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Biomedical subjects

M Castagna

Publications and source records attributed to M Castagna.

At least 19 recordsLinked to original sources
PubMed1992

Bile acids, non-phorbol-ester-type tumor promoters, stimulate the phosphorylation of protein kinase C substrates in human platelets and colon cell line HT29.

Protein kinase C (PKC) is the target for a number of tumor promoters. The mechanism underlying the promoting effects of bile acids in colorectal cancer is not understood. We report that sodium deoxycholate (DOC) triggered activation of PKC in physiological conditions. The biphasic effects of DOC upon PKC activation were Ca(2+)-stimulated and did not require phosphatidylserine (PtdSer) as phospholipid co-factor. The optimal rate of activation was obtained at 0.4 mM DOC and reached approximately half the maximal rate of activation obtained in the presence of PtdSer. Similarly to PtdSer, DOC supported diacylglycerol- as well as phorbol-ester-mediated PKC activation. The reciprocal effects of PtdSer and DOC upon PKC in either 0.5 mM CaCl2 or 0.5 mM EGTA suggest that DOC interacts with the phospholipid-binding domain to elicit PKC activation. DOC-supported enzyme activation exhibited substrate specificity different from that of PtdSer-supported enzyme activation. All tested primary and secondary bile acids activated PKC to various extents, with DOC being the most potent. We suggest that amphipathic bile acids acting in a PtdSer-like manner provide the hydrophobic environment required for PKC activation. Treatment of 32P-labeled platelets and colonic cells HT29 Cl.19A with DOC enhanced the phosphorylation of endogenous substrates for PKC. Colonic cells responsive at 50 microM DOC, appeared to be 10-fold more sensitive than platelets. We suggest that direct or indirect activation of PKC by bile acids may account for the promoting effects of these non-phorbol-ester-type tumor promoters.

Bile Acids and Salts↗
PubMed1992

Tumorigenicity-associated expression of protein kinase C isoforms in rhabdomyosarcoma-derived cells.

Cell sublines were derived from Ni-induced rat rhabdomyosarcoma which differed in their degree of tumorigenicity. We compared the expression of protein kinase C isoforms alpha, beta, gamma and epsilon in three sublines developing tumors in syngeneic rats (Sy+) and in two sublines devoid of tumorigenicity in these animals (Sy-), with that of normal skeletal muscle. Northern and Western blotting experiments showed that PKC alpha was dramatically overexpressed in Sy- cells whereas it was underexpressed in Sy+ cells. Southern blot analysis provided evidence for a 3-fold increase of PKC alpha-related DNA fragments in Sy- cells. Steady-state levels of the PKC epsilon-related transcript were also markedly decreased in Sy+ cells. However, the expression of PKC beta-related RNA was increased in these cells. Our data support the concept that the differential expression of the PKC isoforms may play a critical role in determining the neoplastic phenotype.

Animals↗
PubMed1992

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.

Amputation, Surgical↗
PubMed1992

Immunohistochemical analysis of the distribution of a breast tumor associated antigen recognized by a monoclonal antibody.

A new monoclonal antibody (MoAb), MM 1-80, recognizing a tumor associated epitope of a breast high molecular weight mucin molecule was tested, using the avidin biotin immunoperoxidase method on normal and pathological mammary tissues. The normal mammary ducts and lobules were negative. Fibroadenomas showed a strong intracytoplasmic staining. In apocrine metaplasia, adenosis, and papillomatosis, scattered cells showed intracytoplasmic, luminal border or secretion reactivity. In lobular and ductal hyperplasia the cells showed intracytoplasmic immunoreactivity which, however, became more intense and homogeneous in atypical lesions, i.e. lobular and ductal in-situ carcinomas. The infiltrating carcinomas of different histotype expressed positivity on 98% of the cases (113/115) and axillary metastatic lymph nodes were always positive (20/20). The MoAb was tested on 175 human neoplasias of different origin which were in the majority of the cases negative with the exception of adenocarcinomas of the lung, ovary and bladder. MM1-80 appears to react preferentially with mammary cells undergoing hyperplastic, metaplastic and neoplastic processes. The 1-80 epitope distribution is different in these lesions starting with a predominant luminal expression in benign lesions and becoming strong and cytoplasmic in the malignant breast cell.

