Insulin enhances luteinizing hormone-stimulated steroidogenesis by porcine theca cells

It has been shown recently that insulin enhances differentiation of rat, pig, and human granulosa cells. The present studies were done to determine if insulin also plays a role in the regulation of theca cell steroidogenesis. Theca cells were obtained from prepubertal gilts and cultured under serum-free conditions for 48 h. Theca cell androstenedione production under basal and luteinizing hormone (LH)-stimulated conditions was significantly increased by adding insulin (1 microgram/ml) to the culture medium. Treatment of basal and LH-stimulated cultures with increasing concentrations of insulin (0.001-10 micrograms/ml) caused dose- and time-dependent increments in androstenedione production, but the effect was independent of the dose of LH employed. The ability of insulin to enhance thecal cell androstenedione production was mimicked by somatomedin C, but not by relaxin. Studies to determine the mechanism(s) of action of insulin showed that insulin action is exerted, at least in part, at a site(s) proximal to cyclic adenosine 3'5'-monophosphate (cAMP) generation, since insulin enhanced both basal and LH-stimulated accumulation of extracellular cAMP in addition to increasing androstenedione production. This effect was further enhanced by 3-isobutyl-1-methyl xanthine, an inhibitor of phosphodiesterase activity. Insulin treatment also caused dose-dependent increments in forskolin- and prostaglandin E2-stimulated accumulation of extracellular cAMP and androstenedione. Insulin also increased both the basal and LH-stimulated production of progesterone and its precursor pregnenolone, in addition to the increases in androstenedione.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antilipolytic effects of N6-phenylisopropyladenosine and prostaglandin E2 in fat-cells of obese volunteers before and during energy restriction.

The antilipolytic effects of N6-phenylisopropyladenosine and of prostaglandin E2 were studied with adipocytes of obese volunteers before and after 4 weeks of severe energy restriction [1250 kJ (300 cal)/day] in the presence and absence of adenosine deaminase (1.6 micrograms/ml, corresponding to 320 m-units/ml). The studies were undertaken to define more clearly the role that local modulators might play in adaptation of lipid mobilization to starvation in humans. Starvation was associated with an approx. 3-fold increase in non-stimulated lipolysis. Removal of endogenous adenosine resulted in a similar increase in basal glycerol release under both conditions, averaging 2 and 2.2 mumol/180 min per 10(6) cells respectively. The sensitivity of the cells to N6-phenylisopropyladenosine and to prostaglandin E2 was not changed by starvation in the presence of adenosine deaminase. These results are discussed in terms of the possible role that local regulators might play during dietary adaption in human fat-cells in vitro.     

Natural occurrence of an inhibitor of mammalian cell growth in human and mouse cells of normal and tumor origin.

N6-(delta2-isopentenyl)adenosine was found both as a component of tRNA and as the cytoplasmic mononucleotide in human leukemic lymphoblasts and myeloblasts from peripheral blood and bone marrow samples. This hypermodified nucleotide was also found in the tRNA and as a mononucleotide in human (MRC-5 and KB) and mouse (A9, FLV, LM, and RAG) cell lines. The relative amounts of this hypermodified nucleotide in the tRNA of the cell lines and the human leukemias were similar (the mean value being 0.06 +/- 0.03 mole % of the total tRNA nucleotide content); whereas the amounts occurring as the free cytoplasmic mononucleotide were more varied but still comparable (the mean value being 0.53 +/- .09 mole % of all cytoplasmic nucleotides) for all cells investigated with the notable exception of all normal, diploid cell lines under study (0.04 mole%). A possible relationship of the free cytoplasmic mononucleotide with the nucleotide in the tRNA for control of mammalian cell protein synthesis in vivo was investigated by addition of N6-(delta2-isopentenyl)adenosine to the culture medium. The exogenously added nucleoside caused inhibition of cell growth within 3 h and cell death within 36 h at concentrations as low as 0.4 muM. No comparable effects were seen when adenosine, adenine, or N6-(delta2-isopentenyl)-adenine were added to the cultures. The simultaneous presence of adenosine in cultures containing N6-(delta2-isopentenyl)adenosine did not alter the detrimental effects of the hypermodified nucleoside on cell growth even when the concentration of adenosine was 50-fold that of N6-(delta2-isopentenyl)adenosine. Addition of N6-(delta2-isopentenyl)adenosine to cell cultures caused within the first 6 h a significant reduction in the rates of RNA and protein synthesis; whereas DNA synthesis continued at a rate comparable to control and adenosine-treated cells for 18 h before decreasing.     

