Binding of Salmonella minnesota R-form glycolipid mR595 to rat fibroblasts and its effect on cell metabolism and cell behaviour

Trypsinized normal rat embryo fibroblasts and untrypsinized and trypsinized transformed rat fibroblasts have two orders of binding sites for bacterial glycolipid mR595. The high order sites fix 1–3 μg glycolipid mR595/10⁵ cells and those of the low order fix about 6 μg glycolipid mR595/10⁶ cells. Ca⁺⁺ is required for the low order glycolipid mR595 binding to be trypsinized but not to the untrypsinized transformed rat fibroblasts. The low order binding but not to the untrypsinized transformed rat fibroblasts. The low order binding is temperature dependent with the transition temperature lying between 25 and 37°C. Exogenously added ganglioside and glycoproteins contained in the fetal calf serum do not inhibit fixation of glycolipid mR595. Only β-lipoprotein at high concentrations is slightly inhibitory. Glycolipid mR595 fixation to transformed fibroblast does not alter their morphology and appears to slightly improve cell attachment to substratum. Glycolipid mR595 fixation results in a lengthening of the S-phase of the cell cycle and a reduction in 2-deoxyglucose uptake. Uptake of inorganic phosphate is not affected. Inhibition of phospholipid synthesis is observed in mR595 fixed fibroblasts whereas synthesis of cell surface glycoproteins and the content of cellular gangliosides is not affected.     

Cell surface mobility of a bacterial R-form glycolipid, mR595, after binding to rat cells, and its effect on the mobility of concanavalin A receptors

Binding of small amounts of glycolipid mR595 to rat cells, followed by sequential incubation of cells at 37 °C with rabbit anti-glycolipid mR595 and fluorescein-conjugated sheep anti-rabbit γ-globulin antisera results in the localization of fluorescence at one pole of the cell surface (capping). Binding of higher amounts of glycolipid mR595 to cells not only inhibits formation of glycolipid caps but those of the ConA receptor-fluorescent ConA complex as well. Glycolipid mR595 binding does not alter [³H]ConA binding to cells but cell agglutination by ConA is inhibited in a competitive fashion. Binding of small amounts of ConA to cells does not affect glycolipid capping. Colchicine and cytochalasin B (CB) treatment of cells inhibits glycolipid cap formation.     

The effect of dibutyryl adenosine 3':5'-cyclic monophosphate on the synthesis of glycolipids by normal and transformed Nil cells

The synthesis of complex glycolipids was examined in two clones (Nil 1Cl and Nil 2Cl) of the Nil 2 line and their hamster sarcoma virus-transformed derivatives. Nil 2Cl contains three neutral glycolipids which increase in concentration as cells become confluent. However, the kinetics of increase differs for each of the three. The Forssman glycolipid increases in parallel with cell growth and reaches a maximal level just before cells reach their saturation density. Synthesis of globoside increases significantly only after the culture reaches saturation density. These “density-dependent” glycolipids are lost or decreased in concentration after transformation by hamster sarcoma virus.The growth of both normal and transformed cells is inhibited by treatment with dibutyryl cyclic AMP. The density at which they stop growth is dependent upon the inoculum density. The glycolipid patterns of normal and transformed cells has been examined in the presence or absence of dibutyryl cyclic AMP. In the presence of the nucleotide transformed cells did not regain the glycolipids or density-dependent synthesis of glycolipids which they lost as a result of transformation.     

Cyclic AMP-binding proteins in normal and virus-transformed fibroblasts.

We examined the notion that altered growth regulation in virally transformed fibroblasts is due to a biochemical lesion affecting cytosol receptor for cyclic AMP. Normal cultured fibroblasts used after two passages and transformed cells from mouse and hamster possess equivalent amounts of total cyclic AMP receptors, with similar apparent affinities for cyclic AMP and similar distribution among isoenzymic forms of cyclic AMP-dependent protein kinase. Furthermore, untransformed 3T3 cells display only a single peak of cyclic AMP-binding activity, as resolved by ion-exchange chromatography. These findings do not support the view that defective binding of cyclic AMP is essential for loss of growth control in vitro.     

