Effect of culture conditions on androgen sensitivity of the human prostatic cancer cell line LNCaP.

Several effects of androgens on LNCaP-FGC prostate tumor cells showed a biphasic pattern. Stimulation of growth and inhibition of secretion of prostatic acid phosphatase (PAP) was observed at low androgen concentrations (below 1 nM of the synthetic androgen R1881), and inhibition of growth and stimulation of PAP secretion was observed at higher concentrations. In contrast, prostate specific antigen (PSA) secretion did not show this biphasic response pattern. Comparable effects were found for two sublines of the LNCaP-FGC cells: an early (passage 20, androgen-dependent) and relatively late (passage 70, androgen-sensitive) passage of the cells. Culturing of both sublines in the presence of a high concentration of androgens (10 nM R1881) resulted initially in a decrease in growth rate, but the cells started to proliferate within 3 weeks. These cells became less sensitive to androgens, lost their biphasic response pattern, and showed reduced androgen receptor levels. Three weeks after removal of the excess of androgens, the passage 70 cells regained a biphasic growth response to androgens. Culture in medium without steroids but with EGF resulted in a decrease of both androgen sensitivity and androgen receptor level. In conclusion, rapid changes of the androgen sensitivity and receptor level of the LNCaP cells occurred under the influence of culture conditions. These changes were partly reversible and, therefore, were most likely due to adaptation of the cells.

Effects of tumor necrosis factor on prostacyclin production and the barrier function of human endothelial cell monolayers.

The endothelium controls the influx of macromolecules into the tissues, a process that may be disturbed at sites of inflammation and in atherosclerotic plaques. In this article, we report our evaluations of the effects of the inflammatory mediator, tumor necrosis factor-alpha (TNF-alpha), on the production of prostacyclin and the barrier function of human endothelial cell monolayers in an in vitro model. TNF-alpha (500 units/ml) had no direct effect on the passage of sucrose, peroxidase, and low density lipoprotein through monolayers of human aortic endothelial cells. On the other hand, during the first hours after addition 500 units/ml TNF-alpha induced a reduction of the permeability of umbilical artery endothelial cell monolayers. Within 10 minutes TNF-alpha induced an increase in prostacyclin production by primary cultures of umbilical artery endothelial cells. However, the reduction in permeability was not caused by a change in prostacyclin production or by a change in cyclic AMP concentration because 1) the effect of TNF-alpha on permeability was not prevented by aspirin, 2) no change in the cellular cyclic AMP concentration could be observed after addition of TNF-alpha, and 3) TNF-alpha was still able to reduce the passage rate in the presence of 25 microM forskolin. The reduction in permeability was accompanied by a decrease of F-actin in stress fibers. With prolonged incubation with TNF-alpha, the permeability of umbilical artery endothelial cell monolayers increased, and F-actin was found again in stress fibers. However, these effects of TNF-alpha were only significant at high concentrations of TNF-alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Norepinephrine and iloprost improve barrier function of human endothelial cell monolayers: role of cAMP.

The barrier function of human artery endothelial cells was improved by addition of agents that increase the cellular adenosine 3',5'-cyclic monophosphate (cAMP) concentration. Together with a decrease in the passage rate of peroxidase, an increase in the transendothelial electrical resistance was observed. A direct correlation was found between the relative increases in cellular cAMP concentration and the relative decrease in peroxidase passage after incubation of the cells with forskolin (0.25 and 2.5 microM), the beta-adrenergic agonist isoproterenol (10 microM), and the stable prostacyclin analogue iloprost (10 microM). Norepinephrine (10 microM) reduced the peroxidase passage to a much larger extent (40% reduction) than might be expected on the basis of a small increase of cAMP concentration. This small increase in cAMP (44%) was the result of interactions of norepinephrine with beta-adrenergic receptors, which increase cAMP, and alpha-adrenergic receptors, which decrease cAMP. The relatively strong reduction in permeability (also found in the presence of the alpha-adrenergic antagonist phentolamine) suggests that an additional cAMP-independent mechanism underlaid the barrier-improving effect of norepinephrine. A marked elevation of cAMP by forskolin was accompanied by a disappearance of F-actin and myosin from stress fibers. They were found diffusely spread over the cell, and F-actin in the cell periphery became prominently visible.

Complete and partial deficiencies of complement factor D in a Dutch family.

