Unlike Esophageal Squamous Cells, Barrett's Epithelial Cells Resist Apoptosis by Activating the Nuclear Factor-κB Pathway Free

Gastresophageal reflux disease (GERD) often causes peptic injury and inflammation of the esophageal squamous epithelium, a condition called reflux esophagitis. In most cases, reflux esophagitis heals through the regeneration of new squamous cells. In some patients, however, reflux esophagitis heals through a metaplastic process in which intestinal-type columnar cells replace reflux-damaged squamous cells (1). This condition is called Barrett's esophagus, and for reasons that are not clear, metaplastic Barrett's epithelium is predisposed to develop neoplasia. GERD and Barrett's esophagus are strong risk factors for esophageal adenocarcinoma, a tumor whose incidence has increased substantially in the United States over the past 30 years (1, 2).

Apoptosis is an important mechanism for maintaining tissue homeostasis and for preventing the proliferation of cells with mutations that could result in malignancy. Normally, mild genomic injury induces p53 expression that triggers cell cycle arrest, which enables the cell to repair its damaged DNA. If the genomic injury is severe and irreparable, however, apoptosis ensues. Failure to undergo apoptosis in the setting of severe DNA injury allows dangerous mutations to persist and contribute to cancer formation. Indeed, resistance to apoptosis is an essential physiologic hallmark of cancer cells (3). Proapoptotic signals can be counterbalanced by the expression of antiapoptotic proteins whose synthesis is mediated by the nuclear factor-κB (NF-κB) gene, a member of the Rel family of transcription factors (4). There are five members of the mammalian Rel family of proteins, with NF-κB/p65 (Rel A) being one of the most important.

Metaplasia seems to be a protective response to inflammation because in a number of organs, the metaplastic tissue is more resistant to the noxious agents causing the chronic inflammation than the native tissue. For example, chronic gastritis due to Helicobacter pylori results in intestinal metaplasia, which is far less susceptible to H. pylori infection than the normal gastric epithelium. Similarly, Barrett's metaplasia seems to be more resistant to acid-peptic injury than the native esophageal squamous mucosa (5, 6). Bile also is present in refluxed gastric juice, and Barrett's metaplasia has been found to be more resistant to bile acid–induced apoptosis than normal esophageal squamous (NES) epithelium (6). This resistance to apoptosis may be one mechanism whereby Barrett's cells survive despite continuous exposure to refluxed gastric juice.

Clinical data suggest that alterations in the susceptibility to apoptosis underlie the neoplastic progression of Barrett's esophagus (7–9). In biopsy samples of Barrett's epithelium, for example, altered staining for the antiapoptotic proteins Bcl-2 and Bcl-xL (downstream NF-κB target genes) has been found as the metaplasia progresses through dysplasia to adenocarcinoma (7, 9, 10). These data suggest a role for the NF-κB pathway in conferring an antiapoptotic phenotype during the neoplastic progression of Barrett's esophagus (11).

Earlier studies found expression of NF-κB in only 13% of biopsy specimens of esophageal squamous epithelium (12). In contrast, NF-κB expression has been found in biopsy specimens of benign Barrett's epithelium in 40% to 60% of cases and, in esophageal adenocarcinoma, in 61% to 80% of cases (12, 13). This suggests the possibility that reflux-mediated activation of the NF-κB pathway, which can increase the expression of NF-κB survival pathway proteins, may enable the metaplastic columnar cells to resist apoptosis and survive, whereas the esophageal squamous cells succumb to GERD. If noxious agents in refluxed gastric juice simultaneously cause DNA injury and NF-κB activation in the Barrett's cells, furthermore, this might enable the survival of cells with cancer-promoting mutations. The present study was designed to explore the contribution of the NF-κB pathway to apoptotic resistance in metaplastic Barrett's epithelial cells.

