Barrett's Adenocarcinoma Cell Line Research Articles

Abstract 2720: Newly established Barrett's adenocarcinoma cell line (TYAE-1).

Abstract Although 10 esophageal adenocarcinoma cell lines have been reported, only 5 were Barrett's adenocarcinoma cell lines. Furthermore, there was only one mouse-inoculated esophageal adenocarcinoma cell line. In this paper, we present a newly established Barrett's esophageal adenocarcinoma cell line (TYAE-1). Material and Method: The Barrett's esophageal adenocarcinoma cells were obtained from the resected tumor of a patient (74 years-old male). The resected tumor was confirmed as well-differentiated intraepithelial Barrett's adenocarcinoma without lymphnode metastasis. The tumor was too small (T1a) for a primary culture, so we transplanted the tumor in the back of a nude mouse. Results: One month later, the tumor grew rapidly and was able to be re-transplanted to other mice. Up to the writing of this report, the tumor (TYAE-1m) was able to be re-transplanted 10 times every month. The histology of the transplanted tumor was slightly different from the primary tumor. The majority of the tumor was cribriform type and there were a few tubular-trabecular type cells. The expression of beta-Catenin was cytoplasmic in the cribriform part and membranous in the tubular part. Both cell types did express VEGF slightly however they did not express HER-2. We also started to perform in vitro cultures from the mice transplanted tumor and established TYAE-1c cell. TYAE-1c cells were able to be transplanted in nude mice and transplanted tumor showed the same histological features as TYAE-1m. TYAE-1m and TYAE-1c cells were sensitive to Gemcitabine treatment. On the other hand, both tumors did not show sensitivity for Docetaxel treatment. In conclusion: Although the tumor was small and had no invasive characteristics by clinicopathological examination, this tumor has aggressive behavior. This in vitro and in vivo Barrett's adenocarcinoma model may useful for understanding the behavior of Barrett's adenocarcinoma. Citation Format: Yutaka Shimada, Makoto Moriyama, Tomoyuki Okumura, Shinichi Sekine, Shigeaki Sawada, Koshi Matsui, Shozo Hojo, Kazuto Shibuya, Isaku Yoshioka, Toru Yoshida, Takuya Nagata, Kazuhiro Tsukada. Newly established Barrett's adenocarcinoma cell line (TYAE-1). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2720. doi:10.1158/1538-7445.AM2013-2720

Bile Acids Down-Regulate Caveolin-1 in Esophageal Epithelial Cells through Sterol Responsive Element-Binding Protein

Bile acids are synthesized from cholesterol and are major risk factors for Barrett adenocarcinoma (BAC) of the esophagus. Caveolin-1 (Cav1), a scaffold protein of membrane caveolae, is transcriptionally regulated by cholesterol via sterol-responsive element-binding protein-1 (SREBP1). Cav1 protects squamous epithelia by controlling cell growth and stabilizing cell junctions and matrix adhesion. Cav1 is frequently down-regulated in human cancers; however, the molecular mechanisms that lead to this event are unknown. We show that the basal layer of the nonneoplastic human esophageal squamous epithelium expressed Cav1 mainly at intercellular junctions. In contrast, Cav1 was lost in 95% of tissue specimens from BAC patients (n = 100). A strong cytoplasmic expression of Cav1 correlated with poor survival in a small subgroup (n = 5) of BAC patients, and stable expression of an oncogenic Cav1 variant (Cav1-P132L) in the human BAC cell line OE19 promoted proliferation. Cav1 was also detectable in immortalized human squamous epithelial, Barrett esophagus (CPC), and squamous cell carcinoma cells (OE21), but was low in BAC cell lines (OE19, OE33). Mechanistically, bile acids down-regulated Cav1 expression by inhibition of the proteolytic cleavage of 125-kDa pre-SREBP1 from the endoplasmic reticulum/Golgi apparatus and nuclear translocation of active 68-kDa SREBP1. This block in SREBP1's posttranslational processing impaired transcriptional activation of SREBP1 response elements in the proximal human Cav1 promoter. Cav1 was also down-regulated in esophagi from C57BL/6 mice on a diet enriched with 1% (wt/wt) chenodeoxycholic acid. Mice deficient for Cav1 or the nuclear bile acid receptor farnesoid X receptor showed hyperplasia and hyperkeratosis of the basal cell layer of esophageal epithelia, respectively. These data indicate that bile acid-mediated down-regulation of Cav1 marks early changes in the squamous epithelium, which may contribute to onset of Barrett esophagus metaplasia and progression to BAC.

