cyclic-gmp and Cell-Transformation--Viral

Many hormones act by combining with cell surface receptors and stimulating adenylate cyclase activity. The cyclic AMP generated is the mediator of a number of cellular metabolic processes. Other processes may be influenced by changes in cyclic GMP levels. Although much evidence from cultured cells suggested that low cellular levels of cyclic AMP and high levels of cyclic GMP are a feature of rapid cell growth and of malignant transformation, review of the data reveals many inconsistencies. Thus in established tumours growing in vivo, for example, cyclic AMP levels appear to be unrelated to tumour growth rates. It seems that tumour cell cyclic AMP is more likely concerned with the regulation of tumour cell function than of growth. This would have implications for therapy, in that drugs which influence cyclic nucleotide metabolism could influence tumour cell function. The control of cyclic nucleotide production in normal and tumour cells is discussed, together with the possible ways in which abnormalities of this may occur.

Topics: Adenylyl Cyclases; Animals; Cell Line; Cell Transformation, Viral; Cells, Cultured; Cyclic AMP; Cyclic GMP; Hormones; Humans; Neoplasms; Neoplasms, Experimental; Prostaglandins E; Protein Kinases; Receptors, Cell Surface

Exposure of normal lymphocytes to phytohaemagglutinin or other lectin mitogens results in increased concentrations of 5-phosphoribosyl-1-pyrophosphate (PP-ribose-P) within minutes. Subsequently, synthesis of purine nucleotides by both the de novo and the salvage pathways is facilitated. This change is prevented by proliferation-inhibiting concentrations of exogenous adenosine. The capacity of lymphocytes to metabolize both adenine and adenosine is increased several-fold by incubation with phytohaemagglutinin but the specific activities of the respective first-step enzymes are not significantly altered. These results suggest that the relatively low quantity of PP-ribose-P available in normal lymphocytes is a major factor limiting the synthesis of purine nucleotides and may be important for the maintenance of the quiescent state. Increased availability of PP-ribose-P may also be associated with proliferative activation of fibroblast-like cells: chick embryo fibroblast cultures released from density-dependent inhibition of growth by insulin, trypsin or serum rapidly increase the rate of adenine incorporation into nucleotides. Chick embryo fibroblasts transformed by Rous sarcoma virus, but not cells infected with the respective non-transforming leukosis virus, show PP-ribose-P concentrations higher than those observed in normal cells.

Topics: Adenine; Adenosine; Animals; Avian Sarcoma Viruses; Calcium; Cell Division; Cell Transformation, Viral; Chick Embryo; Cyclic GMP; DNA; Fibroblasts; Glycine; Humans; Hypoxanthines; Lectins; Lipopolysaccharides; Lymphocytes; Mitogens; Phosphoribosyl Pyrophosphate; Purine Nucleotides; Ribose-Phosphate Pyrophosphokinase; Trypsin

A caprine luteal cell line (tsCLC-D) that synthesizes progesterone (P4) was established following by transformation with a temperature-sensitive A209 (tsA209) mutant of simian virus 40 (SV40). The transformed cells have temperature-sensitive for morphology, cell propagation and progesterone steroidogenesis. At the permissive temperature of 34 degrees C, these cells were spindle-shaped and grew with a similar rapidity as tumor cells. However, at the nonpermissive temperature of 40 degrees C, the cells have exhibited a round shape and ceased to proliferate because the gene for maintenance of transformation was not expressed. The tsCLC-D cell line responds to 8-Br-cyclic AMP, 22-hydroxycholesterol and pregnenolone treatment with an increase in progesterone biosynthesis. This cell line still express StAR protein, 3beta-HSD and P450scc enzyme of three kinds of steroidogenic protein and enzymes, this characteristic is similar to normal luteal cell. However, the addition of any doses oLH did not increase progesterone secretion. We speculate that tsCLC-D might lose the responsiveness to gonadotropins during the immortalization process, while retaining steroidogenic enzyme activity and progesterone production. To our knowledge, this is the first report of a stable cell line derived from corpus luteum of ruminant. The tsCLC-D retains steroidogenic capacity, which will make this cell line useful for the studies of regulation of steroidogenesis.

