Monique Castagna

Further characterization of tumor-promoter-mediated activation of protein kinase C

Tumor promoting phorbol esters are able to activate Ca2+-sensitive, phospholipid-dependent protein kinase (protein kinase C) in a reconstituted system. Indol alkaloid teleocidin, a tumor promoter, has been found to be as potent as tumor promoters from the series of phorbol esters and mezerein in activating the mouse brain enzyme. Chemically unrelated tumor promoters such as tetrachlorodibenzo-p-dioxin, anthralin and phenobarbital are devoid of effect. Diacylglycerol 1,2 diolein strongly activated the enzyme whereas 1,3 diolein like 1,2 distearin were poor activators and 1,3 distearin was inactive. Although tumor-promoter-or diacylglycerol-mediated activation of protein kinase C was observed in the presence of 0.5mM EGTA, the reaction requires traces of Ca2+. Tumor promoters did not prevent inhibitory action of antipsychotic phenothiazines and local anesthetics but appear to increase IC50 of these drugs.

Arachidonic acid and related methyl ester mediate protein kinase C activation in intact platelets through the arachidonate metabolism pathways

Unlike unsaturated fatty acids, which almost fully activated purified brain protein kinase C in a phosphatidylserine- and Ca2(+)-free reaction, related methyl esters were poorly active in vitro. In contrast, methyl arachidonate was revealed to be as potent as arachidonic acid in activating protein kinase C in intact platelets. Arachidonic acid-mediated activation peaked at 20 s while methyl arachidonate-mediated activation plateaued at 2 min when both lipids were added at 50 microM. At concentrations higher than 0.3 mM, all tested unsaturated fatty acids and related methyl esters were weak activators of the enzyme, with the exception of linolenic acid and methyl linolenate which evoked strong enzyme activation. However, inhibitors of arachidonate metabolism blocked both arachidonic-acid and methyl-arachidonate-induced responses. At 5 microM arachidonic acid and methyl arachidonate, protein kinase C activation was due to a cyclooxygenase product(s) whereas at 50 microM the lipoxygenase pathway was mostly involved in the reaction. Therefore, arachidonic acid and its methyl ester activate protein kinase C in platelets mainly through action of their metabolites and eicosanoid synthesis. It is suggested that such indirect protein kinase C activation may account for the tumor-promoting activity of unsaturated fatty acids and related methyl esters.

Tumor promoter chloroform is a potent protein kinase C activator

The major interaction site for tumor-promoting phorbol esters is the calcium-activated, phospholipid-dependent protein kinase (protein kinase C), a key-element in signal transduction. Binding of phorbol esters results in enzyme activation which mediates, at least in part, the action of these agents. We have investigated the effects of tumor promoter chloroform on protein kinase C activity. Like thrombin and 12-O-tetradecanoylphorbol-13-acetate (TPA), chloroform was able to activate protein kinase C in intact rabbit platelets. In addition, chloroform stimulated enzyme activity as well as TPA binding capacity in cell-free system. Scatchard analysis of the data has shown that chloroform increased the number of phorbol ester binding sites. Structurally related compounds, carbon tetrachloride and methylene chloride, activated the enzyme similarly.

Growth-rate-related and hydroxysterol-induced changes in membrane fluidity of cultured hepatoma cells: Correlation with 3-hydroxy-3-methyl glutaryl CoA reductase activity

3-hydroxy-3-methylglutaryl-coenzyme A reductase (EC 1.1.1.3.4.) activity and cell membrane fluidity measured by fluorescence polarization using 1,6 diphenyl, 1,3,5-hexatriene as fluorescent probe have been concomitantly examined in HTC hepatoma cells, both in relation to growth rate and in response to treatment with hydroxylated sterols. A high level of HMG-CoA reductase activity was observed in cells at log phase of growth which progressively decreased to reach a sustained low level at stationary phase. Similarly, membrane fluidity markedly decreased in relation to growth rate. Hydroxylated sterols such as 7 beta-hydroxycholesterol or 25-hydroxycholesterol strongly inhibited HMG-CoA reductase activity whereas a water-soluble derivative of 7 beta-hydroxycholesterol sodium 3,7-bishemisuccinate had no effect. Within the same range of concentrations 7 beta-hydroxycholesterol and 25-hydroxycholesterol strongly decreased membrane fluidity when the water-soluble derivative was ineffective. Thus, the present results provide evidence for a correlation between the two tested parameters and suggest a dependency of HMG-CoA reductase activity on cell membrane fluidity.

A simultaneous protein-binding assay for adenosine 3′:5′-monophosphate and guanosine 3′:5′-monophosphate in biological materials

Adenosine 3′:5′-monophosphate (cyclic AMP) and guanosine 3′:5′-monophosphate (cyclic GMP) have been determined simultaneously by combining individual protein binding assays using different isotopically labeled cyclic nucleotides. Preparations of cyclic AMP-binding protein from beef adrenal cortex and cyclic GMP-binding protein from the fat body of silkworm pupae (Bombyx mori) have been used for the assay. The method allows the analysis of cyclic AMP and cyclic GMP levels in crude extracts without any purification. The assay has been applied to hormone-stimulated Mouse liver and phorbol ester-treated Rat embryo cells.

