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Esophageal adenocarcinoma (EAC) is strongly associated with Barrett's esophagus (BE), a pre-malignant condition resulting from gastric reflux. Esophageal squamous cell carcinoma (ESCC), the other major subtype of esophageal cancer, shows strong association with smoking and alcohol intake and no association with gastric reflux. In this study, we constructed and validated gene expression signatures of EAC vs. ESCC tumors using publicly available datasets, and subsequently assessed the enrichment levels of these signatures in commonly used EAC and ESCC cell lines, normal esophageal tissues and normal esophageal cell lines, and primary BE tissues and BE cell lines. We found that unlike ESCC cell lines which were quite similar to primary ESCC tumors, EAC cell lines were considerably different from primary EAC tumors but still more similar to EAC tumors than ESCC tumors, as the genes up in EAC vs. ESCC (EAC hi ) had considerably lower expression in EAC cell lines than EAC tumors. However, more surprisingly, unlike various normal cell lines (EPC2, Het-1A) which were very similar to various tissues from normal esophagus, BE cell lines (BAR-T, CP-A) were extremely different from primary BE tissues, as BE cell lines had substantially lower levels of EAC hi and substantially higher levels of ESCC hi gene expression. This ESCC-like profile of the BAR-T remained unaltered even after prolonged exposure to an acidic bile mixture in vitro resulting in malignant transformation of this cell line. However, primary BE tissues had EAC-like gene expression profiles as expected. Only one EAC case from the Cancer Genome Atlas resembled BE cell lines, and while it had the clinical profile and some mutational features of EAC, it had some mutational features, the copy number alteration profile, and the gene expression profile of ESCC instead. These incomprehensible changes in gene expression patterns may result in ambiguous changes in the phenotype and warrants careful evaluation to inform selection of appropriate in vitro tools for future studies on esophageal adenocarcinoma.

Esophageal carcinoma (EC) is one of the most pervasive cancers in the world, with upwards of 500,000 new diagnoses, annually. Despite its prominence, advancements in the detection and treatment of EC have been marginal over the past 30 years and the survival rate continues to stay below 20%. This is due to the uncommonly heterogeneous presentation of EC which presents unprecedented challenges in improving patient survival and quality of care. However, distinct epigenetic alterations to the DNA methylome may provide an avenue to drastically improve the detection and treatment of EC. Specifically, the creation of novel biomarker panels that consist of EC-specific methylation markers have shown promise as a potential alternative to the more invasive, contemporary diagnostic methods. Additionally, growing insight into the biological and clinical properties of EC-specific methylation patterns have opened a window of opportunity for enhanced treatment; of growing interest is the application of "DNMT inhibitors" - a class of drugs which inhibit excessive methylation and have been shown to re-sensitize chemoresistant tumors. Here we provide a comprehensive review of the current advancements in EC DNA methylation to underscore a potential approach to its detection and treatment., Competing Interests: None., (AJCR Copyright © 2021.)

Background: Esophageal adenocarcinoma (EA) arises from Barrett's epithelium (BE), and chronic gastroesophageal reflux disease is considered the strongest risk factor for disease progression. All BE patients undergo acid suppressive therapy, surveillance, and BE removal by surgery or endoscopic ablation, yet the incidence of EAC continues to increase. Despite the known side effects and mortality, the one-size-fits-all approach is the standard clinical management as there are no reliable methods for risk stratification. Methods: Paired-end Illumina NextSeq500 RNA sequencing was performed on total RNA extracted from 20-week intervals (0, 20, 40, and 60 W) of an in vitro BE carcinogenesis (BEC) model to construct time series global gene expression patterns (GEPs). The cells from two strategic time points (20 and 40 W) based on the GEPs were grown for another 20 weeks, with and without further acid and bile salt (ABS) stimulation, and the recurrent neoplastic cell phenotypes were evaluated. Results: Hierarchical clustering of 866 genes with ≥ twofold change in transcript levels across the four time points revealed maximum variation between the BEC20W and BEC40W cells. Enrichment analysis confirmed that the genes altered ≥ twofold during this window period associated with carcinogenesis and malignancy. Intriguingly, the BEC20W cells required further ABS exposure to gain neoplastic changes, but the BEC40W cells progressed to malignant transformation after 20 weeks even in the absence of additional ABS. Discussion: The transcriptomic gene expression patterns in the BEC model demonstrate evidence of a clear threshold in the progression of BE to malignancy. Catastrophic transcriptomic changes during a window period culminate in the commitment of the BE cells to a "point of no return," and removal of ABS is not effective in preventing their malignant transformation. Discerning this "point of no return" during BE surveillance by tracking the GEPs has the potential to evaluate risk of BE progression and enable personalized clinical management., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor declared a past co-authorship with one of the authors AP., (Copyright © 2021 Panda, Shin, Cheng and Bajpai.)

