Kathy Hormi-Carver

Acid has antiproliferative effects in nonneoplastic Barrett's epithelial cells.

OBJECTIVES:For patients with Barretts esophagus, physicians commonly prescribe antisecretory medications in dosages above those required to heal reflux esophagitis because acid has been shown to have proproliferative and antiapoptotic effects on Barretts cancer cells and on Barretts mucosal explants. For a number of reasons, these model systems may not be ideal for determining the effects of acid on benign Barretts epithelial cells, however. We studied the effects of acid on proliferation and apoptosis in a nonneoplastic, telomerase-immortalized Barretts epithelial cell line.METHODS:Barretts cells were treated with two 3-minute exposures to acidic media. Cell growth was determined using cell counts, proliferation was studied by flow cytometry, cell viability was determined by trypan blue staining, and apoptosis was assessed by TUNEL and Annexin V. The expression levels of p53 and p21 were determined by Western blotting. p53 siRNA was used to study the effect of p53 inhibition on total cell numbers after acid exposure.RESULTS:Acid exposure significantly decreased total cell numbers at 24 h without affecting either cell viability or apoptosis. Acid exposure resulted in cell cycle prolongation that was associated with greater expression of p53, but not p21. The acid-induced decrease in total cell numbers was abolished by p53 RNAi.CONCLUSIONS:Acid exposure has p53-mediated, antiproliferative effects in nonneoplastic Barretts epithelial cells. These findings contradict the results of prior in vitro and ex vivo studies. We speculate that the prescription of antisecretory medications in dosages beyond those required to heal gastroesophageal reflux disease (GERD) symptoms and endoscopic signs could be detrimental. Controlled, prospective clinical trials are needed to determine the optimal level of acid suppression for patients with Barretts esophagus.

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

Apoptosis is an important mechanism for maintaining tissue homeostasis and for preventing the proliferation of cells with mutations that could result in malignancy. Barretts epithelium has been reported to be more resistant to apoptosis than normal esophageal squamous epithelium. We have explored the contribution of the nuclear factor-kappaB (NF-kappaB) pathway to apoptotic resistance in non-neoplastic, telomerase-immortalized esophageal squamous (NES) and Barretts (BAR-T) epithelial cell lines. We exposed these cells to UV-B irradiation in doses known to cause DNA damage and to induce apoptosis in normal cells, and studied apoptosis as well as the expression of phospho-H2AX, NF-kappaB, Bcl-2, XIAP, cIAP-1, and survivin proteins. We also used Bay 11-7085 and siRNAs to NF-kappaB and Bcl-2 to assess the effects of NF-kappaB and Bcl2 inhibition on apoptosis. UV-B irradiation at low doses (50 and 100 J/m(2)) caused DNA damage in both NES and BAR-T cells but significantly increased apoptosis only in NES cells. UV-B irradiation caused a decrease in the levels of NF-kappaB, Bcl-2, cIAP-1, XIAP, and survivin in NES cells but increased the levels of those proteins in BAR-T cells. The resistance of BAR-T cells to apoptosis induced by low-dose UV-B irradiation was abolished by Bay 11-7085 and by siRNA for NF-kappaB and was decreased significantly by siRNA for Bcl-2. We conclude that the ability of Barretts epithelial cells to activate the NF-kappaB pathway when they have sustained DNA damage allows them to resist apoptosis. This capacity to avoid apoptosis despite genotoxic damage may underlie the persistence and malignant predisposition of Barretts metaplasia.

Differences in activity and phosphorylation of MAPK enzymes in esophageal squamous cells of GERD patients with and without Barrett's esophagus

We hypothesized that, in esophageal squamous epithelial cells, there are differences among individuals in the signal transduction pathways activated by acid reflux that might underlie the development of Barretts esophagus. To explore that hypothesis, we immortalized nonneoplastic, esophageal squamous cells from patients with gastroesophageal reflux disease (GERD) with (NES-B3T) and without (NES-G2T) Barretts esophagus and used those cells to study acid effects on MAPK proteins. During endoscopy in patients with GERD with and without Barretts esophagus, we took biopsy specimens from the distal squamous esophagus to study MAPK proteins before and after esophageal perfusion with 0.1 N HCl. We used immunoblotting and Western blotting to study MEK1/2 phosphorylation at two activating sites (serines 217/221), MEK1 phosphorylation at an inhibitory site (threonine 286), and MEK1/2 activity. After acid exposure, both cell lines exhibited increased MEK1/2 phosphorylation at the activating sites; the NES-B3T cells had higher levels of MEK1 phosphorylation at the inhibitory site, however, and only the NES-G2T cells showed an acid-induced increase in MEK1/2 activity. Similarly, in the squamous epithelium of patients with GERD with and without Barretts esophagus, acid perfusion increased MEK1/2 phosphorylation at the activating sites in both patient groups; the Barretts patients had higher levels of MEK1 phosphorylation at the inhibitory site, however, and only the patients without Barretts demonstrated an acid-induced increase in ERK1/2 phosphorylation. In esophageal squamous cell lines and biopsies from patients with GERD with and without Barretts esophagus, we have found differences in MAPK pathways activated by acid exposure. We speculate that these differences might underlie the development of Barretts metaplasia.

Molecular Markers and Genetics in Cancer Development

This article focuses on the conceptual basis underlying the acquisition of each of the physiologic cancer hallmarks by metaplastic Barretts cells. The acquired genetic alterations that have shown the most promise as potential molecular biomarkers to predict neoplastic progression in patients with Barretts esophagus are reviewed. Moreover, the role of stem cells and stem cell markers in Barretts carcinogenesis is addressed.

W1966 Unlike Deoxycholic Acid, Ursodeoxycholic Acid Does Not Cause DNA Damage or Activate the NF-κB Pathway in Barrett's Metaplasia In Vitro and In Vivo

namely Hi-maize 958 and Hi-maize 260. Probiotic; control diet plus lyophilized cultures (1x10^11 cfu/g) of Bifidobacterium lactis at 1% by weight. Synbiotic; to each of the RS diets was added Bifidobacterium lactis at 1%. After 4 weeks on these nutritionally balanced diets, colonic neoplasms were induced by 2 weekly injections of azoxymethane (15 mg/kg B.W). Colons were resected after 30 weeks of feeding for evaluation of neoplasm formation, short chain fatty acids (SCFA), and epithelial biology. Results: Rats fed the synbiotic combination of RS with Bifidobacterium lactis significantly lowered the incidence and multiplicity of colonic neoplasms (P<0.01, using two-way factorial Poissonmodel) by over 50% compared to the Control group. There was a trend for protection by either RS alone (P=0.07) while no protection against colorectal cancer was seen in the group supplemented with only Bifidobacterium lactis. Fermentation events (SCFA, pH) were altered by the inclusion of RS into the diet while the inclusion of Bifidobacterium lactis into the diet had no significant effect on the fermentation parameters. Conclusion(s): The synbiotic combination of resistant starch and Bifidobacterium lactis significantly protects against the development of colorectal cancer in the rat-azoxymethane model to a degree not seen by the components. This suggests matching of prebiotic to probiotic might be important to maximise health benefit. (1) Le Leu, RK. et al.. Synbiotic combination of resistant starch and bifidobacterium lactis can facilitate apoptotic deletion of carcinogen-damaged cells in the rat colon. (2005) J. Nutr. 135 996-1001