Genomic evolution in Barrett's adenocarcinoma cells: critical roles of elevated hsRAD51, homologous recombination and Alu sequences in the genome

A prominent feature of most cancers including Barrett’s adenocarcinoma (BAC) is genetic instability, which is associated with development and progression of disease. In this study, we investigated the role of recombinase (hsRAD51), a key component of homologous recombination (HR)/repair, in evolving genomic changes and growth of BAC cells. We show that the expression of RAD51 is elevated in BAC cell lines and tissue specimens, relative to normal cells. HR activity is also elevated and significantly correlates with RAD51 expression in BAC cells. The suppression of RAD51 expression, by short hairpin RNA (shRNA) specifically targeting this gene, significantly prevented BAC cells from acquiring genomic changes to either copy number or heterozygosity (P<0.02) in several independent experiments employing single-nucleotide polymorphism arrays. The reduction in copy-number changes, following shRNA treatment, was confirmed by Comparative Genome Hybridization analyses of the same DNA samples. Moreover, the chromosomal distributions of mutations correlated strongly with frequencies and locations of Alu interspersed repetitive elements on individual chromosomes. We conclude that the hsRAD51 protein level is systematically elevated in BAC, contributes significantly to genomic evolution during serial propagation of these cells and correlates with disease progression. Alu sequences may serve as substrates for elevated HR during cell proliferation in vitro, as they have been reported to do during the evolution of species, and thus may provide additional targets for prevention or treatment of this disease.

Abstract 2320: N-cadherin down-regulation has antitumor effects in esophageal cancer

Abstract Tumor microenviroment plays a critical role for cancer and their malignancy. Dynamic changes in tumor microenvironment have been observed in many tumors including esophageal cancer. And during the drastic change, some of tumor cells show EMT (epithelial mesenchymal transition) which means that epithelial cells lose their epithelial feature and acquire mesenchymal properties, as invasiveness and also communication with other kinds of mesenchymal cells. During those change, one of the marker of epithelial feature, E-cadherin is switch to N-cadherin, it has been known as “cadherin switch”. And it is thought that those changes have relations with cancer malignancy. Previously we reported that esophageal cancer cells secrete TGF-b1 and activate mesenchymal fibroblasts, and those activated fibroblasts guided more endothelial cells into tumor and induced angiogenesis. In the other hand, we hypothesis cell-cell direct communication like cell-cell contact is also important role for cancer progression. So in this report, firstly we analyze the expression of cadherins in surgical specimens in immunohistochemistry and determined expression patterns of N-cadherin in esophageal squamous cell carcinoma and Barrett's adenocarinoma. Some of those cancers showed N-cadherin expression and specifically expressed in their invasive front. Secondary we analyzed cancer cell lines as well, TE-1, TE-4, TE-10, OE19 and OE33. Those cell lines are squamous cell carcinoma cell lines and Barrett's adenocarcinoma cell lines, and those showed expression of E-cadherin and some of them showed expression of N-cadherin. And we used those cell lines and explored the behavioral differentiations of each cell lines. Here, we report and show analysis of E- or N- cadherin expression in surgical specimens and cancer cell lines. And also we could show the potential of regulation of cadherins in esophageal cancer as a new therapeutic target, which is well recognized as one of the most aggressive malignancies in the world. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2320.

