Abnormal response of glycolipid synthesis to temperature in transformed BHK cells thermosensitive for growth control.

In DMN4B cells, a line of chemically mutagenized BHK hamster cells which exhibit transformed behavior at 38.5°C but not at 32°C, [¹⁴C]-palmitate incorporation into mono-, di-, and trihexosylceramides was unimpaired at 32°C when compared with incorporation rates in untransformed BHK cells. At 38.5°C, labeling of these glycolipids increased greatly in the BHK cells, but failed to increase comparably in the DMN4B cells. Assay of cell-free preparations of the galactosyltransferase which catalyzes trihexosylceramide synthesis revealed a stimulatory effect of increased temperature on activity of the BHK enzyme, but not the DMN4B enzyme. The results suggest that transformation can result from a mutation affecting glycolipid synthesis.     

In vitro tumoricidal activity of macrophages against virus-transformed lines with temperature-dependent transformed phenotypic characteristics.

The susceptibility of targets to destruction by tumoricidal rat and mouse macrophages was studied with virus-transformed cell lines in which various elements of the transformed phenotype are only expressed at specific temperatures. BHK cells transformed by the ts3 mutant of polyoma virus, rat embryo 3Y1 cells transformed by a temperature-sensitive A cistron mutant of simian virus 40 (SV40) and the ts-H6-15 temperature-sensitive line of SV40-transformed mouse 3T3 cells were killed in vitro by macrophages at both the permissive (33 °C) or nonpermissive (39 °C) temperatures for expression of the transformed phenotype. 3T3, 3Y1 and BHK cells transformed by wild-type SV40 or polyoma virus were also destroyed by tumoricidal macrophages at both 33 and 39 °C, but untransformed 3T3, 3Y1, and BHK cells were not. Thus, transformed cells are killed by macrophages regardless of whether or not they express cell surface LETS protein or Forssman antigen, display surface changes which permit agglutination by low doses of plant lectins, express SV40 T antigen, have a low saturation density, or exhibit density-dependent inhibition of DNA synthesis.     

Hexose sugar transport activity in a mouse fibroblast cell line temperature sensitive for expression of the transformed phenotype

A temperature-sensitive mouse fibroblast cell line was used to examine the relationship between hexose sugar uptake rates and the control of cell growth. The cell line used (ts-H6-15) is a derivative of SV-3T3 cells, exhibiting a transformed phenotype at 32°C and a normal phenotype at 39°C. For cells actively growing at either temperature, a marked decrease in the rate of 3-0-methyl-D-glucose (3-0-MeG) transport is observed as cell population density increases. At all cell population densities tested, 3-0-MeG transport rates (at a common assay temperature) were greater in H6-15 cells grown at 32°C than at 39°C, with the enhancement being maximal at the lowest cell densities. The effect of low serum-arrest on H6-15 cells revealed that cells growing at 39°C arrest in G1, while cells at 32°C stop more randomly throughout their cycle. Under conditions of low serum-arrest the rate of 3-0-MeG transport remained as high as in actively growing cells at both 32°C and 39°C. However, 2-deoxyglucose uptake rates were growth state-dependent at 39°C, indicating perhaps metabolic as well as membrane-level control of sugar accumulation. These results further demonstrate that rates of hexose sugar transport by themselves are not always absolutely correlated with rates of cell proliferation and, thus, may not be reliable predictors of cell growth potential.     

Production of non-defective and defective oligomers of viral DNA in mouse 3T3 cells transformed by a thermosensitive mutant of polyoma virus

Cultures of three lines of mouse 3T3 cells transformed independently by the thermosensitive ts-a mutant of polyoma virus yield virus upon lowering their incubation temperature to 31°C. At 31°C, the internal pools of DNA of all three lines contain not only superhelical viral monomers, but also a small proportion of viral oligomers.From one of these three cell lines, several sublines of different clonal morphology were isolated at 38.5°C. The viral DNA synthesized at 31°C by each different subline displayed a unique oligomer pattern which has been stable through many cell passages and further reclonings. In contrast to the parental line, the monomer in most of these sublines is a minor component of the viral DNA pool. In one subline, more than 80% of the viral DNA consists of superhelical molecules about 1.6-times the size of a monomer. The specific infectivity of these molecules is only about one-tenth that of monomers, whereas the efficiency in transforming hamster (BHK21) cells is about twice that of monomers.     

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.     

