BBA - Molecular Basis of Disease (v.1536, #1)
Enhanced expression of mRNAs of antisecretory factor-1, gp96, DAD1 and CDC34 in human hepatocellular carcinomas by Kenji Tanaka; Nobuo Kondoh; Masahiro Shuda; Osamu Matsubara; Nobuo Imazeki; Akihide Ryo; Toru Wakatsuki; Akiyuki Hada; Narihide Goseki; Toru Igari; Kazuo Hatsuse; Tsukasa Aihara; Sankichi Horiuchi; Naoki Yamamoto; Mikio Yamamoto (1-12).
To identify differentially expressed genes in hepatocarcinogenesis, we performed differential display analysis using surgically resected hepatocellular carcinoma (HCC) and adjacent non-tumorous liver tissues. We identified four cDNA fragments upregulated in HCC samples, encoding antisecretory factor-1 (AF), gp96, DAD1 and CDC34. Northern blot analysis demonstrated that these mRNAs were expressed preferentially in HCCs compared with adjacent non-tumorous liver tissues or normal liver tissues from non-HCC patients. The expression of these mRNAs was increased along with the histological grading of HCC tissues. These mRNA levels were also high in three human HCC cell lines (HuH-7, HepG2 and HLF), irrespective of the growth state. We also demonstrate that sodium butyrate, an inducer of differentiation, downregulated the expression of AF and gp96 mRNAs, supporting in part our pathological observation. Immunohistochemical analysis revealed that gp96 and CDC34 proteins were preferentially accumulated in cytoplasm and nuclei of HCC cells, respectively. Overexpression of these genes could be an important manifestation of HCC phenotypes and should provide clues to understand the molecular basis of hepatocellular carcinogenesis.
Keywords: Hepatocellular carcinoma; Differential gene expression; Differential display; Northern blot analysis; Sodium butyrate; Immunohistochemistry;
Androgen induced cell death in SHSY5Y neuroblastoma cells expressing wild-type and spinal bulbar muscular atrophy mutant androgen receptors by Andrew J Grierson; Christopher E Shaw; Christopher C.J Miller (13-20).
Spinal bulbar muscular atrophy (SBMA) is one of a family of inherited neurodegenerative diseases caused by expansion of CAG encoding polyglutamine repeats; in SBMA the affected gene is the androgen receptor. To understand further the mechanisms that lead to neuronal cell death in SBMA, we generated SHSY5Y neuroblastoma cell lines that stably express identical levels of wild-type (19 polyglutamine repeat) or SBMA (52 polyglutamine repeat) androgen receptor. Parental SHSY5Y cells do not express detectable levels of the androgen receptor. In the absence of androgen, the transfected cell lines have similar phenotypes and growth characteristics to parental SHSY5Y cells. However, upon treatment with androgen, both cell lines undergo a marked dose-dependent loss of viability; this loss was significantly greater in cells expressing the SBMA receptor. Morphological analyses of the androgen treated cells revealed that cell death bore hallmarks of apoptosis involving altered nuclear morphology and cleavage of poly(ADP-ribose) polymerase and of caspase 3 in both wild-type and SBMA cell lines. The caspase inhibitor VAD-fmk was able to decrease loss of viability of both cell lines on exposure to androgen.
Keywords: Kennedy’s disease; Apoptosis; Polyglutamine; CAG repeat;
Xanthine oxidase-derived reactive oxygen metabolites contribute to liver necrosis: protection by 4-hydroxypyrazolo[3,4-d]pyrimidine by Shakir Ali; G Diwakar; Sonica Pawa; M.R Siddiqui; M.Z Abdin; F.J Ahmad; S.K Jain (21-30).
Xanthine oxidase (XO) generates reactive oxygen metabolites (ROM) as a by-product while catalyzing their reaction. The present study implicates these ROM in the pathogenesis of liver necrosis produced in rats by the intraperitoneal administration of thioacetamide (TAA; 400 mg/kg b.wt.). After 16 h of TAA administration, the activity of rat liver XO increased significantly compared to that of the control group. At the same time, the level of serum marker enzymes of liver necrosis (aminotransferases and alkaline phosphatase) and tissue malondialdehyde content also increased in TAA treated rats. Tissue malondialdehyde concentration is an indicator of lipid peroxidation and acts as a useful marker of oxidative damage. Pretreatment of rats with XO inhibitor (4-hydroxypyrazolo[3,4-d]pyrimidine; allopurinol (AP)) followed by TAA could lower the hepatotoxin-mediated rise in malondialdehyde level as well as the level of marker enzymes associated with liver necrosis. The survival rate also increased in rats given AP followed by the lethal dose of TAA. In either case, the effect of AP was dose-dependent. Results presented in the paper indicate that increased production of XO-derived ROM contributes to liver necrosis, which can be protected by AP.