Cell culture. We used two telomerase-immortalized, esophageal squamous cell lines created from endoscopic biopsy specimens of NES mucosa taken from the distal esophagus of patients who had GERD with (NES-B3T) and without (NES-G2T) Barrett's esophagus. We also used three telomerase-immortalized, Barrett's epithelial cell lines (BAR-T, BAR-T 9, and BAR-T 10) that were created from endoscopic biopsy specimens of nondysplastic Barrett's specialized intestinal metaplasia taken from three patients with long segment (>3 cm) Barrett's esophagus (14–16). As controls, we used normal rat intestinal epithelial cells (IEC-6) and normal mouse intestinal epithelial cells (MSIE; refs. 17, 18). All cell lines were cultured in their respective growth medium as previously described (14, 15, 17, 18). MSIE cells are derived from the Immortomouse and, when cultured at 33°C with 5% CO_2, are immortal (18, 19). All other cell lines were maintained in monolayer culture at 37°C in humidified air with 5% CO₂. The telomerase-immortalized NES and Barrett's cell lines were cocultured with a fibroblast feeder layer as previously described (20). Before UV-B irradiation, subconfluent cells were placed in minimal medium (no serum and lacking growth factors) overnight at 37°C and 5% CO₂.

UV-B irradiation. Genomic damage caused by UV-B irradiation is a well-established inducer of apoptosis and p53 expression (21). We used UV-B irradiation to induce apoptosis in our cells, which was carried out with a homemade box built with four UV-B bulbs (280–320 nm wavelength; Philips Lighting). The applied UV-B dose was measured using a radiometer (IL-1400A Radiometer; International/Photometer). Equally seeded wells of cells at 70% confluence were irradiated with 50, 100, 200, and 400 J/m² of UV-B. Twenty-four hours later, cells were collected for analysis.

Detection of apoptosis. Apoptosis rates were assessed quantitatively using Annexin V (BD Biosciences) as previously described (22). Briefly, cells were harvested by trypsinization, stained with Annexin V-FITC, and 50 μg/mL of propidium iodide, then immediately analyzed by flow cytometry (FACScaliber; Becton Dickson); cells staining only with Annexin V were considered to be apoptotic (22). Experiments were performed in triplicate, and a total of 10,000 cells were analyzed in each individual experiment.

Western blotting. The cells were harvested by scraping into lysis buffer containing 150 mmol/L NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 50 mmol/L Tris-HCl (pH 7.4), 0.1 mmol/L phenylmethylsulfonyl fluoride, and 1 Protease Inhibitor Cocktail Tablet per 50 mL of lysis buffer (Roche Applied Science). Equal amounts of protein were separated by SDS-PAGE; protein concentrations were determined using the BCA-200 Protein Assay kit (Pierce). After separation and transfer to nitrocellulose membrane, membranes were incubated with 1:1,000 dilutions of rabbit polyclonal antibodies anti-human, anti–phospho-H2AX, anti–NF-κB, anti-Bcl2, and antibodies against the cIAPS: survivin, XIAP, and cIAP-1 (Cell Signaling, Inc.) Horseradish peroxidase secondary antibody (Cell Signaling) was used at 1:2,000, and chemiluminescence was determined using the ECL detection system (Pierce). Membranes were then stripped using Restore Stripping Buffer (Pierce) and were reprobed with β-actin (Sigma) to confirm equal loading.

Inhibition of the NF-κB signaling pathway. Bay11-7085 (Sigma), is a pharmacologic inhibitor that blocks IκBα phosphorylation. Cells were treated with Bay11-7085 at concentrations ranging from 1 to 10 μmol/L for 1 h as described, followed by UV-B irradiation (23). Because pharmacologic agents can have nonspecific effects, we confirmed the role of NF-κB/p65 in mediating effects on apoptosis using a specific NF-κB/p65 siRNA. We also used a specific siRNA against Bcl-2, a downstream NF-κB target gene implicated in apoptotic resistance of Barrett's esophagus (9, 24). Cells were plated at 50% confluence in 6-well tissue culture plates. After 24 h, the cells were transfected using the SignalSilence NF-κB p65 or Bcl-2 siRNA (Cell Signaling Technology), per the manufacturer's instructions. Forty-eight hours after transfection, cells were exposed to UV-B irradiation. As a control, cells were transfected with an siRNA oligonucleotide against lamin A/C (Millipore).

