Lack of effect of dihydroergotamine on endothelial and smooth muscle cell proliferation and endothelial cell prostanoid production

The most important effect of dihydroergotamine is venoconstriction, but certain metabolic effects and changes in vessel prostanoid activity have also been suggested. In this study endothelial cell production of 6-keto PGF1 alpha and TxB2 was quantitated in vitro. No evidence of altered prostanoid production was noted after incubation with dihydroergotamine (exposure ranging from 5 x 10(-3) to 5 x 10(-7) g/l). Similarly, no effect of dihydroergotamine on the growth rates of endothelial cells or smooth muscle cells in vitro was documented.     

Fatty acid modulation of antiestrogen action and antiestrogen-binding protein in cultured lymphoid cells

Nonsteroidal antiestrogens reversibly and specifically inhibited the proliferation of two estrogen receptor-negative lymphoid cell lines (EL4 and Raji) in a dose-dependent manner. [3H]Thymidine incorporation of concanavalin A-stimulated primary splenocytes was also inhibited by 10(-6) M clomiphene (1-[4-(2-diethylaminoethoxy)phenyl]-1,2-diphenyl-2-chloroethylene). The antiproliferative effect could be prevented by the simultaneous presence in the growth medium of 10(-5) M linoleic acid or 10(-5) M arachidonic acid but not by 10(-6) M estradiol. Both lymphoid cell lines had high affinity antiestrogen-binding sites whose affinity could be altered by conditions of growth. Growth of EL4 cells in RPMI 1640 medium supplemented with charcoal-pretreated 5% fetal calf serum (charcoal-stripped medium) resulted in significantly higher affinity (Kd 0.54 nM +/- 0.11 nM; n = 6) than growth in medium supplemented with untreated serum (complete medium) (Kd = 1.68 nM +/- 0.48 nM; n = 6) (p less than 0.001). This change in affinity was partly due to removal of fatty acids from the growth medium by charcoal pretreatment, since addition of 10(-5) M linoleic acid or 10(-5) M gamma-linolenic to charcoal-stripped medium decreased the affinity of the antiestrogen-binding protein. In contrast, growth in 10(-5) M stearic acid or 10(-5) M oleic acid did not significantly alter the affinity of the antiestrogen-binding protein, whereas 10(-5) M palmitic acid significantly increased its affinity. The same fatty acids were also tested for their intrinsic effects on EL4 cell proliferation. Oleic, linoleic and gamma-linolenic acids were growth stimulatory while stearic and palmitic acids were not. Thus linoleic and gamma-linolenic acids whose presence in the growth medium was associated with decreased affinity of [3H]tamoxifen (1-[4-(2-dimethylaminoethoxy)phenyl]-1,2-diphenylbut-1(Z)-ene) binding to the intracellular antiestrogen-binding protein were also growth stimulatory. Unsaturated fatty acids have previously been shown to inhibit binding of [3H]tamoxifen to the antiestrogen-binding protein in a cell-free system. The present observations demonstrate that unsaturated fatty acids also modify the affinity of the antiestrogen-binding protein in intact cells.     

JNK1-dependent antimitotic activity of thiazolidin compounds in human non-small-cell lung and colon cancer cells

We recently identified two thiazolidin compounds, 5-[(4-methylphenyl)methylene]-2-(phenylamino)-4(5H)-thiazolone (MMPT) and 5-(2,4-dihydroxybenzylidene)-2-(phenylimino)-1,3-thiazolidin (DBPT), that inhibit the growth of human non-small-cell lung and colon cancer cells independent of P-glycoprotein and p53 status. Here we further investigated the mechanism by which these thiazolidin compounds mediate their anticancer effects. Treatment of cancer cells with MMPT and DBPT led to a time-dependent accumulation of cells arrested in the G2/M phase with modulation of the expression of proteins such as cyclin B1, cdc25C, and phosphorylated histone H3. Moreover, treatment with MMPT and DBPT increased M-phase arrest with abnormal spindle formation. DBPT-mediated G2/M phase arrest and phosphorylation of cdc25C and histone H3 were abrogated when JNK activation was blocked either with SP600125, a specific JNK inhibitor, or a dominant-negative JNK1 gene. Moreover, DBPT-mediated microtubule disruption was also blocked by SP600125 treatment. Our results demonstrate that thiazolidin compounds can effectively induce G2/M arrest in cancer cells and that this G2/M arrest requires JNK activation.     