Topics: Animals; Cell Line, Transformed; Cell Separation; Cell Transformation, Viral; Cyclic GMP; Female; Goats; Hormones; Luteal Cells; Simian virus 40; Steroids; Temperature

Cells isolated from the trabecular meshwork (TM) of a male glaucoma patient were transformed by transfection with an origin defective mutant of SV40 virus. Transformation dramatically increased the growth rate of these cells (designated HTM-3 cells), allowing biochemical and pharmacological characterization. The HTM-3 cells had cytoskeletal components that were reported to be present in TM tissue and non-transformed TM cells. Vimentin, tubulin and smooth muscle specific alpha-actin, but not desmin, were localized in these cells by immunocytochemistry. The extracellular matrix components collagen types I, III and IV, fibronectin and laminin were found in HTM-3 cells as well as their non-transformed parental cells. As predicted, the protein profile of the HTM-3 cells revealed by two-dimensional gel electrophoresis was different from that of the non-transformed cells, probably due to the enhanced growth characteristics of these cells. Furthermore, HTM-3 cells had various intracellular second messenger systems that responded to pharmacological agents. Forskolin, prostaglandin E2, beta-adrenergic and adenosine A2 agonists stimulated the adenylyl cyclase in these cells, whereas muscarinic, serotonergic, dopaminergic and other agonists were ineffective. Sodium nitroprusside increased the intracellular concentration of cGMP, demonstrating the presence of a functional guanylyl cyclase. Phospholipase C activity in these cells was also detected. Muscarinic agonists, histamine and bradykinin, but not adrenergic, serotonergic agonists or prostaglandins, increased phosphoinositide turnover. These drug responses of HTM-3 cells agree with published data on primary TM cells and TM tissues, suggesting that the transformed cells may be a valid substitute for certain pharmacological studies of TM.

Topics: Aged; Cell Division; Cell Transformation, Viral; Cells, Cultured; Cyclic GMP; Cytoskeletal Proteins; Extracellular Matrix Proteins; Glaucoma, Open-Angle; Humans; Male; Second Messenger Systems; Simian virus 40; Trabecular Meshwork; Transfection; Type C Phospholipases

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.

Topics: Animals; Bucladesine; Cell Division; Cell Transformation, Viral; Cells, Cultured; Cyclic AMP; Cyclic GMP; Fibroblasts; Mice; Simian virus 40; Theophylline

As part of our study of antiherpetic acyclonucleosides, we synthesized a cyclic GMP analog, 9-[(2-hydroxy-1,3,2-dioxaphosphorinan-5-yl)oxymethyl]guanine P-oxide, sodium salt (2'-nor-cGMP), and discovered its potent and broad spectrum anti-DNA-viral activities. 2'-Nor-cGMP inhibits the replication of many DNA viruses, including herpes simplex virus, human cytomegalovirus, vaccinia, SV40, and adenovirus, but does not inhibit RNA viruses. In plaque reduction studies this potent antiviral agent is also approximately 10-fold more potent than 9-(1,3-dihydroxy-2-propoxymethyl)guanine (2'NDG) against varicella-zoster virus and inhibits cell transformation by bovine papilloma virus. Unlike 2'NDG, the potent activity of 2'-nor-cGMP against herpes virus is not dependent upon the action of virus-specified thymidine kinase. Intercellular metabolism of 2'-nor-cGMP produced small amounts of 2'NDG triphosphate which were insufficient to account for the antiviral activity observed, implying that this potent anti-DNA-viral agent operates by a mechanism different from that of known acyclonucleosides.

Topics: Acyclovir; Animals; Antiviral Agents; Cell Transformation, Viral; Cyclic GMP; DNA Viruses; Female; Ganciclovir; Guanine; Herpes Genitalis; Herpesvirus 3, Human; Male; Mice; Mice, Inbred ICR; Organophosphorus Compounds; Simplexvirus; Thymidine Kinase; Virus Replication

A murine cell line transformed with HSV TK (LH-1) exhibits a greatly enhanced cytotoxicity to the nucleoside analog 9-[(2-hydroxy-1-(hydroxymethyl)ethoxy) methyl]guanine (2'-NDG) as compared to the parental LM cell line (I50 LH-1 = 0.4 microM; I50 LM = 44.4 microM). Toxicity of 2'-NDG for LH-1 and LM is reversed only by the addition of 100 microM thymidine (dThd), indicating that 2'-NDG is a substrate for the viral and cellular TK. In LM(TK-) cells--murine cells expressing no TK activity, 2'-NDG cytotoxicity is partially reversed only with dGuo. A cyclic phosphate derivative of 2'-NDG, 2'-nor-cGMP, contains a phosphodiester bond, is also taken up by cells, and does not depend on viral TK for activation. LH-1 cells and LM(TK-) cells are inhibited by similar concentrations of this analog (5.1 and 4.1 microM, respectively). In all three cell lines (LM, LH-1, LM(TK-], the toxicity of 2'-nor-cGMP is significantly reversed with dGuo or cyclic dGMP. This pattern of reversal differs significantly from that observed with 2'-NDG, suggesting that 2'-nor-cGMP is metabolized as a guanosine analog, similar to acyclovir, in LM and LM(TK-) cells. These results indicate that a cyclic monophosphate analog of 2'-NDG can be activated independently of viral TK expression and that cellular metabolic pathways resulting in elevated dGTP concentrations are important for reversal of toxicity induced by guanosine-like nucleoside analogs.

