Lois Ramsey

Increased Expression and Secretion of Interleukin-6 in Patients with Barrett’s Esophagus

Purpose: Barrett’s esophagus (BE) is a common premalignant lesion of the distal part of the esophagus that arises as a consequence of chronic duodenogastroesophageal reflux. Interleukin (IL)-6 is a pleiotropic cytokine that regulates immune defense mechanisms and hematopoiesis. In addition, IL-6 may also be involved in malignant transformation and tumor progression. IL-6 has been shown to inhibit apoptosis. The major aim of this study was to evaluate expression of IL-6 in BE at the protein and mRNA levels. In addition, we tested whether proteins that are associated with IL-6 signaling, phosphorylated signal transducer and activator of transcription 3 and two antiapoptotic proteins, Bcl-xL and Mcl-1, are also expressed in the same tissues. Experimental Design: Biopsies of duodenum, BE, and squamous epithelium were evaluated by using a human cytokine protein array, ELISA, real-time PCR, and immunohistochemistry. Results: Increased IL-6 levels were found to be secreted from BE tissue compared with duodenum or squamous epithelium from sites adjacent or 5 cm away from the BE lesion. IL-6 mRNA was also elevated in BE compared with duodenum or squamous epithelium in five of seven patients. Immunohistochemical studies confirmed IL-6 expression in intestinal glandular epithelium in BE tissue. Activated signal transducer and activator of transcription 3, Mcl-1, and Bcl-xL are present at higher levels in BE glands, with lower levels being found in duodenum or squamous epithelium Conclusions: These data, taken together, suggest that elevated IL-6 levels in BE may contribute to the development of apoptosis resistance, thereby placing this epithelium at higher risk of developing malignancy.

Biomarker studies in reversed Barrett's esophagus.

Objective:The purpose of this study was to use biomarkers to assess for cancer risk in Barretts esophagus patients with either squamous islands or complete reversal of their intestinal metaplasia to squamous epithelium.Methods:The biomarkers included proliferation characteristic using Ki-67, p53 abnormalities using immunohistochemical methods with two antibodies, DO-1 and DO-7, and ornithine decarboxylase (ODC) activity.Results:Eleven patients had complete reversal produced by a combination of acid suppression and thermal injury (multipolar electrocoagulation). Ki-67 staining was indistinguishable from that of normal squamous esophageal epithelium, i.e., basal layer staining only. All 11 cases were negative for p53. ODC activity was low and in the range for normal squamous epithelium. Fourteen patients had squamous islands (partial reversal) after prolonged proton pump inhibitor therapy. Multilayer Ki-67 staining occurred in nine cases (64%), and six (43%) had areas of positive p53 staining.Conclusion:Initial biomarker studies suggest that completely reversed squamous epithelium is biologically similar to normal squamous epithelium and of low cancer risk. In contrast, partial reversal, manifest as squamous islands, is accompanied by biomarker abnormalities.

Activation of the Interleukin-6/STAT3 Antiapoptotic Pathway in Esophageal Cells by Bile Acids and Low pH: Relevance to Barrett's Esophagus

Objectives: The molecular factors contributing to the development of Barretts esophagus (BE) are unclear. Our previous studies showed that BE tissues secrete interleukin-6 (IL-6) and express proteins associated with IL-6 signaling, including IL-6 receptor, activated signal transducer and activators of transcription 3 (STAT3), and antiapoptotic proteins Bcl-xL and Mcl-1. Here, we test the hypothesis that bile acids and gastric acids, two components of refluxate associated with gastresophageal reflux disease, activate the IL-6/STAT3 pathway. Materials and Methods: Immunohistochemistry was used to assess levels of phosphorylated STAT3 in esophageal tissue samples from BE patients with different grades of dysplasia. Seg-1 esophageal adenocarcinoma cells were evaluated for STAT3 activation and IL-6 and Bcl-xL expression by molecular biology techniques, including Western blot, reverse transcription–PCR, and ELISA after exposure to control media (pH 7.4), media supplemented with a 0.1 mmol/L bile acid cocktail with media at pH 4 or media at pH 4 with bile acid cocktail. Results: Immunohistochemical analysis showed that activated, phosphorylated STAT3 is expressed in nuclei of dysplastic BE and cancer tissues. Treatment of Seg-1 cells with media containing bile acid cocktail and acidified to pH 4 resulted in increased activation of STAT3, IL-6 secretion, and increased expression of Bcl-xL. Inhibition of the STAT3 pathway using STAT3 small interfering RNA or Janus-activated kinase inhibitor resulted in increased apoptosis. Conclusions: The IL-6/STAT3 antiapoptotic pathway is induced by short exposure to bile acid cocktail and low pH. This alteration, if persistent in vivo, may underlie the development of dysplastic BE and tumor progression.

