Abstract Objectives: The molecular factors contributing to the development of Barrett's 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.
Although measures of colonic cell proliferation are being used as potential intermediate markers in chemoprevention studies, measurement standardization is still ongoing. This study was designed to assess the reproducibility of the labeling index quantification, as measured by bromodeoxyuridine, across four laboratories experienced in its use. Each institution submitted 10 slides, with one circled area of each slide to be scored. Each site followed its standard procedures for scoring colonic crypts; no attempts to standardize these procedures were made. There was high concordance among the laboratories on whether scorable crypts were present on a particular slide, but only two pairs of laboratories demonstrated agreement statistically greater than that predicted by chance. The overall difference among the sites on the number of scorable crypts was marginally significant (P = 0.083), and there was a highly significant overall difference in the magnitude of the labeling index (P < 0.0001). Sites 1 and 2 tended to have similar results, as did sites 3 and 4, most likely due to common training. Even with these discrepancies, high correlation (r > 0.75) was observed among the reported labeling index values for each pair of laboratories. Without standardized training, these laboratories may differ in the crypts considered appropriate for counting and in whether cells are counted as labeled or unlabeled. These results suggest that standardized training in scoring across all sites performing labeling index determinations is required to assure reproducibility across sites or studies. These results may also help explain discrepancies in the average values of the labeling index reported in the literature.
Abstract 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.
Cancers often arise within an area of cells (e.g. an epithelial patch) that is predisposed to the development of cancer, i.e. a "field of cancerization" or "field defect." Sporadic colon cancer is characterized by an elevated mutation rate and genomic instability. If a field defect were deficient in DNA repair, DNA damages would tend to escape repair and give rise to carcinogenic mutations.To determine whether reduced expression of DNA repair proteins Pms2, Ercc1 and Xpf (pairing partner of Ercc1) are early steps in progression to colon cancer.Tissue biopsies were taken during colonoscopies of 77 patients at 4 different risk levels for colon cancer, including 19 patients who had never had colonic neoplasia (who served as controls). In addition, 158 tissue samples were taken from tissues near or within colon cancers removed by resection and 16 tissue samples were taken near tubulovillous adenomas (TVAs) removed by resection. 568 triplicate tissue sections (a total of 1,704 tissue sections) from these tissue samples were evaluated by immunohistochemistry for 4 DNA repair proteins. Substantially reduced protein expression of Pms2, Ercc1 and Xpf occurred in field defects of up to 10 cm longitudinally distant from colon cancers or TVAs and within colon cancers. Expression of another DNA repair protein, Ku86, was infrequently reduced in these areas. When Pms2, Ercc1 or Xpf were reduced in protein expression, then either one or both of the other two proteins most often had reduced protein expression as well. The mean inner colon circumferences, from 32 resections, of the ascending, transverse and descending/sigmoid areas were measured as 6.6 cm, 5.8 cm and 6.3 cm, respectively. When combined with other measurements in the literature, this indicates the approximate mean number of colonic crypts in humans is 10 million.The substantial deficiencies in protein expression of DNA repair proteins Pms2, Ercc1 and Xpf in about 1 million crypts near cancers and TVAs suggests that the tumors arose in field defects that were deficient in DNA repair and that deficiencies in Pms2, Ercc1 and Xpf are early steps, often occurring together, in progression to colon cancer.
The proliferative activity of tumors has been shown to have prognostic significance in a variety of solid tumors. The standard method for assessing the proliferating fraction has been the measurement of the tritiated thymidine labeling index (TLI). Ki-67 is a monoclonal antibody that labels proliferating cells. In this study, the authors compared the proliferative activity of seven solid tumor specimens, using both methods simultaneously, on comparable sections from the same specimen. As expected, in every case the Ki-67 labeling index (KiLI) was greater than the TLI. The mean TLI ranged from 4.4 ± 0.4% to 16.7 ± 10.2%. The KiLI from these same tumors ranged from 25.7 ± 8.6% to 41.2 ± 12.2%. The mean coefficient of variation for measurements from different sections within the same specimen was 28.8 for TLI and 16.4 for KiLI. Therefore, KiLI shows less variability within different sections of the same specimen and is, therefore, a more reliable marker for proliferative activity than is TLI.
