Loyal G. Tillotson

Oncogenic ras induces gastrin gene expression in colon cancer

BACKGROUND & AIMS The expression of gastrin, as a tumor growth factor, is significantly increased in some colon cancers compared with the low levels found in normal mucosa. The aim of this study was to elucidate the transcriptional mechanisms of gastrin induction in colon cancer. METHODS Gastrin messenger (mRNA) levels and K-ras genotype were determined in colon cancer cell lines and surgical specimens. Colon cancer cells were transfected with oncogenic ras expression vectors, and transcriptional activity was assayed with gastrin-luciferase reporter genes. RESULTS Colon cancer cell lines and tissues with K-ras mutations all had significantly higher gastrin mRNA levels than those that were ras wild type. Treatment of several ras mutant cell lines with PD98059, an inhibitor of mitogen-activated protein kinase kinase, resulted in a decrease in endogenous gastrin mRNA levels. The effects of ras on gastrin expression appeared to be mediated through the gastrin promoter because transfection of oncogenic ras and activated raf expression vectors both induced gastrin-promoter, luciferase-reporter genes. The inductive effects of oncogenic ras could be blocked by the coexpression of dominant negative forms of raf and extracellular regulated kinase. CONCLUSIONS Oncogenic ras induces gastrin gene expression through activation of the Raf-MEK-ERK signal transduction pathway.

RIN ZF, a novel zinc finger gene, encodes proteins that bind to the CACC element of the gastrin promoter.

Expression of gastrin, a gut hormone and growth factor, has tissue-specific transcriptional regulation and can be induced in some tumors. Previous studies have shown that a CACCcis-regulatory element is important for transcriptional activation in pancreatic insulinoma cells. To identify CACC-binding proteins, a λ phage cDNA library derived from a rat insulinoma cell line, RIN 38A, was screened by a Southwestern method. A novel member of the Cys2-His2 zinc finger gene family was cloned and designated RIN ZF, having a cDNA sequence of 3.8 kilobases. One full-length and a shorter splice variant were sequenced and had predicted protein masses of 91.6 and 88.7 kDa. Expression of both splice forms were ubiquitous in fetal and adult rat tissues. Recombinant RIN ZF protein exhibited sequence-specific binding to the gastrin CACC element in a gel mobility shift assay. In transient transfections, both splice variants appeared to have only weak activating effects on gastrin-luciferase reporter gene transcription. Furthermore, RIN ZF coexpression with Sp1 appeared to block the strongly activating effects of Sp1 mediated through the CACC element. These findings suggest that a novel set of zinc finger proteins may help regulate gastrin gene expression by interfering with Sp1 transactivation.

Inhibition of Na+-stimulated glucose transport function and perturbation of intestinal microvillus membrane vesicles by ethanol and acetaldehyde

Abstract The effects of ethanol and acetaldehyde on rat intestinal microvillus membrane integrity and glucose transport function were examined in vitro with purified membrane vesicles. Ethanol could influence glucose transport function by alterations in the conformation of the carrier, the lipid environment surrounding the carrier, or in the transport driving force (Na+ electrochemical gradient). Due to the rapid nature of glucose uptake, transport was assayed with the use of an apparatus that permitted uptake measurements as early as 1 s. Ethanol (340 m m ) partially and acetaldehyde (44 m m ) completely inhibited concentrative glucose uptake throughout the 1-min time course. Their inhibitory effects were reversible and irreversible, respectively. Kinetic measurements made during the initial rate of uptake (at 2 s) with various concentrations of glucose (0.05–8 m m ) showed that ethanol and acetaldehyde both caused a decrease in V. Although ethanol did not substantially alter the transport Km, acetaldehyde increased the Km almost 50%. To determine whether ethanol or acetaldehyde directly interfered with glucose carrier function, uptake was measured in the presence of equilibrated Na+. Only acetaldehyde had a significant inhibitory effect under these conditions. Membrane permeability, as determined by efflux of preloaded 6-carboxyfluorescein dye, increased upon exposure of the vesicles to ethanol or acetaldehyde. Membrane fluidity measurements by fluorescence polarization showed that only acetaldehyde had a significant fluidizing effect. These results indicate that ethanol and acetaldehyde acted to perturb membrane integrity and inhibited glucose uptake indirectly by allowing the Na+ gradient to dissipate. Acetaldehyde, which had a stronger inhibitory effect than ethanol, appeared also to directly inhibit carrier function.