Catecholestrogens inhibit basal and luteinizing hormone-stimulated androgen production by porcine thecal cells

It has been shown recently that catecholestrogens are produced by cultured porcine granulosa and thecal cells, and that they influence porcine granulosa cell steroidogenesis in a similar manner to estradiol-17 beta (E2). The present studies were performed to determine if catecholestrogens also play a role in the regulation of porcine thecal cell steroidogenesis and to compare their actions to those of E2. Thecal cells were obtained from prepubertal gilts and cultured in a serum-free medium for 48 h. Thecal cell androstenedione production under basal and luteinizing hormone (LH)-stimulated conditions was significantly inhibited by adding E2 or catecholestrogens to the culture medium. Treatment of basal and LH-stimulated cultures with increasing concentrations of E2 or catecholestrogens (0.1-10 micrograms/ml) caused a dose-and time-dependent inhibition of androstenedione production. The inhibitory effect of the catecholestrogens, but not of E2, was enhanced when the cultures contained the catechol-O-methyl transferase inhibitor, U-0521. Studies to determine the mechanism(s) of action of the catecholestrogens showed that E2 and catecholestrogen actions are exerted at a site(s) distal to cyclic adenosine 3'5' monophosphate (cyclic AMP) generation, because neither agent affected the basal or LH-stimulated accumulation of extracellular cyclic AMP, while causing a significant inhibition of androstenedione production. E2 or catecholestrogen treatment also inhibited androstenedione production stimulated by prostaglandin E2 and dibutyryl cyclic AMP. In addition, both E2 and catecholestrogen treatment significantly decreased basal and LH-stimulated 17 alpha-hydroxyprogesterone production, while significantly increasing pregnenolone production. Progesterone production in the presence of E2 or catecholestrogens showed small but statistically insignificant increases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytotoxicity and genotoxicity testing of sodium fluoride on Chinese hamster V79 cells and human EUE cells.

The cytotoxic effects of sodium fluoride (NaF) on hamster V79 cells and human EUE cells were studied by measuring the cloning efficiency and DNA, RNA and protein synthesis in cells cultured in the presence of NaF. Potential mutagenicity of NaF was followed on the basis of induced 6-thioguanine-resistant mutants in treated Chinese hamster V79 cells. The results showed that the addition of 10-150 micrograms of NaF per ml of culture medium induced 10-75% cytotoxic effect on hamster V79 cells but had no toxic effect on human EUE cells. NaF was cytotoxic to human EUE cells at considerably higher concentrations (200-600 micrograms/ml). Growth of both cell types with 100 and 200 micrograms of NaF per ml caused inhibition of 14C-thymidine, 14C-uridine and 14C-L-leucine incorporation. This means that NaF inhibits macromolecular synthesis whereby damaging effects were less drastic in human EUE cells. The results of detailed mutagenicity testing on hamster V79 cells showed that NaF did not show any mutagenic effect after long-term (24-h) incubation of hamster cells in the presence of 10-400 micrograms of NaF per ml of culture medium.     

Human platelet factor 4 is a direct inhibitor of human osteoblast-like osteosarcoma cell growth.

The effect of purified human platelet factor 4, a platelet alpha-granule protein, on the growth of the human osteoblastic osteosarcoma cell lines Saos-2 and G-292 was investigated. Platelet factor 4 (20 ng/ml to 2 micrograms/ml) caused a significant, dose-dependent inhibition of human osteoblast-like osteosarcoma cell proliferation. Platelet factor 4 exerted its inhibitory effect under all growth conditions tested: serum-free, serum-stimulated and thrombin-stimulated. The platelet factor 4-induced cell inhibition was not associated with a cytotoxic effect on the cells (assessed by lactate dehydrogenase release). The inhibitory effect of platelet factor 4 was not affected by the presence of indomethacin in the cultures, indicating that the effect was prostaglandin-independent. These results suggest that platelet factor 4 has direct antitumor effects and that it may be important in pathological and physiological processes of bone.     