Effects of sera types and cell density on the concentration of cyclic nucleotides in normal and simian virus transformed mouse fibroblasts

The effects of serum and cell density on the concentration of cyclic AMP, cyclic GMP in normal mouse fibroblasts cells (3T3 cells) and their Simian Virus 40 transformed derivative (SV3T3 cells) were studied. 3T3 cells grown in 10% foetal bovine serum exhibit density dependent inhibition of growth and associated with this in an increase in the concentration of cyclic AMP, a decrease in the concentration of cyclic GMP and an increase in the ratio (cyclic AMP/cyclic GMP) of the cyclic nucleotides. 3T3 cells grown in 10% newborn calf serum exhibit a higher saturation density and this is associated with a low concentration of cyclic AMP and a high concentration of cyclic GMP. SV3T3 cells grown in either 10% foetal bovine serum or 10% newborn calf serum show high saturation densities and this is associated with a low and decreasing concentration of cyclic AMP and a high concentration of cyclic GMP. When the level of the cyclic AMP in both cell lines was artificially raised by adding dibutyryl cyclic AMP and theophylline to the growth media, the cells grew to low densities.     

Uncoupling of the Glucagon Receptor-Adenylate Cyclase System by Glucagon in Cloned Differentiated Rat Hepatocytes

Abstract

The ability of glucagon to induce a state of desens it ization to glucagon responsiveness has been examined in a cloned line of normal, differentiated, diploid rat hepatocytes (RL-PR-C). These cells, which respond to glucagon with increased production of cyclic AMP, become refractory to further stimulation of cyclic AMP synthesis following a 4 hour exposure period of the cells to the hormone. Refractoriness to glucagon was demonstrated over a wide range of hormone concentrations (10^?12 to 10^?6 M). In desensitized cells that were subsequently washed free of the hormone, recovery from refractoriness was complete by 20 hours. The mechanism underlying this desensitization does not appear to involve decreased receptor numbers, increased efflux of cyclic AMP from the cells, increased degradation of cyclic AMP by phosphodtesterase, or an alteration in the catalytic unit of the adenylate cyclase enzyme system. By elimination, the diminished cellular cyclic AMP responsiveness to glucagon in normal RL-PR-C hepatocytes may involve a reversible uncoupling of glucagon receptors from adenylate cyclase. In addition, late passage, spontaneously transformed RL-PR-C hepatocytes were found to exist in a state in which glucagon receptors are permanently uncoupled from adenylate cyclase.     

Ethanolamine base exchange enzymatic activities in spontaneous transformed glial cell lines. Effect of dibutyryl cyclic AMP treatment

Cultured astrocytes derived from neonatal rats (normal cells) displayed maximal ethanolamine base exchange enzymatic activity (EBEE) when cultures reached confluency and cells almost ceased to divide. At this stage, ethanolamine phosphotransferase (EPT) and choline base exchange enzyme (CBEE) activities reached a plateau. In spontaneously transformed glial cells, no differential activity variation either between EPT and CBEE, or between EPT and EBEE was observed. The EBEE activity was mainly localized in the microsomal fraction and was completely absent from plasma membranes. Dibutyryl cyclic AMP (db-cAMP) treatment of the transformed cells reversed the pattern of these activities to that of normal cells. Moreover, treatment of the transformed cells with medium conditioned by normal astroblasts markedly increased EBEE activity. This study demonstrates that (i) variation of EBEE activity during cell growth differs in normal and in transformed cultured glial cells. (ii) EBEE activity may be modulated via both db-cAMP and normal cell conditioned medium. Our findings suggest a possible implication of EBEE in the maturation and contact inhibition of cell growth.     

Cyclic AMP phosphodiesterase in human lymphocytes and lymphoblasts.

Cyclic nucleotide phosphodiesterase activities were examined in lymphocytes from 12 transformed human B cell lines, two T cell lines, six patients with lymphocytic leukemia, and 10 normal donors. A consistent difference bwtween cells from the normal and leukemic state was observed. The cyclic AMP phosphodiesterase activity from normal lymphocytes is inhibited greater than 80% by muM cyclic GMP while this concentration of nucleotide has little or no effect on the enzyme from transformed lymphocytic cell lines or from lymphocytic cells of leukemia patients. The reported lack of cyclic GMP phosphodiesterase in human lymphocytes from several sources is confirmed. The apparent absence of a cyclic GMP degradation mechanism and of cyclic GMP control of cyclic AMP hydrolysis may be related to defective lymphocyte growth control.     

