Transforming growth factors and the regulation of cell proliferation

The number of different growth regulatory molecules which have been isolated and characterized is continuing to increase. As more information is obtained, it has become apparent that the cooperative actions of many factors with distinct activities is necessary for appropriate proliferative responses. An interplay of both growth stimulatory and growth inhibitory factors is essential for normal growth. Of crucial importance, therefore, is the appropriate regulation of growth factors. Unregulated expression, synthesis, posttranslational processing or activation of either positive or negative growth signals may contribute to neoplastic transformation (Fig. 3). Altered responses to normally positive or negative signals by transformed cells have been demonstrated by several investigators [64, 79, 84]. While altered growth factor responses in transformed cells are well documented, the mechanisms responsible for the loss of growth control are poorly understood and are likely to be both complex and numerous. Continued efforts to dissect and comprehend fully growth factor action on normal cells will be necessary before an understanding of neoplastic transformation can be achieved.     

Control of the pyridine nucleotide-linked Ca release from mitochondria by respiratory substrates

Oxidation of mitochondrial pyridine nucleotides followed by their hydrolysis promotes Ca²⁺ release from intact liver mitochondria. In most of the previous studies oxidation was achieved with pro-oxidants which were added to mitochondria respiring on succinate in the presence of rotenone, a site I-specific inhibitor of the respiratory chain. Here we investigate pro-oxidant dependent and independent Ca²⁺ release from mitochondria when respiration is supported either by the NAD⁺-linked substrate β-hydroxybutyrate, or by succinate. In the presence, as well as in the absence, of the pro-oxidant t-butylhydroperoxide mitochondria retain Ca²⁺ much better with succinate than with β-hydroxybutyrate, as respiratory substrate. When Ca²⁺ release is induced by t-butylhydroperoxide succinate-supported Ca²⁺ retention is impeded by rotenone. Ca²⁺ release (pro-oxidant dependent or independent) is paralleled by oxidation and hydrolysis of intramitochondrial pyridine nucleotides, and Ca²⁺ retention is paralleled by reduction of pyridine nucleotides. It is concluded that the pyridine nucleotide-linked Ca²⁺ release from mitochondria can be controlled by respiratory substrates which regulate the intramitochondrial hydrolysis of oxidized pyridine nucleotides.     

Function of reduced pyridine nucleotide-ferredoxin oxidoreductases in saccharolytic Clostridia

The physiological function of the clostridial NADH- and NADPH-ferredoxin oxidoreductases was investigated with Clostridium pasteurianum and Clostridium butyricum.The NADH-ferredoxin oxidoreductases are concluded to be catabolic enzymes required for the reduction of ferredoxin by NADH. The conclusion is based on the finding that during the entire growth phase the fermentation of glucose can be formally represented by the weighted sum of Eqns 1 and 2, Glucose + 2 H₂O → 1 butyrate^? + 2 HCO₃^? + 3 H⁺ + 2 H₂ (1) Glucose + 4 H₂O → 2 acetate^? + 2 HCO₃^? + 4 H⁺ + 4 H₂ (2) and that in these redox processes NADH rather than NADPH is specifically formed during glyceraldehyde phosphate dehydrogenation. This NADH can be consumed by substrate reduction in Process 1 only, while it must be reoxidized in Process 2 by the ferredoxin-dependent proton reduction to hydrogen which involves the NADH-ferredoxin oxidoreductases.The kinetic and regulatory properties of these enzymes are in line with their catabolic role: they are found with high specific activities typical for other catabolic enzymes; essentially they catalyze electron flow from NADH to ferredoxin only because the back reaction is very effectively inhibited by low concentrations of NADH. These enzymes have a key role in the coupling of the two partial processes and in regulating the overall thermodynamic efficiency of the fermentations.The NADPH-ferredoxin oxidoreductases are concluded to participate in anabolism; they are required for the regeneration of NADPH. The conclusion is based on the finding that in the two clostridia all catabolic oxidations-reductions are specific for NAD(H) and that the usual NADPH-producing processes such as the glucose 6-phosphate dehydrogenase or malate enzyme reactions are absent. The kinetic properties of the enzymes are in agreement with their anabolic function: the NADPH-ferredoxin oxidoreductases are found with sufficient specific activities; they preferentially catalyze electron transfer from reduced ferredoxin to NADP⁺.     

