Activation of casein kinase II in ML-1 human myeloblastic leukemia cells requires IGF-1 and transferrin.

Casein kinase II (CK II), a key enzyme involved in the regulation of cell growth, has been variously reported to be activated by diverse mitogens, including insulin-like growth factor 1 (IGF-1) and epidermal growth factor (EGF). Activation of the enzyme is generally carried out in the presence of serum, and we examined the question whether serum components participate in the activation process. We demonstrated previously that ML-1 cells require IGF-1 plus transferrin (TF) for growth and transforming growth factor beta or tumor necrosis factor alpha plus TF for differentiation. We now found that CK II is activated only when the cells are exposed to both IGF-1 and TF or when TF is replaced in this combination with relatively high levels of iron salts. Induction of differentiation with TGF-beta and TF did not result in CK II activation. These results show that CK II activation in ML-1 cells requires the application of both components of the growth signal, IGF-1 and TF, demonstrating that the growth factor alone is incapable of enhancing the activity of the enzyme.

Differential effect of growth- and differentiation-inducing factors on the release of eicosanoids and phospholipids from ML-1 human myeloblastic leukemia cells.

In the absence of serum, growth of ML-1 human myeloblastic leukemia cells is induced by the insulin-like growth factor-1 (IGF1) together with transferrin (Tf), whereas monocytic differentiation is initiated by the transforming growth factor-beta (TGF-beta) in combination with Tf. Initiation of growth was followed by the rapid release of arachidonic acid (AA), hydroxyeicosatetraenoic acids (HETEs) and phospholipids into the culture medium. In contrast, induction of differentiation occurred without the release of these lipids beyond the level present in control. Inhibitors of enzymes involved in the formation of AA and of HETEs, including phospholipase A2 and lipoxygenases, caused interference with growth but not with differentiation, and an inhibitor of the cyclooxygenase path affected neither growth nor differentiation. These results indicate that the initiation of ML-1 cell growth but not of cell differentiation is dependent upon the increased formation of AA and its derivatives formed primarily via the lipoxygenase path.

Aging modulates calcium-dependent phosphatidylinositol degradation by cerebral cortex synaptic plasma membrane phospholipases.

The synaptic plasma membrane (SPM) and cytosol fractions from cerebral cortex of adult (4-mo-old) and aged (27-mo-old) rats were used as a source of phospholipase A2 (PLA2) and phospholipase C (PLC). The activity of PLC acting on [3H-inositol]phosphatidylinositol ([3H]PtdIns) was investigated in the presence of endogenous and 2 mM Ca2+. Arachidonic acid (AA) release was studied in the same conditions, using 1-stearoyl-[2-14C]arachidonyl-sn-glycerophosphoinositol ([14C]PtdIns) as a substrate. In the presence of endogenous Ca2+ (i.e., no added Ca2+) SPM-bound PLC and PLA2 or diacylglycerol (DAG) lipase of aged brain exert significantly higher activity in degradation of PtdIns as compared to their activities in adult brain. Moreover, these enzymes of aged brain are less or not further activated by 2 mM Ca2+, contrary to the enzymes isolated from adult brain. The activity of cytosolic enzymes involved in degradation [3H]PtdIns and [14C]PtdIns and their regulation by Ca2+ ions are not significantly changed in senescent cerebral cortex as compared to the adult. The intracellular calcium concentration ([Ca2+]i), measured with fura-2, is lower in aged brain compared to adult brain, which may suggest the modification in Ca2+ ion redistribution in aged brain and probably its higher concentration in membranes. These results indicate that aging modifies significantly the activity of membrane-bound, Ca(2+)-dependent phospholipase(s) degrading PtdIns, which may be connected with alteration of Ca2+ ion redistribution and may influence the formation and accumulation of very potent lipid messengers as diacylglycerol, lysophospholipid, and arachidonic acid, known to be involved in neurotransmission processes.

Inhibition of human ovarian carcinoma cell- and hexosaminidase- mediated degradation of extracellular matrix by sugar analogs.

