Transport of potassium, amino acids, and glucose in cells transformed by Rous sarcoma virus

Transport rates of a number of nutrients and ions have been surveyed in chicken embryo fibroblasts that were density inhibited, growing exponentially, or transformed by Rous sarcoma virus. All the transport systems examined displayed changes associated with changes in growth rate. The rate of ouabain-sensitive potassium transport declined in density-inhibited cells, and increased rapidly in response to serum stimulation. This transport system was regulated both by changes in the activity of the transporters and by the number of transporters in the cell membrane. The rate of transport of the amino acid analog alpha-aminoisobutyric acid declined when cells became density inhibited, but also showed alterations in regulation that were associated with malignant transformation. The rate of glucose transport displayed both growth state-related and transformation-specific changes. The increased rate of glucose transport seen in transformed cells is due to an increase in the number of glucose transporters in the cell membrane. Increased glucose transport is necessary for subsequent changes in glycolysis, and temporally precedes some of the changes in activity of glycolytic enzymes.     

Inhibition of protease activity in cultures of rous sarcoma virus-transformed cells: effect on the transformed phenotype.

We have examined the role of proteolytic activity in the genesis and maintenance of the transformed phenotype by growing cultures of chick embryo fibroblasts transfromed by Rous sarcoma virus either in medium containing plasminogen-free serum or in medium to which protease inhibitors were added. Alterations in morphology, adhesiveness, and hexose transport were used as markers for the transformed state. Addition of the trypsin inhibitors NPGB or Soy Bean Trypsin Inhibitor at concentrations which inhibited transformation-associated fibrinolysis restored adhesiveness and morphology to near normal, but did not affect the rate of hexose transport. Growth of Rous-infected cells in plasminogen-free medium blocked the appearance of morphological and adhesive alterations, but allowed the rate of hexose transport to increase to the transformed level. Thus we were able to separate the appearance of transformation-specific changes in morphology and adhesiveness (which apparently require fibrinolytic activity) from the increased rate of hexose transport (which is independent of fibrinolytic activity). Another trypsin inhibitor, TLCK, although it did not inhibit fibrinolysis, was very effective at restoring adhesiveness and morphology as well as hexose transport to normal. This raises the possibility that there is another, perhaps earlier, protease involved in the genesis of the transformed phenotype.     

Cell surface changes and Rous sarcoma virus gene expression in synchronized cells

We have investigated whether cell surface changes associated with growth control and malignant transformation are linked to the cell cycle. Chicken embryo cells synchronized by double thymidine block were examined for cell-cycle-dependent alterations in membrane function (measured by transport of 2-deoxyglucose, uridine, thymidine, and mannitol), in cell surface morphology (examined by scanning electron microscopy), and in the ability of tumor virus gene expression to induce a transformation-specific change in membrane function. We reach the following conclusions: (a) The high rate of 2-deoxyglucose transport seen in transformed cells and the low rates of 2-deoxyglucose and uridine transport characteristic of density-inhibited cells do not occur in normal growing cells as they traverse the cell cycle. (b) Although there are cell cycle-dependent changes in surface morphology, they are not reflected in corresponding changes in membrane function. (c) Tumor virus gene expression can alter cell membrane function at any stage in the cell cycle and without progression through the cell cycle.     

Increased membrane transport of 2-deoxyglucose and 3-O-methylglucose is an early event in the transformation of chick embryo fibroblasts by Rous sarcoma virus.

Transformation of chicken embryo fibroblasts with Rous sarcoma virus results in cells with an enhanced rate of hexose uptake. We have examined transport of the glucose analogs 2-deoxyglucose and 3-O-methylglucose in cells infected with a temperature sensitive variant of the virus. In cells shifted from restrictive to permissive conditions for transformation, increased transport of the non-phosphorylatable analog 3-O-methylglucose occurs at the same time as that of 2-deoxyglucose, a phosphorylatable analog. This enhanced rate of transport can be observed within three hours of the temperature shift. There is a corresponding decrease in the transport rate of both analogs following shift to the restrictive temperature. These results suggest that increased transport is likely to be the primary event in causing transformation-specific changes in sugar metabolism. We have also examined uptake into the internal pools of both the phosphorylated and non-phosphorylated forms of 2-deoxyglucose in normal cells and in cells transformed by the wild-type virus. These data indicate a corresponding increase in the rate of accumulation of the free sugar in transformed cells and point to transport as the rate limiting step in the accumulation of 2-deoxyglucose in both normal and transformed chicken embryo cells.     