Prostaglandin EP2 receptor expression is increased in Barrett’s oesophagus and oesophageal adenocarcinoma

Accumulating evidence suggests that cyclooxygenase-2 (COX-2)-derived prostaglandin E2 (PGE2) is involved in oesophageal adenocarcinogenesis. PGE2 exerts its biological action by binding to specific receptors (EP1, EP2, EP3 and EP4). To investigate which PGE2 receptor subtypes regulate PGE2 signals in the oesophageal adenocarcinoma sequence. Expression was determined in oesophageal biopsies from 85 patients with oesophagitis, Barrett's metaplasia, intraepithelial neoplasia, oesophageal adenocarcinoma and normal oesophagus. Levels of mRNA and protein expression were determined by quantitative PCR, immunohistochemistry and western-blot. Expression of EP receptors was also determined in response to acid and bile exposure in the Barrett's adenocarcinoma cell line OE33. All four EP receptors subtypes were expressed in human oesophageal tissues. COX-2 and, especially, EP2 were increased in the Barrett's metaplasia-intraepithelial neoplasia-adenocarcinoma sequence. Expression of the EP4 receptor protein was increased in oesophageal adenocarcinoma. In contrast, expression levels of COX-1 and EP3 receptor were decreased along the sequence. No differences in EP1 expression were found. Treatment with the bile acid deoxycholate increased COX-2, EP1, EP2 and EP4 expression in OE33 cells. Our data suggest that in addition to COX-2, EP2 and EP4 receptors could be a selective target in the prevention and/or treatment of the Barrett's-associated adenocarcinoma.

Physiological and molecular analysis of acid loading mechanisms in squamous and columnar-lined esophagus

Barrett's esophagus (BE) may be an adaptive cellular response to repeated acid exposure. The aims of this study were to compare intracellular acid loading in BE cells with normal squamous esophageal cells. Primary squamous and BE cells were obtained endoscopically and cultured for up to 24 h. Barrett's adenocarcinoma cell lines TE7 and OE-33 were compared with a normal esophageal (NE) cell line OE-21. Extracellular pH was lowered to 6.0 using HCl; specific ion exchangers were blocked pharmacologically and pH microfluorimetry was performed using 2'7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. The effect of prolonged acid preincubations and repeated acid exposure on acid loading and recovery were examined. Acid loading was greater in primary BE than NE cells (DeltapHi -0.22 +/- 0.08 vs.-0.13 +/- 0.01) and maximal in the BE carcinoma cell line TE7 (DeltapHi -0.30 +/- 0.01). Whereas TE7 cells were able to recover fully from repeated acid exposure, OE-21 cells remained profoundly acidic. BE primary and transformed cells utilize DIDS inhibitable sodium-independent chloride/bicarbonate exchange as well as sodium/hydrogen ion exchange for acid loading. In contrast, SE only requires sodium-independent chloride/bicarbonate exchange for acidification. The degree of acid loading is greater in BE than NE cells and it occurs via dual ion exchangers similar to gastric mucosa. Only Barrett's epithelial cells can maintain a physiological pHi following prolonged and repeated reflux exposure, which may confer a teleological advantage.

899 Transformation of Benign Barrett's Epithelial Cells By Repeated Acid and Bile Exposure Over 22-65 Weeks: An in-Vitro Model of Metaplasia to Neoplasia By Environmental Factors