Topics: Acyclovir; Animals; Cell Division; Cell Line; Cell Survival; Cell Transformation, Viral; Cyclic GMP; Deoxycytidine; Deoxyguanosine; Ganciclovir; Guanine; Mice; Organophosphorus Compounds; Simplexvirus; Thymidine; Thymidine Kinase

Two proteins, termed P85gag-mos and P58gag, are encoded by the temperature-sensitive transformation mutant, ts110 Moloney murine sarcoma virus (MuSV). Based on temperature-shift studies, P85gag-mos is believed to be important for the transforming potential of ts110 MuSV and has been found to be associated with a thermolabile kinase activity that phosphorylates both P85gag-mos and P58gag in immune complexes. Modifications of the original kinase assay conditions are reported here that have allowed a 30-fold increase in the specific activity of P85gag-mos phosphorylated in vitro. The in vitro P85gag-mos-phosphorylating activity was found to be unresponsive to 10 microM-cAMP or 10 microM-cGMP. Addition of 1 mM-pyrophosphate, a known phosphatase inhibitor, to the reaction mixture resulted in an increased yield of phosphorylated P85gag-mos and P58gag; the molar phosphate incorporation per mole of P85gag-mos increased from 0.032 to 0.9, whereas the specific activity of in vitro-phosphorylated P58gag increased 18-fold, from 0.013 to 0.234. pH curves of the in vitro kinase reaction further confirmed the presence of phosphatase activity; in the absence of pyrophosphate, a sharp optimum at pH 4 to 5 was observed, whereas it shifted broadly to pH 7.0 in the presence of pyrophosphate. Under the latter conditions, several experiments were performed in order to determine if the kinase was associated with either gag or mos sequences of P85gag-mos. Antisera directed against p15, p12 and p30 sequences of the gag protein region of P85gag-mos yielded immune complexes that allowed phosphorylation in vitro of P85gag-mos. No phosphorylating activity was detected in immune complexes containing MuSV-124-encoded P62gag. An anti-mos serum generated against a synthetic peptide representing the predicted v-mos amino acid residues 37 to 55 recognizes P85gag-mos and allowed phosphorylation of P85gag-mos in vitro in the absence of P58gag. Peptide mapping of both phosphorylated P85gag-mos and P58gag, by using a combination of Cleveland and Western/immunoperoxidase techniques, demonstrated that P85gag-mos became phosphorylated not only on gag sequences, but also at the N-terminal portion of v-mos. Phosphoamino acid analyses of P85gag-mos and P58gag phosphorylated in vitro under these modified conditions yielded predominantly phosphoserine and lesser amounts of phosphothreonine. Metabolically 32P-labelled P85gag-mos and P58gag were also found to contain phosphoserine and phosphothreonine.

Topics: Antigen-Antibody Complex; Cell Transformation, Viral; Cyclic AMP; Cyclic GMP; Diphosphates; Gene Products, gag; Moloney murine sarcoma virus; Mutation; Phosphorylation; Phosphoserine; Phosphothreonine; Protein Kinases; Quercetin; Retroviridae Proteins; Sarcoma Viruses, Murine; Temperature

Topics: Adenosine Triphosphate; Animals; Cell Division; Cell Transformation, Viral; Cells, Cultured; Cyclic AMP; Cyclic GMP; Guanosine Triphosphate; Mice; Mice, Inbred Strains; Phosphorylation; Protein Kinases; Simian virus 40

Normal and Rous sarcoma virus (RSV)-transformed chick embryo fibroblasts growing on plastic dishes were incubated with ATP (gamma 32P) 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 trypsin 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 phosphorylated in normal and transformed cells and at least two of these were transformation-specific. These data suggest that changes in external cell surface protein phosphorylation are associated with RSV transformation and thus could play a role in the formation of the transformed cell phenotype.

Topics: Adenosine Triphosphate; Animals; Avian Sarcoma Viruses; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Chick Embryo; Cyclic AMP; Cyclic GMP; Fibroblasts; Membrane Proteins; Phosphorylation; Protein Kinases

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; 3',5'-Cyclic-GMP Phosphodiesterases; AKR murine leukemia virus; Animals; Cell Transformation, Viral; Cells, Cultured; Cyclic AMP; Cyclic GMP; Phosphoric Diester Hydrolases; Rats; Rats, Inbred Strains; Thymus Gland

