Effect of hyperthermia on growth of Ehrlich ascites tumor cells

Hyperthermic treatment at 43 degrees C suppressed the growth of Ehrlich ascites tumor (EAT) cells in vitro. Incubation of EAT cells at 43 degrees C for as little as 1.5 h totally abolished the transplantability of the tumor. At the same time, the rate of cellular glucose uptake, the density of glucose transporter on the cells as well as the extent of thymidine, uridine and leucine incorporation were significantly reduced.     

Glucose transport in developing Ehrlich ascites tumor cells: parallel changes in the rate of glucose uptake and cytochalasin B binding activity during tumor development and methotrexate treatment

The ability of Ehrlich ascites tumor cells to take up glucose increased progressively during the course of tumor development. Simultaneously as the rate of uptake rose, the density of a class of glucose-reversible binding sites for cytochalasin B on the cell surface also increased. In its stereospecificity requirement toward competing sugars and in its sensitivity to phloretin and diethylstilbestrol, this class of binding sites resembled the putative glucose carriers identified in various other cell systems and may represent the glucose transporter in Ehrlich ascites cells. Work with methotrexate (MTX) substantiated this view. Methotrexate arrested tumor growth, inhibited glucose uptake, and reduced the number of cytochalasin B binding sites. In both MTX-treated and untreated cells, the magnitude of changes in number of cytochalasin B binding sites closely paralleled and sufficiently accounted for the magnitude of changes in glucose uptake. Qualitative changes in the turnover and affinity for substrate of the putative glucose carrier need not be invoked.     

Purification of insulin-dependent exocytic vesicles containing the glucose transporter

In muscle and fat, insulin causes the cellular redistribution of glucose transporters and insulin-like growth factor II receptors from an intracellular pool of membranes (low density microsomes) to the plasma membrane. This translocation is a major mechanism by which insulin stimulates cellular glucose uptake. Our aim was to purify and characterize the insulin-regulatable exocytic intracellular membranes that are enriched in glucose transporter. Low density microsome and plasma membrane fractions were isolated from basal and insulin-stimulated rat adipocytes by differential centrifugation. In cells exposed to insulin, glucose transporters were decreased in the low density microsomes and correspondingly increased in the plasma membranes as determined by immunoblotting and cytochalasin B binding. Low density microsomes were further fractionated by sucrose density gradient centrifugation. Membranes containing glucose transporters were separated from the major protein-containing peaks and from plasma membranes, Golgi, and endoplasmic reticulum. Further fractionation was achieved by agarose gel electrophoresis. Overall, the intracellular membranes enriched in transporter were purified 9-fold compared to low density microsomes. These purified membranes had the following characteristics: 1) uniformly sized vesicles, diameter 60-100 nm; 2) insulin-regulatable protein composition, one constituent being an Mr 43,000 protein that co-migrated with immunoblotted glucose transporters; 3) enrichment in insulin-like growth factor II receptors, but of a lesser degree than the enrichment in transporters. Thus, using a three-step procedure, insulin-sensitive translocatable vesicles from adipocytes have been highly purified. These are similar in size and density to endosomes, and the glucose transporter is a major constituent of this distinct vesicle population.     

Human tumour necrosis factor-α inhibits glucose transport in cultured ehrlich ascites tumour cells

Human recombinant tumour necrosis factor-α (rhTNF-α) arrested the growth of Ehrlich ascites tumour (EAT) cells in vitro. It suppressed cellular glucose uptake and decreased the membrane density of glucose transporters as measured by glucose-reversible cytochalasin B binding. The glucose transporters' affinity for substrate was also reduced. However, rhTNF-α treatment exerted no effect on the phosphoribosyl pyrophosphate level in EAT cells. The role of rhTNF-α on the inhibition of glucose transport of tumour cells is discussed.     

