51 results on '"Adenine Nucleotide Translocator 2 genetics"'
Search Results
2. Adenine nucleotide translocase 2 (Ant2) is required for individualization of spermatogenesis of Drosophila melanogaster.
- Author
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He Z, Fang Y, Zhang F, Liu Y, Cheng X, Wang J, Li D, Chen D, and Wu F
- Subjects
- Animals, Male, Testis metabolism, Adenine Nucleotide Translocator 2 metabolism, Adenine Nucleotide Translocator 2 genetics, Spermatids metabolism, Drosophila melanogaster genetics, Drosophila melanogaster metabolism, Drosophila melanogaster growth & development, Spermatogenesis, Drosophila Proteins genetics, Drosophila Proteins metabolism
- Abstract
Successful completion of spermatogenesis is crucial for the perpetuation of the species. In Drosophila, spermatid individualization, a process involving changes in mitochondrial structure and function is critical to produce functional mature sperm. Ant2, encoding a mitochondrial adenine nucleotide translocase, is highly expressed in male testes and plays a role in energy metabolism in the mitochondria. However, its molecular function remains unclear. Here, we identified an important role of Ant2 in spermatid individualization. In Ant2 knockdown testes, spermatid individualization complexes composed of F-actin cones exhibited a diffuse distribution, and mature sperms were absent in the seminal vesicle, thus leading to male sterility. The most striking effects in Ant2-knockdown spermatids were decrease in tubulin polyglycylation and disruption of proper mitochondria derivatives function. Excessive apoptotic cells were also observed in Ant2-knockdown testes. To further investigate the phenotype of Ant2 knockdown in testes at the molecular level, complementary transcriptome and proteome analyses were performed. At the mRNA level, 868 differentially expressed genes were identified, of which 229 genes were upregulated and 639 were downregulated induced via Ant2 knockdown. iTRAQ-labeling proteome analysis revealed 350 differentially expressed proteins, of which 117 proteins were upregulated and 233 were downregulated. The expression of glutathione transferase (GstD5, GstE5, GstE8, and GstD3), proteins involved in reproduction were significantly regulated at both the mRNA and protein levels. These results indicate that Ant2 is crucial for spermatid maturation by affecting mitochondrial morphogenesis., (© 2023 Institute of Zoology, Chinese Academy of Sciences.)
- Published
- 2024
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3. ANT2 functions as a translocon for mitochondrial cross-membrane translocation of RNAs.
- Author
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Wang P, Zhang L, Chen S, Li R, Liu P, Li X, Luo H, Huo Y, Zhang Z, Cai Y, Liu X, Huang J, Zhou G, Sun Z, Ding S, Shi J, Zhou Z, Yuan R, Liu L, Wu S, and Wang G
- Subjects
- Animals, Humans, Mice, Immunity, Innate, RNA Transport, HEK293 Cells, Mice, Inbred C57BL, Adenine Nucleotide Translocator 2 metabolism, Adenine Nucleotide Translocator 2 genetics, RNA, Double-Stranded metabolism, Mitochondria metabolism, Mitochondrial Membranes metabolism
- Abstract
Bidirectional transcription of mammalian mitochondrial DNA generates overlapping transcripts that are capable of forming double-stranded RNA (dsRNA) structures. Release of mitochondrial dsRNA into the cytosol activates the dsRNA-sensing immune signaling, which is a defense mechanism against microbial and viral attack and possibly cancer, but could cause autoimmune diseases when unchecked. A better understanding of the process is vital in therapeutic application of this defense mechanism and treatment of cognate human diseases. In addition to exporting dsRNAs, mitochondria also export and import a variety of non-coding RNAs. However, little is known about how these RNAs are transported across mitochondrial membranes. Here we provide direct evidence showing that adenine nucleotide translocase-2 (ANT2) functions as a mammalian RNA translocon in the mitochondrial inner membrane, independent of its ADP/ATP translocase activity. We also show that mitochondrial dsRNA efflux through ANT2 triggers innate immunity. Inhibiting this process alleviates inflammation in vivo, providing a potential therapeutic approach for treating autoimmune diseases., (© 2024. The Author(s) under exclusive licence to Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences.)
- Published
- 2024
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4. The mitochondrial adenine nucleotide transporters in myogenesis.
- Author
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Flierl A, Schriner SE, Hancock S, Coskun PE, and Wallace DC
- Subjects
- Animals, Mice, Mitochondria metabolism, Mitochondrial Membrane Transport Proteins genetics, Mitochondrial Membrane Transport Proteins metabolism, Mitochondrial Permeability Transition Pore, Muscle Development genetics, Adenine Nucleotide Translocator 2 genetics, Adenine Nucleotide Translocator 2 metabolism, Adenine Nucleotides metabolism
- Abstract
Adenine Nucleotide Translocator isoforms (ANTs) exchange ADP/ATP across the inner mitochondrial membrane, are also voltage-activated proton channels and regulate mitophagy and apoptosis. The ANT1 isoform predominates in heart and muscle while ANT2 is systemic. Here, we report the creation of Ant mutant mouse myoblast cell lines with normal Ant1 and Ant2 genes, deficient in either Ant1 or Ant2, and deficient in both the Ant1 and Ant2 genes. These cell lines are immortal under permissive conditions (IFN-γ + serum at 32 °C) permitting expansion but return to normal myoblasts that can be differentiated into myotubes at 37 °C. With this system we were able to complement our Ant1 mutant studies by demonstrating that ANT2 is important for myoblast to myotube differentiation and myotube mitochondrial respiration. ANT2 is also important in the regulation of mitochondrial biogenesis and antioxidant defenses. ANT2 is also associated with increased oxidative stress response and modulation for Ca
++ sequestration and activation of the mitochondrial permeability transition (mtPTP) pore during cell differentiation., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2022
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5. Genistein regulates adipogenesis by blocking the function of adenine nucleotide translocase-2 in the mitochondria.
- Author
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Ikeda T, Watanabe S, and Mitani T
- Subjects
- Animals, Mice, Adenosine Triphosphate metabolism, Phosphorylation drug effects, CCAAT-Enhancer-Binding Protein-beta metabolism, CCAAT-Enhancer-Binding Protein-beta genetics, Adipocytes metabolism, Adipocytes drug effects, Gene Knockdown Techniques, Adipogenesis drug effects, Genistein pharmacology, Mitochondria metabolism, Mitochondria drug effects, AMP-Activated Protein Kinases metabolism, 3T3-L1 Cells, Adenine Nucleotide Translocator 2 metabolism, Adenine Nucleotide Translocator 2 genetics
- Abstract
Genistein exerts antiadipogenic effects, but its target molecules remain unclear. Here, we delineated the molecular mechanism underlying the antiadipogenic effect of genistein. A pulldown assay using genistein-immobilized beads identified adenine nucleotide translocase-2 as a genistein-binding protein in adipocytes. Adenine nucleotide translocase-2 exchanges ADP/ATP through the mitochondrial inner membrane. Similar to the knockdown of adenine nucleotide translocase-2, genistein treatment decreased ADP uptake into the mitochondria and ATP synthesis. Genistein treatment and adenine nucleotide translocase-2 knockdown suppressed adipogenesis and increased phosphorylation of AMP-activated protein kinase. Adenine nucleotide translocase-2 knockdown reduced the transcriptional activity of CCAAT/enhancer-binding protein β, whereas AMP-activated protein kinase inhibition restored the suppression of adipogenesis by adenine nucleotide translocase-2 knockdown. These results indicate that genistein interacts directly with adenine nucleotide translocase-2 to suppress its function. The downregulation of adenine nucleotide translocase-2 reduces the transcriptional activity of CCAAT/enhancer-binding protein β via activation of AMP-activated protein kinase, which consequently represses adipogenesis., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)
- Published
- 2022
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6. Differential Expression of ADP/ATP Carriers as a Biomarker of Metabolic Remodeling and Survival in Kidney Cancers.
