In Non-neoplastic Barrett’s Epithelial Cells, Acid Exerts Early Antiproliferative Effects through Activation of the Chk2 Pathway Hui-Ying Zhang, 1 Xi Zhang, 1 Kathy Hormi-Carver, 1 Linda A. Feagins, 1 Stuart J. Spechler, 1 and Rhonda F. Souza 1,2 1 Department of Medicine, Dallas Veterans Affairs Medical Center and the University of Texas Southwestern Medical School, and 2 Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas Abstract Acid exerts pro-proliferative effects in Barrett’s-associated esophageal adenocarcinoma cells. In non-neoplastic Barrett’s epithelial (BAR-T) cells, in contrast, we have shown that acid exposure has antiproliferative effects. To explore our hypothesis that the acid-induced, antiproliferative effects are mediated by alterations in the proteins that regulate the G 1 -S cell cycle checkpoint, we exposed non-neoplastic Barrett’s cells to acidic media (pH 4.0) and analyzed G 1 -S checkpoint proteins’ expression, phosphorylation, and activity levels by Western blot. We studied acid effects on growth (by cell counts), proliferation (by flow cytometry and bromodeoxyur- idine incorporation), cell viability (by trypan blue staining), and apoptosis (by annexin V staining), and we used caffeine and small interfering RNA to assess the effects of checkpoint kinase 2 (Chk2) inhibition on G 1 -S progression. Acid exposure significantly decreased cell numbers without affecting cell viability and with only a slight increase in apoptosis. Within 2 h of acid exposure, there was a delay in progression through the G 1 -S checkpoint that was associated with increased phosphorylation of Chk2, decreased levels of Cdc25A, and decreased activity of cyclin E–cyclin-dependent kinase 2; by 4 h, a continued delay at G 1 -S was associated with increased expression of p53 and p21. Caffeine and Chk2 siRNA abolished the acid-induced G 1 -S delay at 2 but not at 4 h. We conclude that acid exposure in non-neoplastic BAR-T cells causes early antiproliferative effects that are mediated by the activation of Chk2. Thus, we have elucidated a mechanism whereby acid can exert disparate effects on proliferation in neoplastic and non-neoplastic BAR-T cells. [Cancer Res 2007;67(18):8580–7] Introduction In the last three decades, the incidence of esophageal adenocarcinoma has increased f600% in the United States (1, 2). The major risk factors for this cancer are gastroesophageal reflux disease (GERD) and Barrett’s esophagus, the condition in which a metaplastic, intestinal-type epithelium replaces esophageal squa- mous epithelium that has been damaged by GERD. This metaplastic epithelium is predisposed to malignancy, and most esophageal adenocarcinomas are thought to arise from Barrett’s esophagus (3, 4). It has been proposed that acid reflux promotes the progression of Barrett’s metaplasia to esophageal adenocarcinoma, based largely on the observations that acid exposure can induce pro- proliferative effects in cultures of Barrett’s biopsy specimens (explants) and in cultures of Barrett’s-associated esophageal adenocarcinoma cells (5–7). Those in vitro model systems are notidealforthestudyoftheearlycarcinogeneticeventsinBarrett’s epithelial (BAR-T) cells, however. Explants of Barrett’s mucosa comprise both epithelial and non-epithelial cell types (e.g., inflammatory and stromal cells), and it is difficult to ascertain which of the diverse cell types proliferate in response to acid. Barrett’s-associated adenocarcinoma cells have numerous unchar- acterized genetic abnormalities, and acid-induced events in those cells may not reflect the effects of acid on non-neoplastic BAR-T cells. To develop an in vitro model for Barrett’s esophagus that avoids the pitfalls of explants and adenocarcinoma cell cultures, we have established cultures of BAR-T cells that are immortalized, but not transformed, through the forced expression of telomerase (8). We have shown that these cells retain a diploid karyotype, express differentiation markers typical of benign Barrett’s epithelium, and exhibit phenotypic features characteristic of non-neoplastic cells, including contact inhibition, anchorage-dependent growth, and the expression of functional cell cycle checkpoint proteins (8). Using cultures of BAR-T cells, we have found that acid exerts antipro- liferative effects (9). Cellular proliferation is a manifestation of passage through the cell cycle, which is regulated at various checkpoints. Genomic damage can activate cell cycle checkpoints to halt progression through the cycle, enabling the cell to repair its DNA damage or, if the damage is extensive and irreparable, to trigger cellular destruction by apoptosis (10). Many crucial cell fate decisions are made at the key checkpoint that regulates the transition from G 1 to S phase. Therefore, a potential mechanism whereby acid exposurecouldinfluenceproliferationandapoptosisinBAR-Tcells is by affecting the proteins that regulate the G 1 -S cell cycle checkpoint. A number of proteins are known to influence passage through the G 1 -S cell cycle checkpoint. For example, Cdc25A can activate the cyclin E–cyclin-dependent kinase 2 (Cdk2) complex, which enables progression through the G 1 -S checkpoint (10, 11). In contrast, progression through the checkpoint can be blocked by activating the p53 pathway, which results in the transcription of genes like p21 whose products inhibit the actions of cyclin-Cdk complexes. G 1 -S progression can also be delayed by activating the checkpoint kinases 1 and 2 (Chk1 and Chk2), which mediate the degradation of Cdc25A (10). To investigate mechanisms whereby acid reflux might influence proliferation and apoptosis in Barrett’s Requests for reprints: RhondaF.Souza,DepartmentofGastroenterology,MC111B1, Dallas Veterans Affairs Medical Center, 4500 South Lancaster Road, Dallas, TX 75216. Phone: 214-857-0301; Fax: 214-857-0328; E-mail: rhonda.souza@UTSouthwestern.edu. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-07-2023 Cancer Res 2007; 67: (18). September 15, 2007 8580 www.aacrjournals.org Research Article Downloaded from http://aacrjournals.org/cancerres/article-pdf/67/18/8580/2575806/8580.pdf by guest on 16 February 2023