Cytological effects of kepone on Chinese hamster cells

Cytological and cytotoxic effects of kepone on Chinese hamster cells (M3-1) were investigated. Cells treated with 2 micrograms, 4 micrograms, and 6 micrograms/ml of kepone did not show any morphological abnormalities. However, cytological observations showed that chromosome breaks, chromatid breaks, dicentric chromosomes, and chromosome interchanges were produced by these treatments. Cytotoxic studies revealed dose response and time response reactions to kepone. Cell toxicity was greater at the 30 micrograms/ml concentration, producing 100 percent cell death within 24 hours.     

Proliferation of guinea-pig uterine epithelial cells in serum-free culture conditions: effect of 17 beta-estradiol, epidermal growth factor and insulin

The effects of 17 beta-estradiol (E2), epidermal growth factor (EGF) and insulin, alone or in association on guinea-pig uterine epithelial cell proliferation were examined in serum-free culture conditions. Primary cultures of epithelial cells were made quiescent by serum depletion, then incubated in a chemically defined medium. In this medium, insulin increased DNA synthesis but not in a dose-dependent manner for concentrations ranging from 0.2 to 10 micrograms/ml. A significant effect of EGF was found only for the highest concentration tested (100 ng/ml). E2 alone or in the presence of insulin (1 microgram/ml) had no effect whatsoever on the concentration tested (10(-10)-10(-5)M). Insulin (10 micrograms/ml) plus EGF (100 ng/ml) exerted on DNA synthesis and cell proliferation a significant additive effect which was identical to the growth stimulation induced by 10% fetal calf serum. The effects of insulin plus EGF were not modified by the addition of E2. These findings suggest that E2 is not directly mitogenic for uterine epithelial cells in defined culture conditions and that the mitogenic response to optimal concentration of insulin plus EGF is independent of E2.     

Homeostatic action of adenosine A3 and A1 receptor agonists on proliferation of hematopoietic precursor cells

Two adenosine receptor agonists, N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA) and N6-cyclopentyladenosine (CPA), which selectively activate adenosine A3 and A1 receptors, respectively, were tested for their ability to influence proliferation of granulocytic and erythroid cells in femoral bone marrow of mice using morphological criteria. Agonists were given intraperitoneally to mice in repeated isomolar doses of 200 nmol/kg. Three variants of experiments were performed to investigate the action of the agonists under normal resting state of mice and in phases of cell depletion and subsequent regeneration after treatment with the cytotoxic drug 5-fluorouracil. In the case of granulopoiesis, IB-MECA 1) increased by a moderate but significant level proliferation of cells under normal resting state; 2) strongly increased proliferation of cells in the cell depletion phase; but 3) did not influence cell proliferation in the regeneration phase. CPA did not influence cell proliferation under normal resting state and in the cell depletion phase, but strongly suppressed the overshooting cell proliferation in the regeneration phase. The stimulatory effect of IB-MECA on cell proliferation of erythroid cells was observed only when this agonist was administered during the cell depletion phase. CPA did not modulate erythroid proliferation in any of the functional states investigated, probably due to the lower demand for cell production as compared with granulopoiesis. The results indicate opposite effects of the two adenosine receptor agonists on proliferation of hematopoietic cells and suggest the plasticity and homeostatic role of the adenosine receptor expression.     

Cytotoxic activity of new racemic and optically active N-phosphonoalkyl bicyclic β-amino acids against human malignant cell lines

The cytotoxic effects of novel racemic and optically active constrained N-phosphonoalkyl bicyclic β-amino acids were tested against a panel of human tumor cell lines. All of the compounds investigated exhibited different concentration-dependent antiproliferative effects against the HT-29, MDA-MB-231, HepG2 and HeLa cell lines after 24?h treatment. The most sensitive cells were the HeLa cells at various concentrations of the four compounds tested. The aminophosphonate 3 exerted the most pronounced antiproliferative effect against the HeLa cells (inhibition of the cell vitality up to 70% at 0.5?mg/ml) and was not toxic to the normal Lep3 cells at lower concentration. Furthermore, the N-phosphonophenyl derivatives 1 and 2 displayed antiproliferative effect against mainly the MDA-MB-231 tumour cells at higher concentration.     

