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Transformation stimulates glucose transporter gene expression in the absence of protein kinase C
Abstract
The rat brain glucose transporter (GT) gene is rapidly activated coincident with the initiation of growth in response to oncogenic transformation or the addition of growth factors to quiescent fibroblasts. The latter response has been shown to be mediated by protein kinase C-dependent and-independent pathways. We studied the role of protein kinase C in the transformation-induced activation of the GT gene. Transformation of fibroblasts by either the v-fps or the Ki-ras oncogene rapidly increased the levels of GT mRNA. Either viral oncogene remained capable of stimulating the GT gene after depletion of cellular protein kinase C by prolonged pretreatment of fibroblasts with phorbol 12-myristate 13-acetate. These data indicate that protein kinase C is not required for the rapid activation of gene transcription by oncogenic transformation.
... Cells were solubilized for 1 h at 4°C in 25 mM Tris-HCl, pH 7.6, 1 mM EGTA, 10 mM NaCl, 1% Triton X-100, containing 20 µg/ml leupeptin. After clearing the extracts by centrifugation, PK-C was partially purified by DEAE-cellulose chromatography (Hiraki et al., 1989). PK-C activity was assayed using either lysine-rich histone or myelin basic protein (MBP) as substrates (0.6 mg/ml in both cases) (Hiraki et al., 1989). ...
... After clearing the extracts by centrifugation, PK-C was partially purified by DEAE-cellulose chromatography (Hiraki et al., 1989). PK-C activity was assayed using either lysine-rich histone or myelin basic protein (MBP) as substrates (0.6 mg/ml in both cases) (Hiraki et al., 1989). Protein concentration was determined by the Bradford method (Bio-Rad Protein Assay, Richmond, CA) with bovine serum albumin (BSA) as standard. ...
Recently developed HT-29-derived cell lines, which display variable differentiated phenotypes provide an invaluable opportunity to analyze the mechanism by which cell differentiation is regulated in the intestine. We have studied the effects of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in the differentiation phenotype of mucus-secreting (HT-29 M6) and absorptive (HT-29 M3) cells. TPA prevented the accumulation of differentiation markers such as dipeptidylpeptidase IV, villin or mucins, down-regulated the expression of these molecules in post-confluent differentiated cell cultures and induced the loss of the functional integrity of the tight junction in the monolayer (i.e. decreased transepithelial resistance and inhibited dome formation). These effects were mediated by activation of protein kinase C (PK-C), as demonstrated using the specific inhibitor GF109203x. Analysis by immunoblotting of the PK-C isoforms present in HT-29 M6 cells revealed that the most abundant TPA-sensitive isoform was PK-C epsilon, although low levels of cPK-C were also detected. Further studies are necessary to elucidate the role of the different PK-C isoforms in the differentiation of HT-29 cells.
... In all four cell lines, the uptake of glucose was fully blocked by Cyto B (Fig. 9d) and was insensitive to phlorizin (τ =31.5±4.5 s and 37±5 s, with and without phlorizin, respectively), suggesting GLUTfacilitated glucose transport. PCR data (Fig. 10) further suggested that GLUT1 is the main glucose transporter in the four cell lines, consistent with the observation that GLUT1 is expressed at high levels in tumor cell lines [9]. Figure 9a illustrates the effects of Cyto B in a HEK cell cultured in high glucose (i.e., the slow glucose uptake phenotype, Table 1) and Fig. 9d in a C2C12 cell cultured in low glucose (i.e. the rapid glucose uptake phenotype). ...
... As expected, we found that GLUT transporters mediate glucose flux in all of these cell types, as glucose influx was completely blocked by cytochalasin B but not by phlorizin. The real-time PCR (RTPCR) data suggest that the dominant GLUT is GLUT1, consistent with previous reports, indicating that cell culture lines express elevated levels of GLUT1 [9]. Our most significant and novel findings, however, relate to the extent to which acute and chronic changes in extracellular glucose dynamically regulate intracellular glucose homeostasis by modulating both glucose transport and metabolism. ...
To study intracellular glucose homeostasis, the glucose nanosensor FLIPglu-600 microM, which undergoes changes in fluorescence resonance energy transfer (FRET) upon interaction with glucose, was expressed in four mammalian cell lines: COS-7, CHO, HEK293, and C2C12. Upon addition of extracellular glucose, the intracellular FRET ratio decreased rapidly as intracellular glucose increased. The kinetics were fast (tau=5 to 15 s) in COS and C2C12 cells and slow (tau=20 to 40 s) in HEK and CHO cells. Upon removal of extracellular glucose, the FRET ratio returned to its initial value at similar rates (tau=15 to 40 s) in all cell types. In all cell types, the glucose uptake FRET signal was blocked by the glucose transporter (GLUTx) inhibitor cytochalasin B and was not affected by the Na/glucose transporter inhibitor phlorizin. Glucose clearance was inhibited by the glycolytic inhibitor iodoacetate. Using beta-escin to permeabilize the cell, we found that the glucose gradient across the membrane was strongly dependent on the rates of glucose uptake versus glucose clearance. With 10 mM extracellular glucose and a high rate of glucose clearance, intracellular glucose level fell below 100 muM when glucose uptake rate was low, whereas it exceeded 0.5 mM when glucose uptake was high. Cells cultured in high glucose maintained lower basal intracellular glucose levels than cells cultured in low glucose, attributed to "reciprocal regulation" of glycolysis and gluconeogenesis. Basal glucose level also increased with elevated temperatures. Experiments performed with C2C12 cells demonstrated a shift from fast glucose uptake to slow glucose uptake in the absence of insulin during differentiation.
... Levels of both GLUT-1 and GLUT-4 mRNA and protein can be regulated by several stimuli (reviewed in references 3 and 9). GLUT-l mRNA expression in rodent fibroblasts, brain, or neuronal cells is increased by growth factors (10)(11)(12)(13)(14), glucose deprivation (15), and cellular transformation (12,16,17). GLUT-4 mRNA and protein decrease in adipocytes (18)(19)(20)(21) and to a lesser extent in muscles (3,19,22) of rats with insulinopenic diabetes; whereas in fasted rats GLUT-4 expression is decreased in adipocytes (20,21,23) and increased in muscle (23). ...
Denervation rapidly (within 24 h) induces insulin resistance of several insulin-responsive pathways in skeletal muscle, including glucose transport; resistance is usually maximal by 3 d. We examined the effect of denervation on the expression of two glucose transporter isoforms (GLUT-1 and GLUT-4) in rat hindlimb muscle; GLUT-4 is the predominant species in muscle. 1 d postdenervation, GLUT-1 and GLUT-4 mRNA and protein concentrations were unchanged. 3 and 7 d postdenervation, GLUT-4 mRNA and protein (per microgram DNA) were decreased by 50%. The minor isoform, GLUT-1 mRNA increased by approximately 500 and approximately 100%, respectively, on days 3 and 7 while GLUT-1 protein increased by approximately 60 and approximately 100%. The data suggest that the insulin resistance of glucose transport early after denervation does not reflect a decrease in total glucose transporter number; however, decreased GLUT-4 expression may contribute to its increased severity after 3 d. Parallel decreases in GLUT-4 mRNA and GLUT-4 protein postdenervation are consistent with pretranslational regulation; GLUT-1 expression may be regulated pre- and posttranslationally. The cell type(s) which overexpress GLUT-1 postdenervation need to be identified. Nervous stimuli and/or contractile activity may modulate the expression of GLUT-1 and GLUT-4 in skeletal muscle tissue.
... To learn whether the effect of glucose deprivation on the abundance of the BBB-GLUT1 was dependent on protein kinase C activation, ECL cells were incubated in control and low glucose conditions in 5% fetal calf serum/DMEM with and without 1 pLM 12-0-tetradecanoylphorbol 13-acetate (TPA) for 24 h to induce depletion of the cellular protein kinase C (Hiraki et al., 1989). Poly(A)+ mRNA was isolated and the abundance of GLUTl mRNA was determined as described above. ...
The absence of neuroglucopenia symptoms in chronic hypoglycemia may be due to up-regulation of the blood-brain barrier glucose transporter type 1 (GLUT1). Therefore, we investigated the effect of glucose deprivation on the abundance of the GLUT1 transcript in bovine brain capillary endothelial cells in tissue culture (ECL). Northern blot analysis performed under high stringency conditions with 4-5 micrograms of ECL poly(A)+ mRNA showed that glucose deprivation (5 mg% glucose) caused a 2.4 +/- 0.2-fold increase in the GLUT1/actin mRNA ratio versus control incubations (100 mg% glucose). This rise was dose and time dependent, and the maximum effect was observed 20-24 h after the hexose deprivation. Nuclear transcription run-on assay showed no changes in either the GLUT1 or actin gene transcription rate 24 h after glucose deprivation. To determine whether the increase in the abundance of the GLUT1 mRNA induced by glucose deprivation was due to increased stability of this transcript, the GLUT1 mRNA half-life was measured in ECL cells incubated with actinomycin D. The levels of the GLUT1 transcript continued to be augmented in glucose-deprived cells compared with controls 2 and 4 h after the transcription inhibitor was added to the media. Glucose deprivation induced a 78% increase in the t1/2 of the GLUT1 mRNA (from 3.6 to 6.4 h). Incubation of ECL cells with the protein synthesis inhibitor, cycloheximide, for 4 h partially reversed the effect of glucose deprivation on the abundance of the GLUT1 transcript. On the other hand, incubation with cycloheximide for 24 h completely blocked the effect of glucose deprivation on the GLUT1 transcript. Desensitization of cellular protein kinase C was performed by incubation of ECL cells with 1 microM phorbol ester for 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)
... However, the possibility that other members of the glucose transporter family are also involved in promoting HeLa cell growth cannot be ruled out, since some mammalian cells express several transporters (Rhoads et al., 1988; Kaestner et al., 1989; Charron et al., 1989; Yamamoto et al., 1990; Bell et al., 1993). In addition, the increased amounts of GLUT1 regulated at the transcriptional level, as described in the oncogene-induced transformation of rodent fibroblasts (Flier et al., 1987; Birnbaum et al., 1987; Hiraki et al., 1989), would also support tumor growth and maintenance. In this context, it is notable that TGF-β1 induces the enhanced expression of GLUT1 mRNA as well as the modulation of GLUT1 glycosylation in mouse 3T3 fibroblasts, and stimulates their growth (Kitagawa et al., 1991; Masumi et al., 1993 Masumi et al., , 1994). ...
Studies of human cell hybrids have provided evidence that the tumorigenicity of a cervical carcinoma (HeLa) is under the control of a putative tumor suppressor on chromosome 11. Using these human cell hybrids, we found a tumor-associated glycosylation change in the glucose transporter GLUT1, which is an N-linked glycoprotein at the plasma membrane. The non-tumorigenic HeLa x fibroblast cell hybrid CGL1 and the normal diploid fibroblast WI38 expressed the 50-55 kDa GLUT1, whereas in a tumorigenic segregant hybrid, CGL4, as well as in parental HeLa cells, GLUT1 glycosylation was altered and its molecular mass was about 70 kDa. However, the altered GLUT1 glycosylation was not observed in SV40-transformed WI38 cells, suggesting a correlation between this glycosylation change and a putative tumor suppressor function. Further investigations using glycosidases, glycosylation inhibitors and lectin-affinity chromatography demonstrated that the tumor-associated glycosylation change in GLUT1 was mainly due to the increase in N-acetyl-lactosamine repeats in the N-linked oligosaccharides. In accordance with the altered glycosylation, affinity for 2-deoxyglucose in the tumorigenic CGL4 cells increased 2-fold, but there was little change in the Vmax. These results suggest there may be a functional role for the modulation by glycosylation of GLUT1 in the tumorigenic behavior of CGL4 and HeLa cells.