Barrett's esophagus (BE) is a premalignant metaplasia in patients with chronic gastroesophageal reflux disease (GERD). BE can progress to esophageal adenocarcinoma (EA) with less than 15% 5-year survival. Chromosomal aneuploidy, deletions, and duplication are early events in BE progression to EA, but reliable diagnostic assays to detect chromosomal markers in premalignant stages of EA arising from BE are lacking. Previously, we investigated chromosomal changes in an in vitro model of acid and bile exposure-induced Barrett's epithelial carcinogenesis (BEC). In addition to detecting changes already known to occur in BE and EA, we also reported a novel recurring chromosomal translocation t(10:16) in the BE cells at an earlier time point before they undergo malignant transformation. In this study, we refine the chromosomal event with the help of fluorescence microscopy techniques as a three-way translocation between chromosomes 2, 10, and 16, t(2:10;16) (p22;q22;q22). We also designed an exclusive fluorescent in situ hybridization for esophageal adenocarcinoma (FISH-EA) assay that detects these chromosomal breakpoints and fusions. We validate the feasibility of the FISH-EA assay to objectively detect these chromosome events in primary tissues by confirming the presence of one of the fusions in paraffin-embedded formalin-fixed human EA tumors. Clinical validation in a larger cohort of BE progressors and non-progressors will confirm the specificity and sensitivity of the FISH-EA assay in identifying malignant potential in the early stages of EA., Competing Interests: MB, HA, and KD have a patent application U.S. Application No. 16/083,055 pending for the FISH-EA probes. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor declared a past co-authorship with one of the authors AP., (Copyright © 2021 Bajpai, Panda, Birudaraju, Van Gurp, Chak, Das, Javidian and Aviv.)

Background: The role of 5-aminosalicylic acid (5-ASA or mesalamine) in the prevention of colorectal cancer in ulcerative colitis (UC) patients was reported, but the effect on molecular targets in UC colon mucosa is unknown., Aim: This observational study evaluates gene expression levels of 5-ASA targets using serial colon biopsy specimens from UC patients undergoing long-term 5-ASA therapy., Methods: Transcript levels were compared between colonoscopic biopsy specimens collected from 62 patients at initial and final follow-up colonoscopy at 2-6 years. All patients had mild-to-moderate UC and were undergoing long-term 5-ASA maintenance. Stepwise multiple linear regression analyses were performed to correlate changes in transcript levels with therapeutic response (Mayo clinical score endoscopy and DAI and/or Nancy histopathology score) and nonclinical variables., Results: The transcript levels of colorectal carcinogenesis-associated known 5-ASA target genes were significantly reduced after prolonged 5-ASA therapy (P < 0.005-0.03). Multiple linear regression models predicted significant association between transcript levels of Ki-67, NF-kB (p65), PPARγ, COX-2 and IL-8, CDC25A, and CXCL10 with duration of drug (5-ASA) exposure (P ≤ 0.05). Ki-67, NF-kB (p65), and CXCL10 transcripts were also correlated with reduced endoscopy sub-score (P ≤ 0.05). COX-2, IL-8, CDC25A, and TNF transcripts strongly correlated with DAI sub-scores (P ≤ 0.05). Only COX-2 and IL-8 transcript levels correlated (P ≤ 0.05) with Nancy histological score., Conclusion: This study provides molecular evidence of changes in carcinogenesis-related targets/pathways in colon tissue during long-term 5-ASA maintenance therapy that may contribute to the observed chemopreventive effects of 5-ASA in UC patients.