Rate-limiting steps of 2-deoxyglucose uptake in rat adipocytes

2-Deoxy[1-¹⁴C]glucose uptake in rat adipocytes was measured as a function of time in the absence and presence of unlabelled glucose or 2-deoxyglucose. Uptake of tracer alone was linear from 2 s to 6 min. At 37°C the rate of uptake in insulin-stimulated cells decreased markedly after a few seconds in the presence of glucose (0.5–10 mM) and after 0.5–2 min in the presence of deoxyglucose (2–10 mM). Similar data were obtained at 22°C. With 10 mM glucose (37°C, 30 s) approx. 80% of the intracellular radioactivity was non-phosphorylated deoxyglucose and with 10 mM deoxyglucose approx. 40% was non-phosphorylated. The results show that deoxy[¹⁴C]glucose uptake after a few minutes is mainly limited by hexokinase in the presence of glucose and at least partially in the presence of deoxyglucose. The data suggest caution in using deoxyglucose uptake as a measure of transport, especially in complex kinetic studies.In addition, the initial velocity of tracer ¹$\text{3-O-}\text{methylglucose}$ was found to be approx. 2-fold higher than that of tracer deoxyglucose even though both sugars inhibited the initial velocity of labelled methylglucose half-maximally at a concentration of 5 mM. These data suggest a fundamental difference between deoxyglucose and methylglucose transport.     

Deoxyglucose uptake by mouse astrocytes: Effects of temperature and retrovirus infection

Deoxyglucose uptake by FVB/N mouse astrocytes was studied before and after infecton by tsl retrovirus which causes a neurodegenerative disease in mice similar to HIV-1 encephalopathy in man. The Michaelis-Menten kinetic parameters, Km and Vmax, of 2-deoxy-D-glucose uptake by brain and cerebellar astrocytes were measured following culture at 34°C where tsl retrovirus replicates optimally, and at 37°C. Compared to astrocytes cultured at 37°C, astrocytes cultured at 34°C had increased Km and decreased deoxyglucose uptake despite increased or unchanged Vmax. Following ts1 retrovirus infection, brain astrocyte deoxyglucose uptake doubled [132%] associated with decreased Km but unchanged Vmax, whereas cerebellar astrocyte deoxyglucose uptake doubled [102%] associated with increased Vmax but unchanged Km. These observations of altered deoxyglucose uptake kinetic parameters following retrovirus infection indicate different neurochemical mechanisms for the regional variation in deoxyglucose uptake observed following retrovirus infection of the CNS in vivo.     

Collagen synthesis in normal BHK cells and temperature-sensitive chemically transformed BHK cells.

Collagen synthesis in normal BHK 21/cl 13 and chemically transformed temperature sensitive BHK 21/cl 13 cells (Me2N4) was assessed by examination of hydroxyproline formation and collagenase-susceptible protein. The Me2N4 cells lost their ability to synthesize collagen at both permissive and nonpermissive temperatures for transformation. These conclusions were confirmed by polyacrylamide-gel eletrophoresis and CM-cellulose chromatography. Prolyl hydroxylase activity was present in both normal and transformed cells even when no collagen could be demonstrated. The production of noncollagen protein, although decreased in the transformed cell, did not change as drastically as the collagen synthesis.     

Glucose-regulated membrane properties of untransformed and virus-transformed BHK21 cells.

BHK21 fibroblasts transformed by hamster sarcoma virus have a higher rate of uptake of hexoses than their untransformed counterparts, and therefore rapidly exhaust glucose from the culture medium. The effects of culturing normal and transformed BHK cells, both in limiting and in excess glucose, on several membrane properties related to malignant transformation have been studied. The increase in the rate of hexose uptake in transformed cells is partially but not entirely dependent on extracellular glucose concentration. Two transformation-increased membrane proteins of molecular weights 95 000 and 78 000 are shown to be regulated by extracellular glucose concentration in both normal and transformed cells. The loss of LETS-protein, the high density of intramembranous particles, the increase in the amount of a 177K integral plasma membrane protein and the increase in the amount of high molecular weight surface glycopeptides in transformed cells, are not related to glucose depletion of the medium. Beside LETS, another iodinated protein, of molecular weight 160 000, is decreased in transformed cells. The exposure of this protein increased in both normal and transformed cells when arrested in G1 by asparagine deprivation.     