Cell shape and hexose transport in normal and virus-transformed cells in culture

The rate of hexose transport was compared in normal and virus-transformed cells on a monolayer and in suspension. It was shown that: (1) Both trypsin-removed cells and those suspended for an additional day in methyl cellulose had decreased rates of transport and lower available water space when compared with cells on a monolayer. Thus, cell shape affects the overall rate of hexose transport, especially at higher sugar concentrations. (2) Even in suspension, the initial transport rates remained higher in transformed cells with reference to normal cells. Scanning electron micrographs of normal and transformed chick cells revealed morphological differences only in the flat state. This indicates that the increased rate of hexose transport after transformation is not due to a difference in the shape of these cells on a monolayer.     

Density-dependent changes in hexose transport, glycolytic enzyme levels, and glycolytic rates, in uninfected and murine sarcoma virus-transformed rat kidney cells

In normal rat kidney (NRK) cell cultures, increased cell density results in a decrease in the rates of hexose transport, glucose utilization, and lactate production and an increase in the level of hexokinase activity. A murine sarcoma virus (Kirsten)-transformed cell line (KNRK) showed little or no density-dependent variation in sugar uptake, glucose consumption, or lactate production. On the other hand, hexokinase, phosphofructokinase, pyruvate kinase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase activities were elevated in dense transformed cultures as compared to sparse or uninfected cultures. In another virus-transformed cell line (ts339/NRK) exhibiting temperature-dependent morphology, growth pattern, and transport of 2-deoxy- -glucose, the levels of glycolytic enzyme activity were related to cell density but not to the culture temperature. The lack of correlation between glycolytic enzyme activity and lactate production by either uninfected or murine sarcoma virus-transformed cultures supports the suggestion that enhanced growth and/or hexose transport capacity rather than elevated glycolytic enzyme activity are responsible for the increased rate of lactate production by virus-transformed NRK cells.     

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

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

Alterations in lipid acyl group composition and membrane structure in cells transformed by Rous sarcoma virus.

The acyl group composition of the phospholipids from normal chick embryo fibroblasts and from cells transformed by Rous sarcoma virus was determined by gas-liquid chromatography. Rous-transformed cells had less arachidonate (20:4) and more oleate (18:1) in membrane lipids than normal, growing cells. Normal density-inhibited cells had the lowest ratio of 18:1/20:4. Associated with the decreased content of 20:4 in the transformed cells was a decreased motional freedom of an incorporated spin-labeled fatty acid analogue. Arrhenius plots for uptake of 2-deoxyglucose revealed an increased apparent activation energy in the transformed cells, suggesting that the hexose transport carriers were sensitive to the changes in membrane composition and structure in fully transformed cells. However, the development of the changes in fatty acid composition occurred relatively slowly in the course of transformation, indicating that the observed compositional alterations are not likely to be a primary cause of the early changes in membrane function associated with malignant transformation.     

Relationship between changes in the calcium dependent regulatory protein and adenylate cyclase during viral transformation.

The levels of the calcium dependent regulatory protein in transformed chicken embryo fibroblasts are higher both in soluble fractions and membrane fractions compared to untransformed cells. The kinetics for changes in the calcium dependent regulatory protein, hexose transport, and adenylate cyclase were compared using a temperature sensitive mutant of Rous sarcoma virus. Decreases in adenylate cyclase activity and increased hexose transport accompanying transformation occurred with half-lives of approximately 7 to 8 hours. Increases in the calcium dependent regulatory protein occurred much slower with a half-life of seventeen hours. It is concluded that the increase in calcium dependent regulatory protein levels is a late event during viral transformation and that the decline in adenylate cyclase activity cannot be due to changes in the amount of calcium dependent regulatory protein.     

