypt cells in ethanol-fed animals compared to controls. The activities of GST and GR were four- to six-folds higher in villus tip cells compared to crypt base cells. The activities of SOD and catalase were five- to seven-fold higher in crypt base cells compared to villus tip cells. Ethanol feeding elevated the activities of SOD (76-190%) and catalase (20-150%) in enterocytes all along the crypt-villus axis compared to the controls. H3 thymidine incorporation into DNA of enterocytes was reduced by half in ethanol-fed rats compared to controls. CONCLUSIONS: There is a gradient in the concentration of lipid peroxides in enterocytes across the crypt-villus axis, being high at the villus tip and low at the crypt base in the rat intestine. Ethanol feeding enhanced lipid peroxidation in both villus and crypt cells.

Inhibition of brush border sucrase by polyphenols in mouse intestine.

The interactions of gallic acid and tannic acid with purified brush border sucrase (EC 3.2.1.48) from mouse intestine have been studied. These findings indicate that both gallic acid and tannic acid inhibit sucrase activity, which is pH dependent. Kinetic analysis revealed that enzyme inhibition by gallic acid is a pure V effect at pH 5.0, which changes to mixed type at pH 7.2, and pure K effect at pH 8.5. In contrast, sucrase inhibition by tannic acid was a pure K effect at acidic pH and uncompetitive type in the alkaline pH range. Far-CD spectroscopic analysis revealed an increase in the helicity of the enzyme at acidic pH in the presence of tannic acid but no change at alkaline pH. Fluorescence spectra revealed a red shift in lambdamax of the enzyme, suggesting that tryptophan residues come to a more hydrophilic environment in the presence of polyphenols. These findings suggest that inhibition of mice sucrase by polyphenols is pH dependent, and is associated with conformational modifications of the enzyme.

Kinetic characteristics of brush border sucrase activation by Na+ ions in mice intestine.

The kinetics of Na+ activation of brush border sucrase (sucrose D-glucosidase E.C. 3.2.1.48) has been studied in mice intestine. At pH 5.0, 50 mM Na+ ions stimulated sucrase activity by 84%. At pH 7.2, enzyme stimulation was reduced to 16%, whereas, atpH 8.5, 10-100 mMNa+ ions produced 18-45% inhibition of enzyme activity. Kinetic studies revealed that at pH 5.0, the enzyme activation by Na+ ions was V-type, which changed to K-type atpH 7.2, whereas at alkaline pH (8.5), Na+ ions inhibited the enzyme activity non-competitively. Using the non-compulsory model of Na+ ion stimulation of brush border sucrase [Mahmood & Alvarado, Arch Bioch Biophys, 168 (1975) 585] various kinetic constants involved in activation of sucrase by Na ions were determined. It is apparent that Na+ stimulation of brush border sucrase is pH dependent, which is similar to that described for rat, rabbit and other mammalian species and conform to identical mechanisms, at least with reference to the affinity type effects, as observed in mice intestine.