... GLUT4 depends on insulin for activation. Several papers suggest that activation of the gene coding for synthesis of the glucose transporter GLUT1 is a major early marker of cellular malignant transformation [15][16][17][18][19][20]. ...
Positron emission tomography (PET) is now primarily used in oncological indication owing to the successful application of fluorine-18 fluorodeoxyglucose (FDG) in an increasing number of clinical indications at different stages of diagnosis, and for staging and follow-up. This review first considers the biological characteristics of FDG and then discusses methodological considerations regarding its use. Clinical indications are considered, and the results achieved in respect of various organs and tumour types are reviewed in depth. The review concludes with a brief consideration of the ways in which clinical PET might be improved.
... 12 Especially, the type 1 glucose transporter (GLUT1) gene is one of the early genes that are activated after transformation of cells with oncogenes such as src, ras or fps. [13][14][15][16][17][18][19][20] An increase of GLUT1 mRNA was found 4-6 h after induction of the p21 c-H-ras oncoprotein, whereas morphologic changes occurred 72-96 h later. 18,21 The facilitative glucose transporter GLUT1 has also been shown to be upregulated in human tumor tissues. ...
In order to achieve tumor-specific targeting of adeno-associated virus (AAV)-mediated gene expression, the promoter of the glucose transporter isoform 1 (GLUT1) gene was cloned upstream of the enhanced green fluorescence protein (EGFP) and the herpes simplex virus thymidine kinase (HSVtk) gene. FACS analysis performed at 48 h after transient infection with rAAV/cytomegalovirus (CMV)egfp viral particles revealed an increase of fluorescence in all the cell lines tested. However, EGFP expression under control of the GLUT1 promoter element (rAAV/GTI-1.3egfp) was limited to the tumor cells and oncogene-transformed cells. Evidence for phosphorylation of the HSVtk substrates ganciclovir (GCV) and 125I-deoxycytidine was found in all transfected tumor cell lines compared to noninfected controls (HCT116: 111%; MH3924A: 130%; HaCaT-RT3: 257% increase), but not in HaCaT and HUVEC cells. Furthermore, tumor cells and the oncogene-transformed (ras) cell line HaCaT-RT3 showed a GCV-induced reduction in cell number (HCT116: -71%; MH3924A: -43% and HaCaT-RT3: -31%). No statistically relevant cytotoxic effect was observed in HaCaT (6% decrease) and HUVEC cells (2% decrease). Furthermore, a reduction of 3H-thymidine incorporation into the DNA was seen after treatment with GCV (HCT116: 38%; MH3924A: 33% and HaCaT-RT3: 37% decrease). In a therapy study of HSVtk-expressing tumors with GCV, we achieved total tumor remission.
Specific, anti-peptide antibodies against the five known mammalian facilitative D-glucose transporter isoforms (GLUT1-5) were used to investigate the transporter content of lactating rat mammary gland. Western blots of gland homogenate showed the apparent presence of GLUT1 and GLUT4. However, in isolated epithelial cells only GLUT1 was present, a result which indicated the mammary GLUT4 is present within adipocytes. The putative GLUT1 glucose transporter was recognised by antibodies against several hydrophilic regions of human erythrocyte GLUT1 and endoglycosidase F digestion decreased its apparent Mr from 50,000 to 42,000, a value essentially identical to that of the deglycosylated human protein. Its identity as a glucose transporter was confirmed by the ability of anti-GLUT1 antibodies to immunoprecipitate a mammary protein of apparent 50,000, which could be photolabelled in a D-glucose-sensitive fashion by the transport inhibitor [4-3H]cytochalasin B. Immunocytochemical staining of sections confirmed that the primary location of GLUT1 in the mammary gland is the epithelial cell. Sub-cellular fractionation experiments showed that the transporter is not only located at the cell surface, but also within the Golgi. However, quantitative Western blotting indicated that GLUT1 could only account for about half of the D-glucose-sensitive cytochalasin B binding sites in Golgi vesicles, suggesting the presence of a second, so far unidentified, glucose transporter isoform. Developmental changes in transporter expression in the gland were investigated both at the level of protein and mRNA. GLUT1 protein levels became detectable in the epithelial cells during the final 24-48hr of pregnancy, rose to a peak immediately following parturition, then fell slightly at the time of peak lactation. No major changes in GLUT1 mRNA were observed, implying a post-transcriptional control of GLUT1 expression. Removal of the litter for 24hr at peak lactation resulted in a total loss of GLUT1, suggesting feed-back inhibition influencing GLUT1 expression. The hormonal basis of these changes was investigated using explant culture of alveoli from mid-pregnant mammary gland. These experiments indicated that GLUT1 expression was dependent upon a lactogenic hormone, prolactin. Insulin and cortisol were also required for explant viability, but neither alone could stimulate synthesis of GLUT1.
After receiving a report of a negative biopsy for cancer, patients are usually pleased and relieved whether it be related to breast, gastrointestinal (GI) tract, lung, or other cancer. However, their pleasure is often shortlived, since many will experience significant postoperative complications in the form of pain, needless impoverishment, and many will learn that they were inadequately staged initially and wish that they had had a better preoperative work-up. Only 20% of solitary lung nodules are benign, so that for every happy patient after excision biopsy, four others will learn that their ‘nodule’ had the capability of metastasis, and a radically different therapeutic course might have been taken from the start. For cancer of the GI tract, a similar situation exists, and the reported numbers are more like one and three. For breast cancer, the numbers are reversed, with three negative dissections for every positive one, but for those three initially happy women, some will bear some functional disorder from the surgery for long periods and they, too, may wish they had had a more definitive less invasive pre-operative diagnostic work-up. All these patients may have undergone examination with computed tomography (CT), magnetic resonance imaging (MRI) or ultrasound (US), all non-invasive and much better than they once were; still, as screening procedures, there are many holes in their nets.
There are so many radiopharmaceuticals now available to the nuclear medicine physician that one must constantly refresh one’s knowledge about their properties. The purpose of this chapter is to serve as a reference structure for doing so.
Blood tumour markers are widely used in the follow up of patients treated for a malignant tumour. In many cases where the tumour associated marker is increasing the clinical and radiological evaluations remain normal. PET and CDET-scan with 18-FDG have been demonstrated as powerful tools in oncology and their use in such situations may give a new appraisal on the development of the disease. Seventy patients with an isolated increasing of a tumour associated marker (ACE ou CA19.9, CA15.3, CA125) were tested. Accuracy and sensitivity of the method were 82.8 and 96.5%, specificity 25%, and positive and negative predictive values 50% and 87%. Focusing on breast carcinomas and CA15.3 as well as ovarian cancers and CA125, the sensitivity and the predictive value are reaching 100%. Patients exhibiting a tumoral target associated to an increasing in blood tumour marker may be treated earlier with dedicated protocols. (C) 2000 Editions scientifiques et medicales Elsevier SAS.
Tumor necrosis factor-alpha (TNF-alpha), 12-O-tetradecanoylphorbol-13-acetate and cAMP stimulate hexose transport in quiescent 3T3-L1 preadipocytes by stabilizing the relatively labile mRNA coding for the basal glucose transporter, GLUT-1. The 3'-UTR of GLUT-1 mRNA contains a single copy of the destabilizing AUUUA motif in the context of an AU-rich region. The adenosine-uridine binding factor (AUBF) is a cytosolic protein which interacts with similar AU-rich regions in a variety of labile cytokine and oncogene mRNAs. Here, we demonstrate that AUBF complexes in vitro with GLUT-1 mRNA through the AU-rich portion of the 3'-UTR. AUBF activity is very low in quiescent preadipocytes, but can be up-regulated by agonists such as TPA, TNF-alpha, cAMP, and okadaic acid, all of which stabilize GLUT-1 mRNA. The time courses of TNF-alpha- and TPA-mediated AUBF up-regulation and GLUT-1 mRNA stabilization are coincident, suggesting a cause and effect relationship.
Cellular glucose uptake is mediated by a family of facilitative glucose transporters (GLUT) exhibiting differences in kinetics, substrate specificity, and tissue-specific expression. GLUT isoform expression has not been comprehensively studied in human leukocytes, which participate in immune and inflammatory responses and are critical for host defense. Therefore, we studied the regulated expression of GLUT 1–5 mRNA and protein in isolated human lymphocytes and monocytes and in human THP-1 macrophages and foam cells. Lymphocytes expressed GLUT 1 and GLUT 3 proteins, and cellular levels of both isoforms were augmented 3.5- to 6-fold following activation by phytohemagglutinin (PHA). Monocytes expressed 8.4-fold more GLUT 3 protein and 88% less GLUT 1 than lymphocytes, and activation by lipopolysaccharide (LPS) led to a 1.9-fold increase in GLUT 1. At the level of mRNA expression, GLUT 3 mRNA was the most prevalent GLUT mRNA species in monocytes, while lymphocytes expressed equal numbers of GLUT 1 and GLUT 3 transcripts. Differentiation of THP-1 monocytes into macrophages was associated with marked induction of GLUT 3 and GLUT 5 protein expression, and high levels of GLUT 1, GLUT 3, and GLUT 5 were maintained after transformation to foam cells. GLUT 5 mRNA was expressed in 2-fold greater abundance in macrophages and foam cells than that observed for GLUT 1 mRNA, while the level of GLUT 3 mRNA was intermediate. This facilitative glucose transporters are differentially expressed and regulated in human leukocytes in a pattern that could facilitate cellular functions. Speculatively, high GLUT 1 and GLUT 3 expression could provide cellular fuel for the immune response, and high levels of high-affinity GLUT 3 in macrophages might allow the cell to compete with pathogens for hexoses, even in the presence of low interstitial glucose concentrations. Ample expression of GLUT 1 and GLUT 3 in foam cells could also provide hexose substrates and promote lipid loading. The role for high levels of the fructose transporter GLUT 5 in macrophages and foam cells is unknown since interstitial and circulating fructose concentrations are low in these cells.
We have studied the PKC isoforms present in HT-29 M6 colon cancer cells, the differentiation of which to mucus-secreting cells is blocked by TPA. In addition to a major 72 kDa band, a 77 kDa PKC isoform was recognized by two different antibodies raised against a C-terminus-specific peptide for the TPA-insensitive isoform, PKCζ. By different criteria (association to the membrane, down-regulation, PKC activity in immunoprecipitates) we conclude that, contrary to the 72 kDa band, the 77 kDa band corresponds to a Ca2+- and TPA-sensitive PKC. These results suggest that antipeptide antibodies directed against the C-terminus of PKCζ react in human cells with a member of the conventional PKC subfamily besides PKCζ. Therefore, the data indicating that PKCζ is sensitive to different agents in various cell lines should be carefully re-evaluated.
