Alan P. Johnstone

Nitric oxide protects human extravillous trophoblast cells from apoptosis by a cyclic GMP-dependent mechanism and independently of caspase 3 nitrosylation.

Apoptosis is thought to play an important regulatory role in placental development and inappropriate trophoblast apoptosis has been implicated in complications of pregnancy such as pre-eclampsia. Here we show that apoptosis of a human extravillous trophoblast-derived cell line (SGHPL-4) can be regulated by nitric oxide (NO). Nitric oxide produced exogenously by the addition of NO donors was able to delay or inhibit apoptosis induced by a combination of tumour necrosis factor alpha and actinomycin D and to suppress the activity of caspase 3. Treatment with hepatocyte growth factor (HGF) stimulated expression of the inducible isoform of NO synthase and was also able to protect SGHPL-4 cells from caspase 3 activation and apoptosis. The inhibition of basal NO production with NO synthase inhibitors was shown to sensitise cells to apoptotic stimuli and to reduce the level of endogenous caspase 3 nitrosylation. The anti-apoptotic effects of NO in these extravillous trophoblast cells appear to be mediated through the production of cyclic GMP as inhibitors of soluble guanylate cyclase inhibited the protective effect of both HGF and NO donors.

MONOCLONAL ANTIBODIES THAT RECOGNIZE THE NATIVE HUMAN THYROTROPIN RECEPTOR

Monoclonal antibodies have been produced that recognize the native human thyrotropin receptor by using a sensitive screening protocol based on flow cytofluorimetry combined with recombinant eukaryotic cells expressing high levels of the full-length functional receptor. The more standard screening method of ELISA preferentially selected antibodies that only reacted with the denatured receptor. Mice were immunized with recombinant receptor produced in either eukaryotic or prokaryotic systems; after screening and cloning, three stable hybridoma lines were established. An IgM antibody (7B5) produced in response to the eukaryotic material recognized only the native receptor (by flow cytofluorimetry) and did not react with denatured material on ELISA or immunoblotting, suggesting that its epitope is conformational. In contrast, two IgG1 antibodies (2C11 and 3B12) produced in response to the prokaryotic material recognized both native and denatured receptor (by flow cytofluorimetry, immunoprecipitation and immunoblotting). The use of different recombinant constructs in the immunoblotting procedure allowed the epitopes for both the IgG1 antibodies to be assigned to the region 125-369. None of the antibodies stimulated production of cAMP by recombinant cells expressing the full-length functional receptor, but one of the IgG1 antibodies (2C11) did inhibit binding of radiolabelled thyrotropin to these same cells. These antibodies, and others that can now be produced with this screening protocol, will help define the relationship between structure and function of this important receptor.

Cytotoxic effects of amiodarone and desethylamiodarone on human thyrocytes

Since recent in vivo evidence suggests that the benzofuran antiarrhythmic drug amiodarone has a direct toxic effect on the human thyroid gland, we have investigated the effects of both amiodarone and its metabolite desethylamiodarone on a novel immortalized functional human thyrocyte line (SGHTL-34 cells). Desethylamiodarone markedly reduced cell number as assessed from both DNA and protein content. Few cells were left after 24 hr exposure to 12.5 micrograms/ml; the concentration producing death of 50% of cells (EC50) was 6.8 +/- 1.1 micrograms/ml (mean +/- SE, N = 15). Amiodarone was much less potent, producing a maximum decrease in cell number of approximately 25% at concentrations up to 50 micrograms/ml. The effect of desethylamiodarone was seen within 24 hr of culture. T3 in concentrations up to 0.75 micrograms/ml had no effect on the action of amiodarone or desethylamiodarone on SGHTL-34 cells. Light microscopy demonstrated vacuolation of SGHTL-34 cells after 4-day culture with either the drug or its metabolite. Studies using primary cultures of human retroorbital fibroblasts demonstrated that the greater cytotoxicity of desethylamiodarone was not confined to thyrocytes. When SGHTL-34 cells were incubated with 2.5 micrograms/ml desethylamiodarone for 4 days, 71.7 +/- 0.9% was taken up by the cells; there was no detectable conversion to amiodarone. Incubation of thyrocytes with 50 micrograms/ml amiodarone for 4 days resulted in the uptake of 80.1 +/- 2.1% by the cells. In addition, 5.0 +/- 0.1% of the amiodarone was converted to material with the same retention time as desethylamiodarone standard; of this material, 72.9 +/- 2.8% was taken up by the cells. We conclude that desethylamiodarone, at concentrations near those found in the plasma of patients on long-term amiodarone therapy, exerts a direct cytotoxic effect on human thyroid cells in short-term culture. This effect may play a role in the aetiology of clinical thyroid disease during amiodarone therapy. We suggest that, since the effect is not restricted to thyrocytes, desethylamiodarone may play a role in the aetiology of amiodarone toxicity which occurs clinically in many tissues.

