High Content Imaging of Barrett's-Associated High-Grade Dysplasia Cells After siRNA Library Screening Reveals Acid-Responsive Regulators of Cellular Transitions.

BACKGROUND & AIMS: Esophageal adenocarcinoma (EAC) develops from within Barrett's esophagus (BE) concomitant with gastroesophageal reflux disease (GERD). Wound healing processes and cellular transitions, such as epithelial-mesenchymal transitions, may contribute to the development of BE and the eventual migratory escape of metastatic cancer cells. Herein, we attempt to identify the genes underlying esophageal cellular transitions and their potential regulation by the low pH environments observed in GERD and commonly encountered by escaping cancer cells. METHODS: Small interfering RNA library screening and high-content imaging analysis outlined changes in BE high-grade dysplasia (HGD) and EAC cell morphologies after gene silencing. Gene expression microarray data and low pH exposures studies modeling GERD-associated pulses (pH 4.0, 10 min) and tumor microenvironments (pH 6.0, constant) were used. RESULTS: Statistical analysis of small interfering RNA screening data defined 207 genes (Z-score >2.0), in 12 distinct morphologic clusters, whose suppression significantly altered BE-HGD cell morphology. The most significant genes in this list included KIF11, RRM2, NUBP2, P66BETA, DUX1, UBE3A, ITGB8, GAS1, GPS1, and PRC1. Guided by gene expression microarray study data, both pulsatile and constant low pH exposures were observed to suppress the expression of GPS1 and RRM2 in a nonoverlapping temporal manner in both BE-HGD and EAC cells, with no changes observed in squamous esophageal cells. Functional studies uncovered that GPS1 and RRM2 contributed to amoeboid and mesenchymal cellular transitions, respectively, as characterized by differential rates of cell motility, pseudopodia formation, and altered expression of the mesenchymal markers vimentin and E-cadherin. CONCLUSIONS: Collectively, we have shown that low pH microenvironments associated with GERD, and tumor invasive edges, can modulate the expression of genes that triggered esophageal cellular transitions potentially critical to colonization and invasion.

Types of cell death and apoptotic stages in Chinese Hamster Ovary cells distinguished by Raman spectroscopy.

Cell death is the ultimate cause of productivity loss in bioreactors that are used to produce therapeutic proteins. We investigated the ability of Raman spectroscopy to detect the onset and types of cell death for Chinese Hamster Ovary (CHO) cells-the most widely used cell type for therapeutic protein production. Raman spectroscopy was used to compare apoptotic, necrotic, autophagic, and control CHO cells. Several specific nucleic acid-, protein-, and lipid-associated marker bands within the 650-850 cm-1 spectral region were identified that distinguished among cells undergoing different modes of cell death; supporting evidence was provided by principal component analysis (PCA) of the full spectral data. In addition to comparing the different modes of cell death, normal cells were compared to cells sorted at several stages of apoptosis, in order to explore the potential for early detection of apoptosis. Different stages of apoptosis could be distinguished via Raman spectroscopy, with multiple changes observed in nucleic acid peaks at early stages whereas an increase in lipid signals was a feature of late apoptosis/secondary necrosis.

High-content screening identifies a role for Na(+) channels in insulin production.

Insulin production is the central feature of functionally mature and differentiated pancreatic ß-cells. Reduced insulin transcription and dedifferentiation have been implicated in type 2 diabetes, making drugs that could reverse these processes potentially useful. We have previously established ratiometric live-cell imaging tools to identify factors that increase insulin promoter activity and promote ß-cell differentiation. Here, we present a single vector imaging tool with eGFP and mRFP, driven by the Pdx1 and Ins1 promoters, respectively, targeted to the nucleus to enhance identification of individual cells in a high-throughput manner. Using this new approach, we screened 1120 off-patent drugs for factors that regulate Ins1 and Pdx1 promoter activity in MIN6 ß-cells. We identified a number of compounds that positively modulate Ins1 promoter activity, including several drugs known to modulate ion channels. Carbamazepine was selected for extended follow-up, as our previous screen also identified this use-dependent sodium channel inhibitor as a positive modulator of ß-cell survival. Indeed, carbamazepine increased Ins1 and Ins2 mRNA in primary mouse islets at lower doses than were required to protect ß-cells. We validated the role of sodium channels in insulin production by examining Nav1.7 (Scn9a) knockout mice and remarkably islets from these animals had dramatically elevated insulin content relative to wild-type controls. Collectively, our experiments provide a starting point for additional studies aimed to identify drugs and molecular pathways that control insulin production and ß-cell differentiation status. In particular, our unbiased screen identified a novel role for a ß-cell sodium channel gene in insulin production.