Background: MicroRNAs (miRNAs) are important post-translational regulators. Elevated levels of miR-206 in ulcerative colitis (UC) were associated with suppression of anti-inflammatory A3 adenosine receptor (A3AR) expression. However, the relationship of miR-206 to histologic remission in UC patients remains unknown. This study correlates expression levels of miR-206 with histologic remission in patients treated via long-term mesalamine treatment to identify a possible mode of action for this mainstay drug for UC., Methods: Expression of miR-206 and its target A3AR were analyzed in HT29 cell line before and after mesalamine treatment (2 mM) at different time points (0, 4, 12, and 24 hours) by qRT-PCR and western blot analysis. Expression of miR-206 and pathological scores of colonoscopic biopsy specimens were studied in 10 UC patients treated with mesalamine treatment for 2 to 6 years., Results: miR-206 transcripts decreased 2.23-fold (P = 0.0001) 4 hours after 2 mM mesalamine treatment in HT29 colon cells compared with untreated controls. However, the mRNA/protein levels of A3AR increased by 4-fold (P = 0.04) and 2-fold, respectively, in same cells. miR-206 relative expression decreased significantly in patients treated with 4.8 g of mesalamine (P = 0.002) but not with 2.4 g (P = 0.35). Tissue assessment of sequential mesalamine-treated colonoscopic biopsies indicate a strong correlation between downregulation of miR-206 and histologic improvement (R = 0.9111)., Conclusion: Mesalamine treatment has an effect on epithelial miRNAs. Downregulation of miR-206 by long-term mesalamine treatment may confer a protective effect in inducing and maintaining histologic remission. Thus, miR-206 expression levels can be utilized as a possible biomarker for therapeutic response to mesalamine treatment.