Adult↗
PubMed1992

Resection of liver gastrinoma leading to persistent eugastrinemia. Case report.

In a 30-year-old man with Zollinger-Ellison syndrome, the only detectable gastrinoma was in the right liver lobe. Removal of the lobe, without additional gastric surgery, was followed by normalization of the gastrin level. Long-term follow-up confirmed the good result. The usefulness of quick intraoperative gastrin assay is stressed.

Adult↗
PubMed1991

Does protein kinase C require Ca2+ for activation?

Ca2+ requirement for protein kinase C activation is a matter of controversy. In this report we have examined Ca2+ dependency of the reaction in different assay systems and shown that the enzyme response to Ca2+, as well as diacylglycerol, depends upon phospholipid species, protein substrate and lipid conformation (micelles or sonicates). These results emphasize that the enzyme characteristics as defined in reconstituted membrane systems may not have a physiological relevance.

Calcium↗
PubMed1990

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.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed1990

Phorbol esters mediate phospholipid-free activation of rat brain protein kinase C.

The present study provides evidence that rat brain protein kinase C elicits a phosphotransferase activity towards histone and undergoes autophosphorylation in the absence of phosphatidylserine. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate binds to and activates protein kinase C in a phospholipid-free reaction. The apparent activation constant (Ka = 2.7 nM) is not modified by the absence of phospholipid but the maximum velocity is greatly decreased. The phosphotransfer reaction to exogenous substrates occurs in 0.5 mM ethylene-bis(oxyethylenenitrilo)tetraacetic acid, although autophosphorylation in these conditions requires the presence of Ca2+. The protein kinase C inhibitor (1-(5-isoquinolinesulfonyl)-2-methylpiperazine inhibits the reaction, whereas the cAMP-dependent protein kinase inhibitor is ineffective. In contrast to diacylglycerol, which is a poor activator, unsaturated fatty acids potently activate the phospholipid-free reaction. Moreover, the substrate specificity is markedly changed, e.g., myelin basic protein and histone types VI-S and VII-S appear to be relatively better substrates in the phospholipid-free reaction. The data presented indicate that protein kinase C (or some individual isoforms) may function, at least partially, without binding to membrane phospholipid and suggest that this novel characteristic of phorbol esters may account for their tumor-promoting activity.

Animals↗
PubMed1990

[Role of protein kinase C in cell regulation and tumor promotion].

The signaling pathways which transfer information into the cell form a tissue-specific network. Protein kinase C (PKC) belongs to the inositol 1, 4, 5 trisphosphate/diacylglycerol (IP3/DG) pathway and plays a key role in cell regulation. The enzyme controls cell growth and functions through a set of negative and positive regulatory mechanisms. Some tumor promoters such as phorbol esters activate PKC by acting at the DG binding site. Others do not compete with DG and either activate PKC in the absence of phospholipids, such as arachidonic acid and bile salts, or require the presence of phospholipids, for instance a number of organic solvents. However, some promoters do not directly activate the enzyme although some of them appear to act elsewhere in the IP3/DG signaling pathway. The possibility that tumor promoters impair the expression of PKC is discussed.

Carcinogens↗
PubMed1989

Membrane-phorbol ester interactions monitored by circular dichroism.

The interaction of phorbol 12,13-dibutyrate (PDBu), 12-O-retinoylphorbol 13-acetate (RPA) and 12-O-tetradecanoylphorbol 13-acetate (TPA) with L-alpha-phosphatidylserine-containing small unilamellar vesicles or erythrocyte ghosts was monitored by circular dichroism (CD). No change in the CD spectra of PDBu was observed upon binding, while RPA and TPA spectra were slowly affected by the interaction. The changes in RPA and TPA spectra were assigned to the embedding of these molecules in the membrane bilayers. In the presence of 10(8) cells/ml, after one minute incubation, about 2 to 5% of the amount of phorbol ester added is embedded in the membrane. It is suggested that either phorbol esters entering the membrane is not a prerequisite for protein kinase C activation or the amount of phorbol esters necessary to activate protein kinase C is very small.

Circular Dichroism↗
PubMed1989

Calmodulin antagonist W-7 inhibits lysosomal sphingomyelinase activity in C6 glioma cells.

N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) is known to be a potent calmodulin antagonist and inhibitor of calmodulin-dependent protein kinases. W-7 and 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine (H-7) are inhibitors of protein kinase C and cyclic nucleotide-dependent protein kinases. In C6 glioma cells, W-7 and not H-7 inhibited dose-dependently acid sphingomyelinase, a result indicating the modulation of this lysosomal enzyme by a calmodulin-dependent system. Other lysosomal enzymes, such as beta-glucosidase, alpha-galactosidase, and arylsulfatase A, were unaffected by W-7 and H-7, a finding indicating a selective effect of W-7 on sphingomyelinase.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1989

Benzene-mediated protein kinase C activation.

Extracellular ligands transfer information into the cell through several pathways that operate in an integrated fashion. Protein kinase C, and enzyme that plays a pivotal role in signal transduction, is the molecular target for tumor promoters from the series of phorbol esters. A number of structurally unrelated tumor promoters also enhance protein kinase C, interacting or not interacting with the phorbol ester binding site. Evidence is provided that benzene potently activate protein kinase C in vitro, as well as in intact platelets. The drug does not compete for the phorbol ester binding site and probably affects the hydrophobic environment requires for full enzyme activation. Toluene is equally active. The relevance of the presented findings in the carcinogenic effects of benzene is discussed.

Animals↗
PubMed1989

Distribution of calcitonin- and somatostatin-containing cells in thyroid lymphoma and in Hashimoto's thyroiditis.

Eleven cases of thyroid lymphoma were studied by the immunoperoxidase avidin-biotin technique with calcitonin and somatostatin rabbit antisera. In 6 cases of non-Hodgkin lymphoma, in thyroid tissue residual to the lymphomatous infiltration, the C cell density was markedly increased and clustering was often observed; the C cells often took part in the follicular lining, frequently with polar distribution; these elements displayed a strong positivity for calcitonin, while the number of somatostatin-containing cells was lower and the staining less intense. In the only case of Hodgkin's lymphoma of the thyroid gland the staining was negative; in other 4 cases of non-Hodgkin lymphoma no residual thyroid tissue was found and the staining was also negative. As Hashimoto's thyroiditis is often associated with thyroid lymphoma, 13 cases of Hashimoto's thyroiditis were also studied; no C cells were observed and both stainings were negative. These data show that an increase in the C cell number may be a hallmark of thyroid lymphoma and that hyperplastic C cells show an intensive positivity for calcitonin. On the other hand, C cell hyperplasia is not present in Hashimoto's thyroiditis, in spite of the close association with thyroid lymphoma. Furthermore, we provide evidence that somatostatin-containing cells are present both in normal thyroid glands and in thyroid lymphoma.

Calcitonin↗
PubMed1989

[The role of calcium, diglyceride ester bindings and a synthetic polypeptide in protein kinase C activation].

Protein kinase C, which plays a significant role in the polyphosphoinositide pathway of transmembrane signaling, is activated by a large class of extracellular ligands including neurotransmitters, hormones and growth factors. Diacylglycerols are the intracellular mediators of protein kinase C activation. Tumor promoting phorbol esters mimic the diacylglycerol action in binding to the same site. Active diacylglycerols have the 1.2 sn configuration and saturated short chain or unsaturated long chain fatty acids. Alkyl analogs of diacylglycerols were devoid of activity when an ether bond was present in position 1, whereas activity of the alkyl analog in position 2 was retained. Protein kinase C activation and 3H-TPA binding to the enzyme occurred in the presence of 0.5 mM EGTA. Moreover it has been shown in vivo that full activation of the enzyme was obtained in the intact platelets loaded with an excess of Quin 2, prior to stimulation by phorbol esters. A peptide (residues 499-513) was synthesized which enhanced the affinity of protein kinase C for histone. It is suggested that it may be the receptor site for another peptide of the enzyme (residues 19 to 36) which behaves as a pseudosubstrate.

Animals↗