Modulation of NADP+-dependent isocitrate dehydrogenase in aging

Abstract

NADPH is an important cofactor in many biosynthesis pathways and the regeneration of reduced glutathione, critically important in cellular defense against oxidative damage. It is mainly produced by glucose-6-phosphate dehydrogenase, malic enzyme, and NADP⁺-specific isocitrate dehydrogenases (ICDHs). Here, we investigated age-related changes in ICDH activity and protein expression in IMR-90 human diploid fibroblast cells and tissues from Fischer 344 rats. We found that in IMR-90 cells the activity of cytosolic ICDH (IDPc) gradually increased with age up to the 46–48 population doubling level (PDL) and then gradually decreased at later PDL. 2′,7′-Dichloro-fluorescein fluorescence which reflects intracellular ROS generation was increased with aging in IMR-90 cells. In ad libitum-fed rats, we noted age-related, tissue-specific modulations of IDPc and mitochondrial ICDH (IDPm) activities and protein expression in the liver, kidney and testes. In contrast, ICDH activities and protein expression were not significantly modulated in diet-restricted rats. These data suggest that modulation of ICDH is an age-dependent and a tissue-specific phenomenon.     

Glycation-induced inactivation of NADP(+)-dependent isocitrate dehydrogenase: implications for diabetes and aging

Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and the cellular defense against oxidative damage is one of the primary functions of NADP(+)-dependent isocitrate dehydrogenase (ICDH), because it supplies NADPH for antioxidant systems. When exposed to reducing sugars such as glucose, glucose 6-phosphate, and fructose, ICDH was susceptible to oxidative modification and damage, which was indicated by a loss of activity and fragmentation of the peptide as well as by the formation of carbonyl groups. The glycated ICDH was isolated and identified by boronate-affinity chromatography and immunoblotting with anti-hexitol-lysine antibody. The active site lysine residue, Lys(212), was identified as one of the major sites of nonenzymatic glycation of ICDH. The structural alterations of modified enzymes were indicated by changes in thermal stability, intrinsic tryptophan fluorescence, and binding of the hydrophobic probe 8-anilino-1-naphthalene sulfonic acid. When we examined the antioxidant role of mitochondrial ICDH against glycation-induced cytotoxicity with HEK293 cells transfected with the cDNA for mouse mitochondrial ICDH in sense and antisense orientations, a clear inverse relationship was observed between the amount of mitochondrial ICDH expressed in target cells and their susceptibility to glycation-mediated cytotoxicity. Mitochondrial ICDH was purified by immunoprecipitation and probed with anti-hexitol-lysine antibody, which revealed increased levels of glycated ICDH in the kidneys of diabetic rats and in the lenses of diabetic patients suffering from cataracts. A decrease in ICDH activity was observed in those tissues. We also found that levels of glycated ICDH increased in IMR-90 cells and rat kidney during normal aging. The glycation-mediated damage to ICDH may result in the perturbation of cellular antioxidant defense mechanisms and subsequently lead to a pro-oxidant condition and may contribute to various pathologies associated with the general aging process and long-term complications of diabetes.     

Inactivation of NADP(+)-dependent isocitrate dehydrogenase by nitric oxide

Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and oxidative damage is one of the primary functions of NADP(+)-dependent isocitrate dehydrogenase (ICDH) through to supply NADPH for antioxidant systems. NO donors such as S-nitrosothiols, diethylamine NONOate, spermine NONOate, and 3-morpholinosydnomine N-ethylcarbamide (SIN-1)/superoxide dismutase inactivated ICDH in a dose- and time-dependent manner. The inhibition of ICDH by S-nitrosothiol was partially reversed by thiol, such as dithiothreitol or 2-mercaptoethanol. Loss of enzyme activity was associated with the depletion of the cysteine-reactive 5,5'-dithiobis-(2-nitrobenzoate) and the loss of fluorescent probe N,N'-dimethyl-N(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethyleneamine accessible thiol groups. Using electrospray ionization mass spectrometry with tryptic digestion of protein, we found that nitric oxide forms S-nitrosothiol adducts on Cys305 and Cys387. These results indicate that S-nitrosylation of cysteine residues on ICDH is a mechanism involving the inactivation of ICDH by NO. The structural alterations of modified enzyme were indicated by the changes in protease susceptibility and intrinsic tryptophan fluorescence. When U937 cells were incubated with 200 microM SNAP for 1 h, a significant decrease in both cytosolic and mitochondrial ICDH activities were observed. Furthermore, stimulation with lipopolysaccharide significantly decreased intracellular ICDH activity in RAW 264.7 cells, and this effect was blocked by NO synthase inhibitor N(omega)-methyl-L-arginine. This result indicates that ICDH was also inactivated by endogenous NO. The NO-mediated damage to ICDH may result in the perturbation of cellular antioxidant defense mechanisms and subsequently lead to a pro-oxidant condition.     