Apoptosis resistance in Barrett's esophagus: Ex vivo bioassay of live stressed tissues

BACKGROUND AND AIMS:Barretts esophagus (BE) is a premalignant lesion of the distal esophagus in which squamous epithelial cells are replaced by metaplastic intestinal-like columnar epithelium that contains goblet cells. The factors that contribute to the progression from normal squamous mucosa to BE, Barretts dysplasia, and adenocarcinoma are not well understood at the molecular level. Since reflux of bile acids is associated with BE development, we speculate that cells with an apoptosis-resistant phenotype are selected after long-term repeated exposure to pulses of bile acids. This will result in the survival of cells with unrepaired DNA damage, and a consequent increase in genomic instability leading to cancer progression. The major goal of this study is to compare sensitivity to apoptosis induced by the bile acid, deoxycholate (DOC), a known inducer of apoptosis, in normal esophageal squamous epithelium, normal colon epithelium, and BE.METHODS:Thirteen patients with a confirmed diagnosis of BE and four patients who had undergone clinically indicated colectomy were included in the present study. Freshly obtained biopsies were incubated with control medium or medium supplemented with 1 mM DOC for 3 h and then evaluated for apoptotic changes using transmission electron microscopy and immunohistochemical staining for two apoptotic markers, cleaved caspase 3 and cleaved cytokeratin 18.RESULTS:Our results indicate that BE is resistant to apoptosis induced by DOC compared to esophageal squamous epithelium and normal colon epithelium. In addition, electron micrographs revealed mitochondrial swelling in squamous epithelial cells treated ex vivo with DOC, which was absent in epithelial cells of BE. Formation of swollen mitochondria is an early marker of apoptotic cell death. Altogether, the data indicate that reduced apoptosis capability in BE tissue may contribute to progression to esophageal adenocarcinoma.

Unique dietary‐related mouse model of colitis

Background: A high‐fat diet is a risk factor for the development of inflammatory bowel disease (IBD) in humans. Deoxycholate (DOC) is increased in the colonic contents in response to a high‐fat diet. Thus, an elevated level of DOC in the colonic lumen may play a role in the natural course of development of IBD. Methods: Wild‐type B6.129 mice were fed an AIN‐93G diet, either supplemented with 0.2% DOC or unsupplemented and sacrificed at 1 week, 1 month, 3 months, 4 months, and 8 months. Colon samples were assessed by histopathological, immunohistochemical, and cDNA microarray analyses. Results: Mice fed the DOC‐supplemented diet developed focal areas of colonic inflammation associated with increases in angiogenesis, nitrosative stress, DNA/RNA damage, and proliferation. Genes that play a central role in inflammation and angiogenesis and other related processes such as epithelial barrier function, oxidative stress, apoptosis, cell proliferation/cell cycle/DNA repair, membrane transport, and the ubiquitin‐proteasome pathway showed altered expression in the DOC‐fed mice compared with the control mice. Changes in expression of individual genes (increases or reductions) correlated over time. These changes were greatest 1 month after the start of DOC feeding. Conclusions: The results suggest that exposure of the colonic mucosa to DOC may be a key etiologic factor in IBD. The DOC‐fed mouse model may reflect the natural course of development of colitis/IBD in humans, and thus may be useful for determining new preventive strategies and lifestyle changes in affected individuals.

Perils of immunohistochemistry: variability in staining specificity of commercially available COX-2 antibodies on human colon tissue.

We assessed the immunohistochemical (IHC) staining patterns of three commercially available COX-2 antibodies in human tissues. The location of positive stain in sequential serial 4-μ sections of formalin fixed, paraffin embedded tissue differed considerably. Staining patterns ranged from diffuse cytoplasmic to occasional perinuclear staining. Thus, marked variability in staining results from use of different antibodies, making it important to consider the antibody used when comparing reports of COX-2 staining from different investigators.

Expression of Bile Acid Transporting Proteins in Barrett's Esophagus and Esophageal Adenocarcinoma

OBJECTIVES:Barretts esophagus (BE) is a metaplastic lesion characterized by replacement of the normal squamous epithelium by columnar intestinal epithelium containing goblet cells. It is speculated that this process is an adaptation to protect cells from components of refluxate, such as gastric acid and bile acids. In contrast to the normal squamous epithelium, enterocytes of the distal ileum are adapted to transport bile acids from the intestinal lumen. Several bile acid transporters are utilized for effective removal of bile acids, including the apical sodium-dependent bile acid transporter (ASBT), the ileal bile acid-binding protein (IBABP), and the multidrug-resistant protein 3 (MRP3). We hypothesized that one of the possible functions of newly arising metaplastic epithelium, in the esophagus, is to transport bile acids. Our major goal was to evaluate the expression of bile acid transporters in normal squamous epithelium, BE with different grades of dysplasia, and esophageal adenocarcinoma (EAC).METHODS:A total of 101 patients were included in this study. Immunohistochemistry (IHC) and reverse transcriptase (RT)–PCR were used to detect the expression of these transporters at the mRNA and protein levels.RESULTS:Our immunohistochemical studies showed that all three bile acid transporters are expressed in BE glands, but not in squamous epithelium. ASBT was found in the apical border in BE biopsies. The highest frequency of ASBT expression was in patients with nondysplastic BE (9 of 15, 60%), and a progressive loss of ASBT was observed through the stages of dysplasia. ASBT was not detected in EAC (0 of 15). IBABP staining was observed in the cytoplasm of BE epithelial surface cells. Expression of IBABP was found in 100% of nondysplastic BE (14 of 14), in 93% of low-grade dysplasia (LGD, 15 of 16), in 73% of high-grade dysplasia (HGD, 10 of 14), and in 33% of EAC (5 of 15). MRP3 was expressed in the basolateral membrane in 93% of nondysplastic BE (13 of 14), in 60% of LGD (10 of 16), and in 86% of HGD (11 of 13). Only weak MRP3 staining was detected in EAC biopsies (5 of 15, 33%). In addition, RT–PCR studies showed increased expression of mRNA coding for ASBT (6.1×), IBABP (9.1×), and MRP3 (2.4×) in BE (N=13) compared with normal squamous epithelium (N=15). Significantly increased mRNA levels of IBABP (10.1×) and MRP3 (2.5×) were also detected in EAC (N=21) compared with normal squamous epithelium.CONCLUSIONS:We found that bile acid transporters expression is increased in BE tissue at the mRNA and protein levels and that expression of bile acid transporter proteins decreased with progression to cancer.