Rat embryo fibroblasts and liver epithelial cell lines normally express two isoforms of protein kinase C (PKC), PKC,, and PKC,. Derivatives of these cells transformed by an activated human c-H-ras oncogene display a several-fold increase in expression of PKC, and a concomitant decrease in PKC,, at both the protein and mRNA levels. Similar changes are seen when the transformed phenotype is induced by Zn2� in cells carrying the activated ras oncogene under the control of a metallothionein promoter. Studies using cell lines that express very high levels of PKC,,, studies using a specific inhibitor of PKC (CGP 41251), and studies in which PKC activity is down-regulated by treatment with a phorbol ester tumor promoter provide evidence that the effects of the ras oncogene on the
Most cells express facilitative glucose transporters. Four isoforms (GLUT1-4) transporting D-glucose across the plasma membrane show a specific tissue distribution, which is the basis for tissue-specific patterns in glucose metabolism. GLUT1 is expressed at high levels in tissue barriers such as the blood-brain barrier, and this isoform has been suggested as an indicator of such barriers. GLUT1 has been found in basal layers of human epidermis where no such tissue barrier is present. To further clarify these issues, we examined the distribution of GLUT1 and GLUT4 in skin, different types of oral mucosa from rat and man, and a human oral careinoma by indirect immunofluorescence microscopy. The results showed that GLUT1 was expressed in the basal and parabasal layers of the different stratified squamous epithelia, with some variations between keratinized and non- keratinized subtypes. GLUT1 was also expressed in ductal- and myoepithelial cells of minor salivary glands and perineural sheath located in the lamina propra, and furthermore in the cells of an oral careinoma. GLUT4 was not expressed in any of the tissues examined. This distribution of GLUT1 does not fit with the idea of GLUT1 as a general indicator of tissue barriers. In contrast, our results support the prevailing, but limited knowledge of glucose metabolism in squamous stratified epithelia, a metabolism believed to depend mostly on glycolysis, especially in the basal layers. High-level expression seemed to be confined to keratinocytes without glycogen stores.
Growth factors and oncogenes promote glucose uptake, but the extent to which increased uptake is regulated at the level of glucose transporter function has not been clearly established. In this paper, we show that interleukin-3 (IL-3), a cytokine growth factor, and the transforming oncogenes ras and abl alter the activation state of glucose transporters by distinct mechanisms. Using bone marrow-derived IL-3-dependent 32Dcl3 (32D clone 3) cells and 32D cells transformed with ras and abl oncogenes, we demonstrated that IL-3 enhanced [3H]-2-deoxyglucose (2-DOG) uptake in parental 32Dcl3 cells by 40–50% at 0.2 mM 2-DOG, and this was associated with a 2.5-fold increase in transporter affinity for glucose (reduced Km). In comparison, ras and abl oncogenes enhanced 2-DOG uptake by 72–112%, associated with a 2-fold greater transporter affinity for glucose. The tyrosine kinase inhibitor genistein reversed the effects of both IL-3 and oncogenes on glucose uptake and reduced transporter affinity for glucose. Likewise, with exponentially growing 32D cells in the presence of IL-3, a protein kinase C inhibitor, staurosporine, and a phosphatidylinositol 3-kinase (PI-3) kinase inhibitor, wortmannin, inhibited 2-DOG uptake and decreased transporter affinity for glucose. In contrast, in oncogene-transformed cells, staurosporine inhibited 2-DOG uptake but failed to decrease transporter affinity for glucose, whereas wortmannin did not affect 2-DOG uptake. Inhibition of protein tyrosine phosphatases with vanadate enhanced 2-DOG uptake and transporter affinity for glucose in parental cells and in ras-transformed cells but had little effect on abl-transformed cells. Consistently, the serine/threonine phosphatase type 2A inhibitor okadaic acid enhanced 2-DOG uptake and transporter affinity for glucose in parental cells but had little effect on ras- or abl-transformed cells. These results demonstrate differences in the regulation of glucose transport in parental and oncogene-transformed 32D cells. Thus, IL-3 responses are dependent upon tyrosine, serine/threonine, and PI-3 kinases, whereas ras and abl effects on glucose transport depend upon tyrosine phosphorylation but are compromised in their dependence upon serine/threonine and PI-3 kinases.
The brain-type glucose transporter (GLUT-1) is a blood-brain barrier (BBB)-specific gene. The isolation of BBB-specific genes from total brain cDNA libraries is difficult because the brain capillary endothelium, which makes up the BBB in vivo, constitutes <0.2% of brain volume. Therefore, the present studies describe the preparation of a bovine brain capillary cDNA library in the lambdagt11 vector and the cloning of the BBB GLUT-1 cDNA. The cDNA sequenced was full length, as confirmed by primer extension analysis. The 5'-untranslated region (UTR) of the bovine GLUT-1 was 67 and 75% homologous with 5'-UTR sequences of the rabbit and human GLUT-1 through nucleotide overlaps of 116 and 159, respectively. A unique proline-rich sequence near the N-terminus of the bovine GLUT-1 was not found in other species and this correlated with marked immunoreactivity of the bovine, but not the human or rat, BBB GLUT-1 protein with an antiserum directed against the 15 amino acids at the N-terminus. In conclusion, these studies describe the cloning of the GLUT-1 cDNA from a BBB cDNA library. The extensive phylogenetic conservation of the 5'-UTR suggests the GLUT-1 gene may be subject to translational control in the regulation of BBB glucose transport.
The in vitro angiogenesis of endothelium obtained from peripheral tissues is stimulated by phorbol esters. The present studies examine the effects of phorbol esters or serum factors on GLUT1 glucose transporter, cytoplasmic actin, and beta-tubulin messenger RNA levels and gene transcription rates in bovine brain capillary endothelial cells grown in tissue culture. Messenger RNA levels were measured by Northern blot analysis and transcription rates were quantified by nuclear run-on assays. Although cytoplasmic actin mRNA levels in cultured brain endothelium were comparable to levels found in isolated capillaries isolated in vivo, there was a profound down-regulation of the GLUT1 glucose transporter mRNA in the cultured endothelium. The GLUT1 mRNA level was increased by exposure to 12-O-tetra-decanoyl-phorbol 13-acetate (TPA). Both serum and TPA enhanced cytoplasmic actin and beta-tubulin mRNA levels in cultured cells; the serum effect on cytoskeletal mRNA persisted through at least 24 h of exposure whereas the TPA stimulation was maximal by 2 h of exposure and lost following 8 h. Both serum and TPA increased cytoplasmic actin mRNA levels approximately 2- to 3-fold greater than the increase in beta-tubulin mRNA levels. GLUT1 and actin transcription rates were measured with the nuclear run-on assay, but no stimulation was observed following 3 h exposure to 200 nM TPA. In conclusion, these studies show that GLUT1 glucose transporter, cytoplasmic actin, and beta-tubulin mRNA levels in bovine brain capillary endothelial cells are regulated by both serum factors and phorbol ester, which activates the protein kinase C pathway, and that the mechanism of the phorbol ester effect is post-transcriptional.
The type 1 glucose transporter (GLUT1) gene encodes an integral membrane glycoprotein responsible for facilitating transfer of glucose across plasma membrane and is rapidly activated by serum, growth factors, and by oncogenic transformation. To elucidate the molecular mechanisms of regulation of GLUT1 gene expression, we isolated and characterized the mouse GLUT1 gene. DNA elements regulating transcription of the gene were analyzed in transient expression assays after transfection of NIH/3T3 cells with a low background chloramphenicol acetyltransferase (CAT) vector system pSVOOCAT. We identified two enhancer elements; the first one is located 2.7 kilobases upstream of the cap site of the gene which contains the homologous sequences with two 12-O-tetradecanoylphorbol-13-acetate-responsive elements (TREs), a serum response element, a cyclic AMP-responsive element (CRE) and three GC boxes, and the second one is located in the second intron of the gene which contains the homologous sequences with two TREs and one CRE. With the promoter alone the transcription of the gene is activated by src, only slightly activated by ras and is not activated by serum and platelet-derived growth factor. When the gene is accompanied by one of these enhancers, the transcription is activated by all these stimuli.
Glucose transport across the plasma membrane of mammalian cells is mediated by a family of homologous proteins. Each glucose transporter isoform has a specific tissue distribution which relates to that tissue's demand for glucose. The beta-cells of pancreatic islets are known to express a distinct glucose transporter isoform, termed GLUT 2, which has a high Km for glucose. In this study, we examined the glucose transporter content of normal rat islets and three beta cell lines, beta-TC, HIT and RIN cells. We show that at the protein level, GLUT 2 is the only detectable transporter isoform in normal islets, and that all three cell lines also express detectable GLUT 2. In contrast, all three cell lines expressed high levels of GLUT 1, but this isoform was not detected in normal islets. Neither the native islets nor any of the cell lines expressed GLUT 3. The insulin-responsive glucose transporter GLUT 4 was detected at very low levels in beta-TC cells; to our knowledge, this is the only non-muscle or adipose cell line which expresses this isoform. We propose that the elevated level of GLUT 1 expression, together with a reduced expression of the high Km transporter GLUT 2, may account for the characteristic aberrant patterns of glucose-stimulated insulin release in cell lines derived from beta-cells.
In mammals, glucose transport is mediated by five structurally related glucose transporters that show a characteristic cell-specific expression. However, the rat brain/HepG2/erythrocyte-type glucose transporter GLUT-1 is expressed at low levels in most cells. The reason for this coexpression is not clear. GLUT-1 is negatively regulated by glucose. Another family of proteins, glucose-regulated proteins (GRPs), is also ubiquitously expressed and stimulated by glucose deprivation and other cellular stresses. We therefore hypothesized that GLUT-1 may be a glucose-regulated stress protein. This was tested by subjecting L8 myocytes and NIH 3T3 fibroblasts to glucose starvation or exposure to the calcium ionophore A23187, 2-mercaptoethanol, or tunicamycin, all known to increase GRP levels. The mRNA for GLUT-1 was augmented by 50-300% in a time-dependent manner, similarly to the changes in GRP-78 mRNA. Ex vivo incubation of rat soleus muscles induced a marked and concomitant rise in the mRNA levels of GLUT-1 and GRP-78. Finally, calcium ionophore A23187 and 2-mercaptoethanol induced a 2- to 3-fold increase in the levels of the GLUT-1 protein and hexose uptake. In all instances in which GRP-78 and GLUT-1 responded to stress, the transcription of the cell-specific muscle/adipocyte-type insulin-responsive glucose transporter (GLUT-4) did not change. Thus, despite the lack of structural similarity, GLUT-1 and GRP-78 expression is regulated similarly, whereas the regulation of GLUT-4, which is structurally related to GLUT-1, is different. We propose that GLUT-1 belongs to the GRP family of stress proteins and that its ubiquitous expression may serve a specific purpose during cellular stress.
Understanding of the fundamental mechanisms underlying the complex regulation of glucose homeostasis has been dramatically transformed recently by the realization that glucose transport in mammalian tissues is mediated by a family of structurally related but genetically distinct glucose-transporter proteins. The regulatory factors and intracellular signaling pathways that influence expression of the genes encoding these proteins are just being identified. Factors that regulate glucose-transporter gene expression in vitro include oncogenes, growth factors, insulin, oral hypoglycemic agents, vanadate, glucocorticoids, ambient glucose levels, and the state of cellular differentiation. In vivo, glucose-transporter gene expression in adipose cells, skeletal muscle, and liver is markedly affected by various altered nutritional and metabolic states. Recent studies have demonstrated that two glucose transporters expressed in the same tissue may be regulated differently in response to the same metabolic perturbation. Furthermore, transporter regulation appears to be tissue specific. These observations lay the groundwork for future studies aimed at unraveling the functional roles of the individual transporter species in different tissues, the molecular processes involved in regulating the expression of these genes, and the impact of dysregulated glucose-transporter gene expression in the pathogenesis of insulin-resistant states such as diabetes.