- Author
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Trisolini L, Laera L, Favia M, Muscella A, Castegna A, Pesce V, Guerra L, De Grassi A, Volpicella M, and Pierri CL
- Subjects
- Amino Acid Sequence genetics, Apoptosis genetics, Biomarkers, Tumor genetics, Cell Line, Tumor, Disease-Free Survival, Female, Gene Expression Regulation, Neoplastic genetics, Humans, Kaplan-Meier Estimate, Kidney metabolism, Kidney pathology, Kidney Neoplasms metabolism, Kidney Neoplasms pathology, Male, Mitochondria genetics, Mitochondria metabolism, Mitochondria pathology, Mitochondrial ADP, ATP Translocases metabolism, Oxidative Phosphorylation, Adenine Nucleotide Translocator 2 genetics, Adenine Nucleotide Translocator 3 genetics, Arylamine N-Acetyltransferase genetics, Isoenzymes genetics, Kidney Neoplasms genetics, Mitochondrial ADP, ATP Translocases genetics
- Abstract
ADP/ATP carriers (AACs) are mitochondrial transport proteins playing a strategic role in maintaining the respiratory chain activity, fueling the cell with ATP, and also regulating mitochondrial apoptosis. To understand if AACs might represent a new molecular target for cancer treatment, we evaluated AAC expression levels in cancer/normal tissue pairs available on the Tissue Cancer Genome Atlas database (TCGA), observing that AACs are dysregulated in most of the available samples. It was observed that at least two AACs showed a significant differential expression in all the available kidney cancer/normal tissue pairs. Thus, we investigated AAC expression in the corresponding kidney non-cancer (HK2)/cancer (RCC-Shaw and CaKi-1) cell lines, grown in complete medium or serum starvation, for investigating how metabolic alteration induced by different growth conditions might influence AAC expression and resistance to mitochondrial apoptosis initiators, such as "staurosporine" or the AAC highly selective inhibitor "carboxyatractyloside". Our analyses showed that AAC2 and AAC3 transcripts are more expressed than AAC1 in all the investigated kidney cell lines grown in complete medium, whereas serum starvation causes an increase of at least two AAC transcripts in kidney cancer cell lines compared to non-cancer cells. However, the total AAC protein content is decreased in the investigated cancer cell lines, above all in the serum-free medium. The observed decrease in AAC protein content might be responsible for the decrease of OXPHOS activity and for the observed lowered sensitivity to mitochondrial apoptosis induced by staurosporine or carboxyatractyloside. Notably, the cumulative probability of the survival of kidney cancer patients seriously decreases with the decrease of AAC1 expression in KIRC and KIRP tissues making AAC1 a possible new biomarker of metabolic remodeling and survival in kidney cancers.
- Published
- 2020
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7. The Effect of Deflazacort Treatment on the Functioning of Skeletal Muscle Mitochondria in Duchenne Muscular Dystrophy.
- Author
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Dubinin MV, Talanov EY, Tenkov KS, Starinets VS, Belosludtseva NV, and Belosludtsev KN
- Subjects
- Adenine Nucleotide Translocator 2 genetics, Adenine Nucleotide Translocator 2 metabolism, Adenosine Triphosphate biosynthesis, Animals, Calcium metabolism, Calcium Channels genetics, Calcium Channels metabolism, Peptidyl-Prolyl Isomerase F genetics, Peptidyl-Prolyl Isomerase F metabolism, Electron Transport Complex III genetics, Electron Transport Complex III metabolism, Electron Transport Complex IV genetics, Electron Transport Complex IV metabolism, Male, Mice, Mice, Inbred C57BL, Mice, Inbred mdx, Mitochondria, Muscle genetics, Mitochondria, Muscle metabolism, Mitochondrial Proton-Translocating ATPases genetics, Mitochondrial Proton-Translocating ATPases metabolism, Muscle, Skeletal metabolism, Muscle, Skeletal pathology, Muscular Dystrophy, Duchenne genetics, Muscular Dystrophy, Duchenne metabolism, Muscular Dystrophy, Duchenne pathology, Gene Expression Regulation drug effects, Mitochondria, Muscle drug effects, Muscle, Skeletal drug effects, Muscular Dystrophy, Duchenne drug therapy, Pregnenediones pharmacology
- Abstract
Duchenne muscular dystrophy (DMD) is a severe hereditary disease caused by a lack of dystrophin, a protein essential for myocyte integrity. Mitochondrial dysfunction is reportedly responsible for DMD. This study examines the effect of glucocorticoid deflazacort on the functioning of the skeletal-muscle mitochondria of dystrophin-deficient mdx mice and WT animals. Deflazacort administration was found to improve mitochondrial respiration of mdx mice due to an increase in the level of ETC complexes (complexes III and IV and ATP synthase), which may contribute to the normalization of ATP levels in the skeletal muscle of mdx animals. Deflazacort treatment improved the rate of Ca
2+ uniport in the skeletal muscle mitochondria of mdx mice, presumably by affecting the subunit composition of the calcium uniporter of organelles. At the same time, deflazacort was found to reduce the resistance of skeletal mitochondria to MPT pore opening, which may be associated with a change in the level of ANT2 and CypD. In this case, deflazacort also affected the mitochondria of WT mice. The paper discusses the mechanisms underlying the effect of deflazacort on the functioning of mitochondria and contributing to the improvement of the muscular function of mdx mice.- Published
- 2020
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8. Dysregulated expression of androgen metabolism genes and genetic analysis in hypospadias.
- Author
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Chen Z, Lin X, Wang Y, Xie H, and Chen F
- Subjects
- 17-Hydroxysteroid Dehydrogenases genetics, 17-Hydroxysteroid Dehydrogenases metabolism, Adenine Nucleotide Translocator 2 genetics, Adenine Nucleotide Translocator 2 metabolism, Androgens metabolism, Cell Cycle Proteins genetics, Cell Cycle Proteins metabolism, Child, Cytochrome P-450 CYP3A genetics, Cytochrome P-450 CYP3A metabolism, Exome, Humans, Hypospadias pathology, Male, Multienzyme Complexes genetics, Multienzyme Complexes metabolism, Progesterone Reductase genetics, Progesterone Reductase metabolism, S100 Calcium Binding Protein A6 genetics, S100 Calcium Binding Protein A6 metabolism, Steroid Isomerases genetics, Steroid Isomerases metabolism, Androgens genetics, Hypospadias genetics, Mutation, Transcriptome
- Abstract
Background: The aberrant expression of genes involved in androgen metabolism and genetic contribution are unclear in hypospadias., Methods: We compared gene expression profiles by RNA sequencing from five non-hypospadiac foreskins, five mild hypospadiac foreskins, and five severe hypospadiac foreskins. In addition, to identify rare coding variants with large effects on hypospadias risk, we carried out whole exome sequencing in three patients in a hypospadias family., Results: The average expression of androgen receptor (AR) and CYP19A1 were significantly decreased in severe hypospadias (p < .01) and mild hypospadias (p < .05), whereas expression of several other androgen metabolism enzymes, including CYP3A4, HSD17B14, HSD3B7, HSD17B7, CYP11A1 were exclusively significantly expressed in severe hypospadias (p < .05). Compound rare damaging mutants of AR gene with HSD3B1 and SLC25A5 genes were identified in the different severe hypospadias., Conclusions: In conclusion, our findings demonstrated that dysregulation of AR and CYP19A1 could play a crucial role in the development of hypospadias. Inconsistent AR expression may be caused by the feedback loop of ESR1 signaling or combined genetic effects with other risk genes. This findings complement the possible role of AR triggered mechanism in the development of hypospadias., (© 2020 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)
- Published
- 2020
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9. Duchenne muscular dystrophy is associated with the inhibition of calcium uniport in mitochondria and an increased sensitivity of the organelles to the calcium-induced permeability transition.