Statistical analyses. The data were collected from at least three independent experiments. Quantitative data are expressed as the mean plus (±) the SE. Statistical analysis was performed using ANOVA and the Student-Newman-Keuls multiple comparison test with the Instat for Windows statistical software package (GraphPad Software). P values of <0.05 were considered significant for all analyses.

Low-dose UV-B irradiation (50 and 100 J/m²) induces DNA damage in NES cells and in Barrett's epithelial (BAR-T) cells but induces apoptosis only in NES cells. Clinical studies suggest that Barrett's cells are more resistant to apoptosis than NES cells. We used UV-B irradiation, a well-established inducer of DNA damage and apoptosis, to verify those clinical data in our benign, telomerase-immortalized cells (21). NES-G2T, NES-B3T, BAR-T, BAR-T9, and BAR-T10 cells all were grown to 70% confluence and placed in minimal medium overnight. Cells were then exposed to UV-B irradiation at doses known to cause DNA damage and to induce apoptosis in normal cells (21). Twenty-four hours later, DNA damage was assessed by expression of phospho-H2AX; apoptosis was determined by Annexin V staining. As expected, we found that UV-B irradiation induced a dose-dependent increase in DNA damage in BAR-T, NES-G2T, and NES-B3T cells (Fig. 1).

We previously reported that UV-B irradiation at doses of 50 and 100 J/m² did not induce apoptosis in BAR-T cells, although apoptosis was observed with higher doses (200 and 400 J/m²; ref. 22). In NES-G2T and NES-B3T cells, we found that UV-B irradiation induced apoptosis in a dose-dependent manner and, in contrast to the BAR-T cells, even doses as low as at 50 J/m² caused apoptosis (Fig. 2). Moreover, the level of apoptosis induced by just 100 J/m² in our NES cell lines (NES-G2T, 57.8% ± 2.5%; NES-B3T, 59.5% ± 0.50%) was significantly higher than that induced by 400 J/m² in the BAR-T cells (41.2% ± 2.7% SE; P < 0.05; ref. 22).

To confirm that these findings were not unique to the BAR-T Barrett's cell line, we exposed two additional telomerase-immortalized Barrett's epithelial cell lines (BAR-T9 and BAR-T10) to UV-B irradiation and determined the effects on DNA damage and apoptosis. Similar to the BAR-T cells, we found that low-dose UV-B irradiation increased phospho-H2AX expression in the BAR-T9 and BAR-T10 cells (Fig. 1). In addition, UV-B irradiation at 200 and 400 J/m², but not at 50 or 100 J/m², induced apoptosis in BAR-T9 and BAR-T10 cells (Fig. 2). Similar to the BAR-T cells, the level of apoptosis induced by just 100 J/m² in our NES cell lines (NES-G2T, 57.8% ± 2.5%; NES-B3T, 59.5% ± 0.50%) was significantly higher than that induced by 400 J/m² in the BAR-T cell lines (BAR-T9, 38.3% ± 0.30% SE; BAR-T10, 39.4% ± 0.21% SE; P < 0.05 for BAR-T9 and BAR-T10) cells. This shows that apoptotic resistance to low-dose UV-B irradiation is not restricted to a single cell line but rather seems to be a common feature of Barrett's epithelial cells.

Resistance to UV-B–induced apoptosis is not due to the intestinal phenotype of Barrett's epithelial cells. To determine whether the differences we observed in resistance to apoptosis in response to low-dose UV-B irradiation were due primarily to differences in cell phenotype (i.e., intestinal-type columnar cells versus squamous cells), we treated normal rat intestinal epithelial cells (IEC-6) and normal MSIEs with UV-B irradiation, and assessed DNA damage and apoptosis in response to genotoxic injury. As expected, both columnar cell lines exhibited an increase in DNA damage after UV-B irradiation (Fig. 3A). Unlike the Barrett's cells, however, the IEC-6 and MSIE intestinal cell lines showed a significant increase in apoptosis even after low-dose UV-B irradiation (Fig. 3B).

