In vitro deregulation of markers characteristic of human prostate epithelial cells

We have screened primary cultures of human prostate for the expression of markers reported to be characteristic of specific cell lineages in vivo, in order to ascertain whether human prostate cells in vitro maintain and reflect their in vivo differentiated phenotypes and to evaluate the homogeneity of the populations of cells that can be derived from this tissue. Using single and dual stain immunofluorescent microscopy to analyse very early organoid and subsequently derived monolayer stage cultures, we have observed that expression of markers characteristic of human prostate epithelial cells in vivo is deregulated within 48h, indicating that dissociation of human prostate tissue and cultivation of prostate epithelial cells in culture can result in promiscuous expression of cell type specific markers of prostate epithelial cells. These observations have important implications for studies of cell lineage and differentiation of prostate cells in vitro.     

KCl stimulation increases norepinephrine transporter function in PC12 cells

The norepinephrine transporter (NET) plays a pivotal role in terminating noradrenergic signaling and conserving norepinephrine (NE) through the process of re-uptake. Recent evidence suggests a close association between NE release and regulation of NET function. The present study evaluated the relationship between release and uptake, and the cellular mechanisms that govern these processes. KCl stimulation of PC12 cells robustly increased [3H]NE uptake via the NET and simultaneously increased [3H]NE release. KCl-stimulated increases in uptake and release were dependent on Ca2+. Treatment of cells with phorbol-12-myristate-13-acetate (PMA) or okadaic acid decreased [3H]NE uptake but did not block KCl-stimulated increases in [3H]NE uptake. In contrast, PMA increased [3H]NE release and augmented KCl-stimulated release, while okadaic acid had no effects on release. Inhibition of Ca2+-activated signaling cascades with KN93 (a Ca2+ calmodulin-dependent kinase inhibitor), or ML7 and ML9 (myosin light chain kinase inhibitors), reduced [3H]NE uptake and blocked KCl-stimulated increases in uptake. In contrast, KN93, ML7 and ML9 had no effect on KCl-stimulated [3H]NE release. KCl-stimulated increases in [3H]NE uptake were independent of transporter trafficking to the plasma membrane. While increases in both NE release and uptake mediated by KCl stimulation require Ca2+, different intracellular mechanisms mediate these two events.     

Histidine residues in the region between transmembrane domains III and IV of hZip1 are required for zinc transport across the plasma membrane in PC-3 cells

The proteins from the ZIP and the CDF families of zinc transporters contain a histidine-rich sequence in a loop domain located between transmembrane domains III and IV for the ZIP family and transmembrane domains IV and V for the CDF family. Topological predictions suggest that these loops are located in the cytoplasm. The loops contain a histidine-rich sequence with a variable number of histidine residues depending on the transporter. The histidine-rich sequence was postulated to serve as an extra-membrane metal binding site in these proteins. hZip1 is a human zinc transporter ubiquitously expressed. The histidine-rich motif located in the large loop of this transporter is composed of the following sequence, H₁₅₈WHD₁₆₁. To determine if this motif is involved in the zinc transport activity of the protein, we performed site directed-mutagenesis to replace the loop histidines with alanines. Results suggest that both histidines are necessary for the zinc transport function and are not involved in the plasma membrane localization of the transporter as has been reported for the Zrt1 transporter in yeast. In addition, two histidine residues in transmembrane domains IV and V are also important in the zinc transport function. The results support an intermolecular exchange mechanism of zinc transport.     

Characterization and serial propagation of mouse prostate epithelial cells in serum-free medium

Epithelial cell cultures derived from the ventral prostate of normal adult mice have been propagated in serum-free medium. The cultures were initiated and maintained in Ham's F-12 nutrient mixture supplemented with insulin (5 micrograms/ml), EGF (10 ng/ml), hydrocortisone (0.5 micrograms/ml), cholera toxin (10 ng/ml), bovine pituitary extract (25 micrograms protein/ml) and antibiotics. The cells exhibited microvilli on cell surfaces, interdigitations and junctional complexes including desmosomes between cells, and cytokeratins in cytoplasm which are characteristic of epithelial cells. In addition, the cells exhibited the tissue-specific markers, prostatic acid phosphatase and prostate-specific antigen.     