Human ovarian carcinoma (HOC) cell beta-N-acetylglucosaminidase (beta-NAG, EC 3.2.1.30) was found to be present in three isoenzymatic forms. All three forms were capable of degrading ECM. Therefore, inhibitors of beta-NAG were sought as potential anti-invasive agents. Two sugar analogs, 2-acetamido-2-deoxy-1,5-gluconolactone (CD80110) and 2-acetamido-1,5-imino-1,2,5-trideoxy-D-glucitol (CD 86022), were evaluated for their ability to inhibit 1) human ovarian carcinoma beta-NAG isoenzyme activities, 2) degradation of radiolabeled ECM mediated by HOC cells and beta-NAG, and 3) cell growth. Both compounds were found to be competitive inhibitors of beta-NAG isoenzyme activities, with Ki values similar for all isoenzymes (2-8 microM). CD 80110 and CD 86022 inhibited HOC cell-mediated degradation of [3H]glucosamine labeled ECM without notable effect on cell growth. Enzyme-mediated degradation of ECM was also inhibited by both sugar analogs. Analysis of degradation products after cell- or enzyme-mediated digestion of ECM revealed a decrease in the amount of both free aminosugars and high molecular material caused by inhibitors. These results support a role for beta-NAG in degradation of extracellular matrix components and suggest the usefulness of hexosaminidase inhibitors as potential antiinvasive agents.

Human ovarian carcinoma beta-N-acetylglucosaminidase isoenzymes and their role in extracellular matrix degradation.

Degradation and invasion of basement membrane by tumor cells involves the cooperative hydrolysis of proteoglycans, collagens, and glycoproteins mediated by a number of enzymes including proteases, collagenases, and glycosidases. In order to study these processes in vitro, a tissue culture system was developed in which bovine corneal endothelial cell extracellular matrix (ECM) serves as a substrate for attachment and degradation by human ovarian carcinoma cells. Using this system, a correlation was observed between solubilization of glycoconjugates present in ECM and extracellular levels of beta-N-acetylglucosaminidase (EC 3.2.1.30). To determine the role of individual isoenzymes of beta-N-acetylglucosaminidase (beta-NAG) in ECM degradation, the cellular and secreted forms of the enzyme were fractionated and characterized. Three intracellular isoenzyme forms A, I, and B, were isolated from invasive human ovarian carcinoma cell line A-121. In cell homogenate, forms A and B corresponded to 65 and 33% of total beta-NAG activity, respectively. Form I was found to be localized in the plasma membrane fraction of these cells. Two secreted forms of beta-NAG (As and Bs) were detected in serum-free medium. The separated intracellular and secreted isoenzymes demonstrated similar Km values, ranging from 1 to 5 mM, with p-nitro-B-N-acetylglucosaminide substrate. Treatment of [3H]glucosamine-labeled ECM with the separated isoenzymes of beta-NAG resulted in time- and concentration-dependent releases of radioactivity with potency of 1 greater than B much greater than A. These results suggest that human ovarian carcinoma cell beta-N-acetylglucosaminidase isoenzymes (forms B and A) contribute to ECM degradation as secreted enzymes and form I as a membrane enzyme.

Effect of atropine and gammahydroxybutyrate on ischemically induced changes in the level of radioactivity in [3H]inositol phosphates in gerbil brain in vivo.

Brain ischemia in gerbils was induced by ligation of both common carotid arteries for 1 min or 10 min. Sham-operated animals served as controls. Intracerebral injection of [3H]inositol into gerbil brain 16 hr before ischemic insult resulted in equilibration of the label between inositol lipids and water-soluble inositol phosphate. A short ischemic period (1 min) resulted in a statistically significant increase in the radioactivity of inositol triphosphate (IP3) and inositol monophosphate (IP), by about 48% and 79%, respectively, with little change in that of the intermediate inositol biphosphate (IP2), which increased by about 16%. When the ischemic period was prolonged (10 min), an increase in the radioactivity of inositol monophosphate exclusively, by about 84%, was observed. The level of radioactivity in inositol phosphates IP2 and IP3 decreased by about 50%, probably as a consequence of phosphatase activation by the ischemic insult. The agonist of the cholinergic receptor, carbachol, injected intracerebrally (40 micrograms per animal) increased accumulation of radioactivity in all inositol phosphates. The level of radioactivity in IP3, IP2, and IP was elevated by about 40, 23, and 147%, respectively. The muscarinic cholinergic antagonist, atropine, injected intraperitoneally in doses of 100 mg/kg body wt. depressed phosphoinositide metabolism in control animals. The level of radioactivity in water-soluble inositol metabolites in the brain of animals pretreated with atropine was evidently about 32% lower than in untreated animals. Pretreatment with atropine decreased the radioactivity of all inositol phosphates in the brain of animals subjected to 1-min ischemia and the radioactivity of IP in the case of 10-min brain ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphatidylinositol degradation in ischemic brain specifically activated by synaptosomal enzymes.

The effect of ischemia on phosphatidylinositol (PI) degrading enzymes in brain homogenate, cytosol and synaptosomes was investigated to evaluate the involvement of cholinergic receptor in PI hydrolysis during ischemia. The activity of phospholipase C in cytosol was not changed by ischemic insult. PI degradation in synaptosomes was significantly (60%) enhanced by ischemia. Atropine and gamma-butyrolactone abolished the ischemic effect on PI degradation, suggesting that the cholinergic receptor system mediates breakdown of PI during ischemia.