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

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

Vinculin and 36 kDa protein are not tyrosine-phosphorylated in Rous sarcoma virus infected cells which have been treated with interferon

The expression of membrane-associated transformation-specific parameters was analyzed in de novo Rous sarcoma virus (strain SR-RSV-D) infected chicken embryo fibroblasts pretreated with homologous interferon. Cellular morphology, hexose transport, microfilament organization, and tyrosine-phosphate content of two primary substrates of the transformation-generating viral kinase, pp60src, were found indistinguishable from non-infected controls. These observations support the hypothesis that vinculin and possibly 36 kDa protein are involved in microfilament organization and that tyrosine-phosphorylation of these structural proteins is a prerequisite for the rearrangement of microfilaments during transformation. In de novo infection, interferon pretreatment reduces viral protein synthesis and pp60src activity as compared to non-treated, SR-RSV-D infected cells. However, the phosphotyrosine content of total cellular proteins as measured under steady state conditions is as high in interferon-pretreated as in nontreated transformed cells.     

Dependence of ion transport across the plasma membrane on the density of the cell culture. I. Ion flows and the potassium and sodium content in 3 Chinese hamster cell lines (CHO)

Cation transport has been investigated in three lines of Chinese ovary cells CHO-K1 during the cell culture growth. With the increase in the cell density potassium and sodium contents decreased from 1.2 to 0.8-0.5 and from 0.5 to 0.15-0.1 mmole/g protein, respectively. The time courses of potassium and sodium changes were different, and the increase in intracellular K/Na ratio from 1.5-2.0 to 5-10 with the increase in cell density was revealed. The rubidium influx was found to decrease during the culture growth mainly due to the decrease in ouabain-inhibitable and (ouabain + furosemide)- non-inhibitable influxes. The changes in cation fluxes and cation contents were observed in transformed cells without contact inhibition of division and were considered as a manifestation of density-dependent alterations of plasma membrane.     

External cell surface protein phosphorylation in normal and Rous sarcoma virus transformed chick embryo fibroblasts

Normal and Rous sarcoma virus (RSV)-transformed chick embryo fibroblasts growing on plastic dishes were incubated with ATP (γ³²P) in situ to detect external cell surface protein kinase activity. Under the conditions employed, ³²P was incorporated exclusively into proteins, specifically those at the external cell surface, as radioactivity was removed by tryspin treatment of labeled whole cells. In addition, exogenous histones were phosphorylated when added to the reaction mixture. Cyclic nucleotides had virtually no effect on ³²P incorporation, suggesting that little or no cyclic nucleotide-dependent protein kinase activity was present at the external cell surface. Cell surface protein kinase activity was higher in transformed than in normal cells, and, using a temperature-sensitive RSV src mutant, this difference was shown to be transformation-specific. Several differences were observed in the cell surface proteins phosphoryllated in normal and transformed cells and at least two of these were transformation-specific. These data suggest that changes in external cell surface protein physphorylation are associated with RSV transformation and thus could play a role in the formation of the transformed cell phenotype.     

Dependence of ion transport across the plasma membrane on cell culture density. II. Active and passive cation transport during the growth of L cell cultures

Rubidium and lithium influxes as well as intracellular potassium and sodium contents were investigated in L cells during the culture growth. In sparse culture over the cell densities 0.5-3 X 10(4) cells/cm2 ouabain-sensitive rubidium influx is small and ouabain-resistant lithium influx in high. With the increase in culture density up to 4-5 X 10(4) cells/cm2 the active rubidium influx, mediated by ouabain-sensitive component, is enhanced, and ion "leakage" tested by lithium influx is diminished. Simultaneously with the exponential growth of culture the intracellular potassium content is increased and the intracellular sodium content is decreased resulting in the higher K/Na ratio in cell. During the further transition to dense culture and in stationary state (10-17 X 10(4) cells/cm2) the sodium content and lithium influx do not change significantly, but the potassium content is decreased. The decrease in intracellular potassium is correlated with that in the portion of cells in S-phase from 27-30 to 12%. Thus, in transformed cells the density-dependent alterations in membrane cation transport are observed.     