Modulation of carcinoembryonic antigen release by HT-29 colon carcinoma line in the presence of different agents

In this study we followed the effects of various differentiating agents on the expression of carcinoembryonic antigen (CEA) released into the medium by a colon carcinoma cell line HT-29. Butyric acid 1 mM markedly increased the level of CEA (12-fold in comparison to control levels). 12-O-tetradecanoyl-phorbol-13-acetate (TPA) 50 ng/ml and 5-azacytidine 4 x 10(-6) M increased the amount of CEA, 2- and 1.5-fold respectively. On the other hand retinoic acid 10(-5) M, N methyl-formamide 1% and N,N hexamethylene bisacetamide 2.5 mM decreased CEA 2-, 4- and 3-fold respectively. Our results emphasize that various differentiating agents affect CEA levels differently. Thus changes in CEA levels appear not to be reliable as a marker of a more differentiated phenotype.     

MAPK uncouples cell cycle progression from cell spreading and cytoskeletal organization in cycling cells

Integrin-mediated cytoskeletal tension supports growth-factor-induced proliferation, and disruption of the actin cytoskeleton in growth factor-stimulated cells prevents the re-expression of cyclin D and cell cycle re-entry from quiescence. In contrast to cells that enter the cell cycle from G0, cycling cells continuously express cyclin D, and are subject to major cell shape changes during the cell cycle. Here, we investigated the cell cycle requirements for cytoskeletal tension and cell spreading in cycling mammalian cells that enter G1-phase from mitosis. Disruption of the actin cytoskeleton at progressive time-points in G1-phase induced cell rounding, FA disassembly, and attenuated both integrin signaling and growth factor-induced p44/p42 mitogen-activated protein kinase activation. Although cyclin D expression was reduced, the expression of cyclin A and entry into S-phase were not affected. Moreover, expression of cyclin B1, progression through G2- and M-phase, and commitment to a new cell cycle occurred normally. In contrast, cell cycle progression was strongly prevented by inhibition of MAPK activity in G1-phase, whereas cell spreading, cytoskeletal organization, and integrin signaling were not impaired. MAPK inhibition also prevented cytoskeleton-independent cell cycle progression. Thus, these results uncouple the requirements for cell spreading and cytoskeletal organization from MAPK signaling, and show that cycling mammalian cells can proliferate independently of actin stress fibers, focal adhesions, or cell spreading, as long as a threshold level of MAPK activity is sustained.     

Influence of cyclic nucleotides on protein synthesis in vascular smooth muscle.

The incorporation of leucine-14C into protein in bovine mesenteric arteries was augmented by cyclic GMP (10-3 M) and decreased by cyclic AMP (10-3 M). There was no effect of 5'AMP (10-3 M). The phosphodiesterase inhibiting drugs theophylline (10-3 M) and papaverine (5 x 10-5 g/ml) both decreased the leucine-14C incorporation.     

Effect of sodium octanoate on leucine incorporation into protein of rat liver slices and of Yoshida ascites hepatoma cells.

7.38 X 10(-4) M octanoate does not significantly modify leucine incorporation into protein of rat liver slices, while in hepatoma cells a 19% inhibition has been noted. 3.69 x 10(-3) M octanoate reduces leucine incorporation to about the same extent (71-76%) in both liver slices and hepatoma cells.     

Drug interactions in intestinal absorption of 3H-digitoxin in rats.