Hexose and amino acid transport by chicken embryo fibroblasts infected with temperature-sensitive mutant of rous sarcoma virus: Comparison of transport properties of whole cells and membrane vesicles

The effect of transformation on hexose and amino acid transport has been studied using whole cells and membrane vesicles of chicken embryo fibroblasts infected with the temperature-sensitive mutant of the Rous sarcoma virus, TS-68. In whole cells, TS-68-infected chicken embryo fibroblasts cultured at the permissive temperature (37 degrees C) had a 2-fold higher rate of 2-deoxy-D-glucose uptake than the same cells cultured at the non-permissive temperature (41 degrees C). However, both the non-transformed and transformed cells had comparable rates of alpha-aminoisobutyric acid transport. Membrane vesicles, isolated from TS-68-infected chicken embryo fibroblasts cultured at 41 degrees C or 37 degrees C, displayed carrier-mediated, intravesicular uptake of D-glucose and alpha-aminoisobutyric acid. Membrane vesicles from TS-68-infected chicken embryo fibroblasts cultured at 37 degrees C had an approx. 50% greater initial rate of stereospecific hexose uptake than the membrane vesicles from fibroblasts cultured at 41 degrees C. The two types of membrane vesicle had similar uptake rates of alpha-aminoisobutyric acid. The results of hexose and amino acid uptake by the membrane vesicles correlated well with those observed with the whole cells. Km values for stereospecific D-glucose uptake by the membrane vesicles from TS-68-infected chicken embryo fibroblasts cultured at 41 and 37 degrees C were similar, but the V value was greater for the membrane vesicles from TS-68-infected cells cultured at 37 degrees C. Cytochalasin B competitively inhibited stereospecific hexose uptake in both types of membrane vesicle. These findings suggest that the membrane vesicles retained many of the features of hexose and amino acid transport observed in whole cells, and that the increased rate of hexose transport seen in the virally-transformed chicken embryo fibroblasts was due to an increase in the number or availability of hexose carriers.

Activation of gastrin gene transcription in islet cells by a RAP1‐like cis‐acting promoter element

Gastrin transcription in islet cells is activated by a cis‐regulatory sequence containing a binding site for the yeast transcription factor RAP1. The DNA—protein interactions between RAP1 protein and the gastrin DNA element determined by methylation interference assays are identical to those of RAP1 and yeast genes. Point mutations in the gastrin RAP1 binding site, which abolished RAP1 binding, decreased transcriptional activation by this sequence. Islet cells revealed a DNA binding protein with RAP1 ‐like binding specificity. These findings support the conclusion that gastrin transcription is activated in mammalian cells by a RAP1 ‐like transcription factor.

Isolation, maintenance, and characterization of human pancreatic islet tumor cells expressing vasoactive intestinal peptide

Tissue from a vasoactive intestinal peptide (VIP)-secreting human tumor has been used to establish and characterize human neuroendocrine primary cell cultures from which permanent, clone-derived cell lines have been established. Viable cells were obtained by enzymatic and mechanical dissociation of freshly resected pancreatic islet tumor and hepatic metastatic tumor tissues. Aliquots of tumor cells were established ex vivo under culture conditions including porous substrata coated with type IV collagen and laminin and a low serum, hormonally defined culture medium. The small (<10 &mgr;m) rounded, grape-like cells had a very slow growth rate of doubling times estimated at several weeks or more. After several passages, morphologically uniform cells were derived that strongly expressed neuroendocrine markers of synaptophysin and synaptobrevin. Although chromogranin A and VIP had somewhat weaker expression, both demonstrated phorbol ester-stimulated secretion. The morphologic and secretory properties were maintained by the cells for nearly 2 years in culture. The establishment of this novel VIP-secreting human neuroendocrine cell line (HuNET) makes available a culture model with which to study a transformed version of this pancreatic islet cell type and offers approaches by which to establish islet tumor cell lines.