Hemopoietic cell transformation is associated with failure to downregulate glucose uptake during the G2/M phase of the cell cycle

Growth factors and cytokines initiate multiple signal transduction pathways that lead to cell survival, cell cycle progression or differentiation. A common feature of these pathways is increased cellular metabolism and glucose uptake. Furthermore, the energy requirements of many cancers and transformed cell lines are met by constitutive upregulation of glucose uptake. Relationships among transforming events, glucose uptake and cell cycle progression are not well understood. Here we investigated the regulation of glucose transport during the cell cycle of growth factor-dependent 32D cells, primary T-cells, src-transformed 32D cells and Jurkat cells. Cells were enriched in the G1, S and G2/M phases of the cell cycle, and glucose transporter expression and 2-deoxyglucose uptake were measured. Glucose transporter expression increased with cell volume as cells progressed through the cell cycle. Growth factor-dependent 32D cells and T-lymphocytes were characterised by increased 2-deoxyglucose uptake from G1 to S and reduced uptake at G2/M, with the highest specific activity of transporters in the S phase. In contrast, src-transformed 32D cells and Jurkat cells showed increased 2-deoxyglucose uptake from S to G2/M, with the highest glucose transporter specific activity in G2/M. Our results show that glucose transport is regulated in a cell cycle-dependent manner and suggest that this regulation may be altered in transformed cells.     

Expression of glucose transporters in cancers

It has been known for 80 years that cancer cell growth in an energy-related process supported by an increased glucose metabolism. This phenomenon suggests a need for a corresponding increased uptake of glucose across the plasma membrane through an enhancement in the glucose transporter proteins, SGLT proteins as well as GLUT proteins. The results of many studies have demonstrated that the expression of glucose transporters, especially GLUT1, is increased in a variety of malignancies. GLUT1 overexpression has been found to be associated with tumor progression. It was found that GLUT1 overexpression is associated with poor overall survival in various malignant tumors.     

Differential regulation of two glucose transporters by chronic growth hormone treatment of cultured 3T3-F442A adipose cells

New methods for the analysis of glucose transporters were used to analyze the molecular mechanisms involved in the insulin-antagonistic effects of growth hormone (GH), which is known as a diabetogenic hormone. The ability of GH to alter the number and mRNA levels of two different glucose transporters in cultured 3T3-F442A adipocytes was investigated using specific antibodies and cDNA probes. At concentrations of GH as low as 0.5 and 5 ng/ml and at incubation times as short as 4 h, GH decreased rates of 2-deoxyglucose uptake in 3T3-F442A adipocytes. 3-O-Methyl-D-glucose uptake was inhibited to an extent similar to that of 2-deoxyglucose uptake (60-80%) after a 24-h incubation with GH (500 ng/ml), indicating that GH inhibits glucose metabolism specifically at the step of glucose transport. To determine whether reduced rates of glucose transport might result from reduced numbers of glucose transporters, whole cell lysates were prepared from GH-treated cells and subjected to immunoblotting using antibodies that identify Glut 1 (HepG2/rat brain) and Glut 4 (muscle/adipose) transporters. GH caused a time- and dose-dependent decrease in the number of Glut 1 transporters in the cell. Northern and slot-blot analyses showed a GH-induced dose-dependent decrease in levels of Glut 1 mRNA. In contrast, levels of Glut 4 transporter and mRNA were unchanged by GH. These data suggest that GH regulates Glut 1 and Glut 4 transporters differentially and that it exerts its inhibitory effect on glucose uptake at least in part by decreasing the synthesis of Glut 1 transporters. These studies provide the first evidence that GH regulates a key gene in metabolic regulation and can interfere with gene expression.     