Effects of cerebral ischemia on [3H]inositol lipids and [3H]inositol phosphates of gerbil brain and subcellular fractions.

Intracerebral injection of [3H]inositol into gerbil brain resulted in labeling of phosphoinositides and inositol-phosphates in various subcellular membrane fractions. Phosphatidylinositol (PI) comprised greater than 90% of the radioactivity of inositol lipids. However, the level of labeled poly-PI (with respect to PI) was higher in synaptosomes than in other membrane fractions. Ischemia induced in gerbils by ligation of the common carotid arteries resulted in a 30% decrease in labeled poly-PI in brain homogenates and this decrease was largely attributed to the poly-PI in synaptosomes (50% decrease). Among the inositol phosphates, the ischemia induction resulted in a decrease in labeling of inositol triphosphate (63%) and inositol biphosphate (38%), but labeling of inositol phosphate (IP) was increased by 59%. The results suggested a rapid turnover of the inositol phosphates in the gerbil brain. In general, changes in inositol lipids and inositol phosphates due to ischemia were attenuated after pretreatment with lithium (3 meq/kg) injected intraperitoneally 5 h prior to ligation. Surprisingly, lithium treatment alone did not cause an increase in IP labeling in the gerbil brain.

Brain lipids of a myelin-deficient rabbit mutant during development.

The paralytic tremor (pt) rabbit, a neurological mutant, exhibits hypomyelination transmitted in X-linked recessive fashion. This rabbit mutant was used for regional lipid analyses of different brain structures during development. There was a significant decrease of myelin-specific lipids, particularly in the cerebroside and sulfatide in pt rabbits. The decrease of phospholipid and cholesterol was relevant to the total lipids depletion. The molar ratio of galactolipid to phospholipid decreased in the pt rabbit brain in each age group examined. The other lipids typical for myelin, such as ethanolamine glycerophospholipids, sphingomyelin, and GM1 ganglioside, were also diminished in the myelin-rich structures, but were not changed in the cortical gray matter of pt rabbits. In contrast, the total amount of gangliosides was near control levels and, therefore, in the mutant rabbits, the white matter and brain stem contained a higher proportion of lipid, as ganglioside, relative to the control animals. This result suggests that neuronal membranes were not involved in this pathology. The characteristic biochemical abnormalities exhibited in the pt rabbit suggest that a defect of oligodendroglial cell function is primarily responsible for the myelin abnormality.

Diacylglycerol kinase and lipase activities in rat brain subcellular fractions.

Phosphatidic acid synthesis via diacylglycerol kinase and free fatty acid release via diacylglycerol lipase were investigated in rat brain subcellular fractions using membrane-bound [I-(14)C]arachidonoyl-diacylglycerol as substrate. Labeled diacylglycerol was generated by incubating brain membranes containing [I-(14)C]arachidonoyl-phosphatidylinositols in the presence of deoxycholate and Ca(2+). Incubation of the prelabeled synaptosomes enriched in [1-(14)C]arachidonoyl-diacylglycerols or incubation of brain subcellular fractions with heat-treated prelabeled membranes resulted in the release of free fatty acids from the diacylglycerols. When incubations were carried out in the presence of ATP, MgCl(2) and NaF, both free fatty acid release and conversion of diacylglycerols to phosphatidic acids were observed. The conversion of diacylglycerols to phosphatidate or their hydrolysis to free fatty acids were linear with time for at least 15 min. In three brain subcellular fractions examined, diacylglycerol kinase activity indicated a pH maximum of 7.4. The free fatty acid release was enhanced slightly by Ca(2+) (1 mM), but Ca(2+) (0.5-4 mM) in the presence of Mg(2+) (10 mM) was inhibitory to the diacylglycerol kinase reaction. Phosphatidate formation was also inhibited by an excessive amount of deoxycholate added to the incubation mixture. Among the brain subcellular fractions, diacylglycerol kinase was more active in synaptic vesicles and cytosol than in the microsomal fraction, whereas diacylglycerol lipase activity was higher in the cytosol fraction than in the membrane fractions. Upon washing the membranes by centrifugation, a substantial portion of the diacylglycerol kinase activity was removed after the first washing, whereas the diacylglycerol lipase activity remained essentially unchanged. The metabolic role of arachidonoyl-diacylglycerols in brain membranes in relation to the biosynthesis of phosphatidate and the release of arachidomic acid is discussed.