Background & Aims: Metaplastic glands buried under squamous epithelium are frequently detected in patients with Barrett esophagus (BE). This subsquamous intestinal metaplasia might be responsible for cancers that develop despite endoscopic surveillance and for metaplasia recurrences after endoscopic ablation. To determine whether reflux induces BE cells to undergo an epithelial-to-mesenchymal transition (EMT) that produces subsquamous intestinal metaplasia, we assessed EMT in BE cells exposed to acidic bile salts and in rat and human esophageal tissues., Methods: We compared markers of EMT and cell motility in trans-well and 3-dimensional organotypic culture systems among dysplastic BE epithelial cell lines, nondysplastic telomerase-immortalized BE cell lines (BAR-T), and BAR-T cells exposed acutely or for 20 weeks to acidic bile salts. Vascular endothelial growth factor (VEGF) A was inhibited with a neutralizing antibody or CRISPR-Cas9n and VEGF receptor 2 was inhibited with SU1498 or shRNA, and cells were analyzed by immunohistochemistry, quantitative polymerase chain reaction, or immunoblotting for markers of VEGF signaling and EMT; cell motility was assessed by trans-well assay. We used immunohistochemistry and quantitative polymerase chain reaction to assess EMT markers in the columnar-lined esophagus of rats with surgically induced reflux esophagitis and in esophagectomy specimens from patients with BE., Results: We detected features of EMT (decreased cadherin 1 [CDH1]; increased fibronectin 1, vimentin, and matrix metalloproteinase 2; and increased motility) in dysplastic BE epithelial cell lines and in BAR-T cells exposed for 20 weeks, but not in unexposed BAR-T cells. Acute acidic bile salt exposure induced expression of zinc finger E-box binding homeobox 1 and 2 (ZEB1/2) in BAR-T cells, which decreased their expression of CDH1 and increased motility; inhibitors of VEGF signaling blocked these effects. Columnar-lined esophagus of rats with reflux esophagitis had increased expression of ZEB1/2 and decreased expression of CDH1 compared with controls. Dysplastic BE tissues also had significantly increased levels of ZEB1 and significantly decreased levels of CDH1 compared with nondysplastic BE tissues., Conclusions: In BE cell lines, acidic bile salts induce EMT by VEGF signaling, which increases expression of ZEB1/2, repressors of CDH1. These observations suggest that reflux induces EMT in metaplastic BE tissues, which promotes development of subsquamous intestinal metaplasia., (Copyright © 2019 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Gastroesophageal reflux disease (GERD) clinically predisposes to columnar Barrett's metaplasia (BM) in the distal esophagus. We demonstrate evidence supporting the cellular origin of BM from reprograming or transcommitment of resident normal esophageal squamous (NES) epithelial cells in response to acid and bile (A + B) exposure using an in vitro cell culture model. The hTERT-immortalized NES cell line NES-B10T was exposed 5 min/day to an A + B mixture for 30 wk. Morphological changes, mRNA, and protein expression levels for the inflammatory marker cyclooxygenase-2; the lineage-determining transcription factors TAp63 (squamous), CDX2, and SOX9 (both columnar); and the columnar lineage markers Villin, Muc-2, CK8, and mAb Das-1 (incomplete phenotype of intestinal metaplasia) were assessed every 10 wk. Markers of columnar lineage and inflammation increased progressively, while squamous lineage-determining transcriptional factors were significantly decreased both at the mRNA and/or protein level in the NES-B10T cells at/after A + B treatment for 30 wk. Distinct modifications in morphological features were only observed at/after 30 wk of A + B exposure. These changes acquired by the NES-B10T 30-wk cells were retained even after cessation of A + B exposure for at least 3 wk. This study provides evidence that chronic exposure to the physiological components of gastric refluxate leads to repression of the discernable squamous transcriptional factors and activation of latent columnar transcriptional factors. This reflects the alteration in lineage commitment of the precursor-like biphenotypic, NES-B10T cells in response to A + B exposure as the possible origin of BM from the resident NES cells. NEW & NOTEWORTHY This study provides evidence of the origins of Barrett's metaplasia from lineage transcommitment of resident esophageal cells after chronic exposure to gastroesophageal refluxate. The preterminal progenitor-like squamous cells alter their differentiation and develop biphenotypic characteristics, expressing markers of incomplete-type columnar metaplasia. Development of these biphenotypic precursors in vitro is a unique model to study pathogenesis of Barrett's metaplasia and esophageal adenocarcinoma.

The number of persons 60 years and older has increased 3-fold between 1950 and 2000. Aging alone does not greatly impact the gastrointestinal (GI) tract. Digestive dysfunction, including esophageal reflux, achalasia, dysphagia, dyspepsia, delayed gastric emptying, constipation, fecal incontinence, and fecal impaction, is a result of the highly prevalent comorbid conditions and the medications with which those conditions are treated. A multidisciplinary approach with the expertise of a geriatrician, gastroenterologist, neurologist, speech pathologist, and physical therapist ensures a comprehensive functional and neurological assessment of the older patient. Radiographic and endoscopic evaluation may be warranted in the evaluation of the symptomatic older patient with consideration given to the risks and benefits of the test being used. Treatment of the digestive dysfunction is aimed at improving health-related quality of life if cure cannot be achieved. Promotion of healthy aging, treatment of comorbid conditions, and avoidance of polypharmacy may prevent some of these digestive disorders. The age-related changes in GI motility, clinical presentation of GI dysmotility, and therapeutic principles in the symptomatic older patient are reviewed here.