Induction of acetyl-LDL receptor activity by phorbol ester in human monocyte cell line THP-1

Human monocytic cell line THP-1 incubated with as little as 10 ng/ml of phorbol myristate acetate bound and metabolized 1-2 micrograms of Ac-LDL over a 5-h period. In the absence of phorbol treatment, no specific metabolism of Ac-LDL occurred. Optimal levels of receptor were reached after 72 h of exposure. Induction of receptor was dependent on protein and RNA synthesis and was partially reversed upon removal of the phorbol. Induction of receptor required activation of the protein kinase C pathway. Metabolism of Ac-LDL by THP-1 cells at 37 degrees C was saturated at 25 micrograms/ml. Binding at 4 degrees C was saturable with an average Kd of 8.0 x 10(-9) M. Cell population studies by fluorescent activated cell sorting indicated that approximately 87% of the THP-1 population was expressing scavenger receptor activity 96 h after phorbol treatment as compared to 99% for murine macrophage cell line P388D1. Uptake of Ac-LDL by THP-1 resulted in an 11-fold increase in the rate of cholesterol esterification which was saturable at 50 micrograms/ml. Incubation of cells for 48 h with 50 micrograms/ml of Ac-LDL resulted in a 60% increase in free cholesterol and a 10-fold increase in the cholesteryl ester content of the cells. Lipid accumulation in THP-1 cells after Ac-LDL uptake was readily visible by Oil Red-O staining. Solubilization of THP-1 cells, before and after phorbol treatment, followed by ligand blotting with Ac-LDL detected the presence of a 250-kDa protein only in cells treated with phorbol. The protein comigrated with the scavenger receptor derived from mouse macrophage cell line P388D1.     

Experimental studies on the pathogenesis of ochronotic arthropathy. The effects of homogentisic acid on adult and fetal articular chondrocyte morphology, proliferative capacity and synthesis of proteoglycans in vitro

Lapine adult and fetal articular chondrocytes in monolayer culture were employed as an experimental model for studying the effects of homogentisic acid on chondrocyte morphology, proliferation and synthesis of proteoglycans. Concentrations of homogentisic acid in the range from 0.1 to 100 micrograms/ml were investigated and a cytotoxic effect was detected with concentrations of 5 micrograms/ml and above. Thus, with adult articular chondrocytes this effect was seen between 36 and 48 h of subculture with 5 micrograms/ml or more of homogentisic acid present from the beginning of subculture. In fetal articular chondrocyte cultures a concentration of 1 microgram/ml elicited a statistically significant reduction (13%) in cell proliferation without altering sulphated macromolecular synthesis. Experiments with 3H-thymidine autoradiography to measure the pulse labeling index (LI) in fetal chondrocytes in vitro showed that the 5 micrograms/ml concentration of homogentisic acid present for 21 h from the beginning of subculture gave a LI of 2%, compared with 25% for controls. Thus, homogentisic acid can reduce chondrocyte proliferative capacity before morphological alterations can be detected by light microscopy. No significant alterations in the synthesis of proteoglycans were detected at concentrations below the cytotoxic level. The homogentisic acid-induced cytotoxicity and reduction of proliferation in chondrocytes represents a possible pathway by which cartilage is damaged in ochronotic arthropathy.     

Clostridium difficile toxin A induces multinucleation in the human leukemic T cell line JURKAT.

Clostridium difficile toxin A is a cytotoxic enterotoxin known to be active on all mammalian cell lines tested up to now. It induces a disruption of the cytoskeleton, particularly the microfilament system, leading to inhibition of cell proliferation. Here, we describe some effects of toxin A on the leukemic T cell line JURKAT. Cells exposed to the toxin did not divide, as cell numbers remained constant for 3 days in the presence of 0.5 to 1.0 micrograms/ml of the toxin. However, these cells were found to become multinucleated, a phenomenon which was time- and dose-dependent. After treatment for 72 h with 0.5 micrograms/ml toxin A, 95% of the cells were multinucleated and had a considerably increased cell diameter. These effects in JURKAT cells were partially reversible upon removal of the toxin within 12 h after the beginning of toxin exposure, but irreversible after 24 h of toxin treatment. These results suggest a continuing nuclear division in the absence of cytoplasmic division, i.e., an effect of toxin A on contractile ring formation. The JURKAT cell is the first cell type reported to respond to toxin A with multinucleation.     

