Zachary Spicer

Gastric achlorhydria in H/K-ATPase-deficient (Atp4a(–/–)) mice causes severe hyperplasia, mucocystic metaplasia and upregulation of growth factors

Background:  Gastric neoplasia is common in humans, yet controversy remains over contributions of chronic achlorhydria, gastrinemia and hyperplasia, to cancer risk. To study this, mice lacking the gastric H/K‐ATPase (Atp4a(–/–) mice) were used to determine whether chronic loss of acid secretion, with attendant hypergastrinemia, predisposes to cancer phenotype.

Bcl-2/Adenovirus E1B 19 kDa Interacting Protein-3 Knockdown Enables Growth of Breast Cancer Metastases in the Lung, Liver, and Bone

The mouse breast cancer cell lines 4T1, 4T07, and 67NR are highly tumorigenic but vary in metastatic potential: 4T1 widely disseminates, resulting in secondary tumors in the lung, liver, bone, and brain; 4T07 spreads to the lung and liver but is unable to establish metastatic nodules; 67NR is unable to metastasize. The Bcl-2/adenovirus E1B 19 kDa interacting protein-3 (Bnip-3) was recently shown to be absent after hypoxia in pancreatic cancer cell lines whereas its overexpression restored hypoxia-induced cell death. We found that Bnip-3 expression increased after 6 hours of hypoxia in all cell lines tested but was highest in the nonmetastatic 67NR cells and lowest in the highly metastatic 4T1 cells. Hypoxia-induced expression of Bnip-3 in the disseminating but nonmetastatic 4T07 cells was intermediate compared with 4T1 and 67NR cells. Cleaved caspase-3, a key downstream effector of cell death, increased after 6 hours of hypoxia in the 67NR and 4T07 cells by 1.9- and 2.5-fold, respectively. Conversely, cleaved caspase-3 decreased by 45% in the highly metastatic 4T1 cells after hypoxia. Small interfering RNA oligonucleotides targeting endogenous Bnip-3 blocked cell death and increased clonigenic survival after hypoxic challenge in vitro and increased primary tumor size and enabled metastasis to the lung, liver, and sternum of mice inoculated with 4T07 cells in vivo. These data inversely correlate the hypoxia-induced expression of the cell death protein Bnip-3 to metastatic potential and suggest that loss of Bnip-3 expression is critical for malignant and metastatic evasion of hypoxia-induced cell death.

In VivoEvidence for Interferon-γ-mediated Homeostatic Mechanisms in Small Intestine of the NHE3 Na+/H+Exchanger Knockout Model of Congenital Diarrhea

Mice lacking NHE3, the major absorptive Na+/H+ exchanger in the intestine, are the only animal model of congenital diarrhea. To identify molecular changes underlying compensatory mechanisms activated in chronic diarrheas, cDNA microarrays and Northern blot analyses were used to compare global mRNA expression patterns in small intestine of NHE3-deficient and wild-type mice. Among the genes identified were members of the RegIII family of growth factors, which may contribute to the increased absorptive area, and a large number of interferon-γ-responsive genes. The latter finding is of particular interest, since interferon-γ has been shown to regulate ion transporter activities in intestinal epithelial cells. Serum interferon-γ was elevated 5-fold in NHE3-deficient mice; however, there was no evidence of inflammation, and unlike conditions such as inflammatory bowel disease, levels of other cytokines were unchanged. In addition, quantitative PCR analysis showed that up-regulation of interferon-γ mRNA was localized to the small intestine and did not occur in the colon, spleen, or kidney. These in vivodata suggest that elevated interferon-γ, produced by gut-associated lymphoid tissue in the small intestine, is part of a homeostatic mechanism that is activated in response to the intestinal absorptive defect in order to regulate the fluidity of the intestinal tract.

Genomic and Physiological Analysis of Oxygen Sensitivity and Hypoxia Tolerance in PC12 Cells

Abstract: The mechanisms by which cells adapt and respond to changes in oxygen tension remain largely unknown. Our laboratory has used the PC12 cell line to study both biophysical and molecular responses to hypoxia. This chapter summarizes our findings. We found that membrane depolarization that occurred when PC12 cells were exposed to reduced O2 was mediated by a specific potassium channel, the Kv1.2 channel. The membrane depolarization leads to increased Ca2+ conductance through a voltage‐sensitive channel, which in turn mediates the release of the neurotransmitters dopamine, adenosine, glutamate, and GABA. In addition, increased intracellular Ca2+ and other signaling systems regulate hypoxia‐induced gene expression, which contributes to the adaptive response to reduced O2+. We identified several critical signaling pathways that regulate a complex gene expression profile in PC12 cells during hypoxia. These include the cAMP‐protein kinase A, Ca2+‐calmodulin, p42/44 mitogen‐activated protein kinase (MAPK), stress‐activated protein kinase (SAPK; p38 kinase), and the phosphatidylinositol 3‐kinase‐AKT as regulators of gene expression. Several of these pathways regulate hypoxia‐specific transcription factors that are members of the hypoxia‐inducible factor (HIF) family. Recently, we have successfully used subtractive cDNA libraries and microarray analysis to identify the genomic profile that mediates the cellular response to hypoxia.