Barrett's epithelium (BE) is a premalignant condition resulting from chronic gastroesophageal reflux that may progress to esophageal adenocarcinoma (EAC). Early intervention holds promise in preventing BE progression. However, identification of high-risk BE patients remains challenging due to inadequate biomarkers for early diagnosis. We investigated the effect of prolonged chronic acid and bile exposure on transcriptome, methylome, and mutatome of cells in an in-vitro BE carcinogenesis (BEC) model. Twenty weeks acid and bile exposed cells from the BEC model (BEC20w) were compared with their naïve predecessors HiSeq Illumina based RNA sequencing was performed on RNA from both the cells for gene expression and mutational analysis. HELP Tagging Assay was performed for DNA methylation analysis. Ingenuity pathway, Gene Ontology, and KEGG PATHWAY analyses were then performed on datasets. Widespread aberrant genetic and epigenetic changes were observed in the BEC20w cells. Combinatorial analyses revealed 433 from a total of 863 downregulated genes had accompanying hypermethylation of promoters. Simultaneously, 690 genes from a total of 1,492 were upregulated with accompanying promoter hypomethylation. In addition, 763 mutations were identified on 637 genes. Ingenuity pathway analysis, Gene Ontology, and KEGG PATHWAY analyses associated the genetic and epigenetic changes in BEC20w cells with cellular and biological functions. Integration of high resolution comparative analyses of naïve BAR-T and BEC20w cells revealed striking genetic and epigenetic changes induced by chronic acid and bile exposure that may disrupt normal cellular functions and promote carcinogenesis. This novel study reveals several potential targets for future biomarkers and therapeutic development., (Copyright © 2013 Wiley Periodicals, Inc.)

The following paper on the molecular biology of Barrett's esophagus (BE) includes commentaries on signaling pathways central to the development of BE including Hh, NF-κB, and IL-6/STAT3; surgical approaches for esophagectomy and classification of lesions by appropriate therapy; the debate over the merits of minimally invasive esophagectomy versus open surgery; outcomes for patients with pharyngolaryngoesophagectomy; the applications of neoadjuvant chemotherapy and chemoradiotherapy; animal models examining the surgical models of BE and esophageal adenocarcinoma; the roles of various morphogens and Cdx2 in BE; and the use of in vitro BE models for chemoprevention studies., (© 2013 New York Academy of Sciences.)

This paper presents commentaries on animal models used for Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC) research; acid- and bile-induced chromosomal instability and clonal selection during the progression of BE to EAC; how the components of gastric refluxate, especially acid and bile salts, promote carcinogenesis in metaplastic BE; genome-wide changes in DNA methylation and transcription involved in BE carcinogenesis; the potential role of miRNA in the development of BE and EAC; the effect of inflammatory cytokines linked to obesity on the activation of cell-death pathways and cell survival in BE and esophageal cancer; and the role of autophagy in esophageal cancer development., (© 2013 New York Academy of Sciences.)

Unlabelled: Barrett's esophagus (BE) is an asymptomatic, pre-malignant condition of the esophagus that can progress to esophageal adenocarcinoma (EAC). BE arises typically in individuals with long-standing gastroesophageal reflux disease (GERD). The neoplastic progression of BE has been extensively studied histologically and defined as a metaplasia- dyplasia- carcinoma sequence. However the genetic basis of this process is poorly understood. It is conceived that preclinical models of BE may facilitate discovery of molecular markers due to ease of longitudinal sampling. Clinical markers to stratify the patients at higher risk are vital to institute appropriate therapeutic intervention since EAC has very poor prognosis. We developed a dynamic in-vitro BE carcinogenesis (BEC) model by exposing naïve Barrett's epithelium cell line (BAR-T) to acid and bile at pH4 (B4), 5min/day for a year. The BEC model acquired malignant characteristics after chronic repeated exposure to B4 similar to the sequential progression of BE to EAC in vivo., Aim: To study cytogenetic changes during progressive transformation in the BEC model., Results: We observed that the BAR-T cells progressively acquired several chromosomal abnormalities in the BEC model. Evidence of chromosomal loss (-Y) rearrangements [t(10;16) and dup (11q)] and clonal selection appeared during the early stages of the BEC model. Clonal selection resulted in a stabilized monoclonal population of cells that had a changed morphology and formed colony in soft agar. BAR-T cells grown in parallel without any exposure did not show any of these abnormalities., Conclusions: Prolonged acid and bile exposure induced chromosomal aberrations and clonal selection in benign BAR-T cells. Since aneuploidy preceded morphological/dysplastic changes in the BEC model, chromosomal aberrations may be an early predictor of BE progression. The [t(10;16) and dup(11q)] aberrations identified in this study harbor several genes associated with cancer and may be responsible for neoplastic behavior of cells. After further validation, in-vivo, they may be clinically useful for diagnosis of BE, progressing to dysplasia/esophageal adenocarcinoma.