Inactivation of NADP(+)-dependent isocitrate dehydrogenase by reactive oxygen species

Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and the cellular defense against oxidative damage is one of the primary functions of NADP(+)-dependent isocitrate dehydrogenase (ICDH) through supply of NADPH for antioxidant systems. When exposed to various reactive oxygen species such as hydrogen peroxide, singlet oxygen generated by photoactivated dye, superoxide anion, and hydroxyl radical produced by metal-catalyzed Fenton reactions, ICDH was susceptible to oxidative modification and damage, which was indicated by the loss of activity, fragmentation of the peptide as well as by the formation of carbonyl groups. Oxidative damage to ICDH was inhibited by antioxidant enzymes, free radical scavengers, and spin-trapping agents. The structural alterations of modified enzymes were indicated by the increase in thermal instability and binding of the hydrophobic probe 8-anilino-1-naphthalene sulfonic acid (ANSA). The reactive oxygen species-mediated damage to ICDH may result in the perturbation of cellular antioxidant defense mechanisms and subsequently lead to a pro-oxidant condition.     

Cytosolic NADP(+)-dependent isocitrate dehydrogenase status modulates oxidative damage to cells

NADPH is an important cofactor in many biosynthesis pathways and the regeneration of reduced glutathione, critically important in cellular defense against oxidative damage. It is mainly produced by glucose 6-phosphate dehydrogenase (G6PD), malic enzyme, and the cytosolic form of NADP(+)-dependent isocitrate dehydrogenase (IDPc). Little information is available about the role of IDPc in antioxidant defense. In this study we investigated the role of IDPc against cytotoxicity induced by oxidative stress by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 3-4-fold higher and 35% lower, respectively, than that in the parental cells carrying the vector alone. Although the activities of other antioxidant enzymes, such as superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase, and G6PD, were comparable in all transformed cells, the ratio of GSSG to total glutathione was significantly higher in the cells expressing the lower level of IDPc. This finding indicates that IDPc is essential for the efficient glutathione recycling. Upon transient exposure to increasing concentrations of H(2)O(2) or menadione, an intracellular source of free radicals and reactive oxygen species, the cells with low levels of IDPc became more sensitive to oxidative damage by H(2)O(2) or menadione. Lipid peroxidation, oxidative DNA damage, and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly over-expressed IDPc exhibited enhanced resistance against oxidative stress, compared to the control cells. This study provides direct evidence correlating the activities of IDPc and the maintenance of the cellular redox state, suggesting that IDPc plays an important role in cellular defense against oxidative stress.     

Cellular defense against UVB-induced phototoxicity by cytosolic NADP(+)-dependent isocitrate dehydrogenase

Ultraviolet (UV) radiation is known as a major cause of skin photoaging and photocarcinogenesis. Many harmful effects of UV radiation are associated with the generation of reactive oxygen species. Recently, we have shown that NADP(+)-dependent isocitrate dehydrogenase is involved in the supply of NADPH needed for GSH production against cellular oxidative damage. In this study we investigated the role of cytosolic form of NADP(+)-dependent isocitrate dehydrogenase (IDPc) against UV radiation-induced cytotoxicity by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 2.3-fold higher and 39% lower, respectively, than that in the parental cells carrying the vector alone. Upon exposure to UVB (312 nm), the cells with low levels of IDPc became more sensitive to cell killing. Lipid peroxidation, protein oxidation, oxidative DNA damage, and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly overexpressed IDPc exhibited enhanced resistance against UV radiation, compared to the control cells. The data indicate that IDPc plays an important role in cellular defense against UV radiation-induced oxidative injury.     