The hypothesis that the GLUT-1 glucose transporter isoform is expressed selectively in brain at the capillary endothelium, i.e. the blood-brain barrier (BBB), was tested by using quantitative Western blotting, cytochalasin B binding, and in situ hybridization in bovine brain cortex. Purified human red cell glucose transporter was used as the standard for quantitative Western blots, because the mobility of the human erythrocyte and BBB glucose transporters in electrophoretic gels was identical. The concentration of immunoreactive glucose transporter in bovine BBB plasma membranes was 10.8 +/- 0.9 pmol/mgp (mean +/- S.E., n = 6). This value was not statistically different from the estimate of the maximal binding sites of D-glucose-displaceable [3H]cytochalasin B binding in the BBB membrane preparations, 11.7 +/- 3.5 pmol/mgp. In situ hybridization experiments using 35S-labeled antisense and sense riboprobes corresponding to nucleotides 385-932 of the GLUT-1 cDNA showed prominent hybridization of the antisense probe over brain microvascular endothelium, but no hybridization over neuropil greater than that found with the 35S-labeled sense probe. These studies are consistent with the following conclusion: (a) essentially 100% of the glucose transporter binding sites at the BBB can be accounted for by the GLUT-1 isoform; (b) in situ hybridization studies confirm previous Northern blot analysis and indicate the GLUT-1 gene is expressed selectively in microvascular endothelium in brain with minimal, if any, expression of this gene in neurons or glial cells in vivo.
The homozygous transgenic mouse line TG.AC contains a v-Ha-ras transgene and rapidly develops epidermal papillomas in response to either wounding or treatment with tumor promoters such as 12-O-tetradecanoylphorbol-13-acetate (TPA). The transgenic v-Ha-ras protein product was detected in all papillomas removed from TPA-treated TG.AC mice but not in vehicle- or TPA-treated TG.AC skin without tumors. In situ hybridization demonstrated that focal expression of the transgene was limited to regions of papilloma development and further localized the expression of the transgene message to the epidermal component of the papillomas, with the strongest signal in the basal epidermoid cells. Cellular proliferation, as indicated by immunohistochemical staining for proliferating-cell nuclear antigen (PCNA), was similarly localized primarily to basal epidermoid cells and, to a lesser extent, stratum spinosum cells in all papillomas analyzed. Cells that stained positively for PCNA were much more common in the papillomas than in the surrounding, normal-appearing skin. The focal nature of papilloma development was also evidenced by protein kinase C activity and hyperplasia after TPA treatment. As early as 18 d after the start of TPA treatment, focal hyperplasias associated with the follicular epidermis were observed in TG.AC but not nontransgenic FVB/N skin; these hyperplasias were assumed to be the precursors of the epidermal papillomas. To explain the development of transgene-expressing tumors from apparently transgene-negative, normal-appearing skin, we hypothesize that the papillomas arise from the clonal expansion of focal areas of epidermal cells that overexpress the transgene. We also propose that the TG.AC line is an excellent model for studying very early events in papillomagenesis.
We have used two experimental approaches to examine the possible role of phosphatidylinositol 3-kinase (PI 3-kinase) in the regulation of glucose transport in oocytes isolated from Xenopus laevis. Incubation of oocytes with the PI 3-kinase inhibitor wortmannin inhibited insulin-like growth factor-1-stimulated deoxyglucose uptake. Half-maximal inhibition was observed at concentrations approximately 20 nM. Conversely, we also examined the effects of microinjection of synthetic peptides designed to interact with Src homology 2 domains of the regulatory subunit of PI 3-kinase on deoxyglucose transport in oocytes. We show that a bifunctional synthetic peptide containing two YMXM consensus sequences for binding to SH2 domains stimulated both PI 3-kinase activity and deoxyglucose transport when both tyrosine residues were phosphorylated. However, non-phosphorylated or bisphosphonotyrosine peptides with the identical amino acid sequence failed to stimulate transport or PI 3-kinase activity. Taken together, these data argue strongly for a role for PI 3-kinase in the regulation of glucose transport in oocytes.
Mitogens and growth factors acutely stimulate glucose transport in all cells to supply energy for their growth and division, but little is known about the signalling mechanism by which these agonists promote sugar uptake. Here we show that the transport of deoxyglucose and 3-O-methylglucose into Xenopus laevis oocytes is stimulated about 2.5-fold when mitogen-activated protein kinase (MAP kinase) is microinjected into these oocytes. We also demonstrate that microinjection of the proto-oncogene product c-Mos (an activator of MAP kinase kinase, which activates MAP kinase in Xenopus oocytes), and purified MAP kinase kinase produce similar increases in deoxyglucose transport. Since the activation of MAP kinase is a general response to almost all mitogens and growth factors, we propose that one of its downstream effects is the stimulation of glucose-transport activity.
The plasma membrane in mammalian cells possesses unique permeability properties serving as a selective permeability barrier as well as transporters for nutrients and ions in maintaining cellular homeostasis. External ATP modulates the permeability barrier in transformed cells. The characteristics and possible mechanism for this permeability change are summarized. Application of this membrane change for cancer chemotherapy was also examined in both in vitro and in vivo. The uptake of D-glucose by mammalian cells was carried out by a facilitated diffusion through a specific transporter protein in the membrane. The control mechanism for glucose transport by growth factors based on the changes in the glucose transporter levels is summarized. Modulation of glycosylation in the transporter protein and its possible role are discussed.
Lysophosphatidic acid (LPA) stimulated the transport of deoxyglucose into oocytes isolated from Xenopus laevis. This stimulation was accounted for entirely by an increase in the Vmax for transport. Various LPAs with different acyl groups in the sn-1 position and phosphatidic acid stimulated deoxyglucose (deGlc) transport in these cells with a rank order potency of 1-oleoyl-LPA > 1-palmitoyl-LPA > phosphatidic acid = 1-stearoyl-LPA > 1-myristoyl-LPA. The phosphatidylinositol 3'-kinase inhibitor LY294002 completely blocked LPA-stimulated deoxyglucose uptake (IC50 approximately 2 microM). In marked contrast, wortmannin, which can completely block both insulin-like growth factor-I (IGF-I)-stimulated deGlc uptake in oocytes and phosphatidylinositol 3'-kinase activation at concentrations as low as 20 nM [Gould, Jess, Andrews, Herbst, Plevin and Gibbs (1994) J. Biol. Chem. 269, 26622-26625], was a relatively poor inhibitor of LPA-stimulated deGlc transport, even at concentrations as high as 100 nM. We further show that LPA stimulates phosphatidylinositol 3'-kinase activity(s) that can phosphorylate both phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate, and that this stimulation is inhibited by LY294002 but is relatively insensitive to wortmannin, again in marked contrast to IGF-I-stimulated phosphatidylinositol 3'-kinase activity. Antibodies against the p85 regulatory subunit of phosphatidylinositol 3'-kinase or antiphosphotyrosine antibodies immunoprecipitated IGF-I-stimulated but not LPA-stimulated phosphatidylinositol 3'-kinase activity. We conclude that LPA stimulates glucose uptake in Xenopus oocytes by a mechanism that may involve activation of a form of phosphatidylinositol 3'-kinase that is distinguished from other isoforms by its resistance to wortmannin and by its substrate specificity. Since the LPA-activated form of phosphatidylinositol 3'-kinase is pharmacologically and immunologically distinct from that which is involved in IGF-I-stimulated glucose transport in these cells, we suggest that distinct isoforms of this enzyme are able to function with the same biological effect, at least in the regulation of sugar transport.
Incubation of HT-29 M6 cells with the phorbol ester phorbol 12-myristate 13-acetate (PMA) induces cell scattering, loss of cellular contacts and inactivation of E-cadherin. We have investigated the involvement of different protein kinase C (PK-C) isoforms in these processes using specific activators. Thymeleatoxin, a derivative of mezerein that activates conventional PK-Cs (cPK-Cs) but not novel PK-Cs (nPK-Cs), promoted effects that were similar to those of PMA, i.e. at concentrations of 200 nM it induced scattering of HT-29 M6 colonies, loss of homotypic contacts and dissociation of E-cadherin from the cytoskeleton. Among the isoforms activated by this compound, only cPK-C alpha was detected in HT-29 M6 cells by Western blot. The specificity of this compound with respect to the rest of the PK-C isoforms present in these cells was determined; thymeleatoxin induced, as did PMA, the translocation of cPK-C alpha from the cytosol to the membrane and the cytoskeleton, and its partial down-regulation. On the other hand, thymeleatoxin did not modify the cellular levels or localization of nPK-C epsilon or atypical PK-C zeta. "In vitro' assays also showed that thymeleatoxin did not activate nPK-C epsilon at the concentrations added to the cell cultures. These results indicate that thymeleatoxin is selective for cPK-C alpha over nPK-C epsilon and show a role for the former enzyme in the regulation of cell-cell contacts and the inactivation of E-cadherin in HT-29 M6 cells.
The exposure of 3T3-L1 fibroblasts to growth factors results in a 2-to-3-fold increase in 2-deoxyglucose transport and a approximately 50% to 80% increase in cell-surface transferrin receptor levels. We sought to determine the role of phosphatidylinositol-3'-kinase and p70 ribosomal S6 kinase in these stimulations, using selective inhibitors of these enzymes. Both basal and growth factor-stimulated deoxyglucose transport are blocked by wortmannin, but with different IC50 values (65 nM vs. 15 nM, respectively), suggesting a functional difference between these two states. This is accompanied by the accumulation of glucose transporters in intracellular locations. Both basal and growth factor-stimulated cell-surface transferrin receptor levels are downregulated by wortmannin, but with identical IC50 values (approximately 15 nM). These two proteins are known to recycle between an intracellular site and the plasma membrane in these cells, thus implying a functional role for phosphatidylinositol-3'-kinase in membrane recycling. In an effort to determine whether the effect of wortmannin was selective for the protein component of this recycling, we examined fluid-phase endocytosis of radiolabeled mannitol. Wortmannin was without effect on the fluid phase accumulation of mannitol, suggesting that the effects on membrane traffic are limited to the protein component of recycling membranes. Rapamycin, an inhibitor of p70 ribosomal S6 kinase, was without effect on any of these parameters, but both rapamycin and wortmannin inhibit growth factor-stimulated p70 ribosomal S6 kinase activity. These data support an important role for phosphatidylinositol-3'-kinase, but not p70 ribosomal S6 kinase, in the regulation of membrane protein traffic. We suggest that this enzyme may be involved in sorting of membrane proteins during trafficking.
2-Deoxyglucose uptake was enhanced in ts371 KiMuSV-NRK cells when growing at the permissive temperature to allow the expression of a transforming p21 ras protein. This change is due to a decrease in the K(m) by approximately 2.5-fold without affecting the V(max) of the transporter. The amount of the GLUT1 glucose transporter dit not increase as deduced from immunoblot experiments on total membranes. Nevertheless, ras-transformed GLUT1 displays a higher molecular mass due to an increased N-glycosylation of the protein. Experiments made in tunicamycin-treated cells indicates that a higher glycosylation is responsible for the increase in 2-deoxyglucose uptake in ras-transformed cells.