The following includes commentaries on how genetic code of Barrett's esophagus (BE) patients, the mechanisms for GERD-induced esophageal expression of caudal homeobox, and the development of Barrett's metaplasia are increasingly better known, including the role of stromal genes in oncogenesis. Additional lessons have been learned in vitro models in nonneoplastic cell lines, yet there are limitations to what can be expected from BE-derived cell lines. Other topics discussed include clonal diversity in Barrett's esophagus; the application of peptide arrays to clinical samples of metaplastic mucosa; proliferation and apoptosis of Barrett's cell lines; tissue biomarkers for neoplasia; and transcription factors associated with BE., (© 2011 New York Academy of Sciences.)

The following on histology and immunohistology of Barrett's esophagus (BE) includes commentaries on the various difficulties remaining in reaching a consensus on the definition of BE; the difficulties in the characterization of intestinal and cardiac mucosa, and in the role of submucosal glands in the development of BE; the importance of a new monoclonal antibody to recognize esophageal intestinal mucosa; the importance of pseudo goblet cells; the best techniques for the endoscopic detection of Barrett's epithelium; and the biomarkers for identification of patients predisposed to the development of BE., (© 2011 New York Academy of Sciences.)

The mechanism by which gastroesophageal reflux promotes metaplasia→dysplasia→carcinoma is unknown. The aim of the study is to determine if repeated exposure to acid and bile confers a tumorigenic phenotype in a telomerase (hTERT)-immortalized benign Barrett's cell line (BAR-T). BAR-T cells were exposed to acid (pH 4) (A) and bile salt (200 μM glycochenodeoxycholic acid) (B) daily for 5 min up to 65+ weeks. The control cells were grown in parallel without any A or B treatment. Cell morphology, proliferation, transformation, and molecular changes in the gene expression for COX-2, TC22, p53 and p53 target genes were analyzed at 8-12 weeks intervals. At 46 weeks BAR-T cells exposed to (A+B) showed distinct phenotypic changes: forming clusters and acini, and at 65 weeks displayed foci in monolayer, and formed distinct colonies in soft agar. Untreated cells did not show any such changes. In A+B-treated BAR-T cells, COX-2 mRNA increased 10- to 20-fold, TC22 mRNA increased by 2- to 3-fold at 22-65 weeks, p53, MDM2, PERP, and p21mRNA increased 2.5-, 6.4-, 4-, and 2.6-fold respectively when compared to untreated cells at 34 weeks. However, at 58 weeks onward, there was a sharp decline of p53 and its target genes to the baseline level. At 65 weeks A+B-treated BAR-T cells formed tumor in nude mice whereas untreated cells did not. We demonstrate a novel in vitro model of transformation of a benign Barrett's cell line following repeated exposure to A+B over the course of 65 weeks., (Copyright © 2010 UICC.)