- Author
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Dubinin MV, Talanov EY, Tenkov KS, Starinets VS, Mikheeva IB, Sharapov MG, and Belosludtsev KN
- Subjects
- Adenine Nucleotide Translocator 2 genetics, Adenine Nucleotide Translocator 2 metabolism, Animals, Cations, Divalent metabolism, Disease Models, Animal, Dystrophin genetics, Dystrophin metabolism, Humans, Ion Transport genetics, Male, Membrane Proteins genetics, Membrane Proteins metabolism, Mice, Mice, Inbred mdx, Microscopy, Electron, Mitochondria, Muscle metabolism, Mitochondria, Muscle ultrastructure, Mitochondrial Membrane Transport Proteins genetics, Mitochondrial Membrane Transport Proteins metabolism, Mitochondrial Permeability Transition Pore, Mitochondrial Proteins genetics, Mitochondrial Proteins metabolism, Muscle, Skeletal cytology, Muscle, Skeletal metabolism, Muscle, Skeletal pathology, Muscle, Skeletal ultrastructure, Muscular Dystrophy, Duchenne genetics, Oxidative Phosphorylation, Calcium metabolism, Mitochondria, Muscle pathology, Mitochondrial Transmembrane Permeability-Driven Necrosis genetics, Muscular Dystrophy, Duchenne pathology
- Abstract
Duchenne muscular dystrophy (DMD) is characterized by a pronounced and progressive degradation of the structure of skeletal muscles, which decreases their strength and lowers endurance of the organism. At muscular dystrophy, mitochondria are known to undergo significant functional changes, which is manifested in a decreased efficiency of oxidative phosphorylation and impaired energy metabolism of the cell. It is believed that the DMD-induced functional changes of mitochondria are mainly associated with the dysregulation of Ca
2+ homeostasis. This work examines the kinetic parameters of Ca2+ transport and the opening of the Ca2+ -dependent MPT pore in the skeletal-muscle mitochondria of the dystrophin-deficient C57BL/10ScSn-mdx mice. As compared to the organelles of wild-type animals, skeletal-muscle mitochondria of mdx mice have been found to be much less efficient in respect to Ca2+ uniport, with the kinetics of Na+ -dependent Ca2+ efflux not changing. The data obtained indicate that the decreased rate of Ca2+ uniport in the mitochondria of mdx mice may be associated with the increased level of the dominant negative subunit of Ca2+ uniporter (MCUb). The experiments have also shown that in mdx mice, skeletal-muscle mitochondria have low resistance to the induction of MPT, which may be related to a significantly increased expression of adenylate translocator (ANT2), a possible structural element of the MPT pore. The paper discusses how changes in the expression of calcium uniporter and putative components of the MPT pore caused by the development of DMD can affect Ca2+ homeostasis of skeletal-muscle mitochondria., (Copyright © 2020 Elsevier B.V. All rights reserved.)- Published
- 2020
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10. Tissue-specific dysregulation of mitochondrial respiratory capacity and coupling control in colon-26 tumor-induced cachexia.
- Author
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Halle JL, Pena GS, Paez HG, Castro AJ, Rossiter HB, Visavadiya NP, Whitehurst MA, and Khamoui AV
- Subjects
- Adenine Nucleotide Translocator 2 genetics, Adenine Nucleotide Translocator 2 metabolism, Animals, Cardiolipins metabolism, Colonic Neoplasms, Liver metabolism, Male, Mice, Muscle, Skeletal metabolism, Oxidative Coupling, Random Allocation, Reactive Oxygen Species, Weight Loss, Cachexia metabolism, Mitochondria, Muscle metabolism, Neoplasms, Experimental metabolism, Oxygen Consumption physiology
- Abstract
In addition to skeletal muscle dysfunction, cancer cachexia is a systemic disease involving remodeling of nonmuscle organs such as adipose and liver. Impairment of mitochondrial function is associated with multiple chronic diseases. The tissue-specific control of mitochondrial function in cancer cachexia is not well defined. This study determined mitochondrial respiratory capacity and coupling control of skeletal muscle, white adipose tissue (WAT), and liver in colon-26 (C26) tumor-induced cachexia. Tissues were collected from PBS-injected weight-stable mice, C26 weight-stable mice and C26 mice with moderate (10% weight loss) and severe cachexia (20% weight loss). The respiratory control ratio [(RCR) an index of oxidative phosphorylation (OXPHOS) coupling efficiency] was low in WAT during the induction of cachexia because of high nonphosphorylating LEAK respiration. Liver RCR was low in C26 weight-stable and moderately cachexic mice because of reduced OXPHOS. Liver RCR was further reduced with severe cachexia, where Ant2 but not Ucp2 expression was increased. Ant2 was inversely correlated with RCR in the liver ( r = -0.547, P < 0.01). Liver cardiolipin increased in moderate and severe cachexia, suggesting this early event may also contribute to mitochondrial uncoupling. Impaired skeletal muscle mitochondrial respiration occurred predominantly in severe cachexia, at complex I. These findings suggest that mitochondrial function is subject to tissue-specific control during cancer cachexia, whereby remodeling in WAT and liver arise early and may contribute to altered energy balance, followed by impaired skeletal muscle respiration. We highlight an under-recognized role of liver and WAT mitochondrial function in cancer cachexia and suggest mitochondrial function of multiple tissues to be therapeutic targets.
- Published
- 2019
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11. Selective elimination of senescent cells by mitochondrial targeting is regulated by ANT2.
- Author
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Hubackova S, Davidova E, Rohlenova K, Stursa J, Werner L, Andera L, Dong L, Terp MG, Hodny Z, Ditzel HJ, Rohlena J, and Neuzil J
- Subjects
- Adenine Nucleotide Translocator 2 genetics, Animals, Apoptosis drug effects, Apoptosis genetics, Cell Proliferation drug effects, Cell Survival drug effects, Cell Survival genetics, Gene Knockdown Techniques, Humans, MCF-7 Cells, Mice, Mice, Inbred NOD, Mice, SCID, Mice, Transgenic, Mitochondria metabolism, Transfection, Xenograft Model Antitumor Assays, Adenine Nucleotide Translocator 2 metabolism, Antineoplastic Agents, Hormonal pharmacology, Cellular Senescence drug effects, Mitochondria drug effects, Tamoxifen pharmacology
- Abstract
Cellular senescence is a form of cell cycle arrest that limits the proliferative potential of cells, including tumour cells. However, inability of immune cells to subsequently eliminate senescent cells from the organism may lead to tissue damage, inflammation, enhanced carcinogenesis and development of age-related diseases. We found that the anticancer agent mitochondria-targeted tamoxifen (MitoTam), unlike conventional anticancer agents, kills cancer cells without inducing senescence in vitro and in vivo. Surprisingly, it also selectively eliminates both malignant and non-cancerous senescent cells. In naturally aged mice treated with MitoTam for 4 weeks, we observed a significant decrease of senescence markers in all tested organs compared to non-treated animals. Mechanistically, we found that the susceptibility of senescent cells to MitoTam is linked to a very low expression level of adenine nucleotide translocase-2 (ANT2), inherent to the senescent phenotype. Restoration of ANT2 in senescent cells resulted in resistance to MitoTam, while its downregulation in non-senescent cells promoted their MitoTam-triggered elimination. Our study documents a novel, translationally intriguing role for an anticancer agent targeting mitochondria, that may result in a new strategy for the treatment of age-related diseases and senescence-associated pathologies.