Differential expression of Rous Sarcoma virus-specific transformation parameters in enucleated cells.

Chicken embryo fibroblasts transformed with the Ta and ts68 mutants of Rous Sarcoma virus (RSV) were enucleated and studied for their capacity to express reversibly the transformed phenotype in response to temperature changes. After shift to the permissive temperature (35 degrees C), the cytoplasts acquired a transformed morphology and displayed characteristic ruffles and microvilli at their surface. As detected by immunofluorescence, they also lost their actin filament cables and exhibited characteristic changes in the pattern of cell surface structures containing LETS protein. Expression of all these transformation parameters was reversible after shiftback to the nonpermissive temperature (41 degrees C). These results indicate that a whole set of changes characteristic for the transformed phenotype can be expressed independently of the cell nucleus. In contrast, ts mutant-infected cytoplasts were no longer able to respond to temperature shifts with changes in their hexose transport rate. Cytoplasts prepared from cells grown at 41 degrees C retained their low rate of hexose uptake after shift to 35 degrees C, whereas cytoplasts from cells grown at 35 degrees C exhibited a high rate of hexose transport even after 10 hr of shift to 41 degrees C. These results are in accordance with the hypothesis that the product of the src gene of RSV represents a multifunctional protein which acts independently on nuclear and extranuclear sites.     

The hexose transporters at the plasma membrane and the tonoplast of transformed plant cells: kinetic characterization of two distinct carriers.

The plasma membrane hexose transporter and the tonoplast hexose transporter from heterotrophically grown transformed Nicotiana tabacum cells have been studied in vitro using membrane vesicles for trans-zero transport studies. In highly purified phase-partitioned outside-out plasma membrane vesicles (PMV) the hexose transporter showed an apparent Km value of 230 microM (substrate: 3-O-methyl-D-glucose (3-OMG); pHi 7.2/pHo 7.2), which was reduced to 120 microM when a pH gradient was imposed (pHo 5.7/pHi 7.2). However, the Vmax value was not affected indicating that no stable pH gradient was formed. Uptake experiments with 14C-labelled acetate supported this interpretation. Transport was insensitive to N-ethylmaleimide (NEM; up to 1 mM concentration) and p-chloromercuribenzene sulfonate (PCMBS; up to 500 microM), whereas the tonoplast hexose transporter (in mixed inside / out and outside / out vesicles) was inhibited by NEM in a substrate-protectable manner, and PCMBS was also inhibitory. Kinetically two components with apparent Km values of 6 and 20 mM could be distinguished for the tonoplast hexose transporter. Substrate specificities of both transporters were similar except for D-galactose and D-fructose. The results indicate structural differences between the tonoplast and plasma membrane hexose transporters in plants.     

Hexose transport in plasma membrane vesicles prepared from L6 rat myoblasts

Hexose transport in plasma membrane vesicles prepared from L6 rat myoblasts was shown to be stereospecific, activated by glucose starvation and occurred by both high and low affinity systems. Transport by the high affinity system was shown to occur by an active transport process. Furthermore, the high affinity system was shown to be defective in vesicles prepared from F72 cells (hexose transport mutant). These results indicate that the high affinity hexose transport system is retained in the plasma membrane vesicles. Thus plasma membrane vesicles could be of value in further characterization of the L6 high affinity hexose transport system, without interference from the various metabolic events occurring in whole cells.     

Heterologous expression of the apple hexose transporter MdHT2.2 altered sugar concentration with increasing cell wall invertase activity in tomato fruit