Specific modifications of hepatoma cell-surface glycoproteins with enzymes : Effects on in vitro growth as investigated by the use of lectins

The effects of enzymic treatment on the interactions between Zajdela's tumor cells and various lectins. Concanavalin A (ConA); Wheat Germ Agglutinin (WGA); Robinia lectin; have been studied. (1) The number of lectin-binding sites and the affinity constants were investigated. (2) The effects of the lectins on cell growth and [3H]thymidine incorporation were studied on untreated and enzyme-treated cells. It was observed that treatment of tumor cells with neuraminidase resulted in a change in the binding characteristics of each lectin. However, additional treatment of the cells with galactose oxidase had no further effect on lectin binding. ConA and Robinia lectin induced a decrease of the untreated tumor cell growth and a stimulation of the [3H]thymidine incorporation. This paradoxal result may be explained as a consequence of the stimulation of the [3H]thymidine uptake observed in the presence of lectins. The enzymatic treatments themselves did not change the cell growth although they did induce a change in the effect of ConA and Robinia lectin on cell growth and [3H]thymidine incorporation. As a result of neuraminidase treatment, the effects of ConA were totally suppressed but those of Robinia lectin only partially. Although WGA interacted with untreated and enzyme-treated cell surfaces, it had no effect on tumor cell growth nor [3H]thymidine incorporation. The results are discussed in terms of lectin transport.     

Functional expression of mammalian glucose transporters in Xenopus laevis oocytes: evidence for cell-dependent insulin sensitivity.

We report the functional expression of two different mammalian facilitative glucose transporters in Xenopus oocytes. The RNAs encoding the rat brain and liver glucose transporters were transcribed in vitro and microinjected into Xenopus oocytes. Microinjected cells showed a marked increase in 2-deoxy-D-glucose uptake as compared with controls injected with water. 2-Deoxy-D-glucose uptake increased during the 5 days after microinjection of the RNAs, and the microinjected RNAs were stable for at least 3 days. The expression of functional glucose transporters was dependent on the amount of RNA injected. The oocyte-expressed transporters could be immunoprecipitated with anti-brain and anti-liver glucose transporter-specific antibodies. Uninjected oocytes expressed an endogenous transporter that appeared to be stereospecific and inhibitable by cytochalasin B. This transporter was kinetically and immunologically distinguishable from both rat brain and liver glucose transporters. The uniqueness of this transporter was confirmed by Northern (RNA) blot analysis. The endogenous oocyte transporter was responsive to insulin and to insulinlike growth factor I. Most interestingly, both the rat brain and liver glucose transporters, which were not insulin sensitive in the tissues from which they were cloned, responded to insulin in the oocyte similarly to the endogenous oocyte transporter. These data suggest that the insulin responsiveness of a given glucose transporter depends on the type of cell in which the protein is expressed. The expression of hexose transporters in the microinjected oocytes may help to identify tissue-specific molecules involved in hormonal alterations in hexose transport activity.     

Biochemistry and regulation of folate and methotrexate transport in Leishmania major

Promastigotes of the protozoan parasite Leishmania major exhibit high affinity uptake of folate (Kt = 0.7 microM) and methotrexate (MTX) (Kt = 1.8 microM) which is saturable and sensitive to metabolic poisons. Influx of folate and MTX is competitively inhibited by 5-formyltetrahydrofolate and p-aminobenzoic acid-glutamate, but not by 4-deoxy-4-amino-10-methylpteroate, biopterin, or pteroate. A single carrier is inferred for both folate and MTX transport, as the Ki of each inhibitor for both folate and MTX influx is the same, and the apparent affinities (Kt) of the substrates folate and MTX are identical to their respective Ki values for inhibition of MTX and folate uptake. Folate influx is specifically regulated according to cellular growth phase, as stationary phase cells exhibit 7% of the Vmax of log phase cells, while energy-dependent glucose uptake is only moderately reduced in stationary phase. Folate influx is also regulated by external folate levels, as cells grown in 5 microM folate exhibit 30% of the Vmax of cells grown in folate-depleted medium. Comparison of bacterial, mammalian, and Leishmania folate transport activities indicates considerable diversity in both biochemical and regulatory properties, and suggests the possibility that selective inhibition or manipulation of folate transport may be exploited in parasite chemotherapy.     