Difference in the Cyclic Adenosine 3′,5′-Monophosphate Levels in Normal and Transformed Cells

CYCLIC 3′5′-adenosine monophosphate (cyclic AMP) regulates many physiological phenomena^1,2. Cellular morphology changes when the dibutyryl derivative of cyclic AMP is added in vitro to the nutrient media of cultured mammalian cells^3–6. Dibutyryl cyclic AMP has also been shown to restore controlled growth to transformed cells³, change the cell's surface architecture^3,7 and induce axon formation⁸ with an accompanied increase in acetylcholinesterase activity⁹ in neuroblastoma cells growing in culture. These effects suggest that the cyclic AMP moiety may have some basic regulatory action on cell growth and cell specialization.     

Cholera toxin treatment stimulates tumorigenicity of Rous sarcoma virus-transformed cells.

Chinese hamster ovary cells transformed by Rous sarcoma virus form tumors poorly in nude mice. Tumorigenicity was markedly stimulated by pretreatment of the cells with cholera toxin, which raises cyclic AMP levels and activates cyclic AMP-dependent protein kinase. Increased tumorigenicity was manifested by a severalfold increase in the rate of tumor formation, as well as earlier appearance and more rapid growth of tumors. In contrast, spontaneously transformed Chinese hamster ovary cells showed decreased tumorigenicity after cholera toxin treatment. The activation of tumorigenic potential in Rous sarcoma virus-transformed Chinese hamster ovary cells by cholera toxin correlated with increased phosphorylation of the viral oncogene product pp60src and stimulation of its tyrosine kinase activity.     

The correlation of plasma membrane microvilli and intracellular cyclic AMP content in a rat epitheloid kidney cell line

Modulation of the intracellular concentration of cyclic AMP has been associated with a regulatory role in cell division, cell morphology, and physical properties of the plasma membrane. Untransformed rat kidney cells in culture exhibit epitheloid morphology, high intracellular cyclic AMP levels, and contact inhibition of growth. Untransformed rat kidney cells transformed with the Kirsten murine sarcoma virus exhibit a low cyclic AMP content, rapid growth rate, and a loss of contact inhibition. Scanning electron microscopy reveals a distinctive difference in the surface structure of the two cell types during G1 of the cell cycle. The surface of the transformed cell is covered with microvilli while its untransformed counterpart is devoid of microvilli. The presence of microvilli can be controlled as a function of temperature by two temperature-sensitive mutants of the Kirsten sarcoma virus (ts6t6 and ts371 cl 5). In the ts6t6 mutant, growth at 32°C results in a low cyclic AMP content and the presence of microvilli, while growth at 39°C results in a high cyclic AMP content and a decrease in microvilli. The oposite effect is seen with the ts371 cl 5 mutant. Correlation of cyclic AMP content with the presence of microvilli suggests that this surface phenomenon is a function of cyclic AMP concentration.     

Cyclic AMP specifically blocks proliferation of rat 3T3 cells transformed by polyomavirus.

Elevated exogenous and intracellular levels of cyclic AMP could totally block proliferation of polyomavirus (PyV) transformants derived from rat 3T3 cells without affecting proliferation of normal cells or simian virus 40 (SV40)-induced transformants. Concanavalin A (ConA) had the opposite effect; it could totally block proliferation of both normal cells and SV40 transformants but reduced proliferation of PyV transformants only twofold. Adenylate cyclase was threefold less active in membranes of PyV transformants, and the number of ConA receptors was similar to that of normal cells. Proliferating PyV transformants contained threefold less cyclic AMP than did proliferating SV40 transformants. The sensitivity to cyclic AMP did not correlate with the degree of transformation: cells transformed by Rous sarcoma virus and tumor cells derived from SV40 transformants were not sensitive to cyclic AMP. The differential effect of cyclic AMP and ConA on proliferation was probably due to the activity of an intact middle t protein. The presence of both large T and small t together with middle t was also required for cyclic AMP sensitivity.     