Purification and properties of NADP(+)-dependent isocitrate dehydrogenase from the corpus luteum

Cytoplasmic NADP(+)-dependent isocitrate dehydrogenase (isocitrate: NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42) was purified 290-fold from the 15,000 x g supernatant fraction of porcine corpora lutea. The major purification step was by anion-exchange chromatography with an FPLC mono P column. Enzyme lability was overcome by including Mg2+, DL-isocitrate, dithiothreitol and glycerol in the elution buffers. The molecular weight of the denatured enzyme was found to be 48,000 by SDS-polyacrylamide gel electrophoresis. The Stokes' radius was estimated to be 3.7 nm by gel filtration and the isoelectric point was 4.8 as determined by chromatofocusing. The purified enzyme had a specific activity of 57.8 units/mg and a broad optimal pH for activity from 7.5 to 9.0. The Km for the substrates DL-isocitrate and NADP+ were 13 and 12 microM, respectively. Polyclonal antibodies were raised against the purified enzyme. Protein (Western) blotting showed an immunological similarity between the cytoplasmic enzyme of the ovary, liver, adrenal gland and heart. A difference was demonstrated between the ovarian enzyme and the heart mitochondrial enzyme. The substrate turnover number and Mr of the ovarian enzyme were similar to those found for the enzyme from the liver and adrenal gland.     

Knockdown of cytosolic NADP(+) -dependent isocitrate dehydrogenase enhances MPP(+) -induced oxidative injury in PC12 cells

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its toxic metabolite 1-methyl-4-phenylpyridium ion (MPP(+)) have been shown to induce Parkinson's disease-like symptoms as well as neurotoxicity in humans and animal species. Recently, we reported that maintenance of redox balance and cellular defense against oxidative damage are primary functions of the novel antioxidant enzyme cytosolic NADP(+) -dependent isocitrate dehydrogenase (IDPc). In this study, we examined the role of IDPc in cellular defense against MPP(+) -induced oxidative injury using PC12 cells transfected with IDPc small interfering RNA (siRNA). Our results demonstrate that MPP(+) -mediated disruption of cellular redox status, oxidative damage to cells, and apoptotic cell death were significantly enhanced by knockdown of IDPc.     

Inactivation of NADP(+)-dependent isocitrate dehydrogenase by singlet oxygen derived from photoactivated rose bengal

Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and the cellular defense against oxidative damage is one of the primary functions of NADP(+)-dependent isocitrate dehydrogenase (ICDH) by supplying NADPH for antioxidant systems. When exposed to a singlet oxygen-producing system composed of rose bengal (RB) and visible light, ICDH was susceptible to oxidative modification and damage as indicated by the loss of activity and by the formation of carbonyl groups. The structural alterations of modified enzyme were indicated by the increase in susceptibility to proteases and the change in intrinsic fluorescence spectra. Upon exposure to photoactivated RB, a significant decrease in both cytosolic and mitochondrial ICDH activities was observed in HL-60 cells. The singlet oxygen-mediated damage to ICDH may result in the perturbation of cellular antioxidant defense mechanisms and subsequently lead to a pro-oxidant condition. When we examined the antioxidant role of cytosolic ICDH against singlet oxygen-induced damage with HL-60 cells transfected with the cDNA for mouse cytosolic ICDH in sense and antisense orientations, a clear inverse relationship was observed between the amount of cytosolic ICDH expressed in target cells and their susceptibility to singlet oxygen-mediated oxidative damage.     

Suppression of mitochondrial NADP(+)-dependent isocitrate dehydrogenase activity enhances curcumin-induced apoptosis in HCT116 cells

Curcumin is a polyphenol derived from the plant Curcuma longa that induces apoptotic cell death in malignant cancer cell lines. It has been shown previously that mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDPm) plays an essential role in defense against oxidative stress by supplying NADPH for antioxidant systems. This study demonstrates that curcumin decreased the activity of IDPm, both as a purified enzyme and in cultured cells. It also shows that curcumin-induced apoptosis in the colon cancer cell line HCT116 is significantly enhanced by suppression of IDPm activity. Transfection of HCT116 cells with an IDPm small interfering RNA (siRNA) markedly decreased activity of IDPm, enhancing cellular susceptibility to curcumin-induced apoptosis, as reflected by DNA fragmentation, cellular redox status, mitochondria dysfunction and modulation of apoptotic marker proteins. Together, these results suggest that application of curcumin together with IDPm siRNA may be an effective combination modality in the treatment of cancer.     

The human PICD gene encodes a cytoplasmic and peroxisomal NADP(+)-dependent isocitrate dehydrogenase.