A young man suffering from recurrent Neisseria infections was shown to lack detectable serum complement factor D hemolytic activity. Addition to the patient's serum of purified factor D to a final concentration of 1 microgram/ml resulted in full restoration of the activity of the alternative pathway. Using an enzyme-linked immunosorbent assay, it was shown that the patient's serum did not contain measurable amounts of factor D antigen either. The sister, the father, as well as the parents of the mother had factor D levels within the normal range, and the factor D level of the mother was decreased. The capacity of the patient's serum, at concentrations up to 5%, to promote phagocytosis of Escherichia coli by normal human granulocytes was low when compared to normal serum. Substitution of the patient's serum with purified factor D resulted in a full restoration of opsonic activity. This study describes the first complete deficiency of factor D, and demonstrates its possible relation to recurrent Neisseria infections.

Passage of low density lipoproteins through monolayers of human arterial endothelial cells. Effects of vasoactive substances in an in vitro model.

The endothelium controls the influx of lipoproteins into the arterial wall, a process that may be disturbed in arteriosclerotic blood vessels. We have used an in vitro model to investigate the characteristics of the passage of low density lipoproteins (LDL) through monolayers of human arterial endothelial cells. Umbilical artery, aorta, or carotid artery endothelial cells were cultured on polycarbonate filters and formed a tight monolayer in which the cells were connected by tight junctions. Passage of 125I-LDL through these monolayers proceeded linearly over a 24-hour period. It was threefold lower through monolayers of aorta or carotid artery cells than through monolayers of umbilical artery cells. The LDL passage process did not show saturation with LDL concentrations up to 800 micrograms/ml LDL-protein (i.e., 1.6 nmol/ml apolipoprotein B) between 2 and 4 hours after addition. However, during the first 30 to 60 minutes after addition of high concentrations of LDL, a reduction of the passage rate of both LDL and peroxidase, resulting in an apparent saturation of the passage process, was observed. The passage rate of the negatively charged acetylated LDL was twofold lower than that of native LDL. Addition of histamine to the endothelial monolayer resulted in a large, but transient, increase in permeability paralleled by a decrease in electrical resistance. The effects of histamine were mediated via an H1 receptor. Thrombin and Ca++ ionophore also induced an increase in permeability of the monolayer, while bradykinin did not. The effects of histamine and thrombin were paralleled by a rapid and marked increase in cytoplasmatic Ca++ concentration of the endothelial cells, while bradykinin induced only a small increase. Although the cyclic adenosine 5'-monophosphate-elevating agent, forskolin, markedly decreased the basal rate of LDL passage through the endothelial cell monolayers, it did not change the relative increase in permeability induced by histamine. Thus, histamine induces small, but significant, increases in the permeability of tight endothelial cell monolayers.

Characterization of an in vitro model to study the permeability of human arterial endothelial cell monolayers.

A model has been developed to study the transport of fluid and macromolecules through human arterial umbilical cord endothelial cell monolayers in vitro. Cells were cultured on fibronectin-coated polycarbonate filters and formed within a few days a tight monolayer, with an electrical resistance of 17 +/- 4 Ohm.cm2. The cells were connected by close cell contacts with tight junctions. The passage-rate of horse radish peroxidase (HRP) through these filters was 20-40 fold lower than through filters without an endothelial monolayer. The continuous presence of 10% human serum was needed to maintain the electrical resistance of the monolayer and its barrier function towards macromolecules. Chelation of extracellular calcium resulted in an increased permeability and a decreased electrical resistance of the monolayer. This process was reversible by re-addition of calcium ions to the cells. The permeation rate of dextrans of various molecular weights (9-480 kD) was inversely related to the molecular mass of the molecule. No difference was measured between the passage rate of dextran of 480 kD and dextran of 2,000 kD. Incubation of the endothelial cell monolayer with 2-deoxy-D-glucose resulted in a decreased permeability but it had no effect on electrical resistance. This suggests that the passage-process is energy-dependent. Fluid permeation through the endothelial cell monolayer on filters was measured in a perfusion chamber under 20 mmHg hydrostatic pressure. It was decreased by the presence of serum proteins and responded reversibly on the chelation and re-addition of extracellular calcium ions.

Detection of low dose effects of psychopharmaca: Application of a semi-Marcov model to rhesus monkey behaviour.

Behavioural testing of psycho-active drugs at lower doses is needed for medical applications and for the study of pharmacological brain mechanisms. Sensitive methods to detect effects on the time structure of behaviour are lacking. We propose a procedure based on a description within the framework of continuous time Marcov chain models. This class of models is generalized to account for constraints on the organisation of a motor pattern including the speed at which it is terminated. This leads to semi-Marcov models with transition rates that quickly increase from zero to a constant level. We describe and compare several models. As an application we consider amphetamine effects on infant rhesus monkeys. One of the models is preferred since, besides giving a good fit, it demonstrates consistent amphetamine effects that can be interpreted as short-term effects on motor constraints. Amphitamine also induced more mother-directed behaviour. We give maximum likelihood procedures for behaviour analysis based on the preferred model.