The role of tumour-derived iNOS in tumour progression and angiogenesis

Background:Progressive tumour growth is dependent on the development of a functional tumour vasculature and highly regulated by growth factors and cytokines. Nitric oxide (NO) is a free radical, produced both by tumour and host cells, and functions as a signalling molecule downstream of several angiogenic factors. Both pro- and antitumourigenic properties have been attributed to NO.Methods:The expression of the inducible isoform of NO synthase (iNOS) was knocked down in the C6 glioma cell line using constitutive expression of antisense RNA, and the effect of tumour-derived NO on tumour progression and angiogenesis was investigated.Results:Tumours in which iNOS expression was decreased displayed significantly reduced growth rates compared with tumours derived from parental C6 cells. Quantitative non-invasive magnetic resonance imaging and fluorescence microscopy of tumour uptake of Hoechst 33342, and haematoxylin and eosin staining, revealed significantly impaired vascular development and function in antisense iNOS tumours compared with control in vivo, primarily associated with the more necrotic tumour core. Decreased iNOS expression had no effect on tumour VEGF expression.Conclusion:Nitric oxide derived from tumour iNOS is an important modulator of tumour progression and angiogenesis in C6 gliomas and further supports the therapeutic strategy of inhibiting iNOS for the treatment of cancer.

ADP-ribosyl transferase, rearrangement of DNA, and cell differentiation

Cell differentiation is the process by which genetic information is selectively expressed to produce cells with various morphologies and functions. The integrated changes necessary for this fundamentally important process have recently been the subject of intense study. This review will summarize data from several laboratories correlating differentiation with the activity of the enzyme ADP-ribosyl transferase and with changes in single-strand DNA breaks in various diverse eukaryotic systems. We will then discuss the implications of these observations for differentiation in general, including the possibility that rearrangement of geneticmaterialisa widespread mechanism for controlling gene expression.

Changes in mRNA levels of poly(ADP-ribose) polymerase during activation of human lymphocytes

The level of mRNA encoding the nuclear enzyme poly(ADP-ribose) polymerase (ADP-ribosyltransferase, EC 2.4.2.30) was found to be very low in quiescent human lymphocytes and to increase at least 10-fold between 1 and 2 dyas after stimulation with the mitogen phytohaemagglutinin, staying high for several days thereafter. This increase was inhibited by 3-methoxybenzamide (a competitive inhibitor of poly(ADP-ribose) polymerase) but was not affected significantly by aphidicolin. Incubation of activated cells with cycloheximide for 2 h increased the expression slightly. These data demonstrate that, during lymphocyte activation, the level of mRNA of the poly(ADP-ribose) polymerase gene correlates with, and hence is presumably responsible for, the increase in poly(ADP-ribose) polymerase protein detectable by enzyme assay or immunochemistry.

NAD metabolism and mitogen stimulation of human lymphocytes

The NAD concentration in eukaryotic cells is an important parameter for many aspects of metabolism including differentiation. As reported by other workers, the NAD content of resting human peripheral blood lymphocytes was low and increased dramatically over a period of 3 days after stimulation with the mitogen phytohemagglutinin (PHA). However, simultaneous measurement of the mean cell volumes showed that the average NAD concentration in fresh quiescent lymphocytes (401 +/- 128 microM) (SD, n = 7) was similar to that observed for other cell types. Furthermore, because of the increase in cell volume which occurred on mitogen stimulation, the NAD concentration in stimulated lymphocytes was only 2-3-fold higher than in fresh resting cells. This increase was also observed in lymphocytes incubated without mitogen and was apparently due to the level of NAD precursors in the culture medium and serum supplement. Hence, the NAD concentration in resting and stimulated lymphocytes is comparable to that of other eukaryotic cells and the variations in NAD content reported earlier have been widely misinterpreted.

Chronic lymphocytic leukaemia and its relationship to normal B lymphopoiesis.

The leukaemic cells from patients with chronic lymphocytic leukaemia have long been thought to represent circulating virgin B lymphocytes. In this article Alan Johnstone discusses the accumulated data from structural and functional analyses which place the leukaemic cells at a stage intermediate between the pre-B cell and mature B lymphocyte, a stage not normally found in the circulation.