Akt maintains cell size and survival by increasing mTOR-dependent nutrient uptake

In multicellular organisms, constituent cells depend on extracellular signals for growth, proliferation, and survival. When cells are withdrawn from growth factors, they undergo apoptosis. Expression of constitutively active forms of the serine/threonine kinase Akt/PKB can prevent apoptosis upon growth factor withdrawal. Akt-mediated survival depends in part on the maintenance of glucose metabolism, suggesting that reduced glucose utilization contributes to growth factor withdrawal-induced death. However, it is unclear how restricting access to extracellular glucose alone would lead to the metabolic collapse observed after growth factor withdrawal. We report herein that growth factor withdrawal results in the loss of surface transporters for not only glucose but also amino acids, low-density lipoprotein, and iron. This coordinated decline in transporters and receptors for extracellular molecules creates a catabolic state characterized by atrophy and a decline in the mitochondrial membrane potential. Activated forms of Akt maintained these transporters on the cell surface in the absence of growth factor through an mTOR-dependent mechanism. The mTOR inhibitor rapamycin diminished Akt-mediated increases in cell size, mitochondrial membrane potential, and cell survival. These results suggest that growth factors control cellular growth and survival by regulating cellular access to extracellular nutrients in part by modulating the activity of Akt and mTOR.     

Acute and long-term effects of insulin-like growth factor I on glucose transporters in muscle cells. Translocation and biosynthesis.

Insulin-like growth factor I (IGF-I) rapidly (less than 10 min) stimulated glucose uptake into myotubes of the L6 muscle cell line, at concentrations that act specifically on IGF-I receptors. Uptake remained stimulated at a steady level for 1-2 h, after which a second stimulation occurred. The first phase was insensitive to inhibition of protein synthesis. Subcellular fractionation demonstrated that it was accompanied by translocation of glucose transporters (both GLUT1 and GLUT4) to the plasma membrane from intracellular membranes. Translocation sufficed to explain the first phase increase in glucose transport, and there was no change in the total cellular content of GLUT1 or GLUT4 glucose transporters. The second phase of stimulation was inhibitable by cycloheximide, and involved a net increase in either GLUT1 or GLUT4 transporter content, which was reflected in an increase in transporter number in plasma membranes. These results define a cellular mechanism of metabolic action of IGF-I in muscle cells; furthermore, they suggest that IGF-I has acute metabolic effects that mimic those of insulin, bypassing action on the insulin receptor.     

Adenosine stimulation of proliferation of breast carcinoma cell lines: evaluation of the [3H]thymidine assay system and modulatory effects of the cellular microenvironment in vitro

The purine nucleoside adenosine is produced at increased levels in the tissues of solid cancers as a result of local hypoxia. Adenosine inhibits the cell-mediated anti-tumor immune response, promotes tumor cell migration and angiogenesis, and stimulates the proliferation of tumor cells. We examined the stimulatory effect of adenosine on DNA synthesis, cell cycle progression, and cell proliferation in MCF7 and T-47D breast carcinoma cell lines in culture, and identified factors that modulate the growth response. The ability of adenosine to stimulate DNA synthesis, as measured by the incorporation of [(3)H]thymidine, was independent of the total radioactivity of the [(3)H]thymidine up to 10 microCi/ml, total thymidine concentrations up to 100 microM, and the labeling interval. It was also not affected by the presence of low-molecular-weight compounds (such as thymidine and adenosine) in the serum used to supplement the medium. Adenosine stimulated DNA synthesis and cell proliferation with an EC(50) of 4-6 microM and a maximum response at 30-100 microM, when given as a single addition. The stimulatory effect of adenosine involved progression through the cell cycle and a genuine increase in cell number, in the absence of significant apoptotic or necrotic cell death. The mitogenic effect of adenosine was dependent upon the culture cell density, with an optimum adenosine response at around 50% of confluent density. The response was also highly dependent upon the form of the serum addition to the growth medium, with the best response elicited in the presence of low concentrations of nonfetal bovine serum, although adenosine was mitogenic under standard culture conditions. The effects of serum supplementation and cell density were not due to differences in the rate of adenosine metabolism by either serum or cellular enzymes, but appeared to result from changes in the sensitivity to adenosine of the cell population in response to environmental cues. We, therefore, find that adenosine is consistently mitogenic for human breast carcinoma cells, and that the [(3)H]thymidine incorporation assay is a valid measure of this response. The data are consistent with the stimulatory effect of adenosine on cell proliferation being modulated by the local cellular environment.     

