Localization of putative tumor suppressor loci by genome-wide allelotyping in human pancreatic endocrine tumors.

Only two tumor suppressor gene loci, one on 3p25 and the MEN1 gene on 11q13, have thus far been implicated in the pathogenesis of sporadic human pancreatic endocrine tumors (PETs). A genome-wide allelotyping study of 28 human PETs was undertaken to identify other potential tumor suppressor gene loci. In addition to those on chromosomes 3p and 11q, frequent allelic deletions were identified on 3q (32%), 11p (36%), 16p (36%), and 22q (29%). Finer deletion mapping studies localized the smallest regions of common deletion to 3q27, 11p13, and 16p12.3-13.11. Potential candidate genes at these loci include WT1 (11p13), TSC2 (16p13), and NF2 (22q12), but no known tumor suppressor gene localizes to 3q27. The mean fractional allelic loss among these human PETs is 0.126, and no correlation was observed between allelic loss and clinical parameters, including age, sex, hormonal subtype, and disease stage. These findings highlight novel locations of tumor suppressor gene loci that contribute to the pathogenesis of human PETs, and several of these on 3p, 3q, and 22q are syntenic with loci on mouse chromosomes 9 and 16 that are implicated in a murine transgenic model of PETs.

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).

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.

Separation and proteolytic mapping of the two [3H]cytochalasin B photoaffinity labeled D-glucose-sensitive proteins in the chicken embryo fibroblast plasma membrane.

Photoaffinity labeling with [3H]cytochalasin B detects two D-glucose-sensitive proteins in the chicken embryo fibroblast (CEF) plasma membrane, which accumulate under conditions of glucose starvation and are probably involved in the glucose transport system (Pessin, J.E., et al. (1982) Proc. Natl. Acad. Sci. U.S. 79, 2286-2290). The two labeled components, designated as peak I (Mr 45,000) and II (Mr 52,000) components, were separated by preparative gel electrophoresis in the presence of sodium dodecyl sulfate. The fractions were digested with S. aureus V8 or papain, and the radioactive products were analyzed by one-dimensional gel electrophoresis. The peptide maps showed that they have different peptide structures. Peptide maps of authentic actin, a possible contaminant of the peak I fractions, were quite different from those of the peak I component. Rous sarcoma virus-transformed CEF have two components similar as to apparent molecular size and peptide maps to those present in glucose-starved cells. The peak I and II components show minimal affinity to agarose-bound Ricinus communis agglutinin which binds the human erythrocyte glucose transporter quite well. The peak II component was more susceptible to proteolysis than the peak I one or the human erythrocyte glucose transporter. However, the peptide maps of the peak II component were similar to those of the human erythrocyte glucose transporter.

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 microm) 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.

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 CACC cis-regulatory element is important for transcriptional activation in pancreatic insulinoma cells. To identify CACC-binding proteins, a lambda 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.

Effect of hypertonicity on hexose transporter regulation in chicken embryo fibroblasts.

The regulation of hexose transporters of cultured fibroblasts was investigated by exposing chicken embryo fibroblasts (CEF) to hypertonic culture medium, a condition known to enhance hexose transport activity. The effects of hypertonicity and the role of protein synthesis were examined with CEF in the basal (glucose fed) and transport enhanced (glucose starved) states. Glucose-fed CEF exposed to hypertonic conditions developed four-fold enhancement of hexose transport activity within 4 hrs; this declined in the following 20 hrs to a level slightly higher than the fed control. Protein synthesis was required in part for this effect, since the presence of cycloheximide during hypertonic exposure of fed CEF blocked the increase in of transport by almost 50%. Although the increased transport produced by glucose starvation was not further enhanced by hypertonicity, hypertonic treatment of starved CEF during glucose refeeding largely prevented the loss of transport activity to the basal, fed state. The hypertonic effects were concentration dependent (240mOsm optimal) and could be elicited with NaCl, KCl, or sucrose. Hypertonic treatment typically led to a greater than 50% decline in the incorporation of [3H]leucine into acid-insoluble fractions. The changes in transport were evident at the plasma membrane level, and studies of membrane vesicles prepared from hypertonically treated fed CEF showed a doubling of both [3H]cytochalasin B binding and the Vmax of D-glucose transport. These findings indicate that exposure of CEF to hypertonic conditions has some effects similar to those produced by glucose starvation and suggest that protein synthesis is to some extent involved in the regulation of hexose transporters in CEF.

Mutually exclusive interactions between factors binding to adjacent Sp1 and AT-rich elements regulate gastrin gene transcription in insulinoma cells.