Oxygen sensing in neuroendocrine cells and other cell types: pheochromocytoma (PC12) cells as an experimental model.

A steady supply of oxygen is an absolute requirement for mammalian cells to maintain normal cellular functions. To answer the challenge that oxygen deprivation represents, mammals have evolved specialized cell types that can sense changes in oxygen tension and alter gene expression to enhance oxygen delivery to hypoxic areas. These oxygen-sensing cells are rare and difficult to study in vivo. As a result, pheochromocytoma (PC12) cells have become a vital in vitro model system for deciphering the molecular events that confer the hypoxia-resistant and oxygen-sensing phenotypes. Research over the last few years has revealed that the hypoxia response in PC12 cells involves the interactions of several signal transduction pathways (Ca2+/calmodulin-dependent kinases, Akt, SAPKs, and MAPKs) and transcription factors (HIFs, CREB, and c-fos/junB). This review summarizes the current understanding of the role these signal transduction pathways and transcription factors play in determining the hypoxic response.

The unique ultrastructure of secretory membranes in gastric parietal cells depends upon the presence of H+, K+-ATPase

Abstract. Ion transporters play a central role in gastric acid secretion. To determine whether some of these transporters are necessary for the normal ultrastructure of secretory membranes in gastric parietal cells, mice lacking transporters for H+, K+, Cl–, and Na+ were examined for alterations in volume density (Vd) of basolateral, apical, tubulovesicular and canalicular membranes, microvillar dimensions, membrane flexibility, and ultrastructure. In mice lacking Na+/H+ exchanger 1 (NHE1) or the Na+-K+-2Cl– cotransporter (NKCC1), the ultrastructure and Vd of secretory membranes and the secretory canalicular to tubulovesicular membrane ratio (SC/TV), a morphological correlate of secretory activity, were similar to those of wild-type mice. In mice lacking Na+/H+ exchanger 2 (NHE2) or gastric H+, K+-ATPase α- or β-subunits, the SC/TV ratio and Vd of secretory membranes were decreased, though canaliculi were often dilated. In H+, K+-ATPase-deficient parietal cells, canalicular folds were decreased, normally abundant tubulovesicles were replaced with a few rigid round vesicles, and microvilli were sparse, stiff and short, in contrast to the long and flexible microvilli in wild-type cells. In addition, microvilli of the H+, K+-ATPase-deficient parietal cells had centrally bundled F-actin filaments, unlike the microvilli of wild-type cells, in which actin filaments were peripherally positioned concentric to the plasmalemma. Data showed that the absence of H+, K+-ATPase produced fundamental changes in parietal cell membrane ultrastructure, suggesting that the pump provides an essential link between the membranes and F-actin, critical to the gross architecture and suppleness of the secretory membranes.

Expression of CD3 ɛ Subunit in Gastric Parietal Cells: A Possible Role in Signal Transduction?

CD3 antigen, formerly thought to be specific for T lymphocytes, has been found on gastric parietal cells in animals and humans. The common anti-CD3 antibodies recognize the epsilon subunit, which has a role in signal transduction. The aim of this study was to immunostain stomach specimens from humans and different animal species for CD3 antigen to determine if CD3 antigen is conserved across species and if CD3 antigen expression is altered in humans by use of certain drugs or the presence of gastritis. Gastric biopsies from 50 humans and necropsy sections from 13 different animals were immunostained using commercial anti CD3 epsilon antibodies on an automated immunostainer. Sections of stomach from four mice lacking the gastric H+,K(+)-ATPase alpha-subunit and four control mice were similarly immunostained. CD3 epsilon antigen expression in cytoplasm and cell membranes of gastric parietal cells was graded subjectively based on the number of positive cells. CD3 epsilon antigen was found on gastric parietal cells in all but one species studied, with varying expression in membranes and cytoplasm. There was a trend toward a decreased frequency of CD3+ cells in biopsies from patients on drugs (n = 23) compared to those on no drugs (n = 27). This trend was most marked in patients on H2 receptor antagonists. There was no correlation between CD3 expression and inflammation or Helicobacter pylori (H. pylori) infection. There was loss of CD3 expression in parietal cells in mice lacking the alpha-subunit of H+,K(+)-ATPase. These findings support previous observations that CD3 antigen is present in gastric parietal cells, and suggest that it may function in signal transduction during acid secretion.