Keywords: Thioacetamide; Necrosis; Oxidative damage; Anti-hepatotoxic; 4-Hydroxypyrazolo[3,4-d]pyrimidine; Xanthine oxidase;
Nuclear cathepsin B-like protease cleaves transcription factor YY1 in differentiated cells by Marie C. Pizzorno (31-42).
Differentiation of pluripotent cells into differentiated cell types involves changes in many aspects of cellular biochemistry. Many of these changes result in alterations of gene expression, which may occur by changing the activity of transcription factors. The cell line NTERA-2 (NT2) can be differentiated into various cell types by incubation with retinoic acid. The differentiated cell type is also permissive for infection with the human herpesvirus cytomegalovirus (CMV). The transcription factor YY1 has been shown to regulate the immediate-early promoter of CMV in a differentiation specific manner by binding to one site at −958 to −950 and to at least two sites in the enhancer. It is demonstrated here that there is a second YY1 site in the modulator between −995 and −987. Levels of YY1 DNA binding activity and protein decrease in NT2 cells as they are differentiated with retinoic acid. This decrease in protein is due to the degradation of YY1 by a cathepsin B-like activity found in nuclear extracts. The cleavage products of YY1 include the intact C-terminal half of the protein, which contains the zinc fingers and the DNA binding activity. This suggests a mechanism that allows expression of the CMV immediate-early promoter in differentiated cells.
Keywords: Cathepsin; Cytomegalovirus; Differentiation; Protein degradation; Teratocarcinoma cell; Transcription factor YY1;
Iron and gallium increase iron uptake from transferrin by human melanoma cells: further examination of the ferric ammonium citrate-activated iron uptake process by D.R Richardson (43-54).
Previously we showed that preincubation of cells with ferric ammonium citrate (FAC) resulted in a marked increase in Fe uptake from both 59Fe-transferrin (Tf) and 59Fe-citrate (D.R. Richardson, E. Baker, J. Biol. Chem. 267 (1992) 13972–13979; D.R. Richardson, P. Ponka, Biochim. Biophys. Acta 1269 (1995) 105–114). This Fe uptake process was independent of the transferrin receptor and appeared to be activated by free radicals generated via the iron-catalysed Haber-Weiss reaction. To further understand this process, the present investigation was performed. In these experiments, cells were preincubated for 3 h at 37°C with FAC or metal ion solutions and then labelled for 3 h at 37°C with 59Fe-Tf. Exposure of cells to FAC resulted in Fe uptake from 59Fe-citrate that became saturated at an Fe concentration of 2.5 μM, while FAC-activated Fe uptake from Tf was not saturable up to 25 μM. In addition, the extent of FAC-activated Fe uptake from citrate was far greater than that from Tf. These results suggest a mechanism where FAC-activated Fe uptake from citrate may result from direct interaction with the transporter, while Fe uptake from Tf appears indirect and less efficient. Preincubation of cells with FAC at 4°C instead of 37°C prevented its effect at stimulating 59Fe uptake from 59Fe-Tf, suggesting that an active process was involved. Previous studies by others have shown that FAC can increase ferrireductase activity that may enhance 59Fe uptake from 59Fe-Tf. However, there was no difference in the ability of FAC-treated cells compared to controls to reduce ferricyanide to ferrocyanide, suggesting no change in oxidoreductase activity. To examine if activation of this Fe uptake mechanism could occur by incubation with a range of metal ions, cells were preincubated with either FAC, ferric chloride, ferrous sulphate, ferrous ammonium sulphate, gallium nitrate, copper chloride, zinc chloride, or cobalt chloride. Stimulation of 59Fe uptake from Tf was shown (in order of potency) with ferric chloride, ferrous sulphate, ferrous ammonium sulphate, and gallium nitrate. The other metal ions examined decreased 59Fe uptake from Tf. The fact that redox-active Cu(II) ion did not stimulate Fe uptake while redox-inactive Ga(III) did, suggests a mechanism of transporter activation not solely dependent on free radical generation. Indeed, the activation of Fe uptake appears dependent on the presence of the Fe atom itself or a metal ion with atomic similarities to Fe (e.g. Ga).
Keywords: Iron; Iron uptake mechanism; Melanoma cell; Transferrin;
Effect of fluvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, on nitric oxide-induced hydroxyl radical generation in the rat heart by Toshio Obata; Akio Ebihara; Yasumitsu Yamanaka (55-63).