The gastrin gene is transiently expressed in fetal pancreatic islets during islet neogenesis but then switched off after birth when islet cells become fully differentiated. Previous studies identified a cis-regulatory sequence between -109 and -75 in the human gastrin promoter which binds islet cell-specific activators and a nonspecific repressor and thus may act as a molecular switch. The present study identified another cis-regulatory sequence (-163ACACTAAATGAAAGGGCGGGGCAG-140) which bound two islet nuclear proteins in a mutually exclusive manner, as defined by gel shift competition, methylation interference, and DNase I foot-printing assays. The general transactivator Sp1 recognized the downstream GGGCGGGG sequence, but Sp1 binding was prevented when another islet factor bound to the adjacent AT-rich sequence (CTAAATGA). This gastrin AT-rich element is nearly identical to the binding site (ATAAATGA) for the islet-specific transcription factor beta TF-1. However, the gastrin AT-binding factor appeared to differ from beta TF-1 in its gel mobility shift pattern. Transfections of rat insulinoma cells revealed that mutations which blocked binding to the AT-rich element but allowed Sp1 binding up-regulated transcriptional activity. These results suggest that the gastrin AT-binding factor blocks transactivation by Sp1 and may have a role in the repression of gastrin transcription seen at the end of islet differentiation.

Regulation of hexose transporters in chicken embryo fibroblasts: stimulation by the phorbol ester TPA leads to increased numbers of functioning transporters.

As has been observed with many types of cultured cells, chicken embryo fibroblasts (CEF) when exposed to the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) develop a 3- to 4-fold increase in hexose transport activity in 4 h. This increase in transport activity occurred despite a modest decline of 20% in [3H]leucine incorporation into acid insoluble fractions. Cycloheximide largely, but not completely, blocked the increase in transport activity during TPA exposure. The effects of TPA were somewhat similar to those of glucose starvation induced enhancement of hexose transport activity. Furthermore, with TPA there was no additive effect to that produced by glucose starvation. Plasma membrane enriched fractions were prepared from CEF treated with or without TPA. Membranes prepared from TPA exposed cells had a two-fold enhancement of stereospecific D-glucose transport activity as well as D-glucose inhibitable [3H]cytochalasin B binding as compared to the membranes from control CEF. There was no effect on transport when membranes were exposed to TPA in vitro. These results provide strong evidence that TPA exposure leads to an increase in the number of functioning transporters, an effect largely requiring protein synthesis.

Regulation of hexose carriers in chicken embryo fibroblasts. Effect of glucose starvation and role of protein synthesis.

Regulation of hexose transport was investigated in chicken embryo fibroblasts (CEF) which develop 4- to 8-fold enhanced hexose transport activity during glucose starvation. The presence of cycloheximide in low (0.5 micrograms/ml) concentrations during starvation largely blocked the enhancement of transport activity. Glucose refeeding of CEF in the starvation state led to a decline in transport to the basal level. This decline was either potentiated or blocked by the presence of cycloheximide in low or high (50 micrograms/ml) concentrations, respectively. Exposure of CEF in the fed state to low concentrations of cycloheximide resulted in a 70% decrease of transport within 6 h, whereas exposure to high concentrations of cycloheximide led to only a modest loss (35% decrease). In the glucose-starved state, CEF had no significant decline of transport when exposed to cycloheximide at either high or low concentrations. The uptake of 3-O-methylglucose by fed, starved, or cycloheximide-treated CEF correlated closely with D-glucose transport activity and [3H]cytochalasin B binding by plasma membranes prepared from CEF exposed to the same conditions. Hexose transport activity of CEF seems to largely depend on the number of functioning carriers in the plasma membrane, which apparently reflect the balance between carrier synthesis and inactivation. These two processes require protein synthesis, but are differentially sensitive to the effects of cycloheximide, such that low concentrations of cycloheximide appear to block primarily synthesis while high concentrations block both processes. Furthermore, during starvation the enhancement of transport appears largely due to decreased carrier inactivation in the face of continued carrier synthesis.

Regulation of hexose transporters of chicken embryo fibroblasts during glucose starvation.