The absorption of 3H-digitoxin from perfused rat small intestine was inhibited by probenecid (1.0 x 10-2 M), ethacrynic acid (0.5 x 10-3 M), and mersalyl (8.0 x 10-3 M) indicating that digitoxin absorption is at least partly an active process.     

Fatty acid modulation of antiestrogen action and antiestrogen-binding protein in cultured lymphoid cells

Summary Nonsteroidal antiestrogens reversibly and specifically inhibited the proliferation of two estrogen receptornegative lymphoid cell lines (EL4 and Raji) in a dose-dependent manner. [³H]Thymidine incorporation of concanavalin A-stimulated primary splenocytes was also inhibited by 10^–6 M clomiphene (1-[4-(2-diethylaminoethoxy)phenyl]-1,2-diphenyl-2-chloroethylene). The antiproliferative effect could be prevented by the simultaneous presence in the growth medium of 10^–5 M linoleic acid or 10^–5 M arachidonic acid but not by 10^–6 M estradiol. Both lymphoid cell lines had high affinity antiestrogen-binding sites whose affinity could be altered by conditions of growth. Growth of EL4 cells in RPMI 1640 medium supplemented with charcoal-pretreated 5% fetal calf serum (charcoal-stripped medium) resulted in significantly higher affinity (K_d 0.54 nM±0.11 nM; n=6) than growth in medium supplemented with untreated serum (complete medium) (K_d=1.68 nM±0.48 nM; n=6) (p<0.001). This change in affinity was partly due to removal of fatty acids from the growth medium by charcoal pretreatment, since addition of 10^–5 M linoleic acid or 10^–5 M gamma-linolenic to charcoal-stripped medium decreased the affinity of the antiestrogen-binding protein. In contrast, growth in 10^–5 M stearic acid or 10^–5 M oleic acid did not significantly alter the affinity of the antiestrogen-binding protein, whereas 10^–5 M palmitic acid significantly increased its affinity. The same fatty acids were also tested for their intrinsic effects on EL4 cell proliferation. Oleic, linoleic and gamma-linolenic acids were growth stimulatory while stearic and palmitic acids were not. Thus linoleic and gamma-linolenic acids whose presence in the growth medium was associated with decreased affinity of [³H]tamoxifen (1-[4-(2-dimethylaminoethoxy)phenyl]-1,2-diphenylbut-1(Z)-ene) binding to the intracellular antiestrogen-binding protein were also growth stimulatory. Unsaturated fatty acids have previously been shown to inhibit binding of [³H]tamoxifen to the antiestrogen-binding protein in a cell-free system. The present observations demonstrate that unsaturated fatty acids also modify the affinity of the antiestrogen-binding protein in intact cells.     

Heterogeneous responses of canine basilar and middle cerebral arteries to serotonin at normal and high CO2 tension.

The responses of basilar arteries (BAs) to serotonin were attenuated by high PCO2 (86 +/- 1 mm Hg) and the pH matched acidotic solution (PCO2 37 +/- 1 mm Hg), whereas the responses of middle cerebral arteries (MCAs) were not. High PCO2 decreased the basal tone of both arteries, and the changes in basal tone due to high PCO2 were not influenced by 3 x 10(-7) M imipramine, 10(-5) M pargyline or 10(-4) M aspirin. The responses of BAs to serotonin were attenuated by high PCO2 in the presence of imipramine, pargyline and aspirin. The responses of MCAs to serotonin were not influenced by high PCO2 in the presence of pargyline and aspirin, but attenuated by high PCO2 in the presence of imipramine.     

Androgen receptors in the cytosol of normal mucosa and of pharyngo-laryngeal epitheliomas in humans]

Specific binding for 5 alpha dihydrotestosterone has been characterized in cytosol fraction from human pharyngeal and laryngeal mucosae. The high binding affinity of the protein receptor (Kd 2 x 10(-10)M) and its low capacity are similar in both sexes, and similar in both normal mucosa and epithelioma.     

Modification of theophylline-induced lipolysis in human fat cells after trypsination.

