Acid induces cell cycle arrest via increases in p53 and p21 in metaplastic Barrett’s cells

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Introduction: Reflux of acidic gastric contents has been established as a risk factor for Barrett’s esophagus and its sequela, esophageal adenocarcinoma. Acid exposure has been shown to induce proliferation in ex vivo cultures of Barrett’s esophagus and in esophageal adenocarcinoma cells in vitro. However, ex vivo cultures were established from biopsy tissues whose molecular alterations were not well characterized. Moreover, Barrett’s-associated cancer cell lines, which have acquired numerous genetic abnormalities, are suboptimal reagents for studies on the effects of acid exposure in promoting early carcinogenic events in Barrett’s esophagus. Therefore, we sought to determine the effects of acid exposure on proliferation and apoptosis in a non-neoplastic, in vitro model of metaplastic Barrett’s cells. Methods: Equally seeded telomerase-immortalized, p16 null, metaplastic Barrett’s cells were placed in acidic media (pH 4) for a single 3 minute exposure or two 3 minute exposures 10, 60, and 120 minutes apart. Cell growth was determined at 24 hours by cell counts. Two 3 minute acid exposures 60 minutes apart were then selected for all subsequent experiments. At 24 hours, cell viability of non-adherent cells was assessed by trypan blue; apoptosis rates of adherent cells were assessed by TUNEL. p53 and p21 expression were determined by Western blot on cell lysates collected at 0, 6, 24, and 48 hours after the acid exposures. Results: Compared to controls, multiple acid exposures significantly decreased cell numbers (p<0.0001); no significant decrease was seen following a single acid exposure. There was a dose-dependent decrease in cell number from a single acid exposure (96.1 + 1.67% SEM) to two exposures 60 minutes apart (83.5 + 2.0% SEM); there was no further decrease in cell number by two acid exposures 120 minutes apart. There was no significant difference in cell viability between controls and cells treated with two acid exposures 60 minutes apart. Under the same conditions, few apoptotic cells were seen in the controls and the acid treated cells by TUNEL. Compared to untreated controls, p53 expression increased by 144% and p21 expression by 333% 6 hours following two acid exposures 60 minutes apart; by 24 hours, p53 and p21 expression were less than that of controls (45% and 79%, respectively). Conclusions: Multiple, but not single acid exposures significantly decrease cell number in metaplastic Barrett’s cells. The decrease in cell number by acid is dose-dependent up to exposures separated by 60 minutes. The decrease in cell number is not due to a decrease in cell viability or to an increase in apoptosis. Increased expression of p53 and p21 suggests that multiple acid exposures induce check-point cell cycle arrest. We speculate that the molecular pathways leading to cell cycle arrest following multiple acid exposures are targeted for inactivation during neoplastic progression of Barrett’s esophagus.