Chicken embryo fibroblasts (CEF) when exposed to glucose-deficient culture medium developed 4- to 10-fold enhanced hexose transport activity within a few hours. Plasma membrane fractions prepared from starved and fed CEF revealed that starved cell membranes had a threefold greater glucose transport activity and [3H]cytochalasin B binding. The close correlation between transport activities of whole CEF and plasma membrane fractions indicates that hexose transport regulation during starvation results primarily in an increase in the number of functioning hexose transporters. The effect of protein synthesis inhibition on the overall process was studied with emetine, an inhibitor of translational elongation. Glucose-fed CEF treated with low concentrations of emetine (0.1 microM) showed a loss of transport greater than 65% within 4 h, but with higher concentrations of emetine (10 microM) there was no significant effect. Emetine treatment (0.1-10 microM) of CEF undergoing starvation virtually blocked any enhancement in transport whereas treatment of starved CEF led to only a slight loss of transport. Starved CEF refed with glucose had a decline of transport that was potentiated by low concentrations of emetine (0.1 microM); however, under these conditions high concentrations of emetine (10 microM) largely prevented loss of transport. Thus hexose transport regulation of CEF seems to reflect a balance between transporter synthesis and turnover. Transporter synthesis appears more sensitive to inhibition by emetine than turnover, whereas with hexose starvation there appears to be a decline in the activity of the transporter turnover process.

Activation of gastrin transcription in pancreatic insulinoma cells by a CACC promoter element and a 70-kDa sequence-specific DNA-binding protein.

Gastrin gene expression in the pancreatic islets is developmentally regulated and occurs largely during fetal life. Deletional analysis of transiently transfected rat insulinoma cells with gastrin 5'-flanking sequences in luciferase reporter genes demonstrated that the gastrin promoter sequence proximal to -111 base pairs (bp) contains the cis-regulatory elements necessary for maximal transcription. Mutational analysis identified the sequence CCCCACCCCA (-109 to -100 bp) as a positive cis-regulatory element (CACC) located 5' to a previously described negative element (-100 to -90 bp) and E-box positive element at -82 bp. Multimers of the CACC element in a heterologous promoter activated transcription independent of the other cis-regulatory elements. CACC binding proteins were purified from insulinoma cell nuclear extracts by cation exchange and affinity chromatography. Southwestern blot of nuclear extracts identified a 70-kDa CACC-binding protein. Mutational analysis of the CACC element showed a close correlation between DNA binding of this protein and transcriptional activation. Transcriptional activation by multimers of the CACC element in a heterologous promoter was detected in a variety of cell lines but was strongest in those of islet lineage. Likewise, the presence of the 70-kDa CACC-binding protein was found in many cell lines but was most abundant in the insulinoma cells. The CACC-binding protein has not been previously identified among the known pancreatic regulatory factors and may have an important role in the developmental expression of gastrin.

Stereospecific hexose transport by membrane vesicles from mouse fibroblasts: membrane vesicles retain increased hexose transport associated with viral transformation.

Membrane vesicles isolated from nontransformed BALB/c 3T3 mouse fibroblasts (3T3) and from those cells transformed by simian virus 40 (SV3T3) displayed carrier-mediated and stereospecific uptake of hexose as measured by the difference between D-[(14)C]glucose or its analogues and L-[(3)H]glucose uptake. Stereospecific uptake appeared to be linear for 5 sec and reached a maximum at 5-10 min. Stereospecific D-[(14)C]glucose uptake, osmotically sensitive and temperature dependent, was inhibited by unlabeled D-glucose or its analogues and was stimulated by the countertransport of accumulated unlabeled D-glucose. As with whole cells, the initial rate of stereospecific uptake by SV3T3 membrane vesicles was approximately 2.5-fold greater than that by 3T3 vesicles. Efflux of preloaded D-[(14)C]glucose was also faster from SV3T3 than from 3T3 membrane vesicles. The K(m) value was 5 mM for both the 3T3 and the SV3T3 membrane vesicles, but the V(max) values were 36 and 86 nmol/mg of protein per min, respectively, suggesting an increase in the number or availability of hexose carriers in transformed cell membranes. Cytochalasin B competitively inhibited stereospecific hexose uptake in both types of membrane vesicles. The binding of cytochalasin B to the SV3T3 membrane vesicles was significantly greater than that to 3T3 vesicles. Thus, the membrane vesicles retained many of the features of the altered hexose transport observed in whole cells in association with viral transformation.

Amino acid and hexose transport by cultured crypt cells from rat small intestine.