- Published
- 2019
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12. Upregulation of miR-137 reverses sorafenib resistance and cancer-initiating cell phenotypes by degrading ANT2 in hepatocellular carcinoma.
- Author
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Lu AQ, Lv B, Qiu F, Wang XY, and Cao XH
- Subjects
- 3' Untranslated Regions, Carcinoma, Hepatocellular therapy, Cell Line, Tumor, Female, Gene Expression Regulation, Neoplastic drug effects, Hep G2 Cells, High-Intensity Focused Ultrasound Ablation, Humans, Male, Neoplastic Stem Cells drug effects, Niacinamide pharmacology, Sorafenib, Adenine Nucleotide Translocator 2 genetics, Carcinoma, Hepatocellular genetics, Drug Resistance, Neoplasm, Liver Neoplasms genetics, MicroRNAs genetics, Niacinamide analogs & derivatives, Phenylurea Compounds pharmacology, Up-Regulation
- Abstract
Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. More than 80% of patients with HCC are not good candidates for curative surgical resection due to advanced liver cirrhosis caused by underlying chronic hepatitis virus (B or C) infection. Sorafenib, an oral multikinase inhibitor, is the only approved agent for the treatment of advanced HCC. Although, sorafenib currently sets the new standard for advanced HCC treatment, tumor response rates are usually quite low. An understanding of the underlying mechanisms for sorafenib resistance is critical. In the present study, we found that adenine nucleotide translocator 2 (ANT2) was upregulated in sorafenib‑resistant HCC Huh7 cells (Huh7-R) and its overexpression promoted sorafenib resistance. ANT2 induced the formation of cancer-initiating cell (CIC) phenotypes and promoted metastasis-associated traits in the Huh7 cells. Silencing of miR-137 upregulated ANT2 protein expression in the Huh7 cells. miR-137 was downregulated in the Huh7-R cells, compared with that in the Huh7 cells and its restoration reversed sorafenib resistance in the Huh7-R cells. Restoration of miR-137 inhibited formation of CIC traits and attenuated the abilities of migration and invasion in the Huh7-R cells. Moreover, we demonstrated that high-intensity focused ultrasound (HIFU) in unresectable HCC upregulated serum miR-137. Combining HIFU and sorafenib may be a wise option for advanced and unresectable HCC.
- Published
- 2017
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13. Mitochondrial ATP transporter depletion protects mice against liver steatosis and insulin resistance.
- Author
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Cho J, Zhang Y, Park SY, Joseph AM, Han C, Park HJ, Kalavalapalli S, Chun SK, Morgan D, Kim JS, Someya S, Mathews CE, Lee YJ, Wohlgemuth SE, Sunny NE, Lee HY, Choi CS, Shiratsuchi T, Oh SP, and Terada N
- Subjects
- Adenine Nucleotide Translocator 2 genetics, Animals, Atractyloside analogs & derivatives, Diet, High-Fat, Disease Models, Animal, Fatty Liver therapy, Female, Glucose Clamp Technique, Hyperinsulinism, Lipid Metabolism, Lipogenesis, Liver metabolism, Liver pathology, Male, Mice, Mice, Knockout, Mitochondrial Membranes metabolism, Non-alcoholic Fatty Liver Disease metabolism, Non-alcoholic Fatty Liver Disease pathology, Non-alcoholic Fatty Liver Disease therapy, Obesity metabolism, Obesity therapy, Pyruvic Acid metabolism, Adenine Nucleotide Translocator 2 metabolism, Adenosine Triphosphate metabolism, Fatty Liver metabolism, Insulin Resistance, Mitochondria, Liver metabolism, Protective Agents metabolism
- Abstract
Non-alcoholic fatty liver disease (NAFLD) is a common metabolic disorder in obese individuals. Adenine nucleotide translocase (ANT) exchanges ADP/ATP through the mitochondrial inner membrane, and Ant2 is the predominant isoform expressed in the liver. Here we demonstrate that targeted disruption of Ant2 in mouse liver enhances uncoupled respiration without damaging mitochondrial integrity and liver functions. Interestingly, liver specific Ant2 knockout mice are leaner and resistant to hepatic steatosis, obesity and insulin resistance under a lipogenic diet. Protection against fatty liver is partially recapitulated by the systemic administration of low-dose carboxyatractyloside, a specific inhibitor of ANT. Targeted manipulation of hepatic mitochondrial metabolism, particularly through inhibition of ANT, may represent an alternative approach in NAFLD and obesity treatment.
- Published
- 2017
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14. Deficiency in the mouse mitochondrial adenine nucleotide translocator isoform 2 gene is associated with cardiac noncompaction.
- Author
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Kokoszka JE, Waymire KG, Flierl A, Sweeney KM, Angelin A, MacGregor GR, and Wallace DC
- Subjects
- Adenine metabolism, Adenine Nucleotide Translocator 2 genetics, Animals, Biological Transport, Cell Proliferation, Embryo, Mammalian, Female, Gene Expression Regulation, Developmental, Genes, Lethal, Heart Defects, Congenital embryology, Heart Defects, Congenital metabolism, Heart Defects, Congenital pathology, Heart Failure embryology, Heart Failure metabolism, Heart Failure pathology, Heart Ventricles abnormalities, Heart Ventricles embryology, Integrases, Male, Mice, Mice, Transgenic, Mitochondria pathology, Mitochondrial Swelling genetics, Myocytes, Cardiac pathology, Organogenesis, Phenotype, Adenine Nucleotide Translocator 2 deficiency, Heart Defects, Congenital genetics, Heart Failure genetics, Heart Ventricles metabolism, Mitochondria metabolism, Myocytes, Cardiac metabolism
- Abstract
The mouse fetal and adult hearts express two adenine nucleotide translocator (ANT) isoform genes. The predominant isoform is the heart-muscle-brain ANT-isoform gene 1 (Ant1) while the other is the systemic Ant2 gene. Genetic inactivation of the Ant1 gene does not impair fetal development but results in hypertrophic cardiomyopathy in postnatal mice. Using a knockin X-linked Ant2 allele in which exons 3 and 4 are flanked by loxP sites combined in males with a protamine 1 promoter driven Cre recombinase we created females heterozygous for a null Ant2 allele. Crossing the heterozygous females with the Ant2(fl), PrmCre(+) males resulted in male and female ANT2-null embryos. These fetuses proved to be embryonic lethal by day E14.5 in association with cardiac developmental failure, immature cardiomyocytes having swollen mitochondria, cardiomyocyte hyperproliferation, and cardiac failure due to hypertrabeculation/noncompaction. ANTs have two main functions, mitochondrial-cytosol ATP/ADP exchange and modulation of the mitochondrial permeability transition pore (mtPTP). Previous studies imply that ANT2 biases the mtPTP toward closed while ANT1 biases the mtPTP toward open. It has been reported that immature cardiomyocytes have a constitutively opened mtPTP, the closure of which signals the maturation of cardiomyocytes. Therefore, we hypothesize that the developmental toxicity of the Ant2 null mutation may be the result of biasing the cardiomyocyte mtPTP to remain open thus impairing cardiomyocyte maturation and resulting in cardiomyocyte hyperproliferation and failure of trabecular maturation. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi., (Copyright © 2016 Elsevier B.V. All rights reserved.)