Human PICD was identified by homology probing the data base of expressed sequence tags with the protein sequence of Saccharomyces cerevisiae Idp3p, a peroxisomal NADP(+)-dependent isocitrate dehydrogenase. The human PICD cDNA contains a 1242-base pair open reading frame, and its deduced protein sequence is 59% identical to yeast Idp3p. Expression of PICD partially rescued the fatty acid growth defect of the yeast idp3 deletion mutant suggesting that PICD is functionally homologous to Idp3p. Kinetic studies on bacterially expressed PICD demonstrated that this enzyme catalyzed the oxidative decarboxylation of isocitrate to 2-oxoglutarate with a specific activity of 22.5 units/mg and that PICD displayed K(M) values of 76 microM for isocitrate and 112 microM for NADP(+). In subcellular fractionation experiments, we found PICD in both peroxisomes and cytoplasm of human and rat liver cells, with approximately 27% of total PICD protein associated with peroxisomes. The presence of PICD in mammalian peroxisomes suggests roles in the regeneration of NADPH for intraperoxisomal reductions, such as the conversion of 2, 4-dienoyl-CoAs to 3-enoyl-CoAs, as well as in peroxisomal reactions that consume 2-oxoglutarate, namely the alpha-hydroxylation of phytanic acid. As for cytoplasmic PICD, the phenotypes of patients with glucose-6-phosphate dehydrogenase deficiency (Luzzatto, L., and Mehta, A. (1995) in The Metabolic and Molecular Bases of Inherited Disease (Scriver, C. R., Beaudet, A. L., Sly, W. S., and Valle, D., eds) Vol. 3, 7th Ed., pp. 3367-3398, McGraw-Hill Inc., New York) suggest that PICD serves a significant role in cytoplasmic NADPH production, particularly under conditions that do not favor the use of the hexose monophosphate shunt (Luzzatto et al.).     

Catalytic mechanism of NADP(+)-dependent isocitrate dehydrogenase: implications from the structures of magnesium-isocitrate and NADP+ complexes

The structures of NADP+ and magnesium isocitrate bound to the NADP(+)-dependent isocitrate dehydrogenase of Escherichia coli have been determined and refined at 2.5-A resolution. NADP+ is bound by the large domain of isocitrate dehydrogenase, a structure that has little similarity to the supersecondary structure of the nucleotide-binding domain of the lactate dehydrogenase-like family of nucleotide-binding proteins. The coenzyme-binding site confirms the fundamentally different evolution of the isocitrate dehydrogenase-like and the lactate dehydrogenase-like classes of nucleotide-binding proteins. In the magnesium-isocitrate complex, magnesium is coordinated to the alpha-carboxylate and alpha-hydroxyl oxygen of isocitrate in a manner suitable for stabilization of a negative charge on the hydroxyl oxygen during both the dehydrogenation and decarboxylation steps of the conversion of isocitrate to alpha-ketoglutarate. The metal ion is also coordinated by aspartate side chains 283' (of the second subunit of the dimer) and 307 and two water molecules in a roughly octahedral arrangement. On the basis of the geometry of the active site, the base functioning in the dehydrogenation step is most likely aspartate 283'. E. coli isocitrate dehydrogenase transfers a hydride stereospecifically to the A-side of NADP+, and models for a reactive ternary complex consistent with this stereospecificity are discussed.     

Regulation of replicative senescence by NADP+ -dependent isocitrate dehydrogenase

The free radical hypothesis of aging postulates that senescence is due to an accumulation of cellular oxidative damage, caused largely by reactive oxygen species that are produced as by-products of normal metabolic processes. Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and the cellular defense against oxidative damage is one of the primary functions of cytosolic (IDPc) and mitochondrial NADP+ -dependent isocitrate dehydrogenase (IDPm) by supplying NADPH for antioxidant systems. In this paper, we demonstrate that modulation of IDPc or IDPm activity in IMR-90 cells regulates cellular redox status and replicative senescence. When we examined the regulatory role of IDPc and IDPm against the aging process with IMR-90 cells transfected with cDNA for IDPc or IDPm in sense and antisense orientations, a clear inverse relationship was observed between the amount of IDPc or IDPm expressed in target cells and their susceptibility to senescence, which was reflected by changes in replicative potential, cell cycle, senescence-associated beta-galactosidase activity, expression of p21 and p53, and morphology of cells. Furthermore, lipid peroxidation, oxidative DNA damage, and intracellular peroxide generation were higher and cellular redox status shifted to a prooxidant condition in the cell lines expressing the lower level of IDPc or IDPm. The results suggest that IDPc and IDPm play an important regulatory role in cellular defense against oxidative stress and in the senescence of IMR-90 cells.     