The characteristics of amino acid and sugar transport in intestinal crypt epithelial cells have been examined by measuring substrate uptake in an established epithelial cell line. These cells (IEC-6 cells) have been characterized as derived from rat small intestinal crypt cells on the basis of morphological criteria (J. Cell. Biol. 80: 248-265, 1979). Amino acid transport appeared to be mediated by both Na+-dependent and Na+-independent systems. Hexose uptake was stereospecific and Na+ independent, and was markedly inhibited by phloretin and cytochalasin B. Since glucocorticoids are known to have profound effects on maturation of the intestinal epithelium in vivo, their effects on transport properties of the cultured crypt cells were studied. Hydrocortisone, while completely inhibiting cell growth, increased the initial uptake rates of various hexoses, while having little or nor effect on the initial rate of amino acid uptake. The increased hexose uptake appeared to be due to a change in Vmax rather than Km. Appearance of the Na+-dependent hexose transport system, which is present in differentiated enterocytes, was not elicited by in vitro treatment with glucocortcoids.

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.

Sodium-stimulated active transport of aminoisobutyric acid by reconstituted vesicles from partially purified plasma membranes of mouse fibroblasts transformed by simian virus 40.

Plasma membrane fractions isolated from mouse fibroblast BALB/c 3T3 cells transformed by simian virus 40 were partially purified by treatment with dimethylmaleic anhydride followed by extraction with 2% cholate. The extracted proteins were combined with exogenous phospholipids and eluted through a Sephadex G50 column. Reconstituted vesicles thus obtained were shown to possess the ability of Na+-stimulated transport of alpha-aminoisobutyric acid. The simultaneous addition of NaSCN and alpha-aminoisobutyric acid to these vesicles produced a transient accumulation above the equilibrium level (overshoot, active transport). The Na+-stimulated transport of alpha-aminoisobutyric acid was sensitive to the accompanying anion and to the temperature of incubation. The results demonstrate that partially purified membrane proteins of mouse fibroblast cells can be incorporated into the liposomes that have the characteristics of Na+-stimulated and electrochemically sensitive active transport of alpha-aminoisobutyric acid.

Photoaffinity labeling of the stereospecific D-glucose transport system with cytochalasin B.

A method has been developed for photoaffinity labeling components of the hexose transport system with [3H]cytochalasin B. We have demonstrated that UV photoirradiation of intact human erythrocytes or ghost membranes with 0.5 microM [3H]cytochalasin B yielded a broad peak with an Mr of 44,000-70,000. Labeling that was insensitive to the presence of 0.5 M D-sorbitol was substantially inhibited by 0.5 M D-glucose. Approximately 80% of labeling in the region of Mr = 49,000-70,000 was inhibited by the presence of 0.5 M D-glucose, whereas labeling of the 44,000- to 49,000-dalton region was inhibited only 30%. Somewhat different results were obtained from photoaffinity labeling of plasma membranes from chicken embryo fibroblasts (CEF). The [3H]cytochalasin B-labeling patterns had two relatively sharp and discrete peaks at 46,000 and 52,000 daltons. Comparison of plasma membranes from glucose-fed and starved CEF revealed that the total D-glucose-sensitive labeling increased approximately 12-fold in the starved cell membranes. Labeling of the 52,000-dalton polypeptide was more sensitive than that of the 46,000-dalton polypeptide to inhibition by the presence of D-glucose. These results indicate that [3H]cytochalasin B photoaffinity labeling has wide applicability for identifying and covalently binding components of the facilitated hexose transport system.

Solubilization and separation of the human erythrocyte D-glucose transporter covalently and noncovalently photoaffinity-labeled with [3H]cytochalasin B.

The D-glucose transporter in the human erythrocyte membranes was photoaffinity-labeled with [3H]cytochalasin B and solubilized with n-octyl beta-D-glucopyranoside (octyl glucoside). [3H]Cytochalasin B-bound proteins were further isolated by using Sephadex G-50 chromatography. The amount of [3H]cytochalasin B associated with the membrane proteins was approximately 10% of the total radioactivity in the octyl glucoside extract. The solubilized photoaffinity-labeled D-glucose transporter was isolated and found to consist of two major peaks by DEAE-Sephacel chromatography. The radioactivity of peak II was considerably greater than that of peak I. The incorporation of [3H]cytochalasin B into both peaks was blocked by the presence of D-glucose during photolysis. With preparative NaDod-SO4/polyacrylamide gel electrophoresis, the radioactivity of peak I could be released, but that of peak II remained with the D-glucose transporter. These results indicate that [3H]cytochalasin B was covalently bound to the D-glucose transporter only in peak II and that peak II could be generated by the photoaffinity labeling of peak I. However, the D-glucose transport activity was associated only with peak I. These findings suggest that the anionic domain of the D-glucose transporter becomes exposed because of conformational changes of the protein as a result of covalent binding with [3H]cytochalasin B by photoaffinity labeling.