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Biomedical subjects

W B Anderson

Publications and source records attributed to W B Anderson.

At least 73 records · Page 4Linked to original sources
PubMed1988

Phorbol ester stimulates the synthesis of sphingomyelin in NIH 3T3 cells. A diminished response in cells transformed with human A-raf carrying retrovirus.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulated the synthesis of sphingomyelin (CerPCho) from a [14C]choline-labelled phosphatidylcholine (PtdCho) pool in NIH 3T3 cells. Maximal stimulation (68%) of CerPCho synthesis, accompanied by an increase (38%) in its cellular content, required only 2 nM TPA. Higher concentrations of TPA (2-100 nM) had progressively less effect on CerPCho synthesis which correlated with increased hydrolysis of precursor PtdCho. In cells transformed with human or mouse A-raf carrying retroviruses TPA-stimulated PtdCho hydrolysis, but not CerPCho synthesis, suggesting independent regulation of these processes by the TPA-stimulated signal transduction system.

Animals↗
PubMed1988

Effect of retinoic acid on cAMP dependent protein phosphorylation in psoriatic fibroblasts.

Retinoic acid treatment of psoriatic fibroblasts increases the activity of cyclic AMP dependent protein kinase. In this study we report that retinoic acid treatment of cultured psoriatic fibroblasts modifies their subsequent cAMP dependent protein phosphorylation. In the soluble fraction of normal fibroblasts cAMP clearly enhances the in vitro phosphorylation of proteins of MW 37,49,54,56,68,83 kD while retinoic acid treatment of the same cells results in a decrease of the cAMP dependent phosphorylation of the first five of the same proteins. In contrast, in psoriatic fibroblasts from psoriatic patients retinoic acid either has no effect or increases the cAMP dependent phosphorylation of some of these proteins. Moreover the phosphorylation of a protein of MW 54 kD, undetectable in untreated psoriatic cells, is more phosphorylated in the presence of cAMP after retinoic acid treatment. The appearance of this phosphorylated proteins is time dependent and dose dependent upon the addition of retinoic acid. These in vitro phosphorylation results suggest that retinoic acid treatment of psoriatic fibroblasts change the level of cAMP dependent phosphorylation of some cytosolic proteins. These specific phosphorylations could be implicated in a variation of cell functions.

Cells, Cultured↗
PubMed1988

Effect of phorbol ester on cyclic adenosine 3':5'-monophosphate-dependent protein kinases in PYS teratocarcinoma-derived cells and counteraction with retinoic acid.

Treatment of PYS cells with the tumor promoter (TPA) has been previously shown to enhance calcium- and phospholipid-dependent protein kinase (PK.C) in the membranes and to decrease its activity in the cytosol. Evidence is presented that 0.1 microM TPA treatment of PYS cells causes an opposite effect on the cyclic AMP-dependent protein kinases (PK.A). Within 10 min TPA led to an increase in PK.A in the cytosol and a concomitant decrease in the membranes, as measured by both the kemptide phosphorylation activity and photoaffinity labeling of RI and RH regulatory subunits of PK.A, with 8-azido-cyclic [32P]AMP. Moreover, the antitumor promoter retinoic acid (RA, 0.1 microM), when added simultaneously with TPA to the PYS cells, completely abolished the TPA effects on PK.A. When RA was added 25 min before TPA, the counteraction was not observed, indicating that RA was counteracting the TPA effect directly. These results suggest that TPA induces a rapid change in the compartmentalization of PK.A between the membrane and the soluble fraction. This possible translocation of PK.A seems to be blocked by RA, suggesting that the early antagonistic effects of RA toward TPA-mediated events occur at the plasma membranes.

Animals↗
PubMed1988

Protein kinase C: subcellular redistribution by increased Ca2+ influx. Evidence that Ca2+-dependent subcellular redistribution of protein kinase C is involved in potentiation of beta-adrenergic stimulation of pineal cAMP and cGMP by K+ and A23187.