Objectives: Tropomyosins (TMs) are cytoskeletal microfilament proteins present in all eukaryotic cells. Human TM isoform 5 (hTM5) is the predominant isoform in colonic epithelial cells. Antibodies against hTM5 are found both in the sera and in the mucosa of patients with ulcerative colitis (UC) but not Crohn's disease (CD). We investigated whether anti-hTM5 autoantibodies are pathogenic., Methods: Normal-appearing colonic mucosal biopsy specimens were incubated with autologous serum. After 45 min, deposition of the complement component C3b was identified by indirect immunofluorescence assay (IFA). Additional specimens were incubated with autologous serum fixed in formalin, and their architecture was examined by hematoxylin and eosin (H&E) staining., Results: For 79% of UC patients, autologous serum caused C3b staining along the colonic epithelium. Recombinant hTM5 or anti-hTM5 monoclonal antibody blocked serum-induced C3b deposition. Immunoglobulin G (IgG) antibody and affinity-purified anti-hTM5 IgG antibody from UC sera with complement caused C3b deposition, indicating specificity of hTM5 as an autoantigen. When analyzed by H&E staining, sera obtained from 71% of UC patients caused a significant loss of epithelium. This process was inhibited by Fc fragments, indicating that it is complement mediated. With medium, normal, or CD serum, there was no C3b deposition or morphological changes of the colonic epithelium, indicating disease specificity. The ileal mucosa was not affected by UC sera, suggesting specificity for the colon. In UC mucosa, expression of hTM5 increased., Conclusions: hTM5 acts as an autoantigen in UC. hTM5-specific IgG autoantibody in sera from UC patients induces C3b deposition and destruction of colonic epithelial cells, suggesting a direct pathogenic effect. If used as a diagnostic test to distinguish UC from CD, IFA would have 79% sensitivity and 100% specificity. Development of blocking antibodies may lead to novel therapies.

Although a protective role for mesalamine against colon cancer in ulcerative colitis has been shown epidemiologically, its molecular mechanism is unknown. We cloned and sequenced a novel human tropomyosin (hTM) isoform, TC22, which is an alternatively spliced variant of normal epithelial hTM isoform 5 (hTM5), identical apart from 25 C-terminal amino acids. TC22 is expressed in 100% of colorectal carcinoma but is not expressed in normal colon epithelial cells. To explore a molecular mechanism of chemoprevention, we examined the effect of mesalamine on TC22 expression using LS180 colon cancer cells. Expression of hTM5 and TC22 was investigated at the protein and gene levels by fluorescence-activated cell sorting and real-time reverse transcription-polymerase chain reaction. Small interference RNA (siRNA) against the TC22 variant were transfected into LS180 colon cancer cells, reducing protein and transcript levels by 45 to 50%. Mesalamine or sulfasalazine (2 mM), but not sulfapyridine, significantly (p < 0.02-0.006) reduced the expression of the TC22 transcript and significantly (p < 0.05 to <0.0002) reduced the expression of TC22 protein in a dose-dependent and reversible manner. Rosiglitazone, a specific peroxisome proliferator-activated receptor-gamma (PPARgamma) agonist, similarly and significantly (p < 0.002) reduced TC22 protein expression. A polymerase chain reaction array of 84 cancer-related genes performed on TC22 siRNA-transfected cells demonstrated a significant (more than two times) change in targets involved in apoptosis, adhesion, angiogenesis, and tissue remodeling. We conclude that mesalamine, sulfasalazine, and rosiglitazone significantly reduced the cellular expression of TC22, implicating PPARgamma in this modulation. Similar suppression of TC22 by siRNA produced gene level changes on several critical carcinogenic pathways. These findings suggest a novel antineoplastic molecular effect of mesalamine.

EPR study of copper ions in diglycine calcium chloride tetrahydrate (DGCCT), [(NH(2)CH(2)COOH)(2).CaCl(2).4H(2)O] single crystals at room temperature is carried out. The spin Hamiltonian parameters of copper ions are determined as: g(x)=2.0238+/-0.0002, g(y)=2.1122+/-0.0002, g(z)=2.2250+/-0.0002, A(x)=(83+/-2)x10(-4)cm(-1), A(y)=(86+/-2)x10(-4)cm(-1) and A(z)=(118+/-2)x10(-4)cm(-1). The optical study of the single crystals at room temperature is also done and the bands are assigned to d-d and charge transfer transitions. Using above data, the nature of bonding in the complex is discussed.