Background and aims: Barrett's esophagus (BE) is regarded as pre-malignant condition of esophagus. The exact pathomechanisms responsible for the development Barrett's adenocarcinoma (BA) are still poorly understood. There is an evidence that the development of BE is a consequence of a chronic duodenogastroesophageal reflux disease (DGER) and ongoing inflammation in injured area of esophageal mucosa. The aims of the study were; 1) analysis of NFκB activity in human BE biopsies and influence of bile acids (DCA and UDCA) on its transactivation in BA cell line (OE-19 cells); 2) Implication of CDX-2 in BE development and the influence of bile acids on CDX-2 expression in OE-19 cell line; 3) Influence of siRNA-RelA (siRNA against NFκB) and a NFκB inhibitor (MG-132) on COX-2 and CDX-2 expressions in OE-19 cell line induced by bile salts. Material and Methods: CDX-2 and COX-2 was analysed in human biopsies from the normal esophagus mucosa and BE at mRNA and protein level by qRT-PCR and Western blot, respectively. Additionally, activity of NFκB in BE was assessed by ELISA. In separate In Vitro experiments, OE-19 cells were incubated with or without bile salts (DCA or UDCA, 100μM or 300μM) in neutral pH in the presence of MG-132 or after transfection with siRNA-RelA. The apoptosis rate was evaluated by FACS. The expression of COX-2 and CDX-2 was analyzed at mRNA and protein level by quantitative RT-PCR and immunoblot. Results: BE demonstrated a significant upregulation of CDX-2 and COX-2 expressions and elevated activity of NFκB as compared to normal esophagus. DCA compared to UDCA stronger stimulated COX-2 and CDX-2 expression in OE-19 cells. Both MG-132 and transfection with siRNA-RelA attenuated the increase in COX-2 and CDX-2 expression after treatment of the cells with DCA and induced higher rate of apoptosis. Conclusions: Bile salts are involved development of Barrett's metaplasia probably via direct stimulation of COX-2 and CDX2. These effects are mediated by increased activation of NFκB Thus, targeted therapy against NFκB may have beneficial preventive effect on the development of Barrett's adenocarcinoma.

Effect of Adiponectin and Ghrelin on Apoptosis of Barrett Adenocarcinoma Cell Line

Obesity is an important risk factor for Barrett adenocarcinoma. However, the role of adiponectin (anti-inflammatory adipokine from adipose tissue) and ghrelin (orexigenic peptide gastric origin) on the progression of Barrett's carcinogenesis has not been investigated so far. The aim of the present study was: (1) to compare the expression of adiponectin and ghrelin receptors in Barrett's esophagus and in normal squamous epithelium; (2) to assess the effect of adiponectin and ghrelin on apoptosis in Barrett's adenocarcinoma cells in vitro; and (3) to investigate the effect of ghrelin on IL-1beta and COX-2 expression in OE-19 cells incubated with TNFalpha. The expression of ghrelin and adiponectin receptors (GHS-R1a, Adipo-R1, Adipo R-2) in biopsies from Barrett's esophagus and in Barrett's adenocarcinoma cell line OE-19 was assessed by quantitative RT-PCR (qRT-PCR). The OE-19 cells were also incubated with adiponectin (5-10 microg/ml), and the apoptosis and proliferation were assessed by FACS and MTT assays. Additionally, effects of adiponectin on the mRNA and protein expression of proapoptotic Bax and antiapoptotic Bcl-2 were assessed by RT-PCR and Western blot, respectively. In two different in vitro models of esophagitis the OE-19 cells were incubated with ghrelin alone or in the presence of TNFalpha or bile acids in the normal or pulse acidified medium, and the expression of IL-1beta and COX-2 as markers for inflammation were assessed by FACS and qRT-PCR, respectively. Adiponectin caused a significant increase in apoptosis, and this affect was accompanied by increased Bax and decreased Bcl-2 expression. In contrast, ghrelin had no effect on apoptosis of OE-19 cells incubated in neutral or acidified medium with or without addition of deoxycholic acid. At the mRNA level, the expression of adiponectin receptors (Adipo-R1, Adipo-R2) was decreased, and the expression of ghrelin receptor (GHS-R1a) was increased in Barrett's mucosa. Ghrelin caused a decrease in TNFalpha-induced COX-2 and IL-1beta expression in OE-19 cells. Adiponectin and ghrelin have an inhibitory effect on Barrett's carcinogenesis by two different mechanisms: (1) by an increase in apoptosis by adiponectin, and (2) by anti-inflammatory actions of ghrelin. The decrease in levels of these two peptides in obesity may explain the progression of Barrett's carcinoma in obese individuals.