Reconstitution and characterization of a sodium-stimulated active aminoisobutyric acid transport system derived from partially purified plasma membranes from mouse fibroblasts transformed by simian virus 40: Comparison of reconstituted vesicles with native membrane vesicles

Abstract A Na + -specific and Na + -stimulated active α-aminoisobutyric acid transport system was reconstituted from plasma membranes isolated from mouse fibroblast BALB/c 3T3 cells transformed by simian virus 40. The plasma membranes were treated with dimethylmaleic anhydride and then extracted with 2% cholate. The cholate-solubilized supernatant proteins were combined with exogenous phospholipids and eluted through a Sephadex G-50 column. This yielded reconstituted vesicles which in the presence of Na + could actively transport α-aminoisobutyric acid as shown by the transient accumulation above the equilibrium level (overshoot). The overshoot was not obtained with other monovalent cations such as K + , Li + , and choline + . The electrochemical effect of the lipophilic anion, SCN − , led to greater α-aminoisobutyric acid uptake as compared to that observed with Cl − or SO 4 2− . The Na + -stimulated transport of a-aminoisobutyric acid was a saturable process with an apparent K m of 2 m m . Studies of the inhibition of α-aminoisobutyric acid transport by other amino acids showed that methylaminoisobutyric acid [specifically transported by A system (alanine preferring)]had a pronounced inhibitory effect on a-aminoisobutyric acid uptake in contrast to the slight inhibitory effect produced by phenylalanine [primarily transported by L system (leucine preferring)]. The results show that the reconstituted vesicles, prepared from partially purified membrane proteins and exogenous phospholipids, regained the same important transport properties of native membrane vesicles, i.e., Na + -specific and Na + -stimulated concentrative α-aminoisobutyric acid uptake.

Facilitated glucose transporter of human erythrocyte: proteolytic mapping of the [3H]cytochalasin B photoaffinity-labeled transporter polypeptide.

The human erythrocyte D-glucose transporter is an integral membrane glycoprotein with an heterogeneous molecular mass spanning a range 45-70 kDa. The protein structure of the transporter was investigated by photoaffinity labeling with [3H]cytochalasin B and fractionating the labeled transporter according to molecular mass by preparative SDS-polyacrylamide gel electrophoresis. Each fraction was digested with either papain or S. aureus V8 proteinase, and the labeled proteolytically derived peptide fragments were compared by SDS polyacrylamide gel electrophoresis. Papain digestion yielded two major peptide fragments, of approx. molecular mass 39 +/- 2 and 22 +/- 2 kDa; treatment with V8 proteinase resulted in two fragments, with mass of 24 +/- 2 and 15 +/- 2. Proteolysis of each transporter fraction produced the same pattern of labeled peptide fragments, irrespective of the molecular mass of the original fractions. The binding characteristics of [3H]cytochalasin-B-labeled transporter to Ricinis communis agglutinin lectin was examined for each transporter molecular mass fraction. It was found that higher-molecular-mass fractions of intact transporter had a 2-fold greater affinity for the lectin than lower-molecular-mass fractions (i.e., 67 kDa greater than 45 kDa fraction). However, proteolytically derived labeled peptide fragments from each fraction had minimal affinity for the lectin. These results suggest that the labeled peptide fragments have been separated from the glycosylated regions of the parent transporter protein. The present findings indicate that, although transporter proteins have an apparently heterogeneous molecular mass, some regions of the protein share a common peptide. Furthermore, the glycosylated regions appear to be located some distance from the [3H]cytochalasin-B-labeled site(s).