Neural differentiation of ectodermal explants of the early gastrula from Rana temporaria under the action of various mitogens and kainic acid

The following mitogens: concanavalin A (con A), phytohemagglutinin (PHA), hydra growth factor (HGF) as well as neurotoxic agent kainic acid, caused neural differentiation (N) effects differed in value and also in character of dependence on concentration of the agent. The lowest effective concentration of con A was 75 micrograms/ml (15% neural differentiation, treatment during 3 h), and the effect reached maximum of 50-60% at 100-200 micrograms/ml. Con A concentration 50 micrograms/ml showed no effect but after 1% rabbit gamma-globulin was added, 17% neural differentiation was detected. N-effects observed after treatment of explants with con A (200 micrograms/ml, 3h) at 2 degrees and 21 degrees were similar (58 and 42% respectively). Minimum PHA concentration used (6 micrograms/ml, 18h) led to neural differentiation in 5% of explants. N-effect of PHA increased along with the concentration of the lectin and was most pronounced at 25 micrograms/ml. However, further increase in concentration (up to 200 micrograms/ml) resulted in decrease of its N-effect to 13%. At 12 micrograms/ml PHA exerted not only neural differentiation, but also lens-inducing (32%) action on the ectoderm. N-effect of HGF (2.5, 25 and 250 micrograms/ml) was lower as compared with the maximum effects of con A and PHA (30-35%). No correlation of HGF inducing action with its concentration was observed. Kainic acid showed weak N-effect (20-30%) at 1 and 10 micrograms/ml. Higher concentration (100 micrograms/ml) had no N-effect, but in 27% of explants "free" lentoids were found. Oubain (10(-3) and 10(-4) M) and HEPES (20 mM) did not affect the differentiation of explants.     

Evidence for negative control of growth by adenosine in the mammalian embryo: Induction of Hmx/+ mutant limb outgrowth by adenosine deaminase

We investigated the growth-regulatory actions of adenosine and adenosine deaminase (ADA) during embryonic limb development in the mouse. Polydactylous outgrowth, an expression of the Hemimelia-extra toe (Hmx/+) mutant phenotype, was experimentally regulated in hindlimb buds explanted into a serum-free in vitro system at stage 18 of gestation. Its expression was promoted by exposure to 0.1 or 0.2 IU/ml exogenous ADA and suppressed by co-exposure to 10 nM (-)-N6-(R-phenylisopropyl)-adenosine (N6-PIA). Evidence that N6-PIA acted as a high-affinity agonist against the external adenosine receptor was provided by experiments in which 100 microM caffeine, a known antagonist, competitively blocked its effect. The endogenous adenosine content was analyzed by reversed-phase high-performance liquid chromatography with fluorometric detection following its conversion to the 1,N6-ethenoadenosine derivative. At stage 18, the adenosine levels were 0.5 pmol/micrograms DNA in whole embryos and 0.08 pmol/micrograms DNA in hindlimb buds. At the same stage, partially purified extracts of the embryonal plasma enriched fraction contained high levels of ADA activity (0.04-0.06 IU/embryo, or 0.7-1.0 IU/mg protein). In contrast, blood cells contained 0.0001 IU/embryo (or 0.01 IU/mg protein). This enzyme occurred as a single kinetic form with a molecular weight of 45000-47000 daltons and an apparent Km of 36-38 microM. Its presence in the embryonal plasma argues against an endocrine mechanism of adenosine secretion in favor of autocrine (self-regulatory) or paracrine (proximate-regulatory) mechanisms. Taken together, our results suggest that the in vitro outgrowth of the prospective polydactylous region is induced upon escape from the local growth-inhibitory influence of extracellular adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of adenosine deaminase on cyclic adenosine monophosphate accumulation, lipolysis, and glucose metabolism of fat cells.