Abnormal Expression of Biomarkers in Incompletely Ablated Barrett’s Esophagus

Objective:The aim of this study was to evaluate expression of cancer risk-associated biomarkers in columnar epithelium at squamocolumnar junctions produced by an ablation procedure and proton pump inhibitors in incompletely ablated Barrett’s esophagus (BE) patients that were nondysplastic prior to ablation. Summary Background Data:Ablation of BE to squamous epithelium is achievable by combining a re-injury method with acid suppression. We previously reported that, when there is complete ablation, the neo-squamous epithelium is normal histologically and in biomarker expression. However, squamous islands observed after prolonged use of PPIs were associated with abnormalities in p53 expression and Ki-67 labeling. Methods:Twenty-one nondysplastic BE cases with incomplete ablation were evaluated for the expression of Ki-67 (proliferation), cyclooxygenase-2 (COX-2), and p53 by immunohistochemistry. Results:Pre-ablation biopsies showed the normal staining patterns in columnar epithelium, ie, normal Ki-67 labeling, rare positive COX-2 staining of interstitial cells, and negative or mild staining for p53 in the majority of patients’ biopsies. However, post-ablation biopsies demonstrated abnormal staining patterns in the glandular area at the new squamocolumnar junctions. In 13 of 21 post-ablation cases (62%), increased Ki-67 staining was seen in BE glands. In 8 of 21 patients (38%), increased COX-2 expression was seen in columnar epithelium. Similarly, in 8 of 21 post-ablation junctions (38%), there was increased p53 staining. Conclusions:Our findings of increased expression of cancer-associated biomarkers in incompletely ablated BE patients raise a cautionary note regarding this procedure. We hypothesize that newly formed junctions contain cells undergoing replication, differentiation, etc, and are thus more susceptible to genomic damage.

Deoxycholate, an Endogenous Tumor Promoter and DNA Damaging Agent, Modulates BRCA-1 Expression in Apoptosis-Sensitive Epithelial Cells: Loss of BRCA-1 Expression in Colonic Adenocarcinomas

Deoxycholate, a bile salt present at high levels in the colonic lumen of individuals on a high-fat diet, is a promoter of colon cancer. Deoxycholate also causes DNA damage. BRCA-1 functions in repair of DNA and in induction of apoptosis. We show that, when cultured cells of colonic origin are exposed to deoxycholate at different concentrations, BRCA-1 expression is induced at a low noncytotoxic concentration (10 μM) but is strongly inhibited at higher cytotoxic concentrations (>100 μM). Indication of phosphorylation of BRCA-1 by deoxycholate (100 μM) at a lower dose was seen by Western blot analysis, whereas, at a higher dose, deoxycholate (200 and 300 μM) caused a complete loss of BRCA-1 expression. We show that BRCA-1 is substantially lower in colon adenocarcinomas from five patients compared with associated non-neoplastic colon tissue from the same patients, suggesting that the loss of BRCA-1 expression contributes to the malignant phenotype. In the non-neoplastic colon tissue, BRCA-1 was localized to the nongoblet cells. Our results imply that reduced expression of BRCA-1 may be associated with carcinoma of the colon.

Tissue culture of epithelial cells from esophageal specialized columnar epithelium (Barrett's esophagus)

A procedure is described for the tissue culture of epithelial cells from Barretts specialized columnar epithelium. The method employs use of nonenzymatic disaggregation techniques and use of a specialized growth medium, M-19. Successful growth can be achieved in 60–70% of cases. Characteristics of four cultures are presented including PAS, Alcian blue, and cytokeratin staining properties. Ultrastructural studies showed the presence of distended rough endoplasmic reticulum, abundant Golgi apparatus, glycogen granules, and cell-cell junctional complexes. Colony formation in anchorage-independent growth in soft agar was not observed in any culture. The cultured cells were not capable of indefinite growth. Thus, they do not behave as fully transformed cells. Availability of this culture system should be of use to the study of the biology of this metaplastic premalignant lesion.