Phenylephrine is known to stimulate translocation of protein kinase C in rat pinealocytes (Sugden, D., Vanecek, J., Klein, D.C., Thomas, T.P., and Anderson, W. B. (1985) Nature 314, 359-361). In the present study, the receptor mediating this effect was found to belong to the alpha 1-adrenoceptor subclass. Activation of this receptor is also known to produce a sustained increase in [Ca2+]i by increasing net influx (Sugden, A. L., Sugden, D., and Klein, D. C. (1985) J. Biol. Chem. 261, 11608-11612), which points to the possible importance of Ca2+ influx in the subcellular redistribution (activation) of protein kinase C in intact cells. This possibility was investigated by reducing extracellular Ca2+ ((Ca2+]o) with EGTA or by inhibiting Ca2+ influx with inorganic Ca2+ blockers. These treatments reduced alpha 1-adrenoceptor-mediated translocation of protein kinase C. This suggested that elevation of Ca2+ influx alone triggers activation of protein kinase C. In support of this, it was found that treatments which elevate Ca2+ influx, including increased extracellular K+ and addition of the Ca2+ ionophore A23187, cause redistribution of protein kinase C. The effect of K+ was blocked by nifedipine and that of A23187 by EGTA, indicating that effects of these agents are Ca2+-dependent. The possible role of phospholipase C activation in these effects was examined by measuring the formation of [3H]diacylglycerol by cells labeled with [3H]arachidonic acid. Although [3H]diacylglycerol formation was easily detected in the presence or absence of an effective concentration of an inhibitor of diacylglycerol kinase, none of the agents which cause rapid translocation of protein kinase C were found to cause a rapid increase in the generation of [3H]diacylglycerol. These findings establish that an increase in Ca2+ influx is sufficient to trigger translocation of protein kinase C. In addition, we found that a very close correlation exists between translocation of protein kinase C by phenylephrine, K+, and A23187 and their ability to potentiate beta-adrenergic stimulation of cAMP and cGMP accumulation. This provides strong support to the proposal that translocation of protein kinase C is required for potentiation of beta-adrenergic stimulation of pinealocyte cAMP and cGMP accumulation.

Adrenergic beta-Antagonists↗
PubMed1988

Phorbol ester-mediated association of protein kinase C to the nuclear fraction in NIH 3T3 cells.

Treatment of intact NIH 3T3 cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) causes a rapid redistribution (stabilization) of protein kinase C to the particulate fraction. Part of the enzyme activity stabilized to the membrane fraction in response to TPA can be recovered associated with nuclear-cytoskeletal components. An apparently pure nuclear fraction prepared from NIH 3T3 cells was found to contain 25-30% of the total membrane-associated protein kinase C activity when isolated in the presence of Ca2+. In untreated control cells, most of this activity found with the nuclear fraction can be extracted by chelators. Phorbol ester (TPA) treatment of NIH 3T3 cells induces the tight association of protein kinase C to the nucleus; this tightly bound activity is not dissociable by chelators and can be recovered only by solubilization with detergent. Nuclei purified from untreated human promyelocytic leukemic HL-60 cells contain higher amounts of chelator-stable, detergent-extractable protein kinase C activity compared with control NIH 3T3 cells. However, TPA treatment of HL-60 cells does not enhance the amount of protein kinase C found tightly associated with the nuclear fraction. Immunohistochemical studies with polyclonal antibodies directed against protein kinase C further indicate that TPA treatment of NIH 3T3 cells does significantly enhance the amount of protein kinase C found tightly associated with the nucleus and cytoskeleton, whereas exposure of HL-60 cells to TPA does not appreciably alter the amount of protein kinase C observed to be associated with the nuclear fraction. The TPA-mediated association (activation) of protein kinase C to the nuclear and cytoskeletal fractions with NIH 3T3 cells is further supported by the enhanced phosphorylation of specific endogenous proteins noted when purified nuclei and cytoskeletal preparations are incubated with [gamma-32P]ATP. These results suggest that tumor promoters may induce association (activation) of protein kinase C with different subcellular components to alter the availability of endogenous substrates. This may result in differential responses by different cell types during exposure to tumor promoters.