Isolation and charactrization of a morphologic variant of chinese hamster (CHO) cells

An epithelioid clone of Chinese hamster (CHO) cells which is spontaneously transformed was exposed to the mutagen N-methyl-N-Nitro-N-Nitrosoguanidine (MNNG), and a fibroblastic variant, clone CHO-F₂, was isolated. This clone is partially reverted in several of the in vitro properties characteristic of transformed cells. When compared to wild type CHO, CHO-F₂ has a longer doubling time, a lower saturation density and less piling up at high cell density, and a higher serum requirement. CHO-F₂ also elaborates less plasminogen activator and has more abundant microtubules and actin cables. On the other hand, both CHO and CHO-F₂ grow in agar suspension (although CHO-F₂ grows with a lower efficiency), both lack detectable LETS protein, and both are tumorigenic in nude mice. Thus, expression of the individual properties frequently associated with transformation and tumorigenicity can be dissociated. The most critical biochemical change in CHO-F₂ appears to bean increase in the intracellular level of cyclic AMP, when compared to CHO, and several growth and morphological properties of CHO-F₂ resemble those induced in wild type CHO exposed to exogenous dibutyryl cyclic AMP. Therole of cyclic AMP in expression of the transformed phenotype and the significance of individual in vitro parameters of transformation with respect to tumorigenicity are discussed.     

Growth control in chick embryo fibroblasts; no evidence for a specific role for cyclic purine nucleotides

Cyclic AMP and cyclic GMP concentrations were measured in cultures of normal chick embryo fibroblasts and those transformed by Rous sarcoma virus under different growth conditions. No significant and reproducible correlation between the nucleotide levels and the rate of proliferation was observed. Neither release of normal cells from density dependent inhibition of growth nor transformation of the cultures by different strains of Rous sarcoma virus affected the concentrations of cyclic AMP or cyclic GMP. Activities of cellular cyclic nucleotide phosphodiesterases, enzymes involved in regulating the level of the nucleotides, were not directly affected by growth-stimulating concentrations of insulin or neuraminidase. Growth stimulation by insulin did not alter the activities of cellular cAMP-dependent protein kinase. These results do not support the hypothesis that cyclic AMP or cyclic GMP has a specific role in the growth control of chick embryo fibroblasts.     

Contact Inhibition of Transformed Cells Incompletely Restored by Dibutyryl Cyclic AMP

INCREASED levels of cyclic AMP have been found in normal cells as compared with malignant cells^1,2. Several types of malignant cells become morphologically similar to untransformed cells when incubated in media containing cyclic AMP or its derivative dibutyryl adenosine 3′:5′-cyclic monophosphate (dibutyryl cyclic AMP)^3,4. Sheppard reported that 3T3 mouse fibroblasts, transformed by polyoma virus, grew to low saturation density and became less agglutinable with wheat germ agglutinin if theophylline and dibutyryl cyclic AMP were added to the medium⁵.     

Factors affecting surface expression of glycolipids: influence of lipid environment and ceramide composition on antibody recognition of cerebroside sulfate in liposomes

The reactivity of the acidic glycolipid cerebroside sulfate (CBS) with antibody was studied as a function of its lipid environment in vesicles and of its ceramide composition. The lipid environment was varied by using phosphatidylcholine of varying chain length with cholesterol in a phosphatidylcholine:cholesterol:cerebroside sulfate molar ratio to glycolipid of 1:0.75:0.1. The ceramide structure of CBS was varied by using synthetic forms containing palmitic acid, lignoceric acid, or the corresponding alpha-hydroxy fatty acids. Reactivity with antibody was determined by measuring complement-mediated lysis of the vesicles containing a spin-label marker, tempocholine chloride. The data were analyzed by a theoretical model which gives relative values for the dissociation constant and concentration of antibodies within the antiserum which are able to bind to the glycolipid. If the phosphatidylcholine chain length was increased, increasing the bilayer thickness, only a small population of high-affinity antibodies were able to bind to cerebroside sulfate, suggesting decreased surface exposure of the glycosyl head group. A larger population of lower affinity antibodies were able to bind to it in a shorter chain length phosphatidylcholine environment. However, if the chain length of the cerebroside sulfate was increased, it could be recognized by more antibodies of lower affinity than the short chain length form, suggesting that an increase in chain length of the glycolipid increased surface exposure. Hydroxylation of the fatty acid inhibited antibody binding; only a smaller population of higher affinity antibodies was able to bind to the hydroxy fatty acid forms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alterations of cAMP metabolism and hormone responsiveness of cloned differentiated rat liver cells (RL-PR-C) upon spontaneous transformation