Bile acids induce overexpression of homeobox gene CDX-2 and vascular endothelial growth factor (VEGF) in human Barrett's esophageal mucosa and adenocarcinoma cell line

Objective. Barrett's esophagus (BE) is an acquired precancerous condition that develops from mucosal injury incurred after chronic gastroesophageal acid and bile reflux. The mechanism of progression of carcinogenesis in BE is still not fully understood. Recently, the role of bile acids and the homeobox gene transcription factor CDX-2 has been suggested in the pathogenesis of BE. The aims of the present study were 1) to compare the mRNA and protein expression of CDX-2 in biopsies obtained from patients with BE and normal squamous epithelium and 2) to study the effect of two different bile salts, ursodeoxycholic acid (UDCA) and deoxycholic acid (DCA), on the mRNA expression of CDX-2 and vascular endothelial growth factor (VEGF) in Barrett's the adenocarcinoma cell line (OE-33). Material and methods. CDX-2 expression was measured in Barrett's mucosa and normal esophageal mucosa obtained from 15 patients with BE histologically diagnosed by immunohistochemistry, Western blot, and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). In in vitro experiments, OE-33 cells were incubated with DCA (100 µM) and UDCA (100 µM) in neutral and shortly acidified media (pulse acidification). The expression of CDX-2 and VEGF was assessed by quantitative RT-PCR. Results. Both mRNA and protein expression of CDX-2 were significantly up-regulated in Barrett's mucosa as compared to normal esophageal mucosa. In neutral medium, OE-33 cells showed an increase in CDX-2 expression after incubation with DCA or UDCA. After short acidification of the medium, expression of CDX-2 in OE-33 cells was significantly higher than that in cells incubated in neutral pH. The addition of DCA and UDCA did not cause any further alteration in CDX-2 expression. In neutral and acidified medium, VEGF mRNA expression was only significantly up-regulated by DCA, but not by UDCA. Conclusions. Bile acids, especially in acidic medium, increase expression of CDX-2. DCA appears to be a stronger stimulant of the expression of VEGF than UDCA in the Barrett's carcinoma cell line, indicating a stronger carcinogenic potential of this bile salt.

Relationship between peroxisome proliferator-activated receptor-γ expression and differentiation of human esophageal squamous cell carcinoma

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma), a member of the nuclear hormone receptor superfamily, is involved in suppressing the growth of several tumors. We showed that PPAR-gamma is expressed in Barrett's adenocarcinoma cell lines and inhibited the growth of these lines through the induction of G1 cell cycle arrest and apoptosis. We examined PPAR-gamma expression in human esophageal squamous cell carcinoma (SCC) in vitro and in vivo and investigated whether PPAR-gamma ligands affect the proliferation and apoptosis of human SCC cell lines. Biopsy specimens (n=46) obtained from human SCC of the esophagus were stained using a monoclonal antibody against human PPAR-gamma. We assessed the effects of PPAR-gamma ligands on the growth of SCC cells by adding 15-deoxy prostaglandin J2 (15d-PGJ2), or troglitazone to six human esophageal SCC cell lines (TE-1, TE-2, TE-3, TE-5, TE-8, and TE-9). Immunohistochemical staining showed that 34 of 46 (73.9%) SCC of the esophagus expressed PPAR-gamma. All SCC cell lines expressed PPAR-gamma mRNA and protein, especially when poorly differentiated (TE-2, TE-5, and TE-9). The PPAR-gamma ligands significantly and dose-dependently inhibited the proliferation of SCC lines, except for well-differentiated TE-1 and TE-3. Apoptosis was induced by 15d-PGJ2 (10 microM) in all tested SCC lines except TE-1, whereas troglitazone (50 microM) was marginally effective in only the TE-2 and TE-3 cell lines. The present findings suggest that PPAR-gamma could be a therapeutic target for treating squamous cell carcinoma of the esophagus, possibly through the induction of apoptosis.

PI3 kinase mediates bile salt induced proliferation in a telomerase-immortalized Barrett’s cell line

Abstract Introduction: Bile exposure is implicated in the malignant progression of Barrett's esophagus. Our previous studies demonstrated that bile salt exposure in a Barrett's adenocarcinoma cell line induces proliferation by activating the PI3 kinase/Akt pathway. However, it is not clear that these findings in cancer cells are applicable to the non-neoplastic cells of Barrett's esophagus. Therefore, we studied the effects of bile on PI3 kinase activation and proliferation in a novel, telomerase-immortalized, non-neoplastic Barrett's cell line. Methods: A human, telomerase-immortalized Barrett's cell line (BAR D2847 hTERT lox) was exposed to the conjugated bile salt glycochenodeoxycholic acid (GCDA) at neutral pH. Proliferation was measured by the MTT incorporation assay and Coulter counter. PI3 kinase activity was inferred from western blots for phosphorylated (active form) and total Akt, the major down-stream effector of PI3 kinase. LY294002, a PI3 kinase inhibitor, was used to block the PI3 kinase/Akt pathway. Results: A 5-minute exposure to the clinically relevant concentration of 200uM GCDA produced a 50% cell number increase (P Conclusions: Conjugated bile salts stimulate proliferation in a non-neoplastic Barrett's cell line in a PI3 kinase dependent fashion. We propose that bile exposure acts as a growth signal in the early stages of non-neoplastic Barrett's, thereby promoting progression to adenocarcinoma.

Bile salt exposure causes phosphatidyl-inositol-3-kinase–mediated proliferation in a Barrett's adenocarcinoma cell line

BackgroundThe mechanisms by which gastroesophageal reflux promotes malignant progression in Barrett's esophagus are poorly understood. The phosphatidylinositol-3-kinase (PI3 kinase)/Akt pathway regulates proliferation and apoptosis. We hypothesized that the PI3 kinase/Akt pathway mediates the pro-proliferative and antiapoptotic effects of bile. MethodsThe Barrett's adenocarcinoma cell line, SEG-1, was exposed to the conjugated bile salt, glycochenodeoxycholic acid (GCDA). Cell number was measured by the MTT incorporation assay and by Coulter counter. PI3 kinase/Akt activity was inferred from Western blots of phosphorylated and total Akt. Proliferation and apoptosis were determined by BrdU incorporation and cell death ELISA. ResultsA dose-dependent cell number increase was seen with a 20-minute exposure to GCDA. On Western blot, 200 μmol/L GCDA caused a 3-fold increase in Akt phosphorylation within 20 minutes, which was inhibited by 90% with the addition of PI3 kinase inhibitor, LY294002. LY294002 produced dose-dependent inhibition of GCDA-induced cell number increases. 200 μmol/L GCDA decreased apoptosis by 25%. Addition of LY294002 did not completely inhibit the antiapoptotic effect of bile. ConclusionsBile salts activate the PI3 kinase/Akt signaling pathway and stimulate cell growth in SEG-1. The majority of this PI3 kinase–mediated effect is secondary to increases in proliferation rather than to decreases in apoptosis.

In vitro acid exposure has a differential effect on apoptotic and proliferative pathways in a Barrett's adenocarcinoma cell line.