In fat cells isolated from the parametrial adipose tissue of rats, the addition of purified adenosine deaminase increased lipolysis and cyclic adenosine 3':5'-monophosphate (cyclic AMP) accumulation. Adenosine deaminase markedly potentiated cyclic AMP accumulation due to norepinephrine. The increase in cyclic AMP due to adenosine deaminase was as rapid as that of theophylline with near maximal effects seen after only a 20-sec incubation. The increases in cyclic AMP due to crystalline adenosine deaminase from intestinal mucosa were seen at concentrations as low as 0.05 mug per ml. Further purification of the crystalline enzyme preparation by Sephadex G-100 chromatography increased both adenosine deaminase activity and cyclic AMP accumulation by fat cells. The effects of adenosine deaminase on fat cell metabolism were reversed by the addition of low concentrations of N6-(phenylisopropyl)adenosine, an analog of adenosine which is not deaminated. The effects of adenosine deaminase on cyclic AMP accumulation were blocked by coformycin which is a potent inhibitor of the enzyme. These findings suggest that deamination of adenosine is responsible for the observed effects of adenosine deaminase preparations. Protein kinase activity of fat cell homogenates was unaffected by adenosine or N6-(phenylisopropyl)adenosine. Norepinephrine-activated adenylate cyclase activity of fat cell ghosts was not inhibited by N6-(phenylisopropyl)adenosine. Adenosine deaminase did not alter basal or norepinephrine-activated adenylate cyclase activity. Cyclic AMP phosphodiesterase activity of fat cell ghosts was also unaffected by adenosine deaminase. Basal and insulin-stimulated glucose oxidation were little affected by adenosine deaminase. However, the addition of adenosine deaminase to fat cells incubated with 1.5 muM norepinephrine abolished the antilipolytic action of insulin and markedly reduced the increase in glucose oxidation due to insulin. These effects were reversed by N6-(phenylisopropyl)adenosine. Phenylisopropyl adenosine did not affect insulin action during a 1-hour incubation. If fat cells were incubated for 2 hours with phenylisopropyl adenosine prior to the addition of insulin for 1 hour there was a marked potentiation of insulin action. The potentiation of insulin action by prior incubation with phenylisopropyl adenosine was not unique as prostaglandin E1, and nicotinic acid had similar effects.     

Cytochalasin D treatment induces meiotic resumption in follicular sheep oocytes

Ovine cumulus-enclosed oocytes collected from antral follicles (3-5 mm in diameter) were cultured in vitro with 2 x 10(6) granulosa cells/ml in the presence or absence of gonadotropins or in the presence of cytochalasin D (CD). The maturation rate was assessed after 24 h of culture. In the control group, in the presence of gonadotropins (follicle-stimulating hormone-luteinizing hormone (FSH-LH; -10 micrograms/ml) 100% of the oocytes reached metaphase II. Whereas intercellular junctions were no longer present after 6-7 h of culture, germinal vesicle breakdown (GVBD) occurred by the same time. In contrast, in the absence of gonadotropin, the majority of the oocytes (59%) remained blocked in GV stage. The inhibition exerted by the granulosa cells on meiotic resumption was overcome when the cumulus-oocyte complexes (COCs) were incubated in CD (5 micrograms/ml) for 6 h at the beginning of the culture. Under these conditions, 85% of the oocytes matured with extrusion of the first polar body. Cytological analysis by cytofluorescence (NBD phallacidin) and electron microscopy showed that, after 6 h of treatment, CD provoked a redistribution of the microfilaments, mainly in the cumulus cells and to a lesser extent in the oocyte cortex. Intercellular junctions disappeared concomitantly with a significant decrease of the intercellular transport of tritiated uridine. The initiation of GVBD occurred at the same time. These results indicate that the resumption of meiosis was correlated with a loss of both junctional complexes (intermediate and gap junctions) between the cumulus cells and the oocyte.     

Catecholamine secretion from the adrenal medulla of the fetus, regulation by hormones