Broken cell preparations of WI-38 and SV40-transformed WI-38 (VA13) fibroblasts were used to compare the cyclic nucleotide phosphodiesterase activities of the two cell strains. The bulk of the cAMP or cGMP phosphodiesterase activity of WI-38 and VA13 homogenates was found in the 100,000 x g fibroblast supernatant fractions. WI-38 and VA13 soluble phosphodiesterase activities showed anomalous kinetic behavior with either cAMP or cGMP as the substrate. At low substrate concentrations, e.g., 0.1 muM, WI-38 supernatant fractions hydrolyzed cGMP much more rapidly than cAMP. At high substrate concentrations, e.g., 100muM, the same enzyme preparations degraded cAMP more than twice as fast as cGMP. In contrast, VA13 soluble phosphodiesterase activity catalyzed the hydrolysis of a wide range of cAMP and cGMP concentrations at similar rates. Phosphodiesterase activity in WI-38 supernatant fractions was generally more sensitive than that of the comparable VA13 enzyme activity to inhibition by MIX and papaverine. The cAMP phosphodiesterase activity of both WI-38 and VA13 supernatant preparations was decreased by cGMP in a concentration-dependent manner. cAMP was an effective inhibitor of cGMP hydrolysis by VA13 soluble phosphodiesterase activity. Yet, the cGMP phosphodiesterase activity of WI-38 supernatant fractions was only slightly reduced in the presence of cAMP. DEAE-cellulose chromatography of WI-38 and VA13 supernatant preparations revealed two major peaks of phosphodiesterase activity for each cell type. WI-38 peak I showed much greater activity with 1muM cGMP than with 1muM cAMP and appeared to be composed of two different phosphodiesterase activities. WI-38 peak Ia included phosphodiesterase activity which could be stimulated by boiled, dialyzed fibroblast homogenates while WI-38 peak Ib coincided with column fractions which contained most of the cyclic GMP hydrolytic activity. VA13 peak I phosphodiesterase activity was eluted from DEAE cellulose columns at the same ionic strength as WI-38 peak Ia and hydrolyzed these two substrates at nearly identical rates. This enzyme activity was also increased in the presence of boiled, dialyzed fibroblast preparations. Peak II phosphodiesterase activities from both WI-38 and VA13 fibroblasts were relatively specific for cAMP as the substrate. Phosphodiesterase activity with the properties of WI-38 peak Ib was not isolated from VA13 supernatant fractions. These results suggested that the dissimilar patterns of cAMP acc

Topics: 1-Methyl-3-isobutylxanthine; 3',5'-Cyclic-AMP Phosphodiesterases; 3',5'-Cyclic-GMP Phosphodiesterases; Cell Line; Cell Transformation, Viral; Chromatography, DEAE-Cellulose; Cyclic AMP; Cyclic GMP; Dose-Response Relationship, Drug; Fibroblasts; Humans; Kinetics; Papaverine; Simian virus 40; Subcellular Fractions

Topics: Animals; Biology; Cell Transformation, Viral; Collagen; Cyclic AMP; Cyclic GMP; Fibroblasts; Glucocorticoids; Humans; Mice; Periodontal Diseases; Periodontium; Rats; Receptors, Cell Surface; Steroids

Plasma membranes isolated from normal and RSV transformed chick embryo fibroblasts were phosphorylated in vitro using endogenous protein kinase and ATP (gamma32P) and the labeled phosphoproteins were analyzed by SDS-PAGE. A number of protein phosphorylation changes were observed following transformation, however in most cases they were relatively small quantitative differences. The four major changes were in proteins of 47,000, 58,000, 75,000 and 135,000 daltons. Decreased phosphorylation of the 47,000 dalton polypeptide was found in transformed cell membranes but this alteration was shown to be due to differences in cell growth rather than transformation. Increase phosphorylation of the 75,000 dalton protein was at least partially related to virus infection. However, increased phosphorylation of the 58,000 and 135,000 dalton polypeptides were entirely transformation specific.

Topics: Adenosine Triphosphate; Animals; Autoradiography; Avian Sarcoma Viruses; Cell Division; Cell Transformation, Viral; Chick Embryo; Cyclic GMP; Electrophoresis, Polyacrylamide Gel; Fibroblasts; Membrane Proteins; Phosphoproteins; Phosphorylation

The effect of varying the intracellular cyclic nucleotide concentration of mouse cells infected with HSV-1 or of duck embryo fibroblasts infected with Marek's disease virus has been studied. It has been shown in each case that agents that elevate the intracellular cAMP levels increase the yield of infectious virus or viral DNA, but inhibit cell division. cGMP enhances synthesis of viral DNA but allows continued synthesis of cell DNA.

Topics: Animals; Cell Transformation, Viral; Cells, Cultured; Cyclic AMP; Cyclic GMP; DNA; DNA, Viral; Herpesviridae; Prostaglandins E, Synthetic; Virus Replication