EPN: a novel epithelial cell line derived from human prostate tissue

This work reports the isolation and characterization of a line of human, nontransformed and differentiated prostate epithelial cells (EPN) in continuous culture. Primary cultures of epithelial prostate cells were set up using normal tissue isolated from a prostate sample collected after radical prostatectomy for cancer. After 70 passages, EPN cells did not undergo "Hayflike crisis" and were free of fibroblast contamination and were thus subcloned and characterized. EPN cells in culture, as prostate epithelial cells in vivo, express high-molecular weight cytokeratin and Pyk2, whereas they do not express desmin. EPN cells are nontransformed because they do not form colonies in semisolid medium and do not form tumors once injected into nude mice. EPN cells express the functional androgen receptor, which can mediate the mitogenic activity of testosterone. Finally, clonal production of the prostate-specific antigen could be detected in EPN cells. The availability of a line of epithelial nontransformed prostate cell in culture will be useful in investigating the complex process regulating normal prostate physiology as well as the development and progression of prostate tumors.     

An aluminum-activated citrate transporter in barley

Soluble ionic aluminum (Al) inhibits root growth and reduces crop production on acid soils. Al-resistant cultivars of barley (Hordeum vulgare L.) detoxify Al by secreting citrate from the roots, but the responsible gene has not been identified yet. Here, we identified a gene (HvAACT1) responsible for the Al-activated citrate secretion by fine mapping combined with microarray analysis, using an Al-resistant cultivar, Murasakimochi, and an Al-sensitive cultivar, Morex. This gene belongs to the multidrug and toxic compound extrusion (MATE) family and was constitutively expressed mainly in the roots of the Al-resistant barley cultivar. Heterologous expression of HvAACT1 in Xenopus oocytes showed efflux activity for (14)C-labeled citrate, but not for malate. Two-electrode voltage clamp analysis also showed transport activity of citrate in the HvAACT1-expressing oocytes in the presence of Al. Overexpression of this gene in tobacco enhanced citrate secretion and Al resistance compared with the wild-type plants. Transiently expressed green fluorescent protein-tagged HvAACT1 was localized at the plasma membrane of the onion epidermal cells, and immunostaining showed that HvAACT1 was localized in the epidermal cells of the barley root tips. A good correlation was found between the expression of HvAACT1 and citrate secretion in 10 barley cultivars differing in Al resistance. Taken together, our results demonstrate that HvAACT1 is an Al-activated citrate transporter responsible for Al resistance in barley.     

Properties of yeast Saccharomyces cerevisiae plasma membrane dicarboxylate transporter

Transport of succinate into Saccharomyces cerevisiae cells was determined using the endogenous coupled mitochondrial succinate oxidase system. The dependence of succinate oxidation rate on the substrate concentration was a curve with saturation. At neutral pH the K(m) value of the mitochondrial "succinate oxidase" was fivefold less than that of the cellular "succinate oxidase". O-Palmitoyl-L-malate, not penetrating across the plasma membrane, completely inhibited cell respiration in the presence of succinate but not glucose or pyruvate. The linear inhibition in Dixon plots indicates that the rate of succinate oxidation is limited by its transport across the plasmalemma. O-Palmitoyl-L-malate and L-malate were competitive inhibitors (the K(i) values were 6.6 +/- 1.3 microM and 17.5 +/- 1.1 mM, respectively). The rate of succinate transport was also competitively inhibited by the malonate derivative 2-undecyl malonate (K(i) = 7.8 +/- 1.2 microM) but not phosphate. Succinate transport across the plasma membrane of S. cerevisiae is not coupled with proton transport, but sodium ions are necessary. The plasma membrane of S. cerevisiae is established to have a carrier catalyzing the transport of dicarboxylates (succinate and possibly L-malate and malonate).     

Serum-free growth of adult human prostatic epithelial cells

Summary Proliferation of adult human prostatic epithelial cells in serum-free medium occurs upon the addition of cholera toxin, epidermal growth factor, pituitary extract, and hydrocortisone to basal medium PFMR-4A. Insulin and selenium enhance proliferation and permit growth at lower cell densities. Reducing the level of calcium in the medium dramatically alters morphology and also seems to increase proliferation. Mortal strains of cells derived from normal central or peripheral zone, benign hyperplasia, or cancer respond similarly to growth factors and calcium, but two populations of cancer cells which have been long-lived and may be immortal lines behave differently. GKC-CA cells require serum proteins or high levels of pituitary extract for optimal growth, and neither GKC-CA cells or cells of another cancer line, WB-CA, proliferate well in medium containing reduced levels of calcium. These observations may, however, be a reflection of attachment phenomena rather than of growth responses per se. Growth of cells in serum-free medium has allowed definitive studies of the effects of androgens, and regardless of cell type no response to androgens of prostate epithelial cells under any experimental conditions has been seen.     

