IntroductionOverexpression of the oxygen-responsive transcription factor hypoxia-inducible factor 1α (HIF-1α) correlates with poor prognosis in breast cancer patients. The mouse mammary tumor virus polyoma virus middle T (MMTV-PyMT) mouse is a widely utilized preclinical mouse model that resembles human luminal breast cancer and is highly metastatic. Prior studies in which the PyMT model was used demonstrated that HIF-1α is essential to promoting carcinoma onset and lung metastasis, although no differences in primary tumor end point size were observed. Using a refined model system, we investigated whether HIF-1α is directly implicated in the regulation of tumor-initiating cells (TICs) in breast cancer.MethodsMammary tumor epithelial cells were created from MMTV-PyMT mice harboring conditional alleles of Hif1a, followed by transduction ex vivo with either adenovirus β-galactosidase or adenovirus Cre to generate wild-type (WT) and HIF-1α-null (KO) cells, respectively. The impact of HIF-1α deletion on tumor-initiating potential was investigated using tumorsphere assays, limiting dilution transplantation and gene expression analysis.ResultsEfficient deletion of HIF-1α reduced primary tumor growth and suppressed lung metastases, prolonging survival. Loss of HIF-1α led to reduced expression of markers of the basal lineage (K5/K14) in cells and tumors and of multiple genes involved in the epithelial-to-mesenchymal transition. HIF-1α also enhanced tumorsphere formation at normoxia and hypoxia. Decreased expression of several genes in the Notch pathway as well as Vegf and Prominin-1 (CD133)was observed in response to Hif1a deletion. Immunohistochemistry confirmed that CD133 expression was reduced in KO cells and in tumorspheres. Tumorsphere formation was enhanced in CD133hi versus CD133neg cells sorted from PyMT tumors. Limiting dilution transplantation of WT and KO tumor cells into immunocompetent recipients revealed > 30-fold enrichment of TICs in WT cells.ConclusionThese results demonstrate that HIF-1α plays a key role in promoting primary mammary tumor growth and metastasis, in part through regulation of TICs. HIF-1α regulates expression of several members of the Notch pathway, CD133 and markers of the basal lineage in mammary tumors. Our results suggest that CD133, which has not been profiled extensively in breast cancer, may be a useful marker of TICs in the PyMT mouse model. These data reveal for the first time that HIF-1α directly regulates breast TIC activity in vivo.

The activity of HCO 3 − transporters contributes to the acid-base environment of the nervous system.In the present study, we used in situ hybridization, immunoblotting, immunohistochemistry, and immunogold electron microscopy to localize electrogenic Na/bicarbonate cotransporter NBCe1 splice variants (-A, -B, and -C) in rat brain. The in situ hybridization data are consistent with NBCe1-B and -C, but not -A, being the predominant NBCe1 variants in brain, particularly in the cerebellum, hippocampus, piriform cortex, and olfactory bulb. An antisense probe to the B and C variants strongly labeled granule neurons in the dentate gyrus of the hippocampus, and cells in the granule layer and Purkinje layer (e.g., Bergmann glia) of the cerebellum. Weaker labeling was observed in the pyramidal layer of the hippocampus and in astrocytes throughout the brain. Similar, but weaker labeling was obtained with an antisense probe to the A and B variants. In immunoblot studies, antibodies to the A and B variants (αA/B) and C variant (αC) labeled ~130-kDa proteins in various brain regions. From immunohistochemistry data, both αA/B and αC exhibited diffuse labeling throughout brain, but αA/B labeling was more intracellular and punctate. Based on co-localization Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. studies with antibodies to neuronal or astrocytic markers, αA/B labeled neurons in the pyramidal layer and dentate gyrus of the hippocampus, as well as cortex. αC labeled glia surrounding neurons (and possibly neurons) in the neuropil of the Purkinje cell layer of the cerebellum, the pyramidal cell layer and dentate gyrus of the hippocampus, and the cortex. According to electron microscopy data from the cerebellum, αA/B primarily labeled neurons intracellularly and αC labeled astrocytes at the plasma membrane. In summary, the B and C variants are the predominant NBCe1 variants in rat brain and exhibit different localization profiles. NIH Public Access

Many cellular processes including neuronal activity are sensitive to changes in intracellular and/or extracellular pH-both of which are regulated by acid-base transporter activity. HCO 3 − -dependent transporters are particularly potent regulators of intracellular pH in neurons and astrocytes, and also contribute to the composition of the cerebrospinal fluid (CSF). The molecular physiology of HCO 3 − transporters has advanced considerably over the past ~14 years as investigators have cloned and characterized the function and localization of many Na-Coupled Bicarbonate Transporters of the Slc4 family (NCBTs). In this review, we provide an updated overview of the function and localization of NCBTs in the nervous system. Multiple NCBTs are expressed in neurons and astrocytes in various brain regions, as well as in epithelial cells of the choroid plexus. Characteristics of human patients with SLC4 gene mutations/deletions and results from recent studies on mice with Slc4 gene disruptions highlight the functional importance of NCBTs in neuronal activity, somatosensory function, and CSF production. Furthermore, energydeficient states (e.g., hypoxia and ischemia) lead to altered expression and activity of NCBTs. Thus, recent studies expand our understanding of the role of NCBTs in regulating the pH and ionic composition of the nervous system that can modulate neuronal activity. Keywordsbrain; electrogenic; astrocyte; genetic; neuron; pHThe regulation of intracellular pH (pH i ) is critical for proper cell function because many cellular processes are sensitive to changes in pH (Roos and Boron, 1981; Bevensee and Boron, 2007). pH-sensitive processes include fertilization (Johnson and Epel, 1976), cell © 2010 IBRO. Published by Elsevier Ltd. All rights reserved.Address correspondence to: Mark O. Bevensee, PhD, Department of Physiology and Biophysics, University of Alabama at Birmingham, 1918 University Blvd., 812 MCLM, Birmingham, AL 35294, Ph: (205) Fax: (205) 975-7679, bevensee@uab.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptNeuroscience. Author manuscript; available in PMC 2011 December 29. Beirne et al., 1987;Roos and Boron, 1981), alterations in cell structure (Parton et al., 1991), and the activity of metabolic enzymes such as phosphofructokinase (Trivedi and Danforth, 1966). In the central nervous system (CNS), which is comprised of cells with high surface-to-volume ratios and an extracellular space that is quite tortuous, the movement of acid-base equivalents across plasma membranes (e.g., by passive diffusion and pH...