- Published
- 2016
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15. Targeting Adenine Nucleotide Translocase-2 (ANT2) to Overcome Resistance to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor in Non-Small Cell Lung Cancer.
- Author
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Jang JY, Kim YG, Nam SJ, Keam B, Kim TM, Jeon YK, and Kim CW
- Subjects
- Adenine Nucleotide Translocator 2 antagonists & inhibitors, Carcinoma, Non-Small-Cell Lung drug therapy, Carcinoma, Non-Small-Cell Lung genetics, Cell Line, Tumor, Cell Proliferation drug effects, Cell Survival drug effects, Drug Synergism, ErbB Receptors metabolism, Gefitinib, Gene Expression Regulation, Neoplastic drug effects, Humans, Lung Neoplasms drug therapy, Lung Neoplasms genetics, Molecular Targeted Therapy, Promoter Regions, Genetic drug effects, Up-Regulation drug effects, Adenine Nucleotide Translocator 2 genetics, Antineoplastic Agents pharmacology, Carcinoma, Non-Small-Cell Lung metabolism, Drug Resistance, Neoplasm drug effects, Lung Neoplasms metabolism, Quinazolines pharmacology, RNA, Small Interfering pharmacology
- Abstract
EGFR tyrosine kinase inhibitor (EGFR-TKI) therapy has achieved favorable clinical outcomes in non-small cell lung cancer (NSCLC) patients with EGFR mutations. However, patients eventually develop resistance to EGFR-TKIs by several mechanisms. Adenine nucleotide translocase-2 (ANT2) is an oncogenic mitochondrial membrane-associated protein. We investigated the therapeutic potential of ANT2 inhibition to EGFR-TKI resistance in NSCLC using gefitinib-sensitive (PC9 and HCC827) and gefitinib-resistant (H1975 and HCC827/GR) NSCLC cell lines. ANT2 was inhibited by transfecting cells with an ANT2-specific shRNA. ANT2 expression was elevated in the H1975 and HCC827/GR cells compared with the PC9 and HCC827 cells. ANT2 upregulation in gefitinib-resistant cells was associated with increased SP1 binding to the ANT2 promoter. ANT2-specific shRNA decreased NSCLC cell viability. Moreover, ANT2-specific shRNA sensitized the H1975 and HCC827/GR cells to gefitinib, accompanied by HSP90 and EGFR downregulation. ANT2-specific shRNA also inactivated the PI3K/Akt signaling pathway in the H1975 and HCC827/GR cells, which was mediated by the suppression of miR-221/222 levels and by the subsequent restoration of PTEN. In EGFR-TKI-treated NSCLC patients, ANT2 expression was higher in patients exhibiting poor responses compared with patients showing excellent responses. Furthermore, ANT2 expression increased in tumor tissues biopsied after acquiring gefitinib resistance compared with tissues before gefitinib treatment. These findings suggest that ANT2 overexpression contributes to EGFR-TKI resistance in NSCLC and that ANT2 targeting may be considered a novel strategy for overcoming this resistance. Mol Cancer Ther; 15(6); 1387-96. ©2016 AACR., (©2016 American Association for Cancer Research.)
- Published
- 2016
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16. ANT2 shRNA downregulates miR-19a and miR-96 through the PI3K/Akt pathway and suppresses tumor growth in hepatocellular carcinoma cells.
- Author
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Baik SH, Lee J, Lee YS, Jang JY, and Kim CW
- Subjects
- Animals, Cell Line, Tumor, Cell Proliferation, Disease Models, Animal, Disease Progression, Gene Expression Regulation, Neoplastic, Heterografts, Humans, Mice, Phosphatidylinositol 3-Kinases, Proto-Oncogene Proteins c-akt metabolism, RNA Interference, Signal Transduction, Adenine Nucleotide Translocator 2 genetics, Carcinoma, Hepatocellular genetics, Carcinoma, Hepatocellular metabolism, Liver Neoplasms genetics, Liver Neoplasms metabolism, MicroRNAs genetics, RNA, Small Interfering genetics
- Abstract
MicroRNAs (miRNAs) are negative regulators of gene expression, and miRNA deregulation is found in various tumors. We previously reported that suppression of adenine nucleotide translocase 2 (ANT2) by short hairpin RNA (shRNA) inhibits hepatocellular carcinoma (HCC) development by rescuing miR-636 expression. However, the tumor-suppressive mechanisms of ANT2 shRNA are still poorly understood in HCC. Here, we hypothesized that miRNAs that are specifically downregulated by ANT2 shRNA might function as oncomiRs, and we investigated the roles of ANT2 shRNA-regulated miRNAs in the pathogenesis of HCC. Our data show that miR-19a and miR-96, whose expression is regulated by ANT2 suppression, were markedly upregulated in HCC cell lines and clinical samples. Ectopic expression of miR-19a and miR-96 dramatically induced the proliferation and colony formation of hepatoma cells in vitro, whereas inhibition of miR-19a and miR-96 reduced these effects. To investigate the in vivo function, we implanted miR-96-overexpressing HepG2 cells in a xenograft model and demonstrated that the increase in miR-96 promoted tumor growth. We also found that miR-19a and miR-96 inhibited expression of tissue inhibitor of metalloproteinase-2. Taken together, our results suggest that ANT2-regulated miR-19a and miR-96 play an important role in promoting the proliferation of human HCC cells, and the knockdown of ANT2 directly downregulates miR-19a and miR-96, ultimately resulting in the suppression of tumor growth.
- Published
- 2016
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17. Mitochondrial ATP transporter Ant2 depletion impairs erythropoiesis and B lymphopoiesis.