Molecular mechanisms of membrane polarity in renal epithelial cells

Exciting discoveries in the last decade have cast light onto the fundamental mechanisms that underlie polarized trafficking in epithelial cells. It is now clear that epithelial cell membrane asymmetry is achieved by a combination of intracellular sorting operations, vectorial delivery mechanisms and plasmalemma-specific fusion and retention processes. Several well-defined signals that specify polarized segregation, sorting, or retention processes have, now, been described in a number of proteins. The intracellular machineries that decode and act on these signals are beginning to be described. In addition, the nature of the molecules that associate with intracellular trafficking vesicles to coordinate polarized delivery, tethering, docking, and fusion are also becoming understood. Combined with direct visualization of polarized sorting processes with new technologies in live-cell fluorescent microscopy, new and surprising insights into these once-elusive trafficking processes are emerging. Here we provide a review of these recent advances within an historically relevant context.     

Sorting of plasma membrane proteins in epithelial cells.

Proteins follow two routes to reach the correct surface (apical or basolateral) of a polarized epithelial cell: direct sorting from the trans-Golgi network and transcytosis from early endosomes. Several signals have been identified recently that control these sorting events, namely a glycosyl-phosphatidylinositol anchor for apical targeting, a 14-residue cytoplasmic segment of the polymeric immunoglobulin receptor for basolateral targeting, and phosphorylation of a Ser residue for transcytosis of this receptor. The machinery involved is still poorly understood.     

Molecular cloning and structural analysis of human norepinephrine transporter gene（NETHG）

A cDNA molecule encoding a major part of the human Norepinephrine transporter(hNET) was synthesized by means of Polymerase Chain Reaction(PCR) technique and used as a probe for selecting the human genomic NET gene.A positive clone harbouring the whole gene was obtained from a human lymphocyte genomic library through utilizing the “genomic walking” technique.The clone,designated as phNET,harbours a DNA fragment of about 59 kd in length inserted into BamH I site in cosmid pWE15.The genomic clone contains 14 exons encoding all amino acid residues in the protein.A single exon encodes a distinct transmembrane domain,except for transmembrane domain 10 and 11,which are encoded by part of two exons respectively,and exon 12,which encodes part of domain 11 and all of domain 12.These results imply that there is a close relationship between exon splicing of a gene and structureal domains of the protein,as is the case for the human γ－aminobutyric acid transporter(hGAT) and a number of other membrane proteins.     

hZip1 (hSLC39A1) regulates zinc homoeostasis in gut epithelial cells

Zinc is an essential trace element required for enzyme catalysis, gene regulation and signal transduction. Zinc absorption takes place in the small intestine; however, the mechanisms by which cells accumulate zinc are not entirely clear. Zip1 (SLC39A1) is a predicted transmembrane protein that is postulated, but not conclusively proven to mediate zinc influx in gut cells. The aim of this study was to investigate a role for hZip1 in mediating zinc uptake in human enterocytes. Both hZip1 mRNA and protein were detected in human intestinal tissue. In non-differentiated Caco-2 human gut cells, hZip1 was partially localised to the endoplasmic reticulum. In contrast, in differentiated Caco-2 cells cultured in extracellular matrix, the hZip1 protein was located in proximity to the apical microvilli. Lack of surface antibody binding and internalisation indicated that hZip1 was not present on the plasma membrane. Functional studies to establish a role for hZip1 in cellular zinc accumulation were carried out using ⁶⁵Zn. In Caco-2 cells harbouring an hZip1 overexpression construct, cellular zinc accumulation was enhanced relative to the control. Conversely, Caco-2 cells with an hZip1 siRNA construct showed reduced zinc accumulation. In summary, we show that the Caco-2 cell differentiation endorses targeting of hZip1 to a region near the apical domain. Given the absence of hZip1 at the apical plasma membrane, we propose that hZip1 may act as an intracellular sensor to regulate zinc homoeostasis in human gut cells.     