Acid, a principal component of refluxate, may contribute to the neoplastic progression of Barrett's esophagus. Brief acid exposure in vivo and in vitro has been shown to increase cell proliferation. The mechanisms underlying the hyperproliferative response are not well elucidated but may include alterations in Na(+)-H+ exchanger activity and MAPK signaling pathways. To ascertain the effects of pulsatile acid exposure on gene expression in a Barrett's adenocarcinoma cell line (SEG-1). SEG-1 cells were exposed to either acidified DMEM at pH 3.5 (0.1 M hydrochloric acid) or pH 7.4 (control) for 20 min followed by neutralization of the medium. Total RNA was extracted before acid exposure and over a 10-h time course (0.5, 2, 4, 6, 8, and 10 hours) and hybridized to Affymetrix human U133A oligonucleotide arrays. Data were analyzed using the Affymetrix statistical expression algorithms. Only alterations in gene expression that were > or = 2 and < or = -2 fold were studied further and a subset was further investigated by reverse transcription polymerase chain reaction (RT-PCR) and densitometry. Apoptosis was assayed in SEG-1 cells by western blot for cleaved caspase 3 and an apoptosis ELISA assay. Changes in expression were identified for 138 genes. Analysis of gene function identified immediate downregulation of genes associated with apoptosis and early upregulation of genes associated with proliferation. The gene expression profiles suggest that MAPK pathways may be involved and suppression of apoptosis may occur via p53-dependent mechanisms. Microarray analysis of gene expression changes in a Barrett's adenocarcinoma cell line has identified cellular pathways that may be disrupted following acid exposure.

L-Type Amino Acid Transporter-1 Overexpression and Melphalan Sensitivity in Barrett's Adenocarcinoma

The L-type amino acid transporter-1 (LAT-1) has been associated with tumor growth. Using cDNA microarrays, overexpression of LAT-1 was found in 87.5% (7/8) of esophageal adenocarcinomas relative to 12 Barrett's samples (33% metaplasia and 66% dysplasia) and was confirmed in 100% (28/28) of Barrett's adenocarcinomas by quantitative reverse transcription polymerase chain reaction. Immunohistochemistry revealed LAT-1 staining in 37.5% (24/64) of esophageal adenocarcinomas on tissue microarray. LAT-1 also transports the amino acid-related chemotherapeutic agent, melphalan. Two esophageal adenocarcinoma and one esophageal squamous cell line, expressing LAT-1 on Western blot analysis, were sensitive to therapeutic doses of melphalan (P < .001). Simultaneous treatment with the competitive inhibitor, BCH [2-aminobicyclo-(2,1,1)-heptane-2-carboxylic acid], decreased sensitivity to melphalan (P < .05). In addition, confluent esophageal squamous cultures were less sensitive to melphalan (P < .001) and had a decrease in LAT-1 protein expression. Tumors from two esophageal adenocarcinoma cell lines grown in nude mice retained LAT-1 mRNA expression. These results demonstrate that LAT-1 is highly expressed in a subset of esophageal adenocarcinomas and that Barrett's adenocarcinoma cell lines expressing LAT-1 are sensitive to melphalan. LAT-1 expression is also retained in cell lines grown in nude mice providing a model to evaluate melphalan as a chemotherapeutic agent against esophageal adenocarcinomas expressing LAT-1.

Acid and bile exposure cause additive MAPK activation and proliferation in a Barrett's adenocarcinoma cell line

Acid and bile exposure cause additive MAPK activation and proliferation in a Barrett's adenocarcinoma cell line

Acid exposure activates the mitogen-activated protein kinase pathways in Barrett's esophagus