- Author
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Cho J, Seo J, Lim CH, Yang L, Shiratsuchi T, Lee MH, Chowdhury RR, Kasahara H, Kim JS, Oh SP, Lee YJ, and Terada N
- Subjects
- Adenine Nucleotide Translocator 2 genetics, Adenine Nucleotide Translocator 2 metabolism, Adenosine Triphosphate metabolism, Anemia genetics, Anemia metabolism, Animals, B-Lymphocytes metabolism, Erythrocytes metabolism, Erythropoiesis genetics, Female, Gene Knockout Techniques, Growth Disorders genetics, Growth Disorders metabolism, Humans, Lymphopoiesis genetics, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Mitochondria genetics, Mitochondria metabolism, Adenine Nucleotide Translocator 2 deficiency, B-Lymphocytes cytology, Erythrocytes cytology, Erythropoiesis physiology, Lymphopoiesis physiology, Mitochondrial ADP, ATP Translocases metabolism
- Abstract
Adenine nucleotide translocases (ANTs) transport ADP and ATP through mitochondrial inner membrane, thus playing an essential role for energy metabolism of eukaryotic cells. Mice have three ANT paralogs, Ant1 (Slc25a4), Ant2 (Slc25a5) and Ant4 (Slc25a31), which are expressed in a tissue-dependent manner. While knockout mice have been characterized with Ant1 and Ant4 genes, which resulted in exercise intolerance and male infertility, respectively, the role of the ubiquitously expressed Ant2 gene in animal development has not been fully demonstrated. Here, we generated Ant2 hypomorphic mice by targeted disruption of the gene, in which Ant2 expression is largely depleted. The mice showed apparently normal embryonic development except pale phenotype along with a reduced birth rate. However, postnatal growth was severely retarded with macrocytic anemia, B lymphocytopenia, lactic acidosis and bloated stomach, and died within 4 weeks. Ant2 depletion caused anemia in a cell-autonomous manner by maturation arrest of erythroid precursors with increased reactive oxygen species and premature deaths. B-lymphocyte development was similarly affected by Ant2 depletion, and splenocytes showed a reduction in maximal respiration capacity and cellular ATP levels as well as an increase in cell death accompanying mitochondrial permeability transition pore opening. In contrast, myeloid, megakaryocyte and T-lymphocyte lineages remained apparently intact. Erythroid and B-cell development may be particularly vulnerable to Ant2 depletion-mediated mitochondrial dysfunction and oxidative stress.
- Published
- 2015
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18. TGF-β/NF1/Smad4-mediated suppression of ANT2 contributes to oxidative stress in cellular senescence.
- Author
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Kretova M, Sabova L, Hodny Z, Bartek J, Kollarovic G, Nelson BD, Hubackova S, and Luciakova K
- Subjects
- Adenine Nucleotide Translocator 2 genetics, Cell Line, Cell Nucleus drug effects, Cell Nucleus metabolism, Cytoprotection drug effects, DNA Damage, Down-Regulation drug effects, Etoposide pharmacology, Humans, Mutation, Promoter Regions, Genetic, Repressor Proteins metabolism, Adenine Nucleotide Translocator 2 metabolism, Cellular Senescence drug effects, NFI Transcription Factors metabolism, Oxidative Stress drug effects, Smad4 Protein metabolism, Transforming Growth Factor beta metabolism
- Abstract
Oxidative stress and persistent activation of DNA damage response (DDR) are causally involved in the development of cellular senescence, a phenomenon implicated in fundamental (patho)physiological processes such as aging, fetal development and tumorigenesis. Here, we report that adenine nucleotide translocase-2 (ANT2) is consistently down-regulated in all three major forms of cellular senescence: replicative, oncogene-induced and drug-induced, in both normal and cancerous human cells. We previously reported formation of novel NF1/Smad transcription repressor complexes in growth-arrested fibroblasts. Here we show that such complexes form in senescent cells. Mechanistically, binding of the NF1/Smad complexes to the NF1-dependent repressor elements in the ANT2 gene promoter repressed ANT2 expression. Etoposide-induced formation of these complexes and repression of ANT2 were relatively late events co-incident with production and secretion of, and dependent on, TGF-β. siRNA-mediated knock-down of ANT2 in proliferating cells resulted in increased levels of reactive oxygen species (ROS) and activation of the DDR. Knock-down of ANT2, together with etoposide treatment, further intensified ROS production and DNA damage signaling, leading to enhanced apoptosis. Together, our data show that TGF-β-mediated suppression of ANT2 through NF1/Smad4 complexes contributes to oxidative stress and DNA damage during induction of cellular senescence., (Copyright © 2014 Elsevier Inc. All rights reserved.)
- Published
- 2014
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19. Combined RNA interference of adenine nucleotide translocase-2 and ganciclovir therapy in hepatocellular carcinoma.
- Author
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Kim JE, Hwang MH, Lee HW, Lee SW, Lee J, and Ahn BC
- Subjects
- Animals, Biological Transport, Carcinoma, Hepatocellular diagnostic imaging, Carcinoma, Hepatocellular drug therapy, Carcinoma, Hepatocellular genetics, Cell Line, Tumor, Cell Survival drug effects, Cell Survival genetics, Cell Transformation, Neoplastic, Ganciclovir metabolism, Ganciclovir therapeutic use, Herpesvirus 1, Human enzymology, Herpesvirus 1, Human genetics, Humans, Liver Neoplasms diagnostic imaging, Liver Neoplasms drug therapy, Liver Neoplasms genetics, Mice, Prodrugs pharmacology, Radionuclide Imaging, Sodium Pertechnetate Tc 99m metabolism, Thymidine Kinase genetics, Adenine Nucleotide Translocator 2 deficiency, Adenine Nucleotide Translocator 2 genetics, Carcinoma, Hepatocellular therapy, Ganciclovir pharmacology, Liver Neoplasms therapy, RNA Interference
- Abstract
Purpose: The purpose of this study was to investigate the anticancer effects of combined RNA interference (RNAi) of the adenine nucleotide translocase-2 (ANT2) gene and ganciclovir (GCV) therapy for treatment of hepatocellular carcinoma cells (Huh 7) in an animal model., Methods: The Huh 7/NTG stable cell line was established by transfection of a vector with the human sodium iodide symporter (hNIS), HSV1-sr39 thymidine kinase (tk), and enhanced green florescent protein (EGFP) fusion gene into Huh 7 cells. mRNA expressions of these genes were evaluated by RT-PCR analysis. The functions of hNIS and HSV1-sr39tk were verified with (125)I uptake and (3)H-penciclovir (PCV) uptake tests. EGFP and hNIS expression was confirmed with confocal microscopy after immunocytochemical staining. We treated the tumor cells with ANT2 shRNA or GCV or both ANT2 shRNA and GCV and treated the in vivo mouse model with a Huh 7/NTG tumor xenograft. The therapeutic effects of the in vivo study were assessed with caliper measurements and gamma camera imaging using (99m)Tc-pertechnetate., Results: Huh 7/NTG cells showed a cell number-dependent increase in (125)I uptake and a 24-fold higher (3)H-PCV uptake compared to parent Huh 7 cells. Huh 7/NTG cells transfected with ANT2 shRNA had lower ANT2 mRNA expression and more impaired proliferation activity than cells transfected with scramble shRNA. Proliferation of Huh 7/NTG cells was also inhibited by GCV treatment. Combined GCV and ANT2 shRNA therapy further inhibited cell proliferation in the in vitro study. The combined therapy with GCV and ANT2 shRNA showed a further decrease in tumor growth in the mouse model., Conclusions: Our results suggest that the combined RNA interference with ANT2 and GCV therapy inhibited hepatocellular carcinoma cell proliferation more than single GCV therapy or ANT2 shRNA therapy in vitro and in vivo. Therefore it could be applied treating incurable hepatocellular carcinoma., (© 2013.)
- Published
- 2013
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20. The mitochondrial solute carrier SLC25A5 at Xq24 is a novel candidate gene for non-syndromic intellectual disability.