To determine the influence of tumor cell proliferation and changes in the genetic program in malignant cells on the fluorodeoxyglucose (FDG) uptake we performed PET studies in several animal tumors: spontaneous mammary fibroadenoma, chemically-induced mammary adenocarcinoma and Dunning prostate adenocarcinoma. The expression of the glucose transporter (GLUT1) and of hexokinase (Hk) was measured using 32P-labeled cDNA probes and densitometry. Furthermore the proliferative activity was determined with one-dimensional flow cytometry. The FDG uptake and the proliferation parameters were not correlated. The normalized amounts of GLUT and Hk mRNA were lower in spontaneous fibroadenomas and prostate tumors than in chemically induced mammary. The FDG uptake was correlated to GLUT1 expression with r = 0.83 and to Hk expression with r = 0.77. Multiple regression analysis revealed a relation of FDG uptake to GLUT1 and HK with r = 0.87. Our results show that the FDG uptake in our study was related not to differences in proliferation, but rather to differences in the transcription of glycolysis associated genes.
The stimulation of glucose transport is one of the early cellular responses to growth factors and is essential for cell proliferation, yet the molecular processes that underlie this response are poorly defined. The aim of this study was to characterize the role of the low-molecular-mass G-proteins, Ras and Rho, and their downstream targets, Raf protein kinase and phosphatidylinositol 3-kinase, in the regulation of glucose transport in Xenopus oocytes by two distinct growth-factor receptors: the insulin-like growth factor I (IGF-I) tyrosine kinase receptor and the heterotrimeric G-protein-coupled lysophosphatidic acid (LPA) receptor. Microinjection of a neutralizing anti-Ras antibody partially blocked IGF-I-stimulated deoxyglucose uptake but was without effect on LPA-stimulated deoxyglucose uptake. In contrast, microinjection of the C3 coenzyme of botulinum toxin, which selectively ADP-ribosylates and inactivates Rho, inhibited LPA-stimulated, but not IGF-I-stimulated, deoxyglucose uptake. Similarly, LPA- but not IGF-I-stimulated deoxyglucose uptake was attenuated in oocytes expressing a dominant negative rho construct. Cells expressing a dominant negative mutant of Raf protein kinase exhibited markedly reduced sensitivity to both LPA and IGF-I, consistent with a role for endogenous Raf in glucose uptake by both growth factors. Furthermore, expression of a constitutively activated form of raf-1 resulted in a growth-factor-independent increase in deoxyglucose uptake. Measurements of phosphatidylinositol 3-kinase activity in microinjected cells support the hypothesis that the IGF-I receptor stimulates glucose transport by a Ras-dependent activation of phosphatidylinositol 3-kinase, whereas the G-protein-coupled LPA receptor controls this response by a pathway that involves Rho-dependent activation of a distinct phosphatidylinositol 3-kinase. Thus we provide evidence for clear differences in the signalling pathways that control glucose transport by G-protein-coupled and tyrosine kinase growth-factor receptors. Furthermore this is the first demonstration that active Rho is involved in the signalling pathways that regulate glucose uptake in response to some growth factors.
Studies on human cell hybrids between a cervical carcinoma cell line, HeLa, and normal fibroblasts have indicated that their tumorigenicity is under the control of a putative tumor suppressor on chromosome 11. We have previously demonstrated that a tumorigenic cell hybrid CGL4 expresses a larger glucose transporter, GLUT1, due to altered glycosylation when compared to the nontumorigenic counterpart CGL1. In this study, we demonstrated this glycosylation change in GLUT1 in gamma-ray-induced tumorigenic mutants (GIMs) isolated from CGL1 cells as expressing a tumor-associated surface antigen, intestinal alkaline phosphatase. In contrast, GLUT1 in the gamma-irradiated nontumorigenic control cells (CONs) did not show this alteration. In accordance with this glycosylation change, affinity to 2-deoxyglucose in these GIM clones was increased by about twofold when compared to the nontumorigenic CONs. These results suggest a close correlation between the glycosylation change in GLUT1 with increased affinity to D-glucose and tumorigenicity of these human cell hybrids.
We used mouse hepatoma (Hepa1c1c7) cells to study the role of the serine/threonine kinase Akt in the induction of GLUT1 gene expression. In order to selectively turn on the Akt kinase cascade, we expressed a hydroxytamoxifen-regulatable form of Akt (myristoylated Akt1 estrogen receptor chimera (MER-Akt1)) in the Hepa1c1c7 cells; we verified that hydroxytamoxifen stimulates MER-Akt1 activity to a similar extent as the activation of endogenous Akt by insulin. Our studies reveal that stimulation of MER-Akt1 by hydroxytamoxifen induces GLUT1 mRNA and protein accumulation to levels comparable to that induced by insulin; therefore, activation of the Akt cascade suffices to induce GLUT1 gene expression in this cell system. Furthermore, expression of a kinase-inactive Akt mutant partially inhibits the response of the GLUT1 gene to insulin. Additional studies reveal that the induction of GLUT1 mRNA by Akt and by insulin reflects increased mRNA synthesis and not decreased mRNA degradation. Our findings imply that the GLUT1 gene responds to insulin at the transcriptional level and that Akt mediates a step in the activation of GLUT1 gene expression in this system.
Glucose utilisation by cancer cells is greatly enhanced when compared with that by normal tissue. Glucose is taken up by cells and then phosphorylated to glucose-6-phosphate. Facilitative hexose uptake is achieved by five transmembrane transporters, termed glut1-5, which are protein products of their respective GLUT genes. Glut types differ in their kinetics, which are tailored to the requirements of the cell type they serve, although more than one glut may be expressed by a particular cell type. Herein are reviewed the results from approximately 30 studies which examined glut expression in human cancer tissue. These studies measured GLUT messenger RNA (mRNA) either using the reverse-transcriptase polymerase chain reaction or by Northern blot analysis, or detected glut proteins using the appropriate antibodies. Tumour tissue is frequently associated with the abnormal and/or over-expression of gluts, especially glut1. Some tumour cells express specific GLUT mRNA but not the respective protein. Some studies have reported associations between glut expression and proliferative indices, whilst others suggest that glut may be of prognostic significance, especially in lung cancer.
Positron emission tomography (PET) with FDG has shown to be of substantial value in differential diagnosis of pulmonary lesions and in the assessment of lymph node involvement with higher sensitivity and specificity than CT. A negative PET scan of the mediastinum suggests that mediastinoscopy is unnecessary and that these patients can proceed directly to thoracotomy. The method is also useful for the visualization of distant metastases. Since changes of treatment may result after identification of distant metastases PET is also cost-effective [Eur J Nucl Med 27(2000)1598; Australas Radiol 45(2001)9]. Furthermore, changes of tumor metabolism can be detected with PET at early stages after treatment, which can be used for therapy monitoring and for the detection of recurrent tumor tissue after completion of treatment.
The Myc gene family which includes c-Myc, N-Myc and L-Myc, are transcription factors that play a role in cell proliferation, apoptosis and in the development of human tumors. Myc amplification and overexpression has been detected in lung cancer of different histologic subtypes. Although the mechanism of Myc action is not yet fully understood, Myc has been proposed to play a role in growth control and cell cycle progression by stimulating and repressing the expression of key cell cycle regulators. This review will focus on the role of Myc in stimulating the G1/S transition of the cell cycle by regulating the levels and activity of cyclins, cyclin dependent kinases (cdk), cdk inhibitors and the pRb-binding transcription factor E2F. It is proposed that both the overexpression of Myc and the deregulation of the pRB/E2F pathway promotes the G1 to S transition in parallel by activating cyclinE/cdk2 complexes in lung cancer cells.
The staging of non-small lung cancer has to be performed in an interdisciplinary approach considering all clinical, radiological and histologic results. The staging using imaging procedures is done according to the TNM classification with T describing the extent of the primary tumor, N the presence and location of metastatic lymph nodes and M the presence or absence of distant metastases. It is important to remember that the individual stages of the TNM classification have undergone numerous revisions and thus need to be considered in their most recent version [Chest 111 (1997) 1718; Chest 111 (1997) 1710]. Noninvasive information about the stage of the disease is important for the planning and optimization of therapy. This may be done with imaging procedures such as, CT, MRT or PET.
Targeted transfer of a functionally active sodium iodide symporter (NIS) into tumour cells may be used for radioiodine therapy of cancer. Therefore, we investigated radioiodine uptake in a hepatoma cell line in vitro and in vivo after transfer of the sodium iodide symporter ( hNIS) gene under the control of a tumour-specific regulatory element, the promoter of the glucose transporter 1 gene (GTI-1.3). Employing a self-inactivating bicistronic retroviral vector for the transfer of the hNIS and the hygromycin resistance genes, rat Morris hepatoma (MH3924A) cells were infected with retroviral particles and hNIS-expressing cell lines were generated by hygromycin selection. (125)I(-) uptake and efflux were determined in genetically modified and wild type hepatoma cells. In addition, the iodide distribution in rats bearing wild type and genetically modified hepatomas was monitored. hNIS-expressing MH3924A cell lines accumulated up to 30 times more iodide than wild type hepatoma cells, with a maximal iodide uptake after 30 min incubation time. Competition experiments in the presence of sodium perchlorate revealed a decrease in the iodide uptake (80-84% decrease). Moreover, ouabain led to a loss of accumulated I(-) (81% decrease) whereas 4,4'-diisothiocyano-2,2'-disulphonic acid stilbene (DIDS) increased the I(-) uptake into cells (87% increase). However, a rapid efflux of the radioactivity (70%) was observed 20 min after (125)I(-)-containing medium had been replaced by non-radioactive medium. Lithium had no significant effect on iodide efflux. In rats, the hNIS-expressing tumours accumulated 22 times more iodide than the contralateral wild type tumour. In accordance with the in vitro data, we also observed a rapid efflux of the radioactivity out of the tumour in vivo. Dosimetric calculations resulted in an absorbed dose of 85 mGy in the wild type tumour and 830 mGy in the hNIS-expressing tumour after administration of 18.5 MBq (131)I. In conclusion, transduction of the hNIS gene under the control of the GLUT1 promoter element induces iodide transport in Morris hepatoma cells in vitro and in vivo. However, for therapeutic application additional conditions need to be defined which inhibit the iodide efflux out of the tumour cells.
Cellular glucose uptake is mediated by a family of facilitative glucose transporters (GLUT) exhibiting differences in kinetics, substrate specificity, and tissue-specific expression. GLUT isoform expression has not been comprehensively studied in human leukocytes, which participate in immune and inflammatory responses and are critical for host defense. Therefore, we studied the regulated expression of GLUT 1-5 mRNA and protein in isolated human lymphocytes and monocytes and in human THP-1 macrophages and foam cells. Lymphocytes expressed GLUT 1 and GLUT 3 proteins, and cellular levels of both isoforms were augmented 3.5- to 6-fold following activation by phytohemagglutinin (PHA). Monocytes expressed 8.4-fold more GLUT 3 protein and 88% less GLUT 1 than lymphocytes, and activation by lipopolysaccharide (LPS) led to a 1.9-fold increase in GLUT 1. At the level of mRNA expression, GLUT 3 mRNA was the most prevalent GLUT mRNA species in monocytes, while lymphocytes expressed equal numbers of GLUT 1 and GLUT 3 transcripts. Differentiation of THP-1 monocytes into macrophages was associated with marked induction of GLUT 3 and GLUT 5 protein expression, and high levels of GLUT 1, GLUT 3, and GLUT 5 were maintained after transformation to foam cells. GLUT 5 mRNA was expressed in 2-fold greater abundance in macrophages and foam cells than that observed for GLUT 1 mRNA, while the level of GLUT 3 mRNA was intermediate. This facilitative glucose transporters are differentially expressed and regulated in human leukocytes in a pattern that could facilitate cellular functions. Speculatively, high GLUT 1 and GLUT 3 expression could provide cellular fuel for the immune response, and high levels of high-affinity GLUT 3 in macrophages might allow the cell to compete with pathogens for hexoses, even in the presence of low interstitial glucose concentrations. Ample expression of GLUT 1 and GLUT 3 in foam cells could also provide hexose substrates and promote lipid loading. The role for high levels of the fructose transporter GLUT 5 in macrophages and foam cells is unknown since interstitial and circulating fructose concentrations are low in these cells.