In carcinogenesis, the "field defect" is recognized clinically because of the high propensity of survivors of certain cancers to develop other malignancies of the same tissue type, often in a nearby location. Such field defects have been indicated in colon cancer. The molecular abnormalities that are responsible for a field defect in the colon should be detectable at high frequency in the histologically normal tissue surrounding a colonic adenocarcinoma or surrounding an adenoma with advanced neoplasia (well on the way to a colon cancer), but at low frequency in the colonic mucosa from patients without colonic neoplasia. Using immunohistochemistry, entire crypts within 10 cm on each side of colonic adenocarcinomas or advanced colonic neoplasias were found to be frequently reduced or absent in expression for two DNA repair proteins, Pms2 and/or ERCC1. Pms2 is a dual role protein, active in DNA mismatch repair as well as needed in apoptosis of cells with excess DNA damage. ERCC1 is active in DNA nucleotide excision repair. The reduced or absent expression of both ERCC1 and Pms2 would create cells with both increased ability to survive (apoptosis resistance) and increased level of mutability. The reduced or absent expression of both ERCC1 and Pms2 is likely an early step in progression to colon cancer. DNA repair gene Ku86 (active in DNA non-homologous end joining) and Cytochrome c Oxidase Subunit I (involved in apoptosis) had each been reported to be decreased in expression in mucosal areas close to colon cancers. However, immunohistochemical evaluation of their levels of expression showed only low to modest frequencies of crypts to be deficient in their expression in a field defect surrounding colon cancer or surrounding advanced colonic neoplasia. We show, here, our method of evaluation of crypts for expression of ERCC1, Pms2, Ku86 and CcOI. We show that frequency of entire crypts deficient for Pms2 and ERCC1 is often as great as 70% to 95% in 20 cm long areas surrounding a colonic neoplasia, while frequency of crypts deficient in Ku86 has a median value of 2% and frequency of crypts deficient in CcOI has a median value of 16% in these areas. The entire colon is 150 cm long (about 5 feet) and has about 10 million crypts in its mucosal layer. The defect in Pms2 and ERCC1 surrounding a colon cancer thus may include 1 million crypts. It is from a defective crypt that colon cancer arises.
Gastric intestinal metaplasia (GIM) is a precursor lesion for gastric cancer. It most frequently involves the antrum and the angularis. At endoscopy, it is not possible to visually distinguish GIM from normal stomach. Furthermore, GIM frequently has a patchy distribution with areas of metaplasia coexisting with adjacent areas of other histologies, including normal stomach. In this study we sought to determine whether a "field defect" could be demonstrated in subjects with GIM, involving the entire region of the stomach. The biologic markers tested were ornithine decarboxylase (ODC) activity and bromodeoxyuridine labeling index (LI). Antral biopsies were obtained from 13 subjects with known GIM and 9 controls (no GIM based on multiple biopsies and absence of methylene blue staining). Three adjacent biopsies were obtained for ODC, LI, and histology. Group I consisted of a set of 3 biopsies from the 9 controls. In the 13 subjects with GIM, 2 sets of 3 biopsies were taken with methylene blue guidance in an attempt to obtain both GIM-free (group II) and GIM-containing (group III) tissue. ODC activities were markedly and statistically significantly (P = 0.0001) elevated in groups II and III versus group I; the mean +/- SDs were 0.075 +/- 0.117 for group I, 1.20 +/- 0.83 for group II, and 1.14 +/- 0.76 for group III. Group II versus Group III values were not different (P = 0.979). LI was less discriminatory with more overlap between the groups. The highest LI was in group II, which was significantly different from group I (P = 0.014) and group III (P = 0.006).(ABSTRACT TRUNCATED AT 250 WORDS)
1405 Objectives: Barrett9s esophagus (BE) is a premalignant condition associated with elevated risk for the development of esophageal adenocarcinoma (ADCA). Previous studies indicated that oxidative damage contributes to the development of ADCA. We tested the hypothesis that bile acids and gastric acid, two components of refluxate, can induce oxidative stress and oxidative DNA damage. We speculate that effects of gastric acid and bile acids may be inhibited by treatment with the cytoprotective bile acid, ursodeoxycholic acid (UDCA). Methods: Oxidative stress was evaluated by staining with an antibody to 8-hydroxy-deoxyguanosine (8-OH-dG) in BE tissues with different degrees of dysplasia. The levels of 8-OH-dG were also evaluated ex vivo in BE tissues incubated for 10 minutes with control medium and medium acidified to pH 4 and supplemented with 0.5mM bile acid cocktail. Furthermore, three esophageal cell lines (BE cells, normal squamous esophageal HET-1A cells and adenocarcinoma Seg-1 cells) were exposed for 1-10 minutes to control media, media containing bile acid cocktail, media acidified to pH 4, and media at pH 4 supplemented with 0.1mM bile acid cocktail and evaluated for induction of reactive oxygen species (ROS). In addition, the cells were treated as above but with 0.1 mM UDCA added to the media. Results: Immunohistochemical analysis showed that 8-OH-dG is present at increased levels in epithelial cells of dysplastic BE. Incubation of BE tissues with a combination of a bile acid cocktail and low pH leads to the increased formation of 8-OH-dG. An increase in ROS occurs after exposure to pH 4 and a bile acid cocktail in all three cell lines and pH 4 treatment alone induced ROS in BE cells and HET-1A cells. These increases in ROS were inhibited by treatment with 0.1mM UDCA. Conclusions: Oxidative DNA damage is present at increased levels in dysplastic BE tissue. Oxidative stress can be induced in esophageal tissues and cells by short exposures to bile acids and low pH. These alterations may underlie the development of BE and tumor progression. UDCA treatment may inhibit oxidative damage induced by cytotoxic bile acids and gastric acid.