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Vandewalle J, Bauters M, Van Esch H, Belet S, Verbeeck J, Fieremans N, Holvoet M, Vento J, Spreiz A, Kotzot D, Haberlandt E, Rosenfeld J, Andrieux J, Delobel B, Dehouck MB, Devriendt K, Fryns JP, Marynen P, Goldstein A, and Froyen G
- Subjects
- Adenine Nucleotide Translocator 2 genetics, Alu Elements, Base Sequence, Brain metabolism, Brain pathology, Child, Preschool, Cohort Studies, DNA Mutational Analysis, Female, Heterozygote, Humans, Infant, Intellectual Disability pathology, Male, Mitochondria genetics, Molecular Sequence Data, Pedigree, X Chromosome Inactivation, Adenine Nucleotide Translocator 2 metabolism, Chromosome Deletion, Chromosomes, Human, X genetics, Intellectual Disability genetics, Mitochondria metabolism
- Abstract
Loss-of-function mutations in several different neuronal pathways have been related to intellectual disability (ID). Such mutations often are found on the X chromosome in males since they result in functional null alleles. So far, microdeletions at Xq24 reported in males always have been associated with a syndromic form of ID due to the loss of UBE2A. Here, we report on overlapping microdeletions at Xq24 that do not include UBE2A or affect its expression, in patients with non-syndromic ID plus some additional features from three unrelated families. The smallest region of overlap, confirmed by junction sequencing, harbors two members of the mitochondrial solute carrier family 25, SLC25A5 and SLC25A43. However, identification of an intragenic microdeletion including SLC25A43 but not SLC25A5 in a healthy boy excluded a role for SLC25A43 in cognition. Therefore, our findings point to SLC25A5 as a novel gene for non-syndromic ID. This highly conserved gene is expressed ubiquitously with high levels in cortex and hippocampus, and a presumed role in mitochondrial exchange of ADP/ATP. Our data indicate that SLC25A5 is involved in memory formation or establishment, which could add mitochondrial processes to the wide array of pathways that regulate normal cognitive functions.
- Published
- 2013
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21. Many faces of mitochondrial uncoupling during age: damage or defense?
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Bellanti F, Romano AD, Giudetti AM, Rollo T, Blonda M, Tamborra R, Vendemiale G, and Serviddio G
- Subjects
- Adenine Nucleotide Translocator 1 genetics, Adenine Nucleotide Translocator 1 metabolism, Adenine Nucleotide Translocator 2 genetics, Adenine Nucleotide Translocator 2 metabolism, Adenosine Triphosphate metabolism, Aging genetics, Animals, Energy Metabolism, Fatty Acids metabolism, Free Radicals metabolism, Glycolysis, Ion Channels genetics, Ion Channels metabolism, Male, Membrane Potential, Mitochondrial, Mitochondrial Proteins genetics, Mitochondrial Proteins metabolism, Oxidative Stress, Protons, RNA, Messenger genetics, RNA, Messenger metabolism, Rats, Rats, Wistar, Uncoupling Protein 2, Uncoupling Protein 3, Aging metabolism, Mitochondria, Heart metabolism, Mitochondria, Liver metabolism
- Abstract
An increased mitochondrial proton leak occurs in aging, but the origin of such modification remains unclear. This study defined the cause of mitochondrial uncoupling in mitotic (liver) and postmitotic (heart) rat tissues during aging and its effects on energy homeostasis and free radical production. Proton leak in old heart mitochondria was dependent on uncoupling proteins' upregulation, whereas it was caused by alterations in the mitochondrial membrane composition in old liver. ATP homeostasis was impaired in both tissues from old animals and was associated to disrupted F0F1-ATPase activity. H2O2 production rate and 4-hydroxy-2-nonenalprotein adducts were higher in old liver mitochondria compared with young liver mitochondria, but they were similar in heart mitochondria from both groups. Moreover, key mitochondrial biogenesis regulators were upregulated in old liver but downregulated in old heart. In conclusion, uncoupling proteins mediate proton leak and avoid oxidative damage in heart, acting as a protective mechanism. This does not occur in liver, where ATP depletion and oxidative stress may stimulate mitochondrial biogenesis and eliminate damaged cells.
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- 2013
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22. The combination of ANT2 shRNA and hNIS radioiodine gene therapy increases CTL cytotoxic activity through the phenotypic modulation of cancer cells: combination treatment with ANT2 shRNA and I-131.
- Author
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Choi Y, Lee HW, Lee J, and Jeon YH
- Subjects
- Animals, Apoptosis, Caspase 3 metabolism, Cell Line, Tumor, Enzyme Activation drug effects, Female, Gene Expression, Genes, MHC Class I, Genetic Therapy, Iodine Radioisotopes pharmacology, Iodine Radioisotopes therapeutic use, L-Lactate Dehydrogenase metabolism, Mice, Mice, Inbred BALB C, Phenotype, RNA, Small Interfering genetics, RNA, Small Interfering pharmacology, Radiopharmaceuticals therapeutic use, fas Receptor genetics, Adenine Nucleotide Translocator 2 genetics, Antineoplastic Combined Chemotherapy Protocols therapeutic use, Colonic Neoplasms drug therapy, RNA, Small Interfering therapeutic use, Symporters genetics, T-Lymphocytes, Cytotoxic
- Abstract
Background: It is important to simultaneously induce strong cell death and antitumor immunity in cancer patients for successful cancer treatment. Here, we investigated the cytotoxic and phenotypic modulation effects of the combination of ANT2 shRNA and human sodium iodide symporter (hNIS) radioiodine gene therapy in vitro and in vivo and visualized the antitumor effects in an immunocompromised mouse colon cancer model., Methods: A mouse colon cancer cell line co-expressing hNIS and the luciferase gene (CT26/hNIS-Fluc, named CT26/NF) was established. CT26/NF cells and tumor-bearing mice were treated with HBSS, scramble, ANT2 shRNA, I-131, and ANT2 shRNA + I-131. The apoptotic rates (%) and MHC class I and Fas gene expression levels were determined in treated CT26/NF cells using flow cytometry. Concurrently, the level of caspase-3 activation was determined in treated cells in vitro. For in vivo therapy, tumor-bearing mice were treated with scramble, ANT2 shRNA, I-131, and the combination therapy, and the anti-tumor effects were monitored using bioluminescence. The killing activity of cytotoxic T cells (CTLs) was measured with a lactate dehydrogenase (LDH) assay., Results: For the in vitro experiments, the combination of ANT2 shRNA and I-131 resulted in a higher apoptotic cell death rate compared with ANT2 shRNA or I-131 alone, and the levels of MHC class I and Fas-expressing cancer cells were highest in the cells receiving combination treatment, while single treatment modestly increased the level of MHC class I and Fas gene expression. The combination of ANT2 shRNA and I-131 resulted in a higher caspase-3 activation than single treatments. Interestingly, in vivo combination treatment led to increased gene expression of MHC class I and Fas than the respective mono-therapies; furthermore, bioluminescence showed increased antitumor effects after combination treatment than monotherapies. The LDH assay revealed that the CTL killing activity against CT26/NF cells was most effective after combination therapy., Conclusions: Increased cell death and phenotypic modulation of cancer cells in vitro and in vivo were achieved simultaneously after combination therapy with ANT2 shRNA and I-131, and this combination therapy induced remarkable antitumor outcomes through improvements in CTL immunity against CT26/NF. Our results suggest that combination therapy can be used as a new therapeutic strategy for cancer patients who show resistance to single therapy such as radiation or immunotherapy.
- Published
- 2013
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23. B-cell translocation gene 2: expression in the rat ovary and potential association with adenine nucleotide translocase 2 in mitochondria.