The NF-κB pathway is activated by low-dose UV-B irradiation in BAR-T cells but not in NES cells. Having found a difference in apoptosis induction by low-dose UV-B irradiation between esophageal squamous and metaplastic Barrett' cells, we next sought to determine the contribution of the NF-κB pathway to that difference. We performed Western blotting for NF-kB/p65 and its target genes products (Bcl-2, cIAP-1, XIAP, and survivin) 24 hours after UV-B irradiation. As shown in Fig. 4, low-dose UV-B irradiation (50 and 100 J/m²) increased the expression of NF-κB, Bcl-2, XIAP, cIAP2, and survivin proteins in a dose-dependent manner in BAR-T cells but decreased their expression in NES-G2T and NES-B3T cells. We also found that low-dose UV-B irradiation increased NF-κB expression in BAR-T9 and BAR-T10 (data not shown). These data suggest that the NF-κB pathway is activated by low-dose UV-B irradiation in BAR-T but not in NES cells. Moreover, we found that 50 J/m² UV-B irradiation virtually abolished NF-κB and XIAP protein expression in NES-B3T cells, but not in NES-G2T cells, which continued to express these proteins at lower levels.

Inhibition of NF-κB sensitizes BAR-T cells to apoptosis induced by low-dose UV-B irradiation. Having found that low-dose UV-B irradiation increases NF-κB expression in BAR-T cells, we next sought to determine whether NF-κB contributes to their resistance to apoptosis. We inhibited NF-κB activity using both Bay 11-7085 (a pharmacologic inhibitor) and a specific siRNA against NF-κB/p65. Cells were treated with concentrations of Bay 11-7085 ranging from 0 to 10 μmol/L and then irradiated with 50 to 400 J/m² of UV-B. In BAR-T cells, the resistance to apoptosis in response to low-dose UV-B irradiation was abolished by treatment with Bay 11-7085 in a dose-dependent fashion; Bay 11-7085 also induced a dose-dependent increase in the rates of apoptosis after 200 and 400 J/m² of UV-B, with the maximal rate of apoptosis found to plateau with 5 μmol/L of Bay 11-7085 (Fig. 5A). We next confirmed our findings by transfecting the cells with a NF-κB/p65 siRNA and determining the rates of apoptosis after low-dose UV-B irradiation. To determine the efficiency of NF-κB/p65 siRNA on inhibiting NF-κB/p65 expression, Western blotting was performed at baseline and after irradiation with 100 J/m², a condition that we found increases NF-κB expression. We found complete loss of NF-κB/p65 expression after transfection with siRNA at both conditions (Fig. 5B). Transfection of the BAR-T cells with siRNA against the p65 subunit of NF-κB resulted in a significant increase in apoptosis after low-dose UV-B irradiation; the siRNA against NF-κB/p65 also significantly increased the rates of apoptosis after 200 and 400 J/m² of UV-B (Fig. 5B). In contrast, low-dose UV-B irradiation did not cause apoptosis in control cells that were transfected with a siRNA targeted against lamin A/C, suggesting that the apoptosis induced by low-dose UV-B was indeed due to inhibition of NF-κB and not due to a nonspecific effect of the siRNA (Fig. 5B).

Inhibition of Bcl-2 sensitizes BAR-T cells to apoptosis induced by low-dose UV-B irradiation. Because Bcl-2 is a downstream target gene of NF-κB and clinical data suggest a role for Bcl-2 expression during the early stages of carcinogenesis in Barrett's esophagus, we next sought to determine whether Bcl-2 expression contributes to resistance to apoptosis of metaplastic Barrett's epithelial cells (9, 24). We transfected BAR-T cells with Bcl-2 siRNA oligonucleotides and determined the rates of apoptosis after UV-B irradiation. To determine the efficiency of siRNA for inhibiting Bcl-2 expression, Western blotting was performed at baseline and after irradiation with 400 J/m², a condition that we found increases Bcl-2 protein expression. We noted complete loss of Bcl-2 expression after transfection with the siRNA both at baseline and after UV-B irradiation (Fig. 6A). We found that transfection of the BAR-T cells with siRNA against Bcl-2 resulted in only a small but statistically significant increase in apoptosis after low-dose UV-B irradiation; the siRNA against Bcl-2 also slightly increased the rates of apoptosis after 200 and 400 J/m² of UV-B (Fig. 6B). Low-dose UV-B irradiation did not cause apoptosis in control cells that were transfected with a siRNA targeted against lamin A/C.

Studies using esophageal biopsy specimens have suggested that Barrett's epithelial cells are more resistant to apoptosis than normal squamous epithelial cells. Biopsy specimens contain inflammatory cells and stromal cells in addition to epithelial cells, and in biopsy specimens, it can be difficult to distinguish direct effects of an agent on epithelial cells from indirect effects that are mediated by inflammatory and stromal cells. Conceivably, the apoptotic resistance reported for Barrett's epithelial biopsy specimens might be mediated by inflammatory and stromal cells rather than by the epithelial cells. To eliminate the potential confounding effects of these nonepithelial cells, we have used cultures of benign, telomerase-immortalized esophageal epithelial cells to study the direct effects of a toxic agent on apoptosis.

To determine whether Barrett's epithelial cells are more resistant to apoptosis than esophageal squamous epithelial cells, we exposed benign, telomerase-immortalized squamous (NES) and Barrett's (BAR-T) cell lines to UV-B irradiation. Although UV-B irradiation clearly is not a physiologically relevant agent in the esophagus, UV-B is an extensively studied and well-established inducer of DNA damage and apoptosis in a number of different cell types (21, 25). As expected, we found that UV-B irradiation in all doses studied (50, 100, 200, and 400 J/m²) induced DNA damage (detected by phospho-H2AX expression) in Barrett's and squamous epithelial cells alike. In the esophageal squamous cell lines (NES-G2T and NES-B3T), we found that UV-B irradiation in all doses studied increased the rate of apoptosis in a dose-dependent fashion. A similar UV-B dose-response on apoptosis has been observed in human keratinocytes (21). In three Barrett's epithelial cell lines, in contrast, we found that low-dose UV-B irradiation (50 and 100 J/m²) did not increase apoptosis, although higher doses (200 and 400 J/m²) did have proapoptotic effects (22). Taken together, these studies show that the epithelial cells of Barrett's esophagus are more resistant to apoptosis than esophageal squamous epithelial cells, and that Barrett's epithelial cells can resist apoptosis in the face of genotoxic injury.

To determine whether the differences we observed in resistance to apoptosis of Barrett's epithelial cells were due primarily to differences in cell phenotype (i.e., intestinal-type columnar cells versus squamous cells), we treated normal rat intestinal epithelial cells (IEC-6) and normal MSIEs with UV-B irradiation, and assessed DNA damage and apoptosis. Both of these columnar cell lines exhibited DNA damage after UV-B irradiation. Unlike the BAR-T intestinal-type columnar cells, however, the IEC-6 and MSIE intestinal cell lines showed apoptosis after low-dose UV-B irradiation. Thus, the apoptotic resistance of Barrett's epithelial cells is not due solely to their intestinal phenotype.

Proapoptotic signals can be counterbalanced by the expression of antiapoptotic proteins whose synthesis is mediated by NF-κB. Clinical studies have suggested a role for NF-κB and its downstream targets Bcl-2 and Bcl-xL in the apoptotic resistance of Barrett's metaplasia (7, 9, 12, 13). Another important family of NF-κB target genes are the inhibitors of apoptosis (IAP), which block the activities of the caspases (26, 27). To our knowledge, there are no published data on the expression of IAP family members in Barrett's esophagus. We found that low-dose UV-B irradiation (50 and 100 J/m²) increased the expression of NF-κB, Bcl-2, XIAP, cIAP-1, and survivin proteins in a dose-dependent manner in BAR-T cells but decreased the expression of those same proteins in NES-G2T and NES-B3T cells. These findings suggest that activation of the NF-κB pathway contributes to apoptotic resistance in Barrett's epithelial cells.