We examined the effect of fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, on the production of hydroxyl radical (⋅OH) generation via nitric oxide synthase (NOS) activation by an in vivo microdialysis technique. The microdialysis probe was implanted in the left ventricular myocardium of anesthetized rats and tissue was perfused with Ringer’s solution through the microdialysis probe at a rate of 1 μl/min. Sodium salicylate in Ringer’s solution (0.5 nmol/μl/min) was infused directly through a microdialysis probe to detect the generation of ⋅OH. Induction of [K+]o (70 mM) or tyramine (1 mM), significantly increased the formation of ⋅OH trapped as 2,3-dihydroxybenzoic acid (DHBA). The application of N G-nitro-l-arginine methyl ester (l-NAME), a NOS inhibitor, significantly decreased the K+ depolarization-induced ⋅OH formation, but the effect of tyramine significantly increased the level of 2,3-DHBA. When fluvastatin (100 μM), an inhibitor of low-density lipoprotein (LDL) oxidation, was administered to l-NAME-pretreated animals, both KCl and tyramine failed to increase the level of 2,3-DHBA formation. The effect of fluvastatin may be unrelated to K+ depolarization-induced ⋅OH generation. To examine the effect of fluvastatin on ischemic/reperfused rat myocardium, the heart was subjected to myocardial ischemia for 15 min by occlusion of the left anterior descending coronary artery (LAD). When the heart was reperfused, a marked elevation of the level of 2,3-DHBA was observed. However, in the presence of fluvastatin (100 μM), the elevation of 2,3-DHBA was not observed in ischemia/reperfused rat heart. Fluvastatin, orally at a dose of 3 mg/kg/day for 4 weeks, significantly blunted the rise of serum creatine phosphokinase and improved the electrocardiogram 2 h after coronary occlusion. These results suggest that fluvastatin is associated with a cardioprotective effect due to the suppression of noradrenaline-induced ⋅OH generation by inhibiting LDL oxidation in the heart.
Keywords: Fluvastatin; 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitor; Low-density lipoprotein; N G-Nitro-l-arginine methyl ester; Hydroxyl radical; Microdialysis;
Glycation decreases calmodulin binding to lens transmembrane protein, MIP by S Swamy-Mruthinti (64-72).
Channels of the major intrinsic protein (MIP) of the lens transport water, thus playing an important role in lens fiber cell homeostasis. Calmodulin (CAM) interacts with MIP and possibly regulates MIP channel permeability. Protein glycation has been implicated in lens opacification. We previously identified sites of glycation of MIP, which are in close proximity to the putative CAM binding site. This study is aimed to show the effect of in vitro and in vivo glycation on CAM binding to MIP. Our results show that MIP and MP20 are the major CAM binding proteins of the lens membrane. In vitro incubation of lens membranes with 1 M glucose decreased CAM binding by 38% (P<0.001). Similarly, there was a progressive decrease in CAM binding to diabetic lens membranes compared to age-matched controls (up to 30% decrease, P<0.01). Mutation of K228 and K238 as well as a triple K mutation (K228N, K238N, K259N) of MIP resulted in a decrease in CAM binding. Thus, post-translational protein modifications of MIP influence CAM binding. Since CAM is the ubiquitous Ca2+ receptor, decreases in CAM binding to the target protein will affect the Ca2+-mediated cellular processes leading to lens opacification in diabetic and aging lenses.
Keywords: Calmodulin; Major intrinsic protein; Aquaporin; Glycation; Cataract;
Possible participation of a JAK2 signaling pathway in recombinant rat interleukin-5-induced prolongation of rat eosinophil survival by Kenji Ishihara; Ikuko Satoh; Suetsugu Mue; Kazuo Ohuchi (73-84).
Recombinant rat interleukin (IL)-5-induced prolongation of rat eosinophil survival in culture was inhibited in a concentration-dependent manner by the protein synthesis inhibitor cycloheximide, the DNA-dependent RNA synthesis inhibitor actinomycin D, and the tyrosine kinase inhibitor herbimycin A when examined 96 h after incubation. The MEK-1 inhibitor PD98059 inhibited IL-5-induced phosphorylation of both p44 and p42 MAP kinases, but the IL-5-induced prolongation of eosinophil survival was not inhibited. In contrast, the JAK2 inhibitor AG490 inhibited the IL-5-induced prolongation of eosinophil survival. Treatment of eosinophils with IL-5 resulted in phosphorylation of STAT5 but not STAT1, and the IL-5-induced phosphorylation of STAT5 was inhibited by AG490. These findings suggest that the activation of JAK2 tyrosine kinase and protein synthesis are required for the prolongation of rat eosinophil survival induced by recombinant rat IL-5. STAT5 phosphorylation might also participate in the IL-5-induced survival of rat eosinophils.
Keywords: Interleukin-5; Eosinophil; Apoptosis; p44 mitogen-activated protein kinase; p42 mitogen-activated protein kinase; Janus protein kinase 2; Signal transducer and activator of transcription 5;