Changes in the uptake of 2-deoxy-D-glucose in BALB-3T3 cells chemically transformed in culture

Balb/3T3 cells transformed in culture by chemical carcinogens were shown to multiply in a medium supplemented with 2% calf serum or with 10% agamma new-born calf serum. The cell lines that multiply well in medium supplemented with 10% agamma serum produced a higher incidence of tumors in X-irradiated weanling mice than the lines that multiply poorly. The difference in 2-deoxy-D-glucose uptake into exponentially growing transformed and un-transformed cells was 50–100%. In crowded cultures untransformed Balb/3T3 cells ceased taking up the sugar, while chemically transformed cells continued at the same rate even at high cell densities; thus, the difference became greater in crowded cultures. When the serum concentration in the media was reduced from 10% to 2%, untransformed Balb/3T3 cells took up the sugar at a reduced rate, while chemically transformed cells were only slightly affected; agamma new born calf serum supplemented medium had no effect on sugar uptake in any of the cells. When the serum concentration was changed from 2% to 10%, untransformed cells increased sugar uptake followed by cell division. The immediacy (within 15 min) of the response in the sugar uptake to 10% serum concentration suggested that the increased uptake rate and the consequent higher concentration of the sugar (D-glucose in normal situation) within Balb/3T3 cells triggered the cell cycle. Chemical carcinogens appear to alter permanently the uptake mechanism for a key nutrient.     

Alterations in glucose metabolism in chick embryo cells transformed by Rous sarcoma virus. Transformation-specific changes in the activities of key enzymes of the glycolytic and hexose monophosphate shunt pathways

Effects of transformation by Rous sarcoma virus of Schmidt-Ruppin strain on the activities of key enzymes of the glycolytic and the hexose monophosphate shunt pathways in chick-embryo cells were investigated. Activities of hexokinase, phosphofructokinase, pyruvate kinase, lactate dehydrogenase, and glucose-6-P dehydrogenase were increased about twofold in the transformed cells, but that of 6-P-gluconate dehydrogenase remained unaltered. The transformation-mediated increase in the activity of hexokinase was confined entirely to the bound form of the enzyme. Cells infected with a temperature-sensitive mutant (Ts-68) of Schmidt-Ruppin strain of Rous sarcoma virus showed the typical increase in the rate of 2-deoxyglucose uptake and the activities of hexokinase, phosphofructokinase, pyruvate kinase, and glucose-6-P dehydrogenase at the permissive temperature (37 °C), but when the infected cells were grown at the nonpermissive temperature (41 °C), the increases in the sugar uptake and activities of these enzymes were abolished. Unlike the regulatory enzymes, lactate dehydrogenase activity was increased at both the permissive and the nonpermissive temperatures.     

Collagen synthesis in normal BHK cells and temperature-sensitive chemically transformed BHK cells

Summary Collagen synthesis in normal BHK 21/cl 13 and chemically transformed temperature-sensitive BHK 21/cl 13 cells (Me₂N4) was assessed by examination of hydroxyproline formation and collagenase-susceptible protein. The Me₂N4 cells lost their ability to synthesize collagen at both permissive and nonpermissive temperatures for transformation. These conclusions were confirmed by polyacrylamide-gel electrophoresis and CM-cellulose chromotography. Prolyl hydroxylase activity was present in both normal and transformed cells even when no collagen could be demonstrated. The production of noncollagen protein, although decreased in the transformed cell, did not change as drastically as the collagen synthesis. This paper was supported in part by a grant from the Public Health Service (AG00001), and by the Medical Research Service of the Veterans Administration.     

Properties of mammalian cells transformed by temperature-sensitive mutants of avian sarcoma virus.

Fibroblasts from European field vole (Microtus agrestis) and from normal rat kidney (NRK) have been infected by avian sarcoma virus mutants which are temperature-sensitive for the maintenance of transformation. These cells are transformed at 33 degrees C, but show normal cell characteristics in morphology, colony formation in agar, saturation density, sugar uptake and membrane proteins at 39 degrees C and 40 degrees C, the nonpermissive temperatures. Ts mutant virus was rescued from most of the ts transformed cell lines. NRK cells infected by avian sarcoma virus ts mutants and kept at the nonpermissive temperature can be transformed by wild-type avian sarcoma virus. The susceptibility of the temperature-sensitive NRK lines to this transformation is higher than the susceptibility of uninfected NRK at either permissive or nonpermissive temperature.     