To show a mechanistic link between NF-κB activation and resistance to UV-B–induced apoptosis in our metaplastic Barrett's cells, we treated them with Bay 11-7085, a pharmacologic inhibitor of NF-κB activity, or with NF-κB/p65 siRNA before UV-B irradiation. We found that both of these inhibitors abolished resistance to apoptosis induced by low-dose UV-B irradiation in BAR-T cells, suggesting that the NF-κB pathway mediates the apoptotic resistance of metaplastic Barrett's cells. Using an siRNA against Bcl-2, we also explored whether this NF-κB downstream antiapoptotic protein played a role in this apoptotic resistance. We found that inhibition of Bcl-2 caused a small but statistically significant increase in apoptosis after low-dose UV-B irradiation. This suggests that Bcl-2 may contribute to apoptotic resistance, but that other NF-κB–dependent antiapoptotic proteins (perhaps the IAP family members) play a role as well. Further studies are warranted to determine the specific proteins involved in this phenomenon. Using our esophageal squamous cell lines, we found that 50 J/m² UV-B irradiation virtually abolished NF-κB and XIAP protein expression in NES-B3T cells (from a patient with Barrett's esophagus) but not in NES-G2T cells (from a patient who had GERD without Barrett's esophagus). This suggests that the esophageal squamous epithelium of Barrett's patients may be more susceptible to apoptosis induced by genotoxic injury than the squamous epithelium of GERD patients who do not develop Barrett's esophagus. We and others have previously reported differences in reflux-induced activation of proproliferative molecular signaling pathways between esophageal squamous cells from GERD patients with and without Barrett's esophagus that might play a role in determining whether reflux esophagitis heals through metaplasia or through squamous cell regeneration (15, 28, 29). If esophageal squamous cells have a similar response to toxic agents in refluxed gastric juice as they do to UV-B irradiation, then it is conceivable that differences in the expression of antiapoptotic proteins might determine whether the squamous epithelium would remain intact or would succumb to apoptosis and require repair, perhaps by metaplastic Barrett's epithelial cells that express NF-κB. Further investigations are needed in this area as well.

In conclusion, we have shown that Barrett's epithelial cells are more resistant to apoptosis than NES epithelial cells in the face of genotoxic injury induced by low-dose UV-B irradiation. Moreover, this apoptotic resistance is a unique property of benign Barrett's epithelial cells, not merely a feature of their intestinal phenotype. We have also found that low-dose UV-B irradiation increases the expression of NF-κB, Bcl-2, cIAP-1, XIAP, and survivin proteins in a dose-dependent fashion in Barrett's cells, but decreases the expression of those proteins in esophageal squamous cells, suggesting that activation of the NF-κB pathway mediates resistance to UV-B–induced apoptosis in Barrett's epithelial cells. In addition, we have found differences in expression of NF-κB and XIAP in response to low-dose UV-B irradiation between our esophageal squamous cell lines from patients with and without Barrett's esophagus, suggesting that differences in apoptotic resistance pathways might contribute to the pathogenesis of Barrett's metaplasia. Finally, the resistance to low-dose UV-B–induced apoptosis in Barrett's cells could be abolished by treatment with inhibitors of NF-κB and Bcl-2. These findings show that activation of the NF-κB pathway plays a mechanistic role in the resistance to apoptosis of Barrett's epithelial cells and suggest that this pathway may be a novel target at which to direct therapies to prevent the development and neoplastic progression of Barrett's esophagus.

No potential conflicts of interest were disclosed.

Grant support: Office of Medical Research, Department of Veteran's Affairs, Dallas, TX (R.F. Souza), the Harris Methodist Health Foundation, Dr. Clark R. Gregg Fund (R.F. Souza and K. Hormi-Carver), the NIH R01-DK63621 (R.F. Souza), and R01-HL067256 and R01-HL61897 (L.S. Terada), and the Vanderbilt University Medical Center's Digestive Disease Research Center NIH DK058404 to (R.H. Whitehead).

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