Background & Aims:To explore mechanisms whereby acid reflux might contribute to carcinogenesis in Barrett's esophagus (BE) we studied: (1) the effects of acid on the mitogen-activated protein kinase (MAPK) pathways, cell proliferation, and apoptosis in a Barrett's adenocarcinoma cell line (SEGA1); and (2) the ability of acid to activate the MAPK pathways in vivo in patients with BE.Methods:SEG-1 cells were exposed to acidic media for 3 minutes, and the activities of 3 MAPKs (ERK, p38, and JNK) were determined. Proliferation was assessed using flow cytometry; cell growth and apoptosis were assessed using cell counts and an apoptosis ELISA assay. MAPK activation was studied in biopsy specimens taken from patients with BE before and after esophageal perfusion for 3 minutes with 0.1N HCl.Results:Acid-exposed SEG-1 cells exhibited a significant increase in proliferation and total cell numbers, and a significant decrease in apoptosis. These effects were preceded by a rapid increase in the activities of ERK and p38, and a delayed increase in JNK activity. PD 98059 abolished the acid-induced increase in GO/G1 and decrease in subG0 phases of the cell cycle. Both SB 203580 and DN-JNK 1/2 inhibited the acid-induced progression from G0/G1 to G2/M. The acid-induced decrease in apoptosis was abolished by inhibition of either ERK or p38. In the patients, acid exposure significantly increased the activity of p38 in the metaplastic epithelium.Conclusions:Acid increases proliferation and survival, and decreases apoptosis in SEG-1 cells by activating the MAPK pathways. Acid also activates the MAPK pathways in BE in vivo. These findings suggest that acid might contribute to carcinogenesis in BE through activation of MAPK pathways.

Induction of glutathione s-transferase-pi in Barrett's metaplasia and Barrett's adenocarcinoma cell lines.

Barrett's metaplasia consists of columnar epithelium that replaces the normal esophageal mucosa in patients with chronic gastroesophageal reflux. Because intestinal-type Barrett's metaplasia is the major risk factor for adenocarcinoma development, understanding the mechanisms that predispose the esophageal mucosa to malignant degeneration is clinically important. Glutathione s-transferase (GST)-pi belongs to a class of protective enzymes whose activity has been shown to be much lower in Barrett's metaplasia than in the normal esophagus, where this form of GST is predominant. In the studies described here, using immunocytochemical analysis, we observed higher levels of cytoplasmic GST-pi protein in normal esophageal mucosa than in Barrett's metaplasia. Using northern blot analysis, we also observed lower GST-pi mRNA levels in Barrett's metaplasia than in normal esophagus or adenocarcinomas from the same patients. Using as model systems three Barrett's adenocarcinoma cell lines and short-term organ culture of freshly resected normal esophagus and Barrett's metaplasia, dose-dependent induction of GST-pi mRNA was observed by using butylated hydroxyanisole and dexamethasone. GST-pi mRNA in Barrett's metaplasia was induced up to 2.5-fold with 60 microM butylated hydroxyanisole and nearly fivefold with 320 nM dexamethasone after 24 h. These studies demonstrate the ability to induce protective GST-pi in Barrett's metaplasia and may suggest a mechanism for future chemoprevention studies in patients with this type of epithelium, which is at high risk for malignant degeneration.

Differential expression of Hsp27 in normal oesophagus, Barrett's metaplasia and oesophageal adenocarcinomas.

The protein expression patterns of normal, metaplastic and malignant oesophageal tissues were analysed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) to identify changes associated with Barrett's metaplasia and transformation to oesophageal adenocarcinoma. Heat-shock protein 27 (Hsp27), a small heat-shock protein which is protective against cytotoxic stresses, was abundant in normal oesophagus. However, Hsp27 expression was markedly lower in Barrett's metaplasia and oesophageal adenocarcinomas. This was confirmed by immunohistochemical analysis. Hsp27 protein was most highly expressed in the upper layers of squamous epithelium and exhibited a pattern of expression that corresponded with the degree of squamous maturation. Northern and Southern analysis demonstrated Hsp27 to be regulated at the level of mRNA transcription or abundance. Normal oesophageal tissues were examined for gender differences in Hsp27 expression. Women expressed fourfold higher levels of Hsp27 mRNA, however, this difference was not appreciable in protein expression. Hsp27 protein was inducible by heat shock in Barrett's adenocarcinoma cell lines and an immortalized oesophageal epithelial cell line (HET-1A), but not by oestradiol. These results demonstrate abundant constitutive expression of the stress-response protein Hsp27 in the normal oesophagus, and suggest that low-level expression in Barrett's metaplasia may be one factor which may influence susceptibility to oesophageal adenocarcinoma development. © 1999 Cancer Research Campaign