The laforin-malin complex negatively regulates glycogen synthesis by modulating cellular glucose uptake via glucose transporters

Lafora disease (LD), an inherited and fatal neurodegenerative disorder, is characterized by increased cellular glycogen content and the formation of abnormally branched glycogen inclusions, called Lafora bodies, in the affected tissues, including neurons. Therefore, laforin phosphatase and malin ubiquitin E3 ligase, the two proteins that are defective in LD, are thought to regulate glycogen synthesis through an unknown mechanism, the defects in which are likely to underlie some of the symptoms of LD. We show here that laforin's subcellular localization is dependent on the cellular glycogen content and that the stability of laforin is determined by the cellular ATP level, the activity of 5'-AMP-activated protein kinase, and the affinity of malin toward laforin. By using cell and animal models, we further show that the laforin-malin complex regulates cellular glucose uptake by modulating the subcellular localization of glucose transporters; loss of malin or laforin resulted in an increased abundance of glucose transporters in the plasma membrane and therefore excessive glucose uptake. Loss of laforin or malin, however, did not affect glycogen catabolism. Thus, the excessive cellular glucose level appears to be the primary trigger for the abnormally higher levels of cellular glycogen seen in LD.     

Thymine and Thymidine Uptake by Haemophilus influenzae and the Labeling of Deoxyribonucleic Acid

The influence of ribo- and deoxyribonucleosides and ribo- and deoxyribonucleotides on the uptake of radiolabeled thymidine and thymine by Haemophilus influenzae during growth was investigated. A nucleoside-degrading enzyme similar to that reported in Escherichia coli was found to break down thymidine unless other nucleosides were present to divert its action. The presence of other nucleosides permitted a nearly quantitative uptake of even low levels of thymidine. This quantitative uptake of thymidine in the presence of an excess of other nucleosides suggests that the uptake mechanism for thymidine is specific in this organism. Under optimal conditions, as much as 50% of the deoxyribonucleic acid (DNA) thymine was derived from exogenous thymidine. Thymine was not taken up by H. influenzae but, in the presence of purine deoxynucleosides, labeled thymine entered the cells, presumably as thymidine. Ribonucleosides or ribonucleotides inhibited thymine conversion to thymidine, but, as noted above, they were degraded by a cellular enzyme. Thus, unless the ribonucleoside level was excessively high, a cell level of growth was reached at which the inhibiting ribonucleoside was broken down and labeled thymine appeared in the cells at an increasing rate. Twenty-five per cent of the DNA thymine of this organism was labeled with exogenous thymine. The information noted above serves as the basis for isotopically labeling the DNA.     

Role of microvillar cell surfaces in the regulation of glucose uptake and organization of energy metabolism

Experimental evidencesuggesting a type of glucose uptake regulation prevailing inresting and differentiated cells was surveyed. This type of regulationis characterized by transport-limited glucose metabolism and depends onsegregation of glucose transporters on microvilli of differentiated orresting cells. Earlier studies on glucose transport regulation and arecently presented general concept of influx regulation for ions andmetabolic substrates via microvillar structures provide the basicframework for this theory. According to this concept, glucose uptakevia transporters on microvilli is regulated by changes in thestructural organization of the microfilament bundle, which is acting asa diffusion barrier between the microvillar tip compartment and thecytoplasm. Both microvilli formation and the switch of glucosemetabolism from "metabolic regulation" to "transportlimitation" occur during differentiation. The formation ofmicrovillar cell surfaces creates the essential preconditions toestablish the characteristic functions of specialized tissue cellsincluding the coordination between glycolysis and oxidativephosphorylation, regulation of cellular functions by external signals,and Ca²⁺ signaling. The proposed concept integrates variousaspects of glucose uptake regulation into a ubiquitous cellularmechanism involved in regulation of transmembrane ion and substrate fluxes.