In the ovine fetus, the adrenal medulla activity secretes catecholamines into the circulation under normal and stress conditions. Little is known regarding the endocrine regulation of adrenal medullary catecholamine secretion in the fetus. The present study was undertaken to investigate the direct effects of the hormones prolactin, angiotensin II and cortisol on catecholamine release from fetal adrenal medulla, and to determine whether the effect of the hormones change during development into adulthood. Adrenal medulla from fetal, newborn and adult pregnant sheep was collected, dispersed into single cells and plated. Following preincubation, the cells were treated with ovine prolactin or angiotensin II at 8, 40 and 200 micrograms/ml; or cortisol at 10(-8), 10(-7) and 10(-6)M for 24 h. Catecholamine release into the medium were measured at 3, 6, 12 and 24 h. Ovine prolactin at 8 to 200 micrograms/ml significantly stimulated the release of total catecholamines after 12 h of incubation. The effect of prolactin was dose-dependent such that the magnitude of the response increased and the response time shortened with increasing concentrations of prolactin. In addition, the release of all three catecholamines--dopamine, norepinephrine and epinephrine--was significantly elevated. In newborn cells, only the highest concentration of 200 micrograms/ml ovine prolactin stimulated total catecholamine release at 6 h and 12 h, with significant increases of the three catecholamines at 12 h. In maternal cells, stimulation of catecholamine release was observed also with the highest concentration of prolactin tested (200 micrograms/ml) and after 12 h of incubation, when only the release of epinephrine was significantly enhanced by 324%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Failure of 1-oleoyl-2-acetylglycerol to mimic the cell-differentiating action of 12-O-tetradecanoylphorbol 13-acetate in HL-60 cells

Treatment of human promyelocytic leukemia cells (HL-60 cells) with 12-O-tetradecanoylphorbol 13-acetate (TPA) results in terminal differentiation of the cells to macrophage-like cells. Treatment of the cells with TPA induced marked enhancement of the phosphorylation of 28- and 67-kDa proteins and a decrease in that of a 75-kDa protein. When the cells were treated with diacylglycerol, i.e. 50 micrograms/ml 1-oleoyl-2-acetylglycerol (OAG), similar changes in the phosphorylation of 28-, 67-, and 75-kDa proteins were likewise observed, indicating that OAG actually stimulates protein kinase C in intact HL-60 cells. OAG (1-100 micrograms/ml), which we used, activated partially purified mouse brain protein kinase C in a concentration-dependent manner. Treatment of HL-60 cells with 10 nM TPA for 48 h caused an increase by about 8-fold in cellular acid phosphatase activity. Although a significant increase in acid phosphatase activity was induced by OAG, the effect was scant compared to that of TPA (less than 7% that of TPA). After 48-h exposure to 10 nM TPA, about 95% of the HL-60 cells adhered to culture dishes. On the contrary, treatment of the cells either with OAG (2-100 micrograms/ml) or phospholipase C failed to induce HL-60 cell adhesion. Ca2+ ionophore A23187 failed to act synergistically with OAG. In addition, hourly or bi-hourly cumulative addition of OAG for 24 h also proved ineffective to induce HL-60 cell adhesion. Our present results do not imply that protein kinase C activation is nonessential for TPA-induced HL-60 cell differentiation, but do demonstrate that protein kinase C activation is not the sole event sufficient to induce HL-60 cell differentiation by means of this agent.     

Cytostatic and cytotoxic effects of Pannon (P-30 Protein), a novel anticancer agent

P-30 Protein is a novel protein, of molecular weight approximately 15 KD, obtained from the extract of a vertebrate tissue showing in vivo antitumour activity. Cytostatic and cytotoxic effects of this product in its purified form (P-30 Protein) or in partially purified extracts (Pannon) were studied in vitro on human leukaemic HL-60, human submaxillary carcinoma A-253, human colon adenocarcinoma Colo 320 CM and murine erythroleukaemia (Friend leukaemia) cell lines. Of these cells, HL-60, A-253 and Colo 320 CM were sensitive and Friend leukaemia resistant to this agent. The effects were time- and concentration-dependent. During the initial 24-48 h of treatment, a slowdown in cell proliferation was apparent but cell death was not extensive. After 24-48 h, there was a reduction in the proportion of cells in S phase of the cell cycle and the cells became preferentially arrested in G1 phase. The G1 cells showed high heterogeneity with respect to RNA content and some cells were characterized by very low RNA content. Progressive cell death occurred in cultures maintained with Pannon for up to 7 d in proportion to its concentration. Reductions of 50 and 90% in clonogenicity of A-253 cells were observed during their growth in the presence of 0.13 and 1.5 micrograms/ml of this protein, respectively. Exponentially growing cells were more sensitive to Pannon compared with cells from confluent cultures. Colonies of A-253 cells growing in the presence of Pannon were much smaller in size compared with control colonies, indicating that the rate of proliferation of clonogens is reduced by this agent. It appears that P-30 Protein induces cytostatic effects via modulation of cell transition to quiescence or differentiation. The mechanism of its cytotoxic activity is unclear.