Animals↗
PubMed1988

Differentiation-dependent production of a platelet-derived growth factor-like mitoattractant by endoderm cells derived from embryonal carcinoma cells.

Retinoic acid-induced differentiation of F9 embryonal carcinoma cells to endoderm provokes the secretion of a protein factor that acts as both a chemoattractant and mitogen for smooth muscle cells. Undifferentiated F9 cells and PSA-5E (visceral endodermlike) cells produced little of this factor. However, PYS-2 (parietal endodermlike) and Dif 5 endoderm cells were found to produce significant amounts of endoderm-derived mitoattractant (EDM) activity. The activity secreted by the Dif 5 cells was partially purified using gel filtration chromatography using chemotaxis and mitogenic assays as markers for biological activity. The partially purified activity competes with [125I]iodo-platelet-derived growth factor (PDGF) for binding to target cells, and the biological activity is neutralized with anti-PDGF IgG, suggesting shared domains in the two molecules. However, the factor appears to be different from PDGF, based on its thermal stability, molecular weight, and charge. The differentiated endoderm cells including retinoic acid (RA)-treated F9, Dif 5, PSA-5E, and PYS-2 cells also exhibit specific [125I]iodo-PDGF binding, and the PSA-5E cells respond to PDGF as a chemoattractant. Conceivably, such a PDGF-like factor may contribute to the regulation of cell growth and migration during the early stages of embryogenesis.

Animals↗
PubMed1987

Susceptibility of protein kinase C to oxidative inactivation: loss of both phosphotransferase activity and phorbol diester binding.

Exposure of protein kinase C to low concentrations of either N-chlorosuccinimide or H2O2 resulted in rapid and parallel loss of phosphotransferase activity and phorbol ester binding. This oxidative inactivation of protein kinase C also occurred in intact cells exposed to a low concentration of H2O2. With H2O2 treatment the rate of inactivation of protein kinase C in the cytosol of MCF-7 cells was rather slower than that which occurred in the cytosol of PYS cells. However, in both cell types, the oxidative inactivation of membrane-associated protein kinase C occurred rapidly in comparison to the enzyme in the cytosol. Prior treatment of cells with phorbol ester to induce membrane association (stabilization) of protein kinase C, followed by exposure to H2O2, resulted in increased inactivation of protein kinase C, suggesting that membrane association of protein kinase C increases its susceptibility to oxidative inactivation.

Animals↗
PubMed1987

Retinoic acid-induced rapid loss of nuclear cyclic AMP-dependent protein kinase in teratocarcinoma cells.

To determine what effect retinoic acid might have in modulating cyclic AMP-mediated events at the nucleus of teratocarcinoma cells, we have investigated the effect of retinoic acid treatment of F9 and PC13 cells on cyclic AMP-dependent protein kinase activity and the amounts of the RI and RII cyclic AMP binding proteins present in the nuclear fraction. Exposure of F9 cells to retinoic acid (0.1 microM) induces differentiation to parietal endoderm, while retinoic acid treatment (3 microM) of PC13 cells induces differentiation to visceral endoderm. In both cell types retinoic acid treatment causes a rapid (within 4 h) and pronounced (by 2-fold) decrease in nuclear cyclic AMP-dependent protein kinase activity. Conversely, as measured by cyclic [8-azido-32P]AMP photoaffinity labeling a similar rapid and pronounced decrease in the RI and RII regulatory subunits is observed at the nucleus. This decrease in nuclear cyclic AMP-dependent protein kinases in at least two cell types may be an early event of retinoid action important in the initiation of differentiation.

Animals↗
PubMed1987

Evidence for a GTP-binding protein coupling thrombin receptor to PIP2-phospholipase C in membranes of hamster fibroblasts.