Deoxyglucose transport of uninfected, murine sarcoma virus-transformed, and murine leukemia virus-infected mouse cells

The initial rates of deoxy-D-glucose transport by cultures of growing and density-inhibited mouse embryo cells and lines of mouse cells transformed spontaneously or after infection by murine leukemia virus or murine sarcoma virus were investigated as a function of the deoxyglucose concentration. The apparent K_m for deoxyglucose transport was about the same for all types of cells (1–2 mM). The V_max of secondary cultures of mouse embryo cells decreased from 6 nmoles/10⁶ cells/minute for sparse cultures to less than 1 nmole/10⁶ cells/minute for density-inhibited cultures. The V_max was about the same whether estimated in monolayer culture or in suspensions of cells dispersed by treatment with trypsin. The V_max for deoxyglucose transport by the established cells, whether transformed spontaneously or by virus infection, was 4 to 25 times higher than that for density-inhibited mouse embryo cells and was independent of the cell density of the cultures. Deoxyglucose transport was competitively inhibited by Cytochalasin B, Persantin, glucose and 3-O-methyl-D-glucose and the apparent K_i values of inhibition were similar for the mouse embryo cells and the various cell lines. Similarly, the sensitivity of the glucose transport systems to inactivation by p-chloromercuribenzoate was about the same for all types of cells. The results suggest that the glucose transport system of the normal mouse embryo cells and the cells of the various established lines is qualitatively the same, but that the number of functional transport sites differs for the various cell lines and decreases markedly in mouse embryo cells with an increase in cell density of the cultures.     

Effects of growth conditions on cyclic nucleotide phosphodiesterases of cultured fibroblasts.

Cyclic nucleotide phosphodiesterase activities of baby hamster kidney cells (BHK) grown in surface cultures were altered by modifying growth conditions. The untransformed BHK cells grown in medium containing 10% fetal calf serum showed non-linear LineweaverBurk plots for cyclic AMP phosphodiesterase activity with apparent Michaelis constants for cyclic AMP of approximately 5 and 30 muM. When these cells were placed in medium containing 1% fetal calf serum, linear kinetic plots for cyclic AMP phosphodiesterase with an apparent Km for cyclic AMP of approximately 20 muM were obtained. Modification of the apparent Km of BHK cell phosphodiesterase was detectable within 20 minutes after dillution of cells grown in 10% serum into fresh medium containing 1% serum. With the BHK cell line transformed with Rous sarcoma virus, differences in enzyme kinetics were not seen when these cells were diluted in 1% or 10% serum. In addition to the serum induced differences in the apparent Km of cyclic AMP phosphodiesterases of BHK cells, total cyclic AMP and cyclic GMP phosphodiesterase activities were also modified by growth conditions. BHK cells grown to high cell densities had three to five-fold higher total cyclic AMP activity than did the cells in less dense cultures. When the dense cell cultures were diluted into fresh medium containing 10% serum, total enzyme activities fell to levels comparable to those found in the rapidly growing cells at low cell densities. The reduction in total enzyme activity after dilution of BHK cells occurred rapidly and was influenced by cell density. A similar reduction of total enzyme activity was also seen in diluted RSV cells; however, the time course of the response differed from that seen in the untransformed cells.     

Sulfated mucopolysaccharides from normal and virus transformed rodent fibroblasts.

The sulfated mucopolysaccharide composition of normal and virus transformed Balb 3T3 and BHK21 cell lines is reported. It is shown that normal 3T3 cells contain mainly chondroitin sulfate B and heparitin sulfate. Relatively higher amounts of chondroitin sulface AC were observed in polyoma virus transformed 3T3 cells, besides an absolute increase of all the three sulfated mucopolysaccharides in the polyoma and SV 40 transformed cells. It is shown also that the three sulfated mucopolysaccharides are at least in part at the cell surface. Similar differences in sulfated mucopolysaccharide composition of normal and virus transformed BHK cell lines were also observed.     