RNA interference targeting cytosolic NADP(+)-dependent isocitrate dehydrogenase exerts anti-obesity effect in vitro and in vivo

A metabolic abnormality in lipid biosynthesis is frequently associated with obesity and hyperlipidemia. Nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) is an essential reducing equivalent for numerous enzymes required in fat and cholesterol biosynthesis. Cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) has been proposed as a key enzyme for supplying cytosolic NADPH. We report here that knockdown of IDPc expression by Ribonucleic acid (RNA) interference (RNAi) inhibited adipocyte differentiation and lipogenesis in 3T3-L1 preadipocytes and mice. Attenuated IDPc expression by IDPc small interfering RNA (siRNA) resulted in a reduction of differentiation and triglyceride level and adipogenic protein expression as well as suppression of glucose uptake in cultured adipocytes. In addition, the attenuation of Nox activity and Reactive oxygen species (ROS) generation accompanied with knockdown of IDPc was associated with inhibition of adipogenesis and lipogenesis. The loss of body weight and the reduction of triglyceride level were also observed in diet-induced obese mice transduced with IDPc short-hairpin (shRNA). Taken together, the inhibiting effect of RNAi targeting IDPc on adipogenesis and lipid biosynthesis is considered to be of therapeutic value in the treatment and prevention of obesity and obesity-associated metabolic syndrome.     

Cytosolic NADP(+)-dependent isocitrate dehydrogenase protects macrophages from LPS-induced nitric oxide and reactive oxygen species

Macrophages activated by microbial lipopolysaccharides (LPS) produce bursts of nitric oxide and reactive oxygen species (ROS). Redox protection systems are essential for the survival of the macrophages since the nitric oxide and ROS can be toxic to them as well as to pathogens. Using suppression subtractive hybridization (SSH) we found that cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) is strongly upregulated by nitric oxide in macrophages. The levels of IDPc mRNA and of the corresponding enzymatic activity were markedly increased by treatment of RAW264.7 cells or peritoneal macrophages with LPS or SNAP (a nitric oxide donor). Over-expression of IDPc reduced intracellular peroxide levels and enhanced the survival of H2O2- and SNAP-treated RAW264.7 macrophages. IDPc is known to generate NADPH, a cellular reducing agent, via oxidative decarboxylation of isocitrate. The expression of enzymes implicated in redox protection, superoxide dismutase (SOD) and catalase, was relatively unaffected by LPS and SNAP. We propose that the induction of IDPc is one of the main self-protection mechanisms of macrophages against LPS-induced oxidative stress.     

Inactivation of NADP+-dependent isocitrate dehydrogenase by lipid peroxidation products

Membrane lipid peroxidation processes yield products that may react with proteins to cause oxidative modification. Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and oxidative damage is one of the primary functions of NADP₊-dependent isocitrate dehydrogenase (ICDH) through to supply NADPH for antioxidant systems. When exposed to lipid peroxidation products, such as malondialdehyde (MDA), 4-hydroxynonenal (HNE) and lipid hydroperoxide, ICDH was susceptible to oxidative damage, which was indicated by the loss of activity and the formation of carbonyl groups. The structural alterations of modified enzymes were indicated by the change in thermal stability, intrinsic tryptophan fluorescence and binding of the hydrophobic probe 8-anilino 1-napthalene sulfonic acid. Upon exposure to 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH), which induces lipid peroxidation in membrane, a significant decrease in both cytosolic and mitochondrial ICDH activities were observed in U937 cells. Using immunoprecipitation and immunoblotting, we were able to isolate and positively identify HNE adduct in mitochondrial ICDH from AAPH-treated U937 cells. The lipid peroxidation-mediated damage to ICDH may result in the perturbation of the cellular antioxidant defense mechanisms and subsequently lead to a pro-oxidant condition.     