EPR study of Mn2+ doped ammonium tartrate single crystals is carried out at room temperature. The spin Hamiltonian parameters are: gx=1.9225+/-0.0002, gy=1.9554+/-0.0002, gz=2.1258+/-0.0002, A=(78+/-2) x 10(-4) cm(-1), B=(75+/-2) x 10(-4) cm(-1), D=(191+/-2) x 10(-4) cm(-1), E=(61+/-2) x 10(-4) cm(-1) and a=(22+/-1) x 10(-4) cm(-1) for site I and gx=1.9235+/-0.0002, gy=1.9574+/-0.0002, gz=2.0664+/-0.0002, A=(78+/-2) x 10(-4) cm(-1), B=(75+/-2) x 10(-4) cm(-1), D=(180+/-2) x 10(-4) cm(-1), E=(57+/-2) x 10(-4) cm(-1) and a=(22+/-1) x 10(-4) cm(-1) for site II, respectively. The observed optical bands are fitted with inter-electronic repulsion parameters (B and C), crystal field parameter (Dq) and Trees correction (alpha) and the values found are B=752, C=2438, Dq=765 and alpha=76 cm(-1). The data obtained are further used to discuss the surrounding crystal field and the nature of metal-ligand bonding in the crystal.

Barrett's epithelium is a precancerous, specialized columnar metaplasia in the distal esophagus. We demonstrate the changes in cellular phenotype in a non-neoplastic Barrett's cell line (BAR-T), following exposure to acid and bile salt, the two important components of gastroesophageal refluxate. Cell phenotypes in BAR-T cell line were quantified by fluorescence-activated cell sorting (FACS) using monoclonal antibodies against markers: cytokeratin 8/18 (CK8/18) for columnar, CK4 for squamous, mAbDas-1 for colonic epithelial cell phenotype and p75NTR for esophageal progenitors. Cells were exposed for 5 min each day to 200 microM glycochenodeoxycholic acid at pH 4, pH 6 and pH 7.4 or only to acid (pH 4) for up to 6 weeks. The BAR-T cell line comprised 35+/-5.2% CK8/18, 32+/-3.5% mAbDas-1, 9.5+/-3% CK4 and 4+/-2.5% p75NTR-positive cells. Single exposure to acid and or bile did not change cell phenotypes. However, chronic treatment for at least 2 weeks significantly enhanced (P<0.05) the expression of colonic phenotype and CK8/18-positive cells, as evidenced by FACS analysis. Bile salt at pH 4 and bile salt followed by acid (pH 4) in succession were the strongest stimulators (P<0.01) for induction of colonic phenotype cells. Squamous (CK4(+)) phenotype did not change by the treatments. Cox-2 expression was induced after acute treatment and increased to twofold during chronic treatment, particularly in response to acidic pH. We conclude that BAR-T cells can be utilized as an 'in vitro' model to study the effect of environmental factors and their influence on the cellular phenotype and molecular changes in the pathogenesis of esophageal cancer.

Ulcerative colitis (UC) is a form of chronic inflammatory bowel disease (IBD) that almost always affects the rectal mucosa and variable length of the colon in continuity and at times mucosa of the entire colon. It is not caused by any specific pathogen. Genetics, environmental factors and altered immune responses to dietary macromolecules, colonic bacteria and cellular proteins have been implicated in the pathogenesis of UC. Autoimmune response against cytoskeletal, microfilament protein tropomyosin (Tm) seems to play an important role in the pathogenesis of UC. The predominant colonic epithelial Tm isoform, hTm5, can induce both humoral (B-cells) and cellular (T-cells) response in patients with UC. Such responses are not seen in normal subjects and disease control subjects, such as patients with Crohn's disease (CD, another type of IBD) and patients with lupus. A novel observation that hTm5 is expressed on colon epithelial cell surface but not on small intestinal epithelial cells provides evidence for presentation to immune effector cells. This surface expression of hTm5 seems to be facilitated by a colon epithelial cell membrane associated protein, CEP, that acts as a chaperone for the trans-migration of hTm5 to the surface and both hTm5 and CEP are then released outside the cell. Both CEP and hTm5 expression are increased with pro-inflammatory cytokine, such as gamma-interferon. hTm5 expression in UC mucosa is also significantly increased compared to normal. Finally, autoantibodies against hTm5 observed both in circulation and in the colon mucosa of patients with UC are pathogenic causing colon epithelial cell destruction by antibody and complement mediated cytolysis.