Differential methylation of mitochondrial and nuclear DNA in cultured mouse, hamster and virus-transformed hamster cells. In vivo and in vitro methylation

Information has been lacking as to whether mitochondrial DNA of animal cells is methylated. The methylation patterns of mitochondrial and nuclear DNAs of several mammalian cell lines have therefore been compared by four methods: (1) in vivo transfer of the methyl group from [methyl-³H]methionine; (2) in vivo incorporation of [³²P]orthophosphate and a combination of (1) and (2); (3) in vivo incorporation of [³H]deoxycytidine; (4) in vitro methylation of DNAs with ³H-labeled S-adenosylmethionine as methyl donor and DNA methylase preparations from L cell nuclei. The cell lines were mouse L cells, $\text{BHK21}\text{C13}$, $\text{C13}\text{B4}$ (baby hamster kidney cells transformed by the Bryan strain of Rouse sarcoma virus), and PyY (BHK cells transformed by polyoma virus). DNA bases were separated chromatographically, using 5-methylcytosine, 6-methylaminopurine and, in some cases, 7-methylguanine as markers.Mitochondrial DNA was found to be significantly less methylated than nuclear DNA with respect to 5-methylcytosine in all cell types studied and by all methods used. The relative advantages and disadvantages of each method have been discussed. The level of 5-methylcytosine in mitochondrial DNA as compared with that in nuclear DNA was estimated as one-fourth to one-fourteenth in various cell lines. The estimated 5-methylcytosine content per circular mitochondrial DNA molecule (mol. wt 10 × 10⁶) was about 12 methylcytosine residues for L cells and 24, 30 and 36 methylcytosine residues for BHK, B4 and PyY cells, respectively. Relative to cytosine residues, the estimate was one 5-methylcytosine per 500 cytosine residues of mitochondrial DNA and one 5-methylcytosine per 36 cytosine residues of nuclear DNA from L-cells. The values for methylcytosine of mitochondrial DNA are presumed to be maximal. PyY cells as compared with other cells had the highest methylcytosine content of both mitochondrial and nuclear DNA as estimated by method (3). No methylation of nuclear DNA was observed in confluent L cells.Evidence for the presence of DNA methylase activity associated with mitochondrial fractions was obtained. This activity could be distinguished from other cellular DNA methylase activity by differential response to mercaptoethanol. Radioactivity from ³H-labeled S-adenosylmethionine was found only in 5-methyl-cytosine of DNA.     

Transport and phosphorylation of hexoses in normal and rous sarcoma virus-transformed chick embryo fibroblasts

Effects of transformation by Rous sarcoma virus on sugar uptake and activity and the subcellular distribution of hexokinase isozymes in chick embryo fibroblasts were examined. Transformation caused a several-fold increase in the maximum velocity for uptake of 2-deoxyglucose without a significant change in K_m. Cytochalasin B (CB), was used to differentiate between the effects of transformation on facilitated diffusion and the nonsaturable (CB-insensitive) mode. Transformation was found to stimulate 2-deoxyglucose transport by both mechanisms, but the increase in transport by the CB-insensitive mode was greater. Transformation enhances the activity of hexokinase, the enhancement being confined to the particulate fraction of the enzyme. Heat-inactivation and electrophoretic mobility studies showed that although hexokinase Type I is the major form in both normal and transformed fibroblasts, there is a significant increase in the proportion of the Type II isozyme in the transformed cells.     

EBV increases phosphoinositide kinase activities in human B cells.

To determine whether EBV affects phosphoinositide kinase activities of human B cells, we compared the activities between EBV- and EBV+ human B cell lymphoma lines. The two types of human B cells contained both phosphatidylinositol (PtdIns) 4-kinase and phosphatidylinositol 4-phosphate (PtdIns(4)P) kinase activities irrespective of the presence of EBV. However, both activities were increased in EBV+ cells compared to EBV- cells. The increases were associated with neither altered Km values for substrates nor altered elution profiles in DEAE-cellulose chromatography. Furthermore, expression of a latent EBV protein, EBV nuclear Ag1 (EBNA1) in BHK cells by the transfection of EBNA1 DNA was accompanied by increased PtdIns 4-kinase and PtdIns(4)P kinase activities. These increases also were not associated with altered Km values for substrates. However, phospholipase C activity was altered in neither EBV+ cells nor in EBNA1-expressing cells. These results indicate that EBV selectively increases the two phosphoinositide kinase activities in human B cells, although the viral gene product has no intrinsic phosphoinositide kinase activity. PtdIns 4-kinase and PtdIns(4)P kinase cooperatively synthesize PtdIns 4,5-bisphosphate, the major source of 1,2-diacylglycerol and inositol 1,4,5-triphosphate, the two second messengers in transducing signals for cell activation. Such increase therefore may play a role in EBV-induced human B cell activation.