Sugar transporters are necessary to transfer hexose from cell wall spaces into parenchyma cells to boost hexose accumulation to high concentrations in fruit. Here, we have identified an apple hexose transporter (HTs), MdHT2.2, located in the plasma membrane, which is highly expressed in mature fruit. In a yeast system, the MdHT2.2 protein exhibited high ¹⁴C‐fructose and ¹⁴C‐glucose transport activity. In transgenic tomato heterologously expressing MdHT2.2, the levels of both fructose and glucose increased significantly in mature fruit, with sugar being unloaded via the apoplastic pathway, but the level of sucrose decreased significantly. Analysis of enzyme activity and the expression of genes related to sugar metabolism and transport revealed greatly up‐regulated expression of SlLIN5, a key gene encoding cell wall invertase (CWINV), as well as increased CWINV activity in tomatoes transformed with MdHT2.2. Moreover, the levels of fructose, glucose and sucrose recovered nearly to those of the wild type in the sllin5‐edited mutant of the MdHT2.2‐expressing lines. However, the overexpression of MdHT2.2 decreased hexose levels and increased sucrose levels in mature leaves and young fruit, suggesting that the response pathway for the apoplastic hexose signal differs among tomato tissues. The present study identifies a new HTs in apple that is able to take up fructose and glucose into cells and confirms that the apoplastic hexose levels regulated by HT controls CWINV activity to alter carbohydrate partitioning and sugar content.     

Ca2+-dependent translocation of hexose carrier in mouse fibroblast Swiss 3T3 cells

Ca2+-induced translocation of hexose carriers from microsomal membrane to plasma membrane was demonstrated in saponin-permeabilized Swiss 3T3 cells by a specific D-glucose-inhibitable cytochalasin B-binding assay. The number of hexose carriers in the plasma membrane and the hexose transport activity in intact cells were also compared. The incubation of permeabilized cells with 10 microM Ca2+ at 37 degrees C rapidly increased the number of D-glucose-inhibitable cytochalasin B-binding sites in the plasma membrane from 13 to 40 pmol/mg protein and concomitantly decreased that in the microsomal membrane from 66 to 36 pmol/mg protein, each with a half-time of approx. 2 min. Furthermore, when Ca2+-stimulated cells were exposed to 50 microM EGTA, the effect of Ca2+ on the translocation of D-glucose-inhibitable cytochalasin B-binding sites was reversed with a half-time of approx. 5 min. The concentration of Ca2+ required for the half-maximal effect was approx 500 nM. The magnitude of the stimulatory effect of D-glucose-inhibitable cytochalasin B-binding sites in the plasma membrane closely correlated with the magnitude of stimulatory action of Ca2+ on 3-O-methylglucose transport in the intact cells. These results suggest that Ca2+ regulates the activity of hexose transport across the plasma membrane through a rapid and reversible translocation of hexose carrier between microsomal and plasma membranes of mouse fibroblast Swiss 3T3 cells.     

Molecular and biochemical events during differentiation of the HD3 chicken erythroblastic cell line

The chicken erythroblast cell line HD3 is transformed by a temperature-sensitive mutant of avian erythroleukemia virus. Upon shift to the non-permissive temperature in the presence of inducers (hemin and butyric acid), HD3 cells differentiate to an erythrocyte phenotype and provide a model system for analyzing events associated with this process. Expression of some cell surface proteins undergoes drastic changes as cells mature to the erythrocyte stage with a selective loss of membrane proteins that appears to be species-specific. Specific changes also occur in the expression and activities of cytosolic enzymes reflecting alterations of metabolism. HD3 differentiation is characterized by increased transferrin receptor (TFR) expression and increased hemoglobin (Hb) synthesis, a marker for the erythrocyte. In parallel, there is a decrease in glucose transport and an increase in nucleoside transport signifying a switch from glycolytic hexose metabolism to metabolism of pentose from nucleoside. Likewise the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAD) declines while glucose-6-phosphate dehydrogenase (G6PDH) activity remains constant. Commitment to the erythrocyte lineage alters expression of specific genes: TFR mRNA level increases while expression decreases for GLUT1 and GLUT3 glucose transporter mRNAs and GAD mRNA. However, the relationship between GAD activity and GAD mRNA was complex indicating modulation of GAD mRNA and protein half-lives. Serine/threonine and tyrosine phosphorylation and cAMP levels were shown to regulate the level of these messages. In this review, we describe how HD3 differentiation involves changes in plasma membrane composition, metabolism and gene expression that are orchestrated at different levels of control by multiple signaling modalities.