Statins block de novo synthesis of cholesterol by inhibiting the enzyme, HMG CoA reductase. The product of this reaction, mevalonic acid, is also a precursor of isoprenoids, molecules required for the activation of signalling G-proteins, such as Ras. Signal transduction pathways involving Ras are important for cell survival and this may be why statins induce apoptotic death of several cell types. Given that statins are used to treat vascular disease, it is surprising that no studies have been conducted on vascular endothelial cells. For this reason, we have tested the effect of fluvastatin (FS) on the endothelial cell line EA.hy 926. Here we show that FS, at concentrations from 1 to 2 µM, blocks growth and induces apoptosis of the endothelial cell line, EA.hy 926. As considerable redundancy exists in cell signalling pathways for cell survival, toxicity of FS under more physiological conditions might be prevented by pathways that do not require Ras, such as those activated by adrenal or sex steroids. To test this hypothesis, first RT-PCR analysis was performed for nuclear receptor mRNA expression. This revealed the presence of mRNA for the androgen receptor (AR) and glucocorticoid receptor (GR). The effect of the AR agonist, dihydrotestosterone (DHT), and the GR agonist, dexamethasone (Dex), was then tested. Whilst DHT (100 nM) had no effect on FS-induced cell death, Dex (1 µM) blocked FS-induced apoptosis. Cell cycle analysis revealed that 24 h exposure to FS prevented cells from leaving G 1 and 24-48 h later a marked sub-G 1 peak was observed. Dex was able to reduce the sub-G 1 peak, but it failed to reduce accumulation of cells in G 1 . Further studies revealed that, in addition to blocking FS-induced apoptosis, Dex was able to block apoptosis of EA.hy 926 cells induced by serum deprivation, tumour necrosis factor-, oxidants, DNA damage and mitochondrial disruption. This study strongly suggests that glucocorticoids have a role to play in preventing vascular injury and they may provide a reason why statins are apparently not toxic to vascular endothelial cells in vivo.
Sesamol is a potent inhibitor of fungal fatty acid biosynthesis. This effect is apparently due to inhibition of malic enzyme and the supply of NADPH that is required for this biosynthetic pathway. It is the ability of sesamol to reduce the synthesis of the coenzyme, NADPH, that makes it attractive for use in studying the effect of oxidants on tumor and vascular endothelial cells. By conducting preliminary studies on the effect of sesamol alone, it was clear that the compound demonstrated marked cytotoxicity. This paper describes the experiments performed.
Development of the long-term culture models of haematopoietic stem cells (HSCs) is one of the important tasks in modern biotechnology. It has been suggested that stromal presence is important for haematopoiesis in vitro and in vivo, but the question remains: whether diffusible factors produced by stromal cells are sufficient for the regeneration of primitive and definitive haematopoietic cells, or direct cell-to-cell contacts of the cultured material with underlying stromal base would be required. During present studies, influence of various feeder layers and feeder layer conditioned media on proliferative, differentiative and clonogenic activity of human AC133+ derived from human umbilical cord blood was investigated. Cell extracts for feeder layers were prepared from 4-6 weeks old human embryos and co-cultured feeder cells. Effects of the conditioned media were also determined. Culture and feeder layer media were additionally supplemented with commonly implemented factors such as GM-CSF, IL-3 and LIF. Estimation of morpho-functional properties of AC133+ cultivated suspension cultures was performed in subculture experiments using semisolid agar culture conditions. Multipotential CFU-MIX (CFU-GEMM) and unipotential progenitor cells CFU-GM, BFU-E and CFU-E were observed and analyzed. Our data suggest that haematopoiesis can be sustained for prolonged cultivation periods in the presence of feeder layer cells or conditioned media supported culture models. Prolonged support of primitive haematopoietic cells and their clonogenic capacity and functional characteristics in feeder layer positive cultures, indicates that diffusible factors are sufficient for haematopoiesis and suggests that direct cell-to-cell contacts may not be exclusively required for successful long-term in vitro haematopoiesis.
The oestrogen receptor is fundamental to the growth and survival of the rat pituitary tumour cell line, GH 3 . Our previous studies have shown that antioestrogens such as RU 58668 and ZM 182780 will reduce the rate of cell division and also induce cell death. Death of these cells in response to antioestrogen treatment appears to be due to a heightened sensitivity to reactive oxygen species (ROS). As part of a study to determine the cross-talk between steroid receptor systems in these cells, we have observed that the glucocorticoid, dexamethasone (Dex), inhibits antioestrogen-induced cell death. Cell death induced by H 2 O 2 is enhanced by ZM 182780 and this effect is also blocked by Dex. As apoptotic cell death in a number of systems involves an early loss of mitochondrial membrane potential ( m ), we have performed detailed studies on the time-course of m loss in relation to the loss in cell membrane function. These studies have indicated that a loss of m parallels a loss of cell membrane functionthis is more characteristic of necrosis than of apoptosis.From microscopic observations of these cells in response to H 2 O 2 , it has been noted that early cell membrane blebbing, induced by H 2 O 2 , is blocked in the presence of ZM 182780. Cell membrane blebbing can precede necrosis as well as apoptosis and it is thought to involve cytoskeletal changes, for which localised glycolytic reactions provide ATP. These observations, together with those showing that removal of glucose, but not inhibition of mitochondrial function, enhances ROS-induced cell death, prompted studies on the glycolytic pathway. As a strong candidate mechanism, it would appear that, via an effect on one of the rate-limiting glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase, Dex is able to overcome the antioestrogen-enhanced loss of glycolytic function following exposure of cells to ROS. This report contributes to the growing body of evidence showing that glucocorticoids provide a survival advantage to both normal and tumour cell types.
As an enzyme implicated in the stress response, we investigated poly(ADP-ribose) polymerase (PARP) in the response of GH3 rat pituitary tumor cells to oxidants. These cells are unusual in that they undergo rapid cell death (90 min) with low doses of the prooxidant, H2O2 (50-200 microm), whereas at higher doses (1 mm), death occurs some hours later (4-5 h). Measurement of PARP activity shows that low doses of H2O2 (50-200 microm) fail to increase the activity of PARP, whereas at 0.5 and 1 mm, the enzyme becomes activated. In parallel with the activation of PARP, cellular ATP concentrations fall at high H2O2 doses and the PARP inhibitors, 3-aminobenzamide and nicotinamide (NIC) partially prevent this fall. Using NIC to inhibit PARP activity, we show that treatment of cells with NIC before the addition of H2O2 (0.5-1 mm), results in rapid cell death (90 min). In contrast, prior exposure to H2O2 (0.5-1 mm) for 1 h, before withdrawal and exposure to 1 mm NIC, allows cell survival for many hours. These data suggest that PARP is involved in blocking rapid death of GH3 cells in response to oxidants. In contrast to other cell types tested here, in which inhibitor studies show that PARP is activated at low H2O2 doses and this decreases the extent of apoptosis, GH3 cells are unable to sufficiently activate PARP to prevent rapid cell death.
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