Structural analogies between protein kinase C activators

Phorbol esters and diacylglycerols activate protein kinase C but specific structural parameters appear to be required for the enzyme activation. We have analyzed the conformation of potent and not potent diacylglycerols and phorbol esters. The orientation of the CH20H group at C3 of 1,2 diolein is remarkably similar to that of the same group at C-20 of 4 beta phorbol didecanoate and crucial for potency in activating the enzyme. Our data suggest that the new conformational approach here described could be used to rationally design specific inhibitors preventing the effects of tumor promoters and to predict the structure of potential tumor promoters.

Oscillating levels of adenylate and guanylate cyclase activities in rat embryo fibroblasts stimulated to divide

Abstract Basal activities of membrane-bound adenylate and guanylate cyclase were determined in confluent rat embryo cells stimulated to proliferate by either the renewal of serum-supplemented growth medium or the addition of a mitogen, the 12-0-tetradecanoyl-phorbol-13-acetate (TPA). A transient increase in guanylate cyclase activity was observed within minutes following either treatment while adenylate cyclase activity either abruptly declined in serum-stimulated cells or remained unaffected in TPA-treated cells. In response to both mitogenic treatment, adenylate and guanylate cyclase activities varied reciprocally throughout the pre-replicative phase up to DNA synthesis. The lower levels of guanylate over adenylate activity ratio occurred prior to the onset of the replicative phase whereas the higher levels were coincident with DNA synthesis. A similar pattern of oscillating levels of sodium-fluoride-stimulated adenylate and lubrol-treated guanylate cyclase activities was observed.

Overview of Promotion as a Mechanism in Carcinogenesis

Considerable evidence has accumulated that cancer is a multifactorial and multistep process. Cancer does not result from a simple exposure to a single exogenous factor but rather from a complex interaction between exogenous and/or endogenous factors. The model of chemical carcinogenesis in mouse skin designed by Berenblum (1) and Mottram (2) has led to the characterization of two stages in carcinogenesis, initiation and promotion, caused by two different classes of agents, the initiators and the promoters. More recently, models of cultured cells have provided evidence that cell transformation by chemical carcinogens, U.V. light or X-rays, requires at least two steps. As a result of probability-based studies J. Little and co-workers have emphasized that the initial step is a frequent event since 100 % of the progeny of methylcholanthrene-treated cells are potentially transformed or “initiated” cells. However, a very small minority of the progeny of initiated cells actually yields transformed colonies, suggesting that a second step occurs which is a very rare event. This second step behaves like a spontaneous mutation in that it has small but constant probability of occurring each time an initiated cell divides (3). The requirement for two genetic events in the process of cancer appears to be in agreement with the results of transfection experiments, which indicate that at least two cooperating oncogenes are needed to convert embryo fibroblasts in primary cultures into tumor cells (4). Likewise, the model of transgenic mice in which oncogenes were expressed from tissue-specific promoters, has allowed to state that the expression of oncogen appears to result in hyperplasia with tumors arising as a rare clonal outgrowth, suggesting also the occurrence of secondary events.

Increased protein kinase C activity in the central nervous system of the newt during limb regeneration

Protein kinase C (PKC) activity was examined in the CNS of the newt Pleurodeles waltlii undergoing regeneration after limb amputation. In the spinal cord and brain of control newts, the level of PKC activity was virtually the same for the cytosolic and the particulate fractions. At days 7 and 14 after amputation of two limbs, a twofold increase in overall PKC activity occurred in the spinal cord and accounted for increased membrane-bound activity, while cytosolic activity was not significantly impaired. In contrast, overall PKC activity was not affected in brain. However, a twofold increase in the brain particulate fraction occurred at day 14 while cytosolic activity decreased proportionately. Similar alterations were observed in newts undergoing one or multiple limb amputations. Such changes in PKC activity neither occurred in the CNS of newt after limb denervation nor in the CNS of limb amputated frog Rana temporaria, an Amphibian which is unable to regenerate. Taken together, these results provide evidence that PKC of the CNS is involved in the regeneration process of newts. Changes in activation-associated PKC distribution proceeded through different mechanisms: long-lasting increase in membrane bound activity with a net increase of overall activity in the spinal cord, and long-term redistribution of enzyme activity to the particulate fraction in brain.

Protein Kinase C and Granule Release in Human Platelets

Upon activation at sites of vascular injury or in response to physiological stimuli, platelets undergo a number of biochemical changes associated with aggregation and release of various secretory granules by exocytosis. A number of proteins and biologically active substances such as thrombospondin, serotonin, ADP and PDGF are secreted.