Two different methods were used to study directly alpha-thrombin modulation of polyphosphoinositide breakdown in membranes prepared from Chinese hamster lung (CHL) fibroblasts. In the first one we labelled the lipid pool by incubating the intact cells with myo-[3H]inositol prior to membrane isolation; in the other we used exogenous [3H]PIP2 with phosphatidylethanolamine (1:10) added as liposomes to freshly isolated membranes. A Ca2+-dependent PIP2 and PIP phospholipase C activity was characterized by measuring the rate of formation of inositol tris- and bisphosphate. Basal phospholipase C activity was stimulated up to 3-fold by GTP or GTP-gamma-S. Of the two mitogens, alpha-thrombin and EGF, known to stimulate DNA synthesis in Chinese hamster fibroblasts, only alpha-thrombin is a potent activator of PIP2 breakdown in intact cells. Consistent with this observation, alpha-thrombin but not EGF potentiated GTP-gamma-S-dependent phospholipase C activity in membrane preparations. These results strongly support the hypothesis that a GTP-binding protein couples alpha-thrombin receptor to PIP2 hydrolysis. Because both methods used to assay phospholipase C gave identical results, we conclude that the coupling is at the level of PIP2-phosphodiesterase activity.

Animals↗
PubMed1987

Altered hormonal responses: markers for embryonal and embryonic carcinoma stem cells and their differentiated derivatives.

Teratocarcinoma cells in culture offer an in vitro system for studying certain aspects of embryonic differentiation. To gain some insight into regulatory systems that might be operative during early development, we have characterized the alterations that occur in the hormonal responsiveness of the membrane adenylate cyclase of different embryonal carcinoma cell lines with differentiation. Each undifferentiated embryonal carcinoma stem cell studied (F9, PCC4, PC13, P19) has an adenylate cyclase system predominantly activated by calcitonin. Of great interest is the fact that cAMP production is also enhanced specifically by calcitonin in an embryo-derived stem cell line. Differentiation of the embryonal carcinoma stem cell population toward parietal endoderm results in a decrease in calcitonin activation with a concomitant appearance of sensitivity to parathyroid hormone. Differentiation toward visceral endoderm is characterized by a lack of response of the adenylate cyclase system to both calcitonin and parathyroid hormone. These results indicate that the changes noted in adenylate cyclase hormonal responsiveness might serve as useful markers during early stages of differentiation.

Adenylyl Cyclases↗
PubMed1987

Retinoid treatment of human psoriatic fibroblasts induces an increase in cyclic AMP-dependent protein kinase activity.

We recently showed a deficiency of cyclic AMP (cAMP)-dependent protein kinases in psoriatic cells. In this work the effects of retinoids on cAMP-dependent protein kinases of fibroblasts from 7 normal subjects and 7 psoriatic patients were studied. The levels of RI and RII (two forms of the cAMP-dependent protein kinases) present in control and retinoic acid-treated cells were quantitated by photoaffinity labeling with [8-azido-32P]cAMP. In psoriatic fibroblasts the levels of RII are decreased or undetectable compared with those of normal fibroblasts both in the cytosolic and membrane fractions. The amount of RI was normal in the cytosol of fibroblasts of 5 out of 7 patients and decreased in 2 patients. Membrane-associated levels of RI were decreased in 5 patients and normal in 2 patients. Retinoic acid treatment induces an increase in the amount of RI and RII regulatory subunits when they are deficient in the cytosolic and membrane fractions of psoriatic fibroblasts. Retinoic acid had no effect on RI and RII in normal fibroblasts. In addition, with in vitro retinoic acid treatment the cAMP-dependent protein kinase activity, measured in the fibroblasts of 4 psoriatic patients, was increased in the cytosol in 2 patients and in the membranes in all 4 patients. In these studies, comparable results were obtained with fibroblasts cultured from involved and uninvolved skin. This in vitro effect of retinoids on cAMP-dependent protein kinases in psoriatic fibroblasts may help to explain some of the in vivo therapeutic effects of retinoids.