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Park JI, Kim SG, Baek MW, Park TJ, Lim IK, Seo YW, and Chun SY
- Subjects
- Adenine Nucleotide Translocator 2 genetics, Adenosine Triphosphate metabolism, Animals, Chorionic Gonadotropin pharmacology, Female, Granulosa Cells drug effects, Granulosa Cells metabolism, HeLa Cells, Humans, Hydrogen Peroxide metabolism, Immediate-Early Proteins chemistry, Immediate-Early Proteins genetics, Luteinizing Hormone pharmacology, Mitochondria drug effects, Neoplasm Proteins genetics, Neoplasm Proteins metabolism, Ovary drug effects, Protein Binding drug effects, Protein Transport drug effects, Rats, Rats, Sprague-Dawley, Sheep, Tumor Suppressor Proteins chemistry, Tumor Suppressor Proteins genetics, Adenine Nucleotide Translocator 2 metabolism, Immediate-Early Proteins metabolism, Mitochondria enzymology, Ovary metabolism, Tumor Suppressor Proteins metabolism
- Abstract
The B-cell translocation gene 2 (Btg2) is an anti-proliferative tumor suppressor gene that behaves as a transcriptional regulator. The present study investigated gonadotropin induction of Btg2 in the rat ovary and the mechanism of Btg2 action as a partner of mitochondrial protein adenine nucleotide translocase 2 (Ant2). Transient induction of Btg2 as well as Btg1 mRNA levels by LH/hCG was observed in ovarian granulosa cells. Btg2 protein levels were also stimulated by LH/hCG. LH-induced gene expression of Btg2 required ERK signal pathway. Studies of deletion mutants in HeLa cells showed that deletion of Btg2 C-terminus (Btg2/ΔC) abolished the interaction with Ant2. In fact, the expression levels of Btg2/ΔC construct were decreased in mitochondrial fraction. Btg2 was also expressed in mitochondria and interacted with Ant2 in preovulatory granulosa cells. Interestingly, a Btg2/ΔC construct inhibited an action of Btg2 wild-type on ATP and H(2)O(2) production. These findings demonstrate the gonadotropin stimulation of Btg2 in the ovary and, the physical interaction of Btg2 with Ant2 in mitochondria., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)
- Published
- 2013
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24. ANT2 suppression by shRNA may be able to exert anticancer effects in HCC further by restoring SOCS1 expression.
- Author
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Jang JY, Jeon YK, Lee CE, and Kim CW
- Subjects
- Adenine Nucleotide Translocator 2 metabolism, Carcinoma, Hepatocellular pathology, Cell Line, Tumor, Cell Proliferation, Gene Expression Regulation, Neoplastic, Humans, Janus Kinase 1 metabolism, Liver Neoplasms pathology, MicroRNAs genetics, RNA, Small Interfering, Signal Transduction, Suppressor of Cytokine Signaling 1 Protein, Suppressor of Cytokine Signaling Proteins metabolism, Adenine Nucleotide Translocator 2 genetics, Carcinoma, Hepatocellular genetics, Liver Neoplasms genetics, Suppressor of Cytokine Signaling Proteins genetics
- Abstract
Suppressor of cytokine signaling 1 (SOCS1) is a negative regulator of Janus kinase and the signal transducer and activation of transcription (Jak-STAT) pathway. SOCS-1 is known to be silenced by aberrant promoter methylation in human hepatocellular carcinoma (HCC) during early tumorigenesis, therefore, a strategy to restore SOCS1 expression can be utilized for cancer therapy. Here, we examined the influence of adenine nucleotide translocase 2 (ANT2) suppression by short-hairpin RNA (shRNA) on SOCS1 expression and its downstream effect in HCC. ANT2 shRNA treatment led to restoration of SOCS1 expression along with its promoter demethylation in Hep3B cells, which was accompanied by decreased DNA methyltransferase 1 (DNMT1) activity through the suppression of Ras/PI3K/Akt signaling. Restoration of SOCS1 by ANT2 knockdown, subsequently, inhibited STAT3 activity and downregulated the expression of miR-21, which has been reported to be an important onco-miR in HCC. Downregulation of miR-21 efficiently suppressed Hep3B cell proliferation in vitro with a comparable level to ANT2 shRNA treatment. ANT2 suppression by shRNA may be able to exert anticancer effects in HCC further by restoring SOCS1 expression.
- Published
- 2013
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25. Apigenin sensitizes prostate cancer cells to Apo2L/TRAIL by targeting adenine nucleotide translocase-2.
- Author
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Oishi M, Iizumi Y, Taniguchi T, Goi W, Miki T, and Sakai T
- Subjects
- Adenine Nucleotide Translocator 2 antagonists & inhibitors, Adenine Nucleotide Translocator 2 genetics, Apigenin chemistry, Apoptosis drug effects, Cell Line, Tumor drug effects, Drug Synergism, Gene Knockdown Techniques, Genistein chemistry, Humans, Male, Protein Binding, RNA, Small Interfering genetics, Receptors, TNF-Related Apoptosis-Inducing Ligand genetics, Receptors, TNF-Related Apoptosis-Inducing Ligand metabolism, Up-Regulation drug effects, Adenine Nucleotide Translocator 2 metabolism, Antineoplastic Agents pharmacology, Apigenin pharmacology, Prostatic Neoplasms drug therapy, TNF-Related Apoptosis-Inducing Ligand pharmacology
- Abstract
Apo2 ligand (Apo2L)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic agent. Recombinant human Apo2L/TRAIL has been under clinical trials, whereas various kinds of malignant tumors have resistance to Apo2L/TRAIL. We and others have shown that several anticancer agents and flavonoids overcome resistance to Apo2L/TRAIL by upregulating death receptor 5 (DR5) in malignant tumor cells. However, the mechanisms by which these compounds induce DR5 expression remain unknown. Here we show that the dietary flavonoid apigenin binds and inhibits adenine nucleotide translocase-2 (ANT2), resulting in enhancement of Apo2L/TRAIL-induced apoptosis by upregulation of DR5. Apigenin and genistein, which are major flavonoids, enhanced Apo2L/TRAIL-induced apoptosis in cancer cells. Apigenin induced DR5 expression, but genistein did not. Using our method identifying the direct targets of flavonoids, we compared the binding proteins of apigenin with those of genistein. We discovered that ANT2 was a target of apigenin, but not genistein. Similarly to apigenin, knockdown of ANT2 enhanced Apo2L/TRAIL-induced apoptosis by upregulating DR5 expression at the post-transcriptional level. Moreover, silencing of ANT2 attenuated the enhancement of Apo2L/TRAIL-induced apoptosis by apigenin. These results suggest that apigenin upregulates DR5 and enhances Apo2L/TRAIL-induced apoptosis by binding and inhibiting ANT2. We propose that ANT2 inhibitors may contribute to Apo2L/TRAIL therapy.
- Published
- 2013
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26. The mammalian proteins MMS19, MIP18, and ANT2 are involved in cytoplasmic iron-sulfur cluster protein assembly.
- Author
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van Wietmarschen N, Moradian A, Morin GB, Lansdorp PM, and Uringa EJ
- Subjects
- Adenine Nucleotide Translocator 2 genetics, Animals, Carrier Proteins genetics, Cytoplasm genetics, HEK293 Cells, Humans, Metallochaperones genetics, Metallochaperones metabolism, Metalloproteins, Mice, Multiprotein Complexes genetics, Nuclear Proteins genetics, Protein Binding physiology, Spindle Apparatus genetics, Spindle Apparatus metabolism, Transcription Factors genetics, Adenine Nucleotide Translocator 2 metabolism, Carrier Proteins metabolