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Biomedical subjects

M Castagna

Publications and source records attributed to M Castagna.

At least 91 records · Page 5Linked to original sources
PubMed1983

Tumor promoter 12-O-tetradecanoylphorbol 13-acetate alters state, fluidity and hydration of 1,2-diacyl-sn-glycero-3-phosphocholine bilayers.

Previous results (Castagna et al. (1979) FEBS Lett. 100, 62-66; Fisher et al. (1979) Biochem. Biophys. Res. Commun. 86, 1063-1068) indicated us that the active tumor promoter TPA (12-O-tetradecanoylphorbol 13-acetate) decreased fluorescence polarisation of diphenylhexatriene in lymphoblastoid and rat embryo cells. In the present study, experiments aimed at examining the molecular interactions of tumor promoters with cell membrane components are performed with fully hydrated multibilayers of 1,2-diacyl-sn-glycero-3-phosphocholine (DPPC) into which increasing amounts of TPA are inserted. The thermotropic behaviour of both the phospholipid bilayers and the interbilayer water was investigated using the differential scanning calorimetry (DSC) and the approach of Ter-Minassian-Saraga et al. ((1982) J. Colloïd Interface Sci. 81, 369-383). The major effects of the tumor promoter are confined to concentrations up to 20% mol fractions of TPA. In this range of concentrations the incorporation of TPA into liposomes decreases the phase-transition temperature but did not affect delta HDPPC. Furthermore TPA increases the hydration of the multibilayers. Above 20% mol fractions of TPA, a different thermal behaviour of the system which might suggest morphological rearrangements was observed. The lipid state in TPA-treated liposomes was monitored by fluorescence polarisation using diphenylhexatriene as a lipophilic fluorescent probe and the phase-transition temperature was calculated. The phase transition temperatures determined by both methods were in good agreement. The lowering of this temperature and the decay of fluorescence anisotropy of diphenylhexatriene were parallel. Those effects are consistent with the "fluidising' effect of TPA on DPPC.

Calorimetry, Differential Scanning↗
PubMed1983

Tumor promoters enhance cap formation in mouse thymocytes.

Potent tumor promoters such as 12-O-tetradecanoylphorbol 13-acetate (TPA) and teleocidin, rapidly evoked a dose-dependent stimulation of concanavalin A (Con A)-induced cap formation in mouse T lymphocytes. The effect was reversible upon removal of the drugs. Weaker tumor promoters, phorbol didecanoate, phorbol dibenzoate and iodoacetic acid stimulated capping to a lower extent. Mezerein, a phorbol-related macrocyclic diterpene derivative, which acts as a second-stage promoter was also active in increasing the number of caps. In contrast, 4 alpha-phorbol didecanoate and phorbol which are devoid of tumor promoting activity, did not affect capping. Anti-promoting glucocorticoids inhibited capping stimulation. Flow cytofluorometric analysis of Con A binding has shown that TPA did not modify the lectin binding to surface receptors. TPA-facilitated capping was energy-dependent. Cytochalasin B prevented the TPA-induced response whereas colchicine was ineffective. Phenothiazines fully inhibited the TPA effect, thus suggesting that tumor-promoter-mediated lectin receptor redistribution may be ascribed to the facilitation of a Ca2+-dependent process involving the submembrane actin filaments.

Animals↗
PubMed1983

Transmembrane signalling systems.

The three major strategies for transmembrane signalling at the plasma membrane level involve either second messengers, ion channels or receptor-mediated endocytosis. Two second-messenger types of transmembrane signalling systems have been analyzed: the adenylate cyclase and protein kinase C systems. Both are triggered by a variety of biological signals and eventually activate a specific protein kinase. The pathogenic stimulation of adenylate cyclase and protein kinase C systems by cholera toxin and tumor promoters, respectively, is described.

Adenylyl Cyclases↗
PubMed1982

Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters.

Tumor-promoting phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) directly activate in vitro Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C), which normally requires unsaturated diacylglycerol. Kinetic analysis indicates that TPA can substitute for diacylglycerol and greatly increases the affinity of the enzyme for Ca2+ as well as for phospholipid. Under physiological conditions, the activation of this enzyme appears to be linked to the receptor-mediated phosphatidylinositol breakdown which may be provoked by a wide variety of extracellular messengers, eventually leading to the activation of specific cellular functions or proliferation. Using human platelets as a model system, TPA is shown to enhance the protein kinase C-specific phosphorylation associated with the release reaction in the total absence of phosphatidylinositol breakdown. Various phorbol derivatives which have been shown to be active in tumor promotion are also capable of activating this protein kinase in in vitro systems.

Animals↗
PubMed1981

Phorbol esters parallel effects on tumor promotion, insulin release and calcium ionophoresis.

The tumor promoters 12-O-tetradecanoylphorbol-13-acetate (TPA) and phorbol 12,13-didecanoate (PDD) both stimulated insulin release from rat pancreatic islets and facilitated ionophore-mediated calcium transport in liposomes, the latter effect not resulting from a change in viscosity of the liposomial matrix. Phorbol and 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD), which fail to cause tumor promotion, also failed to stimulate insulin release and ionophore-mediated calcium transport. An alteration of calcium transport may represent a fundamental event in both the early (insulin release) and late (tumor promotion) biological responses to phorbol esters.

Animals↗
PubMed1981

Variation with embryonic development and regional localization of specific [3H]phorbol 12,13-dibutyrate binding to brain.

We have previously characterized specific binding of the phorbol ester tumor promoter [20-3H]phorbol 12,13-dibutyrate to several tissues, including mouse skin and brain. We report here that specific binding activity in chicken brain increases dramatically during development. In whole embryonic chicken brain, binding increased from 2.0 to 6.8 to 10.6 pmol/mg of protein at 7, 14, and 20 days, respectively. Adult chicken brain bound 15 pmol/mg of protein. In contrast to specific binding activity, binding affinity remained constant. Substantial regional localization of binding activity within the brain was found. For calf brain, representative values ranged from 4.6 pmol/mg for medulla to 38.1 pmol/mg for frontal lobe. Binding affinity did not vary. Inhibition of binding by postulated neurotransmitters and antagonists was examined. D-Propranolol and quinidine, membrane-stabilizing drugs, inhibited binding competitively, although at mM concentrations.

Animals↗
PubMed1981

Kinetics and subcellular localization of specific [3H]phorbol 12, 13-dibutyrate binding by mouse brain.

The specific binding of [3H]phorbol 12,13-dibutyrate ([3H]-PDBU) to particulate preparations from mouse brain has been further characterized. Kinetic analysis, using a filtration assay to measure binding, yielded a second-order rate constant at 23 degrees of 3.75 X 10(7) M-1 min-1 and a first-order dissociation rate constant of 0.21 min-1. The Kd of 5.6 nM calculated from the kinetic data agreed well with the value determined previously in equilibrium binding studies. The Kd for [3H]PDBU binding varied only slightly with temperature. From its temperature dependence, [3H]PDBU binding appeared to be associated with a small increase in enthalpy (delta H degrees = +0.4 kcal/mol) and a large increase in entropy (delta S degrees = +38 e.u.). Such values are characteristic for hydrophobic interactions. The dissociation rate constant for binding, in contrast to the Kd, varied dramatically with temperature. The half-time for release ranged from 1.75 min at 30 degrees to 62 min at 4 degrees. The Kd for binding was Ca2+ sensitive; chelation of Ca2+ by ethyleneglycolbis(beta-aminoethyl ether)N,N'-tetraacetic acid increased the Kd 2.4-fold. Upon subcellular fractionation, the specific [3H]PDBU binding activity was exclusively particulate; no binding to cytosol was detectable. Binding clearly did not correlate with nuclear or mitochondrial markers. On the other hand, a broader distribution of binding activity was seen on sucrose density gradients than for either Na+-K+-adenosine triphosphatase activity or binding of quinuclidinyl benzilate (a muscarinic cholinergic antagonist). The localization of specific [3H]PDBU binding to the plasma membrane therefore remains uncertain.

Animals↗
PubMed1980

Correlation between growth rate, cell density, and intracellular concentrations of cyclic nucleotides in chemostat cultures of mouse L1210 cells.

Constant intracellular concentrations of both adenosine 3',5' cyclic monophosphate (cyclic AMP) and guanosine 3',5' cyclic monophosphate (cyclic GMP) were obtained when mouse leukemia L1210 cells were cultivated under steady-state conditions in the chemostat. When L1210 cells were maintained at a constant growth rate in the chemostat, the mean steady-state intracellular concentration of cyclic AMP decreased with increasing cell density, while the concentration of cyclic GMP remained unchanged. When cell growth rate was increased by three-fold, independently of cell density, in the chemostat, then the mean steady-state intracellular concentration of cyclic GMP increased by 3.6-fold, whereas that of cyclic AMP increased by only 22%. The ratio of cyclic AMP to cyclic GMP was found to decrease with increasing cell growth rate in the chemostat. Our results show that a close correlation exists between the steady-state intracellular concentrations of cyclic nucleotides and changes in both cell growth rate and cell density in chemostat cultures of L1210 tumour cells.

Animals↗
PubMed1980

Tumor-promoting phorbol diesters inhibit in vitro antibody synthesis.

The tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate prevents the synthesis of antibodies directed against sheep red blood cells in an in vitro system. The inhibitory effect of 3 phorbol diesters on this immune response was positively correlated with their tumor-promoting activity. The effect did not appear to be mediated through the inhibition of cell proliferation. Results suggest that the tumor promoter may alter the differentiation of the precursor cells to antibody-producing cells.

Animals↗
PubMed1980

Insulinotropic effect of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate in rat pancreatic islets.

In isolated rat pancreatic islets, the tumor-promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA), when used in the 2.10(-9) to 2.10(-7) M range, was found to stimulate insulin release both in the absence and presence of glucose. The non-tumor-promoting agent 4-methylphorbol-12,13-didecanoate failed to stimulate insulin release. The insulinotropic capacity of TPA was enhanced by glucose in a dose-related fashion. In the absence of glucose, the TPA-stimulated release of insulin was a slowly induced and not rapidly reversible phenomenon. It was inhibited by antimycin A, by epinephrine, at low temperatures, and in the absence of extracellular Ca2+ or the presence of cytochalasin B, was unaffected by the organic calcium antagonist D600 or indomethacin, and was potentiated by theophylline. No obvious effect of TPA upon 86Rb or 32P efflux and 45Ca net uptake could be detected in the isolated islets. However, TPA caused a progressive increase in both 45Ca fractional outflow rate and cyclic adenosine 3':5'-monophosphate content in the islets. It is proposed that the insulinotropic action of TPA may be due, in part at least, to interference with the transport of calcium by native ionophores.

Animals↗
PubMed1979

Modifications of the activities of key enzymes and intracellular levels of cyclic nucleotides, in correlation with the glyogen deposition in a cultured hepatoma cell line.

Glycogen accumulation in growing cultures of ZHC cells (originally derived from the Zajdela ascitic hepatoma) is accompanied by an increase in glycogen synthetase (E.C. 2.4.1.11) and phosphorylase (E.C. 2.4.1.1) activities. Essentially the synthetase b and the phosphorylase a are involved in this process. The glycogen accumulation in ZHC cells us preceeded by a noticeable peak of cAMP, whereas cGMP rises early after replating and then decreases simultaneously with the growth rate. The present results suggest that these cultured hepatoma cells undergo throughout every passage an induction process involved in glycogen synthesis storage. Since the original ascites cells growing in vivo (which lack glycogen) and the cultured ZHC cells exhibit similar glycogen synthetase and phosphorylase activities, the resurgence of the glycogenic function (Staedel and Beck, 1978) in the in vitro cultureed cells does not seem related to a change in these two enzymes. By contrast, the high cyclic nucleotide levels in the cultured cells, as compared to those in the ascites cells, offer a possible explanation.

Animals↗
PubMed1979

Tumour-promoting phorbol diester induces substrate-adhesion and growth inhibition in lymphoblastoid cells.

The treatment of human lymphoblastoid cell cultures with the phorbol diester, 12-O-tetradecanoyl-phorbol-13-acetate, a tumour promoter, caused at nM concentration surface structural changes associated with altered adhesion properties. The effect was observed in several cell lines from normal or leukaemic origin. In addition, the tumour promoter induced an early and transitory growth inhibition which was observed in all tested B-characteristic cells. The phorbol diester, 4-O-methyl-phorbol-12,13-didecanoate, which is devoid of tumour-promoting activity, was much less effective in altering cell adhesion properties and cell growth than the active derivative. These observations suggest that lymphoblastoid cells may be a useful model for studying the molecular alterations at the membrane level resulting from the action of tumour-promoting phorbol diesters.

Animals↗