The aim was to assess the requirements for a positron emission tomography (PET) cancer imaging service. The UK was used as an example to create a mathematical model for calculating the number of dedicated PET scanners and cyclotron/radiochemistry production facilities required to support the demand for PET studies in lung cancer. This was then extended to all oncological indications for PET and comparison was made with present infrastructure in the UK and Europe. A clinical algorithm for the use of PET in lung cancer management was created and built into a comprehensive computer model with variable parameters. From lung cancer incidences, data reported in the literature and local data, the proportion of patients following each algorithmic path was determined and used to calculate the number of PET scans and hence PET scanners required for lung cancer, and all cancer indications. Substituting lung cancer incidences, the PET infrastructure required for each European country was assessed. From this analysis, 29,886 PET scans per year for lung cancer investigation (provision of 12 scanners) and 121,589 PET scans (2,026.5 per million population) for all indications [provision of 49 scanners (0.82 per million population)] are required in the UK; at present there are seven scanners, and thus 42 new scanners are required. Results reported here demonstrate considerable lack of investment in PET in Europe, with marked variation; Belgium has the most sufficient infrastructure (197.80% of requirements), and excluding France, which is soon to see extensive development, the UK has the least sufficient infrastructure (14.39% of requirements). Considerable investment is required so that cancer management can gain the clinical and cost-effective benefit of this functional imaging technique, which has been established.
Activation of Ras promotes oncogenesis by altering a multiple of cellular processes, such as cell cycle progression, differentiation, and apoptosis. Oncogenic Ras can either promote or inhibit apoptosis, depending on the cell type and the nature of the apoptotic stimuli. The response of normal and transformed colonic epithelial cells to the short chain fatty acid butyrate, a physiological regulator of epithelial cell maturation, is also divergent: normal epithelial cells proliferate, and transformed cells undergo apoptosis in response to butyrate. To investigate the role of k-ras mutations in butyrate-induced apoptosis, we utilized HCT116 cells, which harbor an oncogenic k-ras mutation and two isogenic clones with targeted inactivation of the mutant k-ras allele, Hkh2, and Hke-3. We demonstrated that the targeted deletion of the mutant k-ras allele is sufficient to protect epithelial cells from butyrate-induced apoptosis. Consistent with this, we showed that apigenin, a dietary flavonoid that has been shown to inhibit Ras signaling and to reverse transformation of cancer cell lines, prevented butyrate-induced apoptosis in HCT116 cells. To investigate the mechanism whereby activated k-ras sensitizes colonic cells to butyrate, we performed a genome-wide analysis of Ras target genes in the isogenic cell lines HCT116, Hkh2, and Hke-3. The gene exhibiting the greatest down-regulation by the activating k-ras mutation was gelsolin, an actin-binding protein whose expression is frequently reduced or absent in colorectal cancer cell lines and primary tumors. We demonstrated that silencing of gelsolin expression by small interfering RNA sensitized cells to butyrate-induced apoptosis through amplification of the activation of caspase-9 and caspase-7. These data therefore demonstrate that gelsolin protects cells from butyrate-induced apoptosis and suggest that Ras promotes apoptosis, at least in part, through its ability to down-regulate the expression of gelsolin.
The proenzyme of a Ca2+-dependent protease-activated protein kinase previously obtained from mammalian tissues (Inoue, M., Kishimoto, A., Takai, Y., and Nishizuka, Y. (1977) J. Biol. Chem. 252, 7610-7616) was enzymatically fully active without limited proteolysis when Ca2+ and a membrane-associated factor were simultaneously present in the reaction mixture. The activation process was reversed by removing Ca2+ with ethylene glycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid. An apparent Ka value for Ca2+ was less than 5 x 10(-5) M. Other divalent cations were inactive except for Sr2+, which was 5% as active as Ca2+. The factor was almost exclusively localized in membrane fractions of various tissues including brain, liver, kidney, skeletal muscle, blood cells, and adipose tissue. It was easily extractable with chloroform/methanol (2:1), and was recovered in the phospholipid fraction. In fact, this membrane factor could be replaced by chromatographically pure phosphatidylinositol, phosphatidylserine, phosphatidic acid, or diphosphatidylglycerol. Phosphatidylethanolamine, phosphatidylcholine, and sphingomyelin were far less effective under the comparable conditions. Ca2+-dependent modulator protein was unable to support enzymatic activity. The enzyme thus activated showed an ability to phosphorylate five histone fractions and muscle phosphorylase kinase, and appeared to possess multifunctional catalytic activities.
The human HepG2/erythrocyte glucose-transporter gene, including the promoter region, has been isolated and characterized. The gene, which is approximately 35,000 base pairs, is interrupted by nine intervening sequences or introns. The sequence of the HepG2 glucose-transporter protein predicted from the gene sequence differs from that determined from the published cDNA sequence in having Leu rather than Phe at position 152. In addition, there are several other nucleotide differences between the gene and cDNA sequences in both the coding region and 3'-untranslated region that do not alter the amino acid sequence of the protein. The sequence of the promoter and the site of transcription initiation have also been determined. The promoter region includes a TATA motif and two binding sites for the transcription factor Spl as well as a sequence that is found in the promoter region of several phorbol ester-inducible genes.
To identify mRNAs with altered expression in Rous sarcoma virus (RSV)-transformed cells, we screened a chicken embryo fibroblast
(CEF) cDNA library by differential hybridization. One clone, designated R1H, showed markedly elevated mRNA expression in RSV-transformed
cells. Nucleotide sequence analysis indicated that R1H mRNA encodes 78-kilodalton glucose-regulated protein (GRP78). Chicken
GRP78 was found to be very highly conserved in comparison with rat GRP78 (96% identity between chicken and rat amino acid
sequences). In contrast to previous observations, we found that GRP78 was induced in RSV-transformed cells in the absence
of glucose deprivation. When cells were grown in glucose-supplemented medium, the level of GRP78 mRNA was approximately fivefold
higher in RSV-transformed CEF than in transformation-defective virus-infected or uninfected CEF. Similar changes in GRP78
protein content were also found. Using a temperature-sensitive mutant of RSV and supplemental glucose, we found a gradual
increase in the level of GRP78 mRNA beginning at 4 h after shiftdown to permissive temperature. Uridine supplementation did
not block the induction seen in CEF infected with a temperature-sensitive mutant. These results indicate that GRP78 is induced
by p60v-src in the absence of glucose deprivation.
A simple enzymatic method for the quantitation of the mass of sn-1,2-diacylglycerol (DAG) present in crude lipid extracts was developed to assess the function of DAGs as intracellular "second messengers" of extracellular agents and of oncogene products. The assay employed Escherichia coli DAG kinase which constituted approximately 15% of the membrane protein of a plasmid-bearing strain and defined mixed micellar conditions to solubilize the DAG present and allow its quantitative conversion to [32P]phosphatidic acid. The assay was proportional with the amount of DAG added over the range of 25 pmol to 25 nmol. The rapid rise of DAG in platelets stimulated with thrombin (210% over basal) and in hepatocytes stimulated with vasopressin (230% over basal) was quantitated and the values agreed with previous measurements. The amounts of DAG in normal rat kidney (NRK) cells grown at 34 and 38 degrees C, respectively, were 0.47 and 0.61 nmol/100 nmol of phospholipid. In K-ras-transformed NRK cells grown at 34 or 38 degrees C, DAG levels were elevated 168 or 138%, respectively. When a temperature-sensitive K-ras NRK cell line was investigated, the amount of DAG present was elevated at the permissive but not at the restrictive temperature. These data are consistent with the K-ras protein functioning in transmembrane signalling by activating phospholipase C. Protein kinase C (Ca2+/phospholipid-dependent enzyme) activation by DAG may play an important role in cellular transformation.
The gene encoding the rat brain facilitated glucose transporter protein was cloned and partially sequenced. The transcribed regions encode 10 exons that span about 30 kilobases of genomic DNA. The intron size is markedly biased, the first two significantly greater in length than the seven others. All of the introns are predicted to occur in regions that encode putative extramembranous domains of the protein, consistent with the proposed topology of 12 alpha-helical membrane-spanning segments. In brain, transcription of the glucose transporter gene initiates at two adjacent adenosine residues located about 30 base pairs 3' to a TATA sequence. In addition, there is at least one minor upstream start site. Both transformation of fibroblasts by the oncogenic retrovirus Fujinami sarcoma virus and stimulation of quiescent fibroblasts with serum increase transcription of the glucose transporter gene from identical initiation sites, which are the same as the predominant start sites in brain. The use of the same promoter for increased transcription under both conditions is consistent with the hypothesis that the regulation of gene expression by normal growth and by oncogenesis is mediated by similar or identical pathways.
The expression of the gene encoding the facilitated glucose transporter (GT) protein was studied in fibroblast cell lines. Addition of 15% calf serum to confluent BALB/c3T3, NIH3T3, or Rat-2 cells rapidly induced a 5-10-fold increase in GT mRNA, as determined by hybridization of size-fractionated total RNA to a rat brain GT cDNA. The rise in GT mRNA was maximal at 3-4 h after stimulation, and then returned to basal values by 16 h. The serum-stimulated increase in GT mRNA was not blocked by the protein synthesis inhibitors cycloheximide (10 micrograms/ml) or anisomycin (100 microM). In BALB/c3T3 cells, fibroblast growth factor (100 ng/ml), platelet-derived growth factor (5 units/ml), and epidermal growth factor (40 ng/ml) stimulated GT mRNA accumulation, although, when added individually, none of these growth factors increased DNA synthesis. The tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), which activates the enzyme protein kinase C, also caused GT mRNA accumulation in BALB/c3T3 and NIH3T3 cells. Prolonged pretreatment of cells with TPA abolished the response to TPA but not fibroblast growth factor. The involvement of GT gene transcription was assessed by the nuclear run-on technique. Treatment of NIH3T3 cells with serum increased transcription at least 10-20-fold by 30 min and returned to near basal levels by 2 h. This rapid activation paralleled that of the c-fos gene, but preceded the increase in c-myc gene transcription. These data indicate the following: 1) serum growth factors increase glucose transporter mRNA levels by a process not requiring intermediary new protein synthesis and clearly dissociable from mitogenesis, 2) the changes in GT mRNA are preceded by a rapid and transient activation of GT gene transcription, and 3) there exist protein kinase C-dependent and independent pathways for regulation of GT gene expression.
Microinjection of purified protein kinase C (PKC) into Swiss 3T3 fibroblasts pretreated with the phorbol ester phorbol-12,13-dibutyrate
restores the mitogenic response of the cells to phorbol-12,13-dibutyrate (G. Pasti, J.C. Lacal, B.S. Warren, S.A. Aaronson,
and P.M. Blumberg, Nature [London] 324:375-377, 1986). Our present studies demonstrate that the mitogenic activity of the
H-ras oncogene in H-ras p21-microinjected quiescent cells is markedly reduced under conditions in which PKC is downregulated
by chronic phorbol ester treatment. The ability to reconstitute the mitogenic response upon microinjection of both H-ras p21
and PKC implies involvement of functional PKC in the mitogenic activity of the H-ras oncogene product.