In normal Rat Liver Primary Culture (RL-PR-C) liver cells, cAMP was low prior to confluency, then rose continuously as cells became contact inhibited. In contrast, spontaneously transformed RL-PR-C cells did not become contact inhibited, and cAMP decreased steadily with increasing cell density. Normal cells released large amounts of cAMP into the extracellular fluid at all densities, while transformed cells did not do so at any density. Neither exogenous db-cAMP nor phosphodiesterase inhibitors reversed the uncontrolled growth of transformed cells, nor did conditioned media from contact-inhibited normal cells.While both normal and transformed RL-PR-C hepatocytes produced large amounts of cAMP in response to epinephrine and cholera toxin, transformed cells were much more sensitive to these agents; however, only normal cells responded to glucagon. Although the plasma membrane adenylate cyclase of transformed hepatocytes responded better than did that of normal cells to epinephrine, cholera toxin and fluoride, the basal cyclase activity of transformed cells was only about half that of normal cells. The adenylate cyclase of transformed cells did not respond to glucagon, although the number of glucagon receptors of such cells far exceeded that of normal cells. The V_max of cyclic nucleotide phosphodiesterase of normal hepatocytes was five times that of transformed cells, although the K_m was unchanged.The data indicate that spontaneous transformation of diploid differentiated RL-PR-C hepatocytes leads to cultural hormone receptor and cAMP changes similar to those seen in undifferentiated fibroblasts and other cells transformed by viruses and chemical carcinogens. Although there are significant changes in various parameters of cAMP metabolism upon transformation, decreased cAMP per se does not seem to be responsible for transformation. Furthermore, it is possible that following transformation, these hepatocytes lose some factor necessary for coupling of the glucagon receptor to adenylate cyclase.     

Transformation-dependent effect of dibutyryl cyclic AMP on surface properties of rat cultured cells

The possible differential effect of dibutyryl cyclic AMP on the surface properties of ts-NT3-KR rat cells that express a normal phenotype at 37 degrees C and a transformed morphology and behavior at 33 degrees C has been studied. Electrophoretic examination of glycosylated macromolecules revealed a 350,000 dalton glycoconjugate in phenotypically normal cells but not in the corresponding samples from phenotypically transformed cells or in phenotypically "normal" cells rounded by exposure to the cyclic nucleotide. A decreased exposure of a major 100,000 dalton surface component characteristic of cells that expressed a transformed phenotype, was observed when the corresponding cultures were exposed to dibutyryl cyclic AMP. No change in the 230,000 dalton fibronectinlike molecule of phenotypically normal cells was apparent even in the corresponding cultures exposed to the cyclic nucleotide.     

Prolonged prostaglandin E1 stimulation of cyclic AMP production in transformed and normal WI-38 fibroblasts.

Long-term (48-hr) incubations of either the fibroblast strain WI-38 or its SV40-transformed counterpart, WI-38-VA13-2RA, in growth medium containing 1 micron prostaglandin E1 (PGE1) resulted in a sustained production and release of cyclic AMP from the cells into the medium. Despite the steady production, intracellular levels of the nucleotide decreased, reaching steady-state values within 4 hr of the initial exposure to PGE1. These values were maintained for the remainder of the 48-hr experimental period. The steady-state levels of intracellular cyclic AMP were higher than those observed in unstimulated cells, and cyclic AMP-dependent protein phosphokinase was in a highly activated state as compared to controls. Under these conditions little change in the growth or morphology of either the normal or transformed cells was observed. In contrast, inhibition of growth, apparent cell death, and unusual morphological changes were observed in both normal and transformed cells when high concentrations of either PGE1 (10 micron) or the phosphodiesterase inhibitor 1-methyl, 3-isobutylxanthine (0.5 mM to 2 mM) were used, which was indicative of toxic effects of the drugs. It was concluded that cyclic AMP-mediated activation of protein phosphokinase does not completely inhibit growth in WI-38 cells or restore normal growth and morphology to the SV40-transformed cells.