Regulation of mitochondrial NADP+-dependent isocitrate dehydrogenase activity by glutathionylation

Recently, we demonstrated that the control of mitochondrial redox balance and oxidative damage is one of the primary functions of mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDPm). Because cysteine residue(s) in IDPm are susceptible to inactivation by a number of thiol-modifying reagents, we hypothesized that IDPm is likely a target for regulation by an oxidative mechanism, specifically glutathionylation. Oxidized glutathione led to enzyme inactivation with simultaneous formation of a mixed disulfide between glutathione and the cysteine residue(s) in IDPm, which was detected by immunoblotting with anti-GSH IgG. The inactivated IDPm was reactivated enzymatically by glutaredoxin2 in the presence of GSH, indicating that the inactivated form of IDPm is a glutathionyl mixed disulfide. Mass spectrometry and site-directed mutagenesis further confirmed that glutathionylation occurs to a Cys(269) of IDPm. The glutathionylated IDPm appeared to be significantly less susceptible than native protein to peptide fragmentation by reactive oxygen species and proteolytic digestion, suggesting that glutathionylation plays a protective role presumably through the structural alterations. HEK293 cells and intact respiring mitochondria treated with oxidants inducing GSH oxidation such as H(2)O(2) or diamide showed a decrease in IDPm activity and the accumulation of glutathionylated enzyme. Using immunoprecipitation with anti-IDPm IgG and immunoblotting with anti-GSH IgG, we were also able to purify and positively identify glutathionylated IDPm from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice, a model for Parkinson's disease. The results of the current study indicate that IDPm activity appears to be modulated through enzymatic glutathionylation and deglutathionylation during oxidative stress.     

Inactivation of mitochondrial NADP+-dependent isocitrate dehydrogenase by hypochlorous acid

Myeoloperoxidase catalyses the formation of hypochlorous acid (HOCl) via reaction of H(2)O(2) with Cl(-) ion. Although HOCl is known to play a major role in the human immune system by killing bacteria and other invading pathogens, excessive generation of this oxidant is known to cause damage to tissue. Recently, it was demonstrated that the control of mitochondrial redox balance and oxidative damage is one of the primary functions of mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDPm) to supply NADPH for antioxidant systems. This study investigated whether the IDPm would be a vulnerable target of HOCl as a purified enzyme and in intact cells. Loss of enzyme activity was observed and the inactivation of IDPm was reversed by thiols. Transfection of HeLa cells with an IDPm small interfering RNA (siRNA) markedly enhanced HOCl-induced oxidative damage to cells. The HOCl-mediated damage to IDPm may result in the perturbation of the cellular antioxidant defense mechanisms and subsequently lead to a pro-oxidant condition.     

The distribution pattern of NADP+-dependent isocitrate dehydrogenase in rat liver

Activities of the NADP+-dependent isocitrate dehydrogenase were measured along the entire sinusoidal path (1) between small portal tracts and central veins and (2) between regions of adjoining septal branches and central veins in the liver of male Wistar rats using a Lowry technique. The measured activities show a slight increase from the periportal to the perivenous end, whereas no such septal-) perivenous gradient could be established. These profiles of enzyme activity give further support to previous studies, suggesting functional heterogeneity of liver sinusoids and their abutting hepatocytes related to morphological differences of the sinusoidal bed.     

Suppression of tumorigenesis in mitochondrial NADP+-dependent isocitrate dehydrogenase knock-out mice

The tumor host microenvironment is increasingly viewed as an important contributor to tumor growth and suppression. Cellular oxidative stress resulting from high levels of reactive oxygen species (ROS) contributes to various processes involved in the development and progress of malignant tumors including carcinogenesis, aberrant growth, metastasis, and angiogenesis. In this regard, the stroma induces oxidative stress in adjacent tumor cells, and this in turn causes several changes in tumor cells including modulation of the redox status, inhibition of cell proliferation, and induction of apoptotic or necrotic cell death. Because the levels of ROS are determined by a balance between ROS generation and ROS detoxification, disruption of this system will result in increased or decreased ROS level. Recently, we demonstrated that the control of mitochondrial redox balance and cellular defense against oxidative damage is one of the primary functions of mitochondrial NADP⁺-dependent isocitrate dehydrogenase (IDH2) that supplies NADPH for antioxidant systems. To explore the interactions between tumor cells and the host, we evaluated tumorigenesis between IDH2-deficient (knock-out) and wild-type mice in which B16F10 melanoma cells had been implanted. Suppression of B16F10 cell tumorigenesis was reproducibly observed in the IDH2-deficient mice along with significant elevation of oxidative stress in both the tumor and the stroma. In addition, the expression of angiogenesis markers was significantly down-regulated in both the tumor and the stroma of the IDH2-deficient mice. These results support the hypothesis that redox status-associated changes in the host environment of tumor-bearing mice may contribute to cancer progression.