Influence of amino acids,mammalian serum,and osmotic pressure on the proliferation of Drosophila cell lines

In the D22 medium of ECHALIER and OHANESSIAN for the culture of Drosophila cell lines lactalbumin hydrolysate could be replaced by a synthetic amino acids mixture. In spite of the presence of yeast extract and fetal calf serum the omission of any one of arginine, asparagine, cysteine, histidine, methionine, proline, serine, or threonine prevented cell proliferation. Of these eight amino acids cysteine had to be added in concentrations higher than 0.1 mM. Without much effect on cell proliferation foetal calf serum could be reduced from 10% to 2% or be replaced by 1% horse serum or 1% porcine serum. Cells could grow in media of osmolarities from 225 mOsm up to 400 mOsm depending on the osmotic agent used. Chloride concentrations up to 80 mM were compatible with proliferation as was a wide range of sodium/potassium ratios.     

FoxO调控细胞增殖、分化与凋亡

与细胞发育和代谢相关的转录因子中,2000年才正式发布并统一命名的Fox家族受到了研究者的高度重视,其广泛存在于从酵母到哺乳类的真核生物中.FoxO转录因子作为Fox家族主要成员,是INS/IGF-1信号通路中的关键因子,通过转录调控和信号转导途径在动物的生理调节、代谢和细胞周期等方面起重要作用.     

Role of interferons in the regulation of cell proliferation,differentiation, and development

There now appears to be evidence to support the view that the type I IFNs are naturally produced negative regulators of growth that also modify cell differentiation. Consistent with this, it appears that the ability to produce and respond to IFN is suppressed in early embryonic development when cell proliferation and differentiation are essential. In the later stages of fetal development, IFN production is de-repressed, and cells show increased sensitivity to IFN, which may be important in regulating cell proliferation and/or differentiation processes or the interaction between fetal and maternal tissues. Interestingly, the IFN system can also be suppressed in disease states such as the development of tumours or in the establishment of a (chronic) viral infection. Therefore, understanding the developmental regulation of the IFN system may be important to understanding and controlling the IFN system in disease. More extensive studies of the developmental stage and tissue-specific expression of type I IFNs and their receptors are necessary, as well as more direct in vivo experiments to further elucidate the role of the IFN system in reproduction and development. © 1994 Wiley-Liss, Inc.     

Endogenous regulation of endothelial cell proliferation

Bovine aortic endothelial cells (BAEC) in culture have the ability to regulate their own proliferation. We have found that a fraction below 100,000 daltons obtained from the media of confluent cultures of BAEC inhibits tritiated thymidine [3H]TdR incorporation as well as their proliferation. The inhibition is dose- and time-dependent; maximum inhibition of [3H]TdR incorporation occurs 8 hr after cells are released from synchronization and the inhibitory fraction is added. Inhibition is evident at concentrations as low as 50 micrograms/ml and reaches a maximum at 600 micrograms/ml. The blockage of [3H]TdR incorporation is reflected in the inhibition of cell proliferation. In the presence of 400 micrograms of endogenous inhibitor per ml of media, added at the time of plating, the average population doubling time increases from 19 to 41 hr. These findings indicate that, in culture, BAEC can regulate their own proliferation by synthesizing an endogenous inhibitor(s) of proliferation.     

The effect of calcium chelators on microsomal pyridine nucleotide-linked dehydrogenases of sugarbeet cells

Cell suspension cultures of Beta vulgaris L., treated with calciumchelators or untreated, were used to characterize pyndine nucleotide-dependentdiaphorases of microsomes. The microsomal activity of NADH-dependentduroquinone reductase from cultures treated with 10 mM Na₂EGTAfor 24 h increased by a factor of 1.8 with respect to controlmicrosomes, and was mainly associated with particles of d=1.17gml^–1. NADPH-duroquinone reductase and NADH-ferricyanidereductase activities showed smaller increases. Bacterial protein-lipopolysaccharidecomplexes (prLPS) also promoted the increase of microsomal diaphorases;CaEGTA was Ineffective. EGTA effects on enzymes of supernatantand mitochondria were negligible, although Na₂EGTA treatmentinduced cell aggregation and strong acidification of the medium. When microsomes from control cultures were solubilized with1% LPC and fractionated in high-efficiency gel permeation columns(FPLC) the diaphorase activities were found associated to threemajor proteins: (i) NADH-specific quinone reductase (NADH-QR)of 340 kDa; (ii) pyndine nucleotide-nonspecific quinone reductase(NAD(P)H-QR) of 85 kDa also having ferricyanide reductase activity;(iii) NADH-specific ferricyanide reductase (NADH-FCR) of 38kDa. The microsomes from EGTA-treated cells also showed a highlyactive NADH-QR having a larger molecular mass (440 kDa) thanin control cells. NAD(P)H-QR also showed increased activity.We conclude that external Ca²⁺ chelation induces changes indehydrogenase components in microsomes. Furthermore, prLPS probablyexert part of their effect on plants through Ca²⁺ chelation. Key words: Beta vulgaris, cell cultures, calcium chelators, diaphorase, NAD(P)H-dehydrogenase, lipopolysaccharide, EGTA, quinone reductase     