Increased angiogenic factor secretion by decidual natural killer cells from pregnancies with high uterine artery resistance alters trophoblast function

STUDY QUESTION Are the concentrations of factors secreted by decidual natural killer (dNK) cells from pregnancies at high risk of poor spiral artery remodelling different to those secreted from pregnancies at low risk? SUMMARY ANSWER Expression levels of PLGF, sIL-2R, endostatin and angiogenin were significantly increased by dNK cells from high-risk pregnancies, and angiogenin and endostatin were found to alter trophoblast function. WHAT IS KNOWN ALREADY During early pregnancy, maternal uterine spiral arteries are remodelled from small diameter, low-flow, high-resistance vessels into larger diameter, higher flow vessels, with low-resistance. This change is essential for the developing fetus to obtain sufficient oxygen and nutrients. dNK cells have been implicated in this process. STUDY DESIGN, SIZE, DURATION dNK cells were isolated from first trimester terminations of pregnancies (obtained with local ethical approval) screened for normal- or high-resistance index, indicative of cases least (<1%) and most (>21%) likely to have developed pre-eclampsia had the pregnancy not been terminated (n = 18 each group). Secreted factors and the effects of these on the trophoblast cell line, SGHPL-4, were assessed in vitro. PARTICIPANTS/MATERIALS, SETTING, METHODS A multiplex assay was used to assess dNK cell-secreted factors. SGHPL-4 cell functions were assessed using time-lapse microscopy, 3D invasion assays, endothelial-like tube formation ability and western blot analysis. MAIN RESULTS AND THE ROLE OF CHANCE The expression levels of PLGF (P < 0.01), sIL-2R (P < 0.01), endostatin (P < 0.05) and angiogenin (P < 0.05) were significantly increased by dNK cells from high-risk pregnancies. Endostatin significantly decreased SGHPL-4 invasion (P < 0.05), SGHPL-4 tube formation (P < 0.05) and SGHPL-4 Aktser473 phosphorylation (P < 0.05). Angiogenin significantly decreased SGHPL-4 invasion (P < 0.05), but increased SGHPL-4 tube formation (P < 0.01) and decreased SGHPL-4 Aktser473 phosphorylation (P < 0.05). LIMITATIONS, REASONS FOR CAUTION The culture of dNK cells and protein concentrations in vitro may not fully represent the in vivo situation. Although SGHPL-4 cells are extravillous trophoblast derived, further studies would be needed to confirm the roles of angiogenin and endostatin in vivo. WIDER IMPLICATIONS OF THE FINDINGS The altered expression of secreted factors of dNK cells may contribute to pregnancy disorders associated with poor spiral artery remodelling. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by the Wellcome Trust (project reference 091550). R.F. was a recipient of a PhD studentship from the Division of Biomedical Sciences, St. Georges, University of London. The authors have no conflict of interests.

Hepatocyte growth factor-induced endothelial cell motility is mediated by the upregulation of inducible nitric oxide synthase expression

OBJECTIVES Hepatocyte growth factor (HGF) is an angiogenic mitogen which stimulates migration in various cell types and has been shown to induce the production of nitric oxide (NO) in epithelial cells. Conflicting data exist on the effect of NO on endothelial cell migration. The aim of this study was to investigate a possible role for NO in HGF-stimulated endothelial cell motility. METHODS The study was performed primarily using an endothelial cell line derived from adult human saphenous vein. Transient transfection experiments were additionally performed using an adult human coronary artery endothelial cell line. Nitric oxide synthase expression was examined by western blot analysis. Time-lapse digital image microscopy was used to measure cell motility. A DNA construct was used in transient transfections to over-express inducible nitric oxide synthase (iNOS) as an N-terminal fusion to enhanced green fluorescent protein (EGFP). RESULTS HGF upregulated the expression of iNOS but not constitutive endothelial nitric oxide synthase (eNOS). Treatment of cells with the specific iNOS inhibitor 1400 W revealed that functional iNOS was required for HGF-stimulated endothelial cell motility. HGF-induced iNOS expression was partially abrogated in the presence of the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor LY294002, but not the Src kinase inhibitor, PP1. Endothelial cell motility increased significantly (P<0.0001) in the presence of the exogenous NO donor spermine-NO and cells expressing the iNOS-EGFP fusion protein exhibited significantly greater (P=0.0038) motility than those expressing EGFP alone. CONCLUSIONS These combined data show that elevated NO production is sufficient to stimulate endothelial cell motility and link HGF and NO, both previously implicated in modulating motility, in a common signalling pathway.