Fibroblasts↗
PubMed1986

Factors influencing chelator-stable, detergent-extractable, phorbol diester-induced membrane association of protein kinase C. Differences between Ca2+-induced and phorbol ester-stabilized membrane bindings of protein kinase C.

One of the early events associated with the treatment of cells by tumor promotor phorbol esters is the tight association of protein kinase C to the plasma membrane. To better understand the factors that regulate this process, phorbol ester-induced membrane binding of protein kinase C was studied using homogenates, as well as isolated membranes and purified enzyme. Addition of 12-O-tetradecanoylphorbol 13-acetate (TPA) to the homogenates of parietal yolk sac cells and NIH 3T3 cells in the presence of Ca2+ resulted in plasma membrane binding of protein kinase C which subsequently remained bound to the membrane independent of Ca2+. Although protein kinase C was activated by TPA in the absence of Ca2+ and by diolein in the presence of Ca2+, both these agents when added to homogenates under these respective conditions had no effect on membrane association of protein kinase C. However, under these conditions relatively weak binding of protein kinase C was found if purified protein kinase C was used with isolated membranes. Binding studies using purified protein kinase C and washed membranes showed that the binding of the TPA-kinase complex to membranes required phospholipids and reached saturation at 0.1 unit (24 ng of protein kinase C)/mg of parietal yolk sac cell membrane protein. Phorbol ester treatment of cells in media with and without Ca2+ showed that the TPA-induced increase in membrane-associated protein kinase C was regulated by Ca2+ levels even in intact cells. TPA-stabilized membrane binding of protein kinase C differs in several aspects from the previously reported Ca2+-induced reversible binding. TPA-stabilized binding of protein kinase C to isolated membranes is temperature dependent, relatively high in the plasma membrane-enriched fraction, saturable at physiological levels of protein kinase C, requires the presence of both membrane protein(s) and phospholipids, and further requires the addition of phospholipid micelles. In contrast, Ca2+-induced reversible binding is more rapid, not appreciably influenced by temperature, not selective for a particular subcellular fraction, not saturable with physiological amounts of protein kinase C, exhibits trypsin-insensitive membrane binding sites, and requires membrane phospholipids but not added phospholipid micelles.

Animals↗
PubMed1986

Direct phosphorylation of the IL-2 receptor Tac antigen epitope by protein kinase C.

Phorbol esters induce a rapid phosphorylation of the antigenic epitope of the human IL-2 receptor identified by anti-Tac monoclonal antibody. The physiological activator of protein kinase C, diacylglycerol also stimulated the phosphorylation of the Tac epitope in intact activated human T lymphocytes. Stable derivatives of cyclic nucleotides had no effect on the stimulation of Tac phosphorylation with cultured lymphocytes. Immunoprecipitated Tac derived from particulate membranes could serve as a direct substrate for purified protein kinase C in vitro. The Ca2+/phospholipid dependency of the in vitro phosphorylation reaction substantiated that the phosphorylation of Tac observed in intact cells stimulated by phorbol ester or diacylglycerol was the result of the physiological activation of protein kinase C.

Antigens, Surface↗
PubMed1986

Retinoic acid effect on cyclic AMP-dependent protein kinases in embryonal carcinoma cells: studies with differentiation-defective sublines.

Retinoic acid induces the differentiation of PCC4.aza 1R and Nulli-SCC1 embryonal carcinoma (EC) cells. In response to retinoic acid treatment, the levels of cyclic AMP (cAMP)-dependent protein kinases are enhanced in the plasma membrane within 17 hours and in the cytosol fractions of these cells within 2 to 3 days, as determined by phosphotransferase activity and by 8-azido-cyclic [32P]AMP binding to the RI and RII regulatory subunits. PCC4 (RA)-1 and Nulli (RA)-1 are mutant EC lines that fail to differentiate in response to retinoic acid. The former line, but not the latter, lacks cellular retinoic acid-binding protein (cRABP). Basal levels of cAMP-dependent protein kinase activities are elevated in PCC4 (RA)-1 cells. When these cells are treated with retinoic acid, neither cAMP-dependent protein kinase activities nor cAMP binding activities are enhanced; rather, there is a decrease in cytosolic kinase activity and RI subunit. On the other hand, Nulli (RA)-1 cells exhibit increases both in cAMP-dependent protein kinase activities and cAMP binding in response to retinoic acid. These results raise the possibility that cRABP mediates the enhancement of regulatory and catalytic subunits of cAMP-dependent protein kinases in both the membrane and the cytosolic fractions of the teratocarcinoma cells. There also might be some effects of retinoic acid on the cAMP-dependent protein kinase that are unrelated to differentiation and to the presence of cRABP.

Animals↗
PubMed1986

Altered activation by calcitonin and inhibition by somatostatin of human embryonal carcinoma cell adenylate cyclase with retinoic acid-induced differentiation.

Human teratocarcinoma cells in culture offer an in vitro system for studying certain aspects of embryonic differentiation. To gain some insight into regulatory systems that might be operative during early human development, we have characterized the alterations that occur in the hormonal responsiveness of human embryonal carcinoma cell adenylate cyclase with differentiation in response to 10 microM retinoic acid. Two cell lines CL12 and CL13, cloned from Tera 2 cells by Dr. C. F. Graham, have been used in these studies. Adenylate cyclase of CL12 and CL13 cells is stimulated in the presence of 10 microM GTP by epinephrine and calcitonin, with calcitonin being the most potent stimulator of cyclic AMP production. Exposure of these cells to retinoic acid leads to an arrest in growth and within 6 days to a differentiated cell population with a stable nonreversible phenotype. No changes in basal, GTP- and fluoride-stimulated adenylate cyclase activities are observed with retinoic acid treatment, but the cyclase of differentiated cells exhibits a greater stimulation by calcitonin (7.5-fold) and the appearance of a somatostatin inhibitory effect. Somatostatin specifically inhibits, by 25%, the hormonal stimulation of adenylate cyclase of cells treated for 5 days with retinoic acid. The increase in calcitonin stimulation of adenylate cyclase activity of the differentiated cells is related to an increase (congruent to 3-fold) in the number of hormonal receptors and not to a significant change in receptor binding affinity (Kd 4.6 X 10(8) M-1). These alterations in calcitonin and somatostatin responsiveness suggest a possible regulatory role for these hormones during embryonic development. Furthermore, the results indicate that changes in adenylate cyclase hormonal responsiveness might serve as useful markers during early stages of human embryonal carcinoma cell differentiation.

Adenylyl Cyclases↗
PubMed1986

Microbial release of 226Ra2+ from (Ba,Ra)SO4 sludges from uranium mine wastes.

226Ra2+ is removed from uranium mine effluents by coprecipitation with BaSO4. (Ba,Ra)SO4 sludge samples from two Canadian mine sites were found to contain active heterotrophic populations of aerobic, anaerobic, denitrifying, and sulfate-reducing bacteria. Under laboratory conditions, sulfate reduction occurred in batch cultures when carbon sources such as acetate, glucose, glycollate, lactate, or pyruvate were added to samples of (Ba,Ra)SO4 sludge. No external sources of nitrogen or phosphate were required for this activity. Further studies with lactate supplementation showed that once the soluble SO4(2-) in the overlying water was depleted, Ba2+ and 226Ra2+ were dissolved from the (Ba,Ra)SO4 sludge, with the concurrent production of S2-. Levels of dissolved 226Ra2+ reached approximately 400 Bq/liter after 10 weeks of incubation. Results suggest that the ultimate disposal of these sludges must maintain conditions to minimize the activity of the indigenous sulfate-reducing bacteria to ensure that unacceptably high levels of 226Ra2+ are not released to the environment.

Bacteria↗