Activity of the Ca2+/phospholipid-dependent protein kinase C has been shown to increase during differentiation of the human promyelocytic leukemia cell line HL-60 by dimethyl sulfoxide and retinoic acid (Zylber-Katz, E., and Glazer, R. I. (1985) Cancer Res. 45, 5159-5164). Antipeptide antibodies were prepared that specifically recognize the alpha, beta, and gamma isozymes of protein kinase C in rat brain cytosol and HL-60 cell extracts. The three isozymes do not share a common tissue distribution pattern. The gamma enzyme is abundant in brain but a relatively minor component in HL-60 cells; the opposite is true for the alpha enzyme. All three isozymes increase at least 2-fold in abundance in HL-60 cells exposed to 1.2% dimethyl sulfoxide for 48 h. The increase in abundance of the alpha and beta isoforms reaches 7- and 5-fold, respectively, by 96 h without further increase in the abundance of the gamma isozyme. Similarly, all three isozymes increase at least 1.5-fold in abundance after 48 h and 3-fold after 96 h with 1 microM retinoic acid. No further increase in the abundance of any of the isozymes is seen between 96 and 144 h of incubation with retinoic acid. The increase in protein kinase C activity is not limited to the cytosolic forms of the enzyme; a parallel increase in membrane-associated protein kinase C is also observed during differentiation. Approximately 10% of total protein kinase C activity is membrane-associated in both control and differentiating cells. These studies provide the first immunochemical evidence that all three protein kinase C isozymes increase during HL-60 cell differentiation, and they suggest that the increase in the isozyme levels may be coordinately regulated.
Subconfluent cultures of NIH-3T3 fibroblasts transformed by the Ha-ras, Ki-v-ras, v-src, and v-fms oncogene proteins all possess elevated steady-state levels of diacylglycerol, the endogenous activator of protein kinase C, as compared to the nontransformed parental lines. These oncogene-transformed fibroblasts also exhibit a significantly decreased level of cellular protein kinase C activity as measured by four different criteria: phorbol ester-stimulated phosphorylation of an endogenous 80-kilodalton (80 kDa) substrate; phorbol ester-stimulated changes in 86Rb uptake; enzymatic assay; and [3H]phorbol ester binding. In all cases, the transformed cells demonstrated an attenuated response to phorbol ester addition and a lower phorbol ester binding capacity as compared to the parental lines. Western analysis of the endogenous 80-kDa substrate of protein kinase C revealed a significantly lower level of this protein in the transformed cells than in the untransformed controls, and this decrease could be mimicked in parental cells by long-term incubation with phorbol esters, suggesting that the level of the 80-kDa protein is regulated by the state of activation of protein kinase C. These effects do not appear to be nonspecific responses to autocrine secretions by the transformed cells. They may represent an unsuccessful attempt by the transformed cells to negatively modulate the constitutive proliferative signals generated by the oncogene products.
The effect of phorbol 12-myristate 13-acetate (PMA) on protein kinase C was studied by metabolically labeling GH3 cells with [35S]methionine and using a polyclonal antibody raised against rat brain protein kinase C to immunoprecipitate the enzyme. PMA accelerates the loss of immunologically reactive protein kinase C from the cells in a time- and dose-dependent manner. The half-life of the enzyme in cells treated with 400 nM PMA was 2 h whereas in control cells 60-70% of the enzyme was still detectable after 24 h. The concentration of PMA required to reduce cellular protein kinase C 50% after 24 h was 130 nM. PMA also induced the translocation of [35S]Met-labeled protein kinase C from the cytosol to the membranes in a concentration-dependent manner. Less protein kinase C was translocated to membranes when cells were treated with 20 nM PMA than when they were exposed to 400 nM PMA. In the latter case, most of the labeled protein kinase C became membrane-associated. Maximal translocation was evident after 15 min of incubation with either concentration of PMA and was followed by degradation of the membrane-associated enzyme. The rate of degradation of membrane-associated protein kinase C was the same with both concentrations of PMA. In cells treated with 20 nM PMA, disappearance of [35S]Met-labeled protein kinase C from the cytosolic fraction occurred in two phases, a rapid decrease characteristic of the membrane-associated enzyme, followed by a slower loss similar to that seen in control cells. The results indicate that turnover of protein kinase C is enhanced by membrane association.
Two temperature-sensitive mutants of Fujinami sarcoma virus were isolated and characterized. Cells infected with the mutants were temperature sensitive in focus formation, colony formation, increased sugar uptake, and synthesis of plasminogen activator. The changes between transformed and nontransformed states of cultures were completely reversible by shifting the temperature. A Fujinami sarcoma virus-specific protein of 130,000 daltons, p130, was synthesized in mutant-infected cells regardless of the temperature, but the immunoprecipitates of p130 from extracts of infected cells were active in protein kinase only when cells had been incubated at the permissive temperature. These results appear to indicate that p130 is the transforming protein of Fujinami sarcoma virus, and that its protein kinase activity plays a crucial role in cell transformation by this virus.
Tumor-promoting phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) directly activate in vitro Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C), which normally requires unsaturated diacylglycerol. Kinetic analysis indicates that TPA can substitute for diacylglycerol and greatly increases the affinity of the enzyme for Ca2+ as well as for phospholipid. Under physiological conditions, the activation of this enzyme appears to be linked to the receptor-mediated phosphatidylinositol breakdown which may be provoked by a wide variety of extracellular messengers, eventually leading to the activation of specific cellular functions or proliferation. Using human platelets as a model system, TPA is shown to enhance the protein kinase C-specific phosphorylation associated with the release reaction in the total absence of phosphatidylinositol breakdown. Various phorbol derivatives which have been shown to be active in tumor promotion are also capable of activating this protein kinase in in vitro systems.
A protocol has been devised to radiolabel proteins secreted by murine fibroblasts in vitro. A radiolabeled polypeptide of molecular weight 35,000 is released into medium in relatively large amounts by transformed cells and in much smaller amounts by nontransformed fibroblasts. This major excreted polypeptide (MEP) is found in the medium of spontaneously transformed mouse cells and in the medium of mouse cells transformed by a DNA tumor virus, RNA tumor viruses, or methylcholanthrene. The appearance of MEP appears to be well correlated with anchorage independence in these transformed cells. MEP can be localized within the cytoplasm of transformed but not untransformed cells by indirect immunofluorescence. The presence of MEP within murine fibroblasts or in their culture medium serves as a novel biochemical marker of transformation. A biological role for this protein has not been assigned.
Intact ribonucleic acid (RNA) has been prepared from tissues rich in ribonuclease such as the rat pancreas by efficient homogenization in a 4 M solution of the potent protein denaturant guanidinium thiocyanate plus 0.1 M 2-mercaptoethanol to break protein disulfide bonds. The RNA was isolated free of protein by ethanol precipitation or by sedimentation through cesium chloride. Rat pancreas RNA obtained by these means has been used as a source for the purification of alpha-amylase messenger ribonucleic acid.
A molecular clone corresponding to a 1.2-kilobase mRNA enriched in Rous sarcoma virus-transformed chicken embryo fibroblasts (CEF) was identified by differential screening of a cDNA library. The induction of the cloned sequence (denoted pCEF-4) in CEF infected by the temperature-sensitive mutant NY72-4 Rous sarcoma virus is rapid and independent of protein synthesis. DNA sequencing of the 1.2-kilobase insert of CEF-4 revealed an open reading frame that predicts an 11-kDa protein. The predicted pCEF-4 gene product is homologous to human connective tissue-activating peptide III (CTAP-III) and platelet factor 4 (PF-4). Serum stimulation of quiescent normal CEF results in a rapid but transient expression of pCEF-4 mRNA. Hence, pCEF-4 mRNA is expressed at the G0-G1 transition and during the first G1 phase of normal CEF reentering the cell cycle. The expression of pCEF-4 mRNA in Rous sarcoma virus-transformed CEF appears to be the result of transcriptional activation and stabilization of the transcript.
The ras oncogenes are implicated in the onset of some human tumours, and in cellular proliferation and terminal differentiation. The ras proteins are plasma membrane bound transducers of signals between the outside of the cell and unknown targets in the cell. Identifying these targets and understanding how they are regulated will have a major impact on our understanding of the molecular basis of transformation. We have already shown that c-Ha-ras and the tumor promoter TPA (12-o-tetradecanoyl phorbol-13-acetate) can activate a transcriptional enhancer. We now report the identification of a short sequence in the polyoma virus (Py) enhancer which mediates Ha-ras activation, and show that this sequence (ras responsive element, RRE) also mediates activation by TPA and serum. This responsive element is a specific binding-site for the mouse transcription factor PEA1 (ref. 4 and below) and for the jun oncogene (ref. 5 and M. Karin, personal communication). These results are in keeping with a role for ras protein in signal transduction from outside the cell to a transcription factor in the nucleus, through protein kinase C. The striking similarity between RRE and DNA sequences present in the promoter regions of a number of transformation-related genes suggests that deregulated activation of RRE is a critical event in transformation.
Transcription from the c-fos promoter and from minimal promoter constructs carrying the phorbol ester-responsive element [12-O-tetradecanoylphorbol-13-acetate (TPA) responsive element (TRE)] corresponding to the sequence in the human collagenase gene is activated by elevated levels of the oncogene products v-src, c-Ha-ras, activated c-Ha-ras, and v-mos, as well as by phorbol ester. Elevated c- or v-fos expression stimulates TRE-dependent transcription but represses the c-fos promoter. Antisense fos sequences abolish basal and induced transcription from TRE constructs and derepress the c-fos promoter. These results establish a key role for fos in signal transduction and implicate the fos protein as a trans-activating and -repressing molecule.
Steady-state cellular levels of phosphatidylinositol-4,5-bisphosphate (PIP2), 1,2-diacylglycerol (DAG), and inositol phosphates
have been measured in two different fibroblast cell lines (NIH 3T3 and NRK cells) before and after transformation with three
different ras genes. At high cell density the ratio of DAG to PIP2 was 2.5- to 3-fold higher in the ras-transformed cells
than in their untransformed counterparts. The sum of the water-soluble breakdown products of the polyphosphoinositides, inositol-1,4-bisphosphate
and inositol-1,4,5-trisphosphate, was also elevated in ras-transformed NRK cells compared with nontransformed NRK cells. These
findings suggest that the ras (p21) protein may act by affecting these levels, possibly as a regulatory element in the PIP2
breakdown pathway.
Antibody raised against the human erythrocyte glucose transporter identified a recombinant lambda gt11 bacteriophage in a cDNA library prepared from immunoselected polysomal RNA from adult rat brain. The cDNA predicts a 492-amino acid protein that demonstrates 97.6% identity to the human hepatoma hexose carrier. The tissue distribution of the transporter mRNA is identical to that of immunologically identifiable protein and transport activity, except in liver in which high levels of transport are associated with little or no transporter mRNA or protein. As assayed by blot-hybridization analysis, mRNA from insulin-responsive and nonresponsive tissues are indistinguishable. These data suggest that a genetically unrelated protein is responsible for hexose transport in normal liver.