Changes of cell surface configuration and regulation of cell proliferation

The electric surface charge configuration of 3T3 and SV40-3T3 cells was characterized by determining the product of electrophoretic mobility of the cells times the viscosity of suspension medium. This quantity could be shown to change with temperature and/or treatment with calf serum or trypsin in close correlation with the effects of these agents on characteristics of cell proliferation. The present results, taken together with those of earlier studies on cell-electrophoresis and characterization of lipid constituents of the cells, support the hypothesis of a lateral phase separation in the plasmamembrane as triggering process in stimulation of proliferation of resting normal cells.     

A large-scale preparative electrophoretic method for the purification of pyridine nucleotide-linked dehydrogenases.

A large-scale preparative polyacrylamide gel electrophoresis (PAGE) method that uses a 1.5- or a 2.0-cm-thick slab gel has been developed for the purification of NAD-dependent dehydrogenases. With the 2.0-cm-thick gel, a maximum volume (up to about 160 ml) of enzyme sample was applied to a gel plate, resulting in the application of a large amount of protein and enzyme. After the electrophoretic run, the enzyme band on the gel was detected by activity staining and recovered from the gel by extraction with a fairly loose-fitting glass-Teflon homogenizer. NAD-dependent alanine dehydrogenase, leucine dehydrogenase, and glycerol dehydrogenase were purified in high yields (more than 80%) by the preparative PAGE method. The method can be carried out using a simple slab gel apparatus, which is modified from the conventional analytical apparatus for the purpose of preparative PAGE under conditions used for routine analytical runs. Thus, the method may be suitable for use in purifying NAD(P)-dependent dehydrogenases and many other enzymes after conventional chromatography such as dye-ligand affinity chromatography or ion-exchange chromatography.     

Activin signaling and its role in regulation of cell proliferation, apoptosis, and carcinogenesis

Activins, cytokine members of the transforming growth factor-beta superfamily, have various effects on many physiological processes, including cell proliferation, cell death, metabolism, homeostasis, differentiation, immune responses endocrine function, etc. Activins interact with two structurally related serine/threonine kinase receptors, type I and type II, and initiate downstream signaling via Smads to regulate gene expression. Understanding how activin signaling is controlled extracellularly and intracellularly would not only lead to more complete understanding of cell growth and apoptosis, but would also provide the basis for therapeutic strategies to treat cancer and other related diseases. This review focuses on the recent progress on activin-receptor interactions, regulations of activin signaling by ligand-binding proteins, receptor-binding proteins, and nucleocytoplasmic shuttling of Smad proteins.     

肿瘤细胞恶性增殖和细胞周期调控改变的分子机制

真核细胞通过复杂的细胞周期调控系统控制细胞的分裂,从而维持有机体的正常代谢和增殖.细胞周期的调控是由一系列重要的信号分子和周期蛋白家族来完成的,这些调节因子发生突变或者表达水平发生改变,将导致细胞周期调控的改变,使细胞增殖能力增强、分化减弱,丧失细胞原有的功能,最终发展成肿瘤细胞.因此细胞周期及其相关调控蛋白和信号机制成为抗肿瘤研究的热点.     

Native gel activity stain and preparative electrophoretic method for the detection and purification of pyridine nucleotide-linked dehydrogenases

An activity stain for the detection of pyridine nucleotide-linked dehydrogenases in polyacrylamide gels is described. Following incubation of the gel with substrate and cofactor, bands are visualized under ultraviolet light, where reduced cofactors fluoresce and oxidized cofactors appear black. The methods described are useful for any NAD- or NADP-linked dehydrogenase; the enzymes can be assayed in either the oxidative or the reductive direction. Also described is a preparative polyacrylamide gel system using the activity stain, which can be used as a general purification method for dehydrogenases. The preparative gels are crosslinked with bisacrylylcystamine. These crosslinks can be broken by the addition of thiols after the bands of interest have been located and excised. The protein of interest is then separated from the solubilized acrylamide by adsorption to a suitable resin.