Phenotypic characterization of telomerase-immortalized primary non-malignant and malignant tumor-derived human prostate epithelial cell lines

In vitro human prostate cell culture models are critical for clarifying the mechanism of prostate cancer progression and for testing preventive and therapeutic agents. Cell lines ideal for the study of human primary prostate tumors would be those derived from spontaneously immortalized tumor cells; unfortunately, explanted primary prostate cells survive only short-term in culture, and rarely immortalize spontaneously. Therefore, we recently have generated five immortal human prostate epithelial cell cultures derived from both the benign and malignant tissues of prostate cancer patients with telomerase, a gene that prevents cellular senescence. Examination of these cell lines for their morphologies and proliferative capacities, their abilities to grow in low serum, to respond to androgen stimulation, to grow above the agar layer, to form tumors in SCID mice, suggests that they may serve as valid, useful tools for the elucidation of early events in prostate tumorigenesis. Furthermore, the chromosome alterations observed in these immortalized cell lines expressing aspects of the malignant phenotypes imply that these cell lines accurately recapitulate the genetic composition of primary tumors. These novel in vitro models may offer unique models for the study of prostate carcinogenesis and also provide the means for testing both chemopreventive and chemotherapeutic agents.     

Markers of prostate region-specific epithelial identity define anatomical locations in the mouse prostate that are molecularly similar to human prostate cancers

Although the basic functions of the prostate gland are conserved among mammals, its morphology varies greatly among species. Comparative studies between mouse and human are important because mice are widely used to study prostate cancer, a disease that occurs in a region-restricted manner within the human prostate. An informatics-based approach was used to identify prostate-specific human genes as candidate markers of region-specific identity that might distinguish prostatic ducts prone to prostate cancer from ducts that rarely give rise to cancer. Subsequent analysis of normal and cancerous human prostates demonstrated that the genes microseminoprotein-beta (MSMB) and transglutaminase 4 (TGM4) were expressed in distinct groups of ducts in the normal human prostate, and only MSMB was detected in areas of prostate cancer. The mouse orthologs of MSMB and TGM4 were then used for expression studies in mice along with the mouse ventrally expressed gene spermine binding protein (SBP). All three genes were informative markers of region-specific epithelial identity with distinct expression patterns that collectively accounted for all ducts in the mouse prostate. Together with the human data, this suggested that MSMB expression defines an anatomical domain in the mouse prostate that is molecularly most similar to human prostate cancers. Computer-assisted serial section reconstruction was used to visualize the complete expression domains for MSMB, SBP, and TGM4 in the mouse prostate. This showed that MSMB is expressed in prostatic ducts that comprise 21% of the mouse dorso-lateral prostate. Finally, the expression of MSMB, SBP, and TGM4 was evaluated in a mouse prostate cancer model created by the prostate epithelium-specific deletion of the tumor suppressor PTEN. MSMB and TGM4 were rapidly and dramatically down-regulated in response to PTEN deletion suggesting that this model of prostate cancer includes a more rapid de-differentiation of the prostatic epithelium than is observed in organ-confined human prostate cancers.     

Cholesterol heterogeneity in the plasma membrane of epithelial cells.

The distribution of cholesterol in the plasma membrane of epithelial cells has been determined using renal brush border vesicles as a model. In brush borders treated with Brevibacterium sp. or Nocardia erythropolis cholesterol oxidases, a significant fraction of the free cholesterol was oxidized rapidly, without glutaraldehyde fixation, and the remaining cholesterol was oxidized at a slower rate. The size of the readily accessible cholesterol pool, however, depended on the enzyme used, varying from 16% of the total in membranes treated with N. erythropolis oxidase, to 27% using the Brevibacterium sp. enzyme. The slowly accessible pool detected by the Brevibacterium oxidase was suppressed upon sphingomyelinase addition. On the other hand, the restricted activity of the Nocardia oxidase might depend on phosphatidylcholine/cholesterol interactions. These results indicate that cholesterol distribution is heterogeneous in intact renal brush border vesicles. They suggest that, as proposed for model system [Demel, R.A. Jansen, J.W.C.M., van Dijck, P.W.M., & van Deenen, L.L.M. (1977) Biochim. Biophys. Acta 465, 1-10], preferential interactions between some classes of phospholipids and cholesterol define cholesterol pools in the plasma membrane of epithelial cells.     

Regulation of divalent metal transporter expression in human intestinal epithelial cells following exposure to non-haem iron

A number of regulatory factors including dietary iron levels can dramatically alter the expression of the intestinal iron transporter DMT1. Here we show that Caco-2 cells exposed to iron for 4h exhibited a significant decrease in plasma membrane DMT1 protein, though total cellular DMT1 levels were unaltered. Following biotinylation of cell surface proteins, there was a significant increase in intracellular biotin-labelled DMT1 in iron-exposed cells. Furthermore, iron-treatment increased levels of DMT1 co-localised with LAMP1, suggesting that the initial response of intestinal epithelial cells to iron involves internalisation and targeting of DMT1 transporter protein towards a late endosomal/lysosomal compartment.