Diacylglycerol (DG) plays a central role in phospholipid metabolism and is an endogenous activator of protein kinase C. We have suggested that constitutive activation of this kinase is one mechanism by which oncogenes transform cells. The ras-encoded proteins are similar to regulatory G-proteins and are candidates for the unknown G-protein that modulates phosphatidylinositol (PI) turnover. Differences in polyphosphoinositide metabolism have been reported for ras-transformed cells. But because these experiments were performed on confluent cultures of established cell lines, the differences are difficult to attribute to ras transformation. Here we show that exponentially growing NIH 3T3 fibroblasts recently transformed by Ha-ras or Ki-ras possess elevated DG concentrations without significant alterations in the levels of other polyphosphoinositide metabolites. The basal phosphorylation of protein kinase C substrate of relative molecular mass (Mr) 80,000 (80K) is significantly increased in all the ras-transformed cell lines. Surprisingly, however, further phosphorylation of this protein on addition of phorbol ester was greatly reduced. Ha-ras cells also show less binding of phorbol ester than control cells, suggesting that elevation of DG causes partial down-regulation in addition to activation of protein kinase C.
Elevation of glucose transport is an alteration common to most virally induced tumors. Rat fibroblasts transformed with wild-type
or a temperature-sensitive Fujinami sarcoma virus (FSV) were studied in order to determine the mechanisms underlying the increased
transport. Five- to tenfold increases in total cellular glucose transporter protein in response to transformation were accompanied
by similar increases in transporter messenger RNA levels. This, in turn, was preceded by an absolute increase in the rate
of glucose transporter gene transcription within 30 minutes after shift of the temperature-sensitive FSV-transformed cells
to the permissive temperature. The transporter messenger RNA levels in transformed fibroblasts were higher than those found
in proliferating cells maintained at the nonpermissive temperature. The activation of transporter gene transcription by transformation
represents one of the earliest known effects of oncogenesis on the expression of a gene encoding a protein of well-defined
function.
A cDNA clone, designated 9E3, was isolated from a chick embryo fibroblast (CEF) cDNA library. 9E3 mRNA was 20-fold higher in CEF following transformation by Rous sarcoma virus because of increased transcription rate. In CEF infected with temperature-sensitive mutants, increased 9E3 mRNA was found within 2 hr of a shift to permissive temperature. Nucleotide sequence and in vitro translation results indicate that 9E3 mRNA encodes an 11 kd polypeptide that is homologous to human connective tissue activating peptide III (CTAP-III), a mitogenic platelet alpha-granule protein, and to beta-thromboglobulin and platelet factor 4. The reported biological activities of CTAP-III suggest that elevated expression of 9E3 may play a role in producing some of the phenotypic features of RSV-transformed cells.
An accelerated rate of glucose transport is among the most characteristic biochemical markers of cellular transformation.
To study the molecular mechanism by which transporter activity is altered, cultured rodent fibroblasts transfected with activated
myc, ras, or src oncogenes were used. In myc-transfected cells, the rate of 2-deoxy-D-glucose uptake was unchanged. However,
in cells transfected with activated ras and src oncogenes, the rate of glucose uptake was markedly increased. The increased
transport rate in ras- and src-transfected cells was paralleled by a marked increase in the amount of glucose transporter
protein, as assessed by immunoblots, as well as by a markedly increased abundance of glucose transporter messenger RNA. Exposure
of control cells to the tumor-promoting phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) for 18 hours had a similar
effect of increasing the rate of glucose transport and the abundance of transporter messenger RNA. For ras, src, and TPA,
the predominant mechanism responsible for activation of the transport system is increased expression of the structural gene
encoding the glucose transport protein.
Transfection of NIH 3T3 cells with plasmids containing rat brain protein kinase C-I (PKC-I) cDNA controlled by strong viral promoter/enhancer elements led to PKC-I gene expression as assessed by Northern analysis, cellular binding of phorbol ester, immunoblotting of cellular PKC, and membrane-associated PKC activity. While transfection did not induce foci, altered growth regulation was observed in established transfectant lines: transfectants displayed reduced dependence on serum for growth, grew to higher saturation densities, and displayed enhanced tumorigenicity when inoculated into nude mice. Continued high-level expression of PKC-I, however, may not be obligatory for the malignant phenotype in vivo. Tumors that retained transfected sequences had lower PKC-I transcript levels than the parental in vitro lines, suggesting an in vivo modulation. Our data show that PKC-I dysregulation leads to altered cell growth regulation and may be functionally equivalent to the action of tumor promoters.
Protein kinase C of normal and ras-transformed NIH 3T3 cells was purified by chromatography on TSK DEAE-5PW, threonine-Sepharose, and TSK phenyl-5PW columns. Comparison of the fibroblast enzyme with several types of rat brain protein kinase C by chromatography on a hydroxyapatite column and by immunoblotting, indicates that both normal and transformed fibroblasts possess only one of the four subspecies of protein kinase C which have been identified in brain tissues. This subspecies presumably has the structure encoded by alpha-sequence or a closely related sequence. No significant difference was seen between those enzymes purified from normal and transformed fibroblasts.
Many external signals (hormones, neurotransmitters, and growth factors) act through receptors which stimulate the hydrolysis of a minor inositol lipid to give diacylglycerol and inositol trisphosphate. The latter functions by mobilizing calcium from intracellular stores, whereas diacylglycerol stimulates protein kinase C to phosphorylate specific proteins, some of which regulate ionic mechanisms such as the Na⁺/H⁺ exchanger and potassium channels. These two second messenger pathways constitute a highly versatile signaling system controlling numerous cellular processes such as secretion, contraction, metabolism, neuronal excitability, and cell growth.
We have generated a series of rat fibroblast cell lines that stably overexpress a full-length cDNA encoding the beta 1 form of protein kinase C (PKC). These cell lines contain a 20- to 53-fold increase in PKC activity and exhibit dramatically enhanced morphologic changes following exposure to the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA). They grow to a high saturation density in monolayer cultures and, when maintained at postconfluence, develop small, dense foci. In contrast to control cells, which display complete anchorage dependence, PKC-overproducing cells form small colonies in soft agar in the absence of TPA and large colonies in the presence of TPA. Thus, the mere overproduction of a single form of PKC is sufficient to confer multiple growth abnormalities in rat fibroblasts. These results provide direct evidence that PKC plays a critical role in growth control and that it mediates several of the cellular effects of the phorbol ester tumor promoters. They also suggest that the activation of PKC may be of central importance in the process of multistage carcinogenesis.
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The amino acid sequence of the glucose transport protein from human HepG2 hepatoma cells was deduced from analysis of a complementary DNA clone. Structural analysis of the purified human erythrocyte glucose transporter by fast atom bombardment mapping and gas phase Edman degradation confirmed the identity of the clone and demonstrated that the HepG2 and erythrocyte transporters are highly homologous and may be identical. The protein lacks a cleavable amino-terminal signal sequence. Analysis of the primary structure suggests the presence of 12 membrane-spanning domains. Several of these may form amphipathic alpha helices and contain abundant hydroxyl and amide side chains that could participate in glucose binding or line a transmembrane pore through which the sugar moves. The amino terminus, carboxyl terminus, and a highly hydrophilic domain in the center of the protein are all predicted to lie on the cytoplasmic face. Messenger RNA species homologous to HepG2 glucose transporter messenger RNA were detected in K562 leukemic cells, HT29 colon adenocarcinoma cells, and human kidney tissue.
A quantitative as well as sequential analysis of the uptake of certain sugars during the process of cell transformation with Rous sarcoma virus has been made. Significant alteration in the kinetics of sugar transport occurs in the infected cells concomitant with changes in cell morphology. These alterations appear to be expressed by the transforming gene(s) of RSV and are not found in cells infected with an avian leukosis virus.
Transformed and nontransformed cells in tissue culture differ in their rate of uptake of certain nutrients, as determined by a polyester-coverslip technique. A 2.5- to 3.5-fold increased rate of uptake of alpha-aminoisobutyric acid, cycloleucine, and 2-deoxy-D-glucose was observed with polyoma virus-transformed baby hamster kidney (BHK) 21 cells and simian virus 40 (SV40)-transformed BALB/3T3 (mouse fibroblast) cells, compared to their nontransformed counterparts. Kinetic analysis suggested that the increased uptake by cells transformed with virus was associated with a 3-fold greater V(max), with no detectable changes in apparent K(m). Limited studies also revealed increased initial rates of uptake by murine sarcoma virus-transformed rat liver cells, as compared to the parental line. Exposure of cells to concanavalin A and wheat-germ agglutinin led to significant reductions in amino-acid uptake by both transformed and nontransformed cells; however, transformed cells showed a greater decrease in uptake after exposure to wheat-germ agglutinin. Increased initial rates of uptake of certain amino acids and sugars may be a feature common to transformed cells, compared to their parental control.
We have examined culture fluids from a variety of Kirsten murine sarcoma virus (KiMSV) transformed rat and mouse cells for the presence of factors which induce normal Rat-1 cells to assume the transformed phenotype. All KiMSV transformants produced transforming factor (TF). Revertants of KiMSV transformed rat or mouse failed to release TF as did normal rat or mouse cells. Cells transformed by a temperature sensitive mutant of KiMSV produced TF at the permissive temperature but not at the nonpermissive temperature. Further, cells from a spontaneous transformant of Rat-1 cells also produced TF. TF is a small polypeptide which competes for the epidermal growth factor receptor. Its effect upon normal cells is reversible and requires de novo RNA and protein synthesis. Cells treated with TF lose the actin fibers observed in normal fibroblasts, assume a transformed cell morphology, become anchorage independent for growth, grow in low concentrations of serum, grow to a high cell density, and have an increased rate of hexose uptake.
The Ca2+-phospholipid-regulated protein kinase has been purified to homogeneity from a 100,000 X g supernatant fluid of rat brain homogenate by a procedure that includes DEAE-cellulose chromatography and successive filtrations on Ultrogel AcA 34 in EGTA and in phosphatidylserine and Ca2+. A more rapid purification consisting of DEAE-cellulose chromatography, Ultrogel AcA 34 gel filtration chromatography, and DEAE-trisacryl chromatography, all in the presence of EGTA, was also developed. Although the enzyme obtained by the latter procedure is not homogeneous, it exhibits properties similar to those of the pure enzyme and is more stable. In addition, the DEAE-trisacryl step permitted resolution of a contaminating Ca2+-inhibitable protein kinase that can interfere with studies of the Ca2+-phospholipid-stimulated enzyme. The homogeneous enzyme, purified about 300-fold, was estimated to have a Mr of 84,000. Its activity was 20- to 30-fold higher in the presence of phospholipid and Ca2+ than in the presence of phospholipid and EGTA, EGTA, or Ca2+ alone. The specific activity of the activated kinase was 852 nmol of P incorporated into histone per min/mg at 20 degrees C. The pure enzyme underwent autophosphorylation in a Ca2+- and phospholipid-dependent manner. This reaction was inhibited in the presence of histones without affecting the kinetic properties of the enzyme. Under optimal assay conditions, the homogeneous enzyme was activated 10-20% by either 10 microM diolein or 100 nM phorbol 12-myristate 13-acetate. Activation of the purified enzyme by diolein or the phorbol ester was far greater (3- to 4-fold) when aggregated instead of freshly sonicated phospholipids were used, suggesting that these compounds affect the interaction of the enzyme with phospholipids and Ca2+. The purified enzyme catalyzed the phosphorylation of the 40S ribosomal subunit protein S6. The Km for S6 was approximately equal to 1 microM and it was estimated that 2 mol of phosphate were incorporated per mol of S6. The observation that protein S6 can be phosphorylated by the purified Ca2+-phospholipid-dependent protein kinase may link recent reports that phorbol ester tumor promoters activate the Ca2+-phospholipid-dependent protein kinase in vitro and stimulate phosphorylation of the ribosomal protein S6 in vivo.
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