Trypsin-treatment of human fat cells results in the potentiation of the lipolytic response and the cAMP accumulation induced by theophylline (5 . 10(-4) M) but not of those induced by theophylline (5 . 10(-3) M). The amount of cAMP formed after exposure to theophylline (5 . 10(-3) M) plus norepinephrine (5 . 10(-6) M) remains, however, 2.6 fold higher in trypsin-treated human fat cells than in the control ones.     

Electrophysiological actions of chlorimipramine on guinea-pig ventricular fibres.

Chlorimipramine (CMI, 1 x 10(-5)M to 7 X 10(-5)M) decreased the amplitude, overshoot and rate of rise of ventricular action potentials and abolished the Ca-mediated action potentials elicited in guinea-pig papillary muscles. These results indicates that CMI inhibits the rise in sodium and calcium conductances during the cardiac action potential.     

Effects of the adenylate cyclase activator forskolin and its inactive derivative 1,9-dideoxyforskolin on insect cytochrome P-450 dependent steroid hydroxylase activity.

The adenylate cyclase activator forskolin and its pharmacologically inactive derivative 1,9-dideoxyforskolin were found to inhibit in a dose-dependent fashion the ecdysone 20-monooxygenase activity associated with wandering stage larvae of Drosophila melanogaster and fat body and midgut from last instar larvae of the tobacco hornworm, Manduca sexta. The concentrations of these labdane diterpenes required to elicit a 50% inhibition of the cytochrome P-450 dependent steroid hydroxylase activity in the insect tissues ranged from approximately 5 x 10(-6) to 5 x 10(-4) M.     

Interaction of polyamines or their precursors with the calcium-controlled secretion of peroxidase by sugarbeet cells

Summary Three polyamines tested (cadaverine, spermidine and spermine) and their 2 precursors (the amino acids arginine and ornithine) inhibit the Ca²⁺-mediated secretion of peroxidases by sugarbeet cells in suspension culture at concentrations ranging from 10^–15 to 10^–5M. In the absence of exogenous Ca²⁺, spermine added at higher concentrations mimics the activatory effect of Ca²⁺, the other polyamines being without effect.Supported by the Belgian FRFC (grant No. 2.9009.75 to T.G.) and the Swiss National Foundation for Scientific Research (grant No. 3.140-0.81 to C.P. and H.G.).     

Monoclonal antibodies to L-asparaginase

Five hybridomas secreting monoclonal antibody to E. coli L-asparaginase were isolated. These monoclonal antibodies were classified into 3 different subclasses; Ig G1 (1 clone), Ig G2 (2 clones) and Ig G3 (2 clones). One of them possessed anti-L-asparaginase neutralizing activity. Four antibodies examined demonstrated a linear Langmuir binding plot and binding affinities, with equilibrium dissociation constant (Kd) ranging between 2.5 X 10(-9) M and 6.3 X 10(-10) M. The monoclonal antibodies should be useful probes for investigation of the enzyme activity.     

Fructose-1,6-diphosphate reduces acute ECG changes due to doxorubicin in isolated rat heart

Doxorubicin (DXR) (0.17 x 10(-4) M) induces an acute cardiotoxicity in isolated rat heart; there is a progressive widening of the S alpha T segment, with a decrease in force derivatives and in the coronary flow. Concurrent perfusion with fructose-1,6-diphosphate (FDP) (10(-5)-10(-4) M) dose-dependently reduces the S alpha T enlargement but fails to affect the reduction in force derivatives and coronary flow. The target of cardiac protection by FDP might be the ionic mechanisms underlying the action potential configuration.     

Retinoic acid enhances the proliferation of smooth muscle cells

Retinoic acid (RA, 10(-5) - 10(-7) M) is shown to enhance the proliferation of cultured rat aortic smooth muscle cells (SMC). This effect is not connected with a synergistic action of RA together with serum mitogens. Moreover, the expression of L1, a surface antigen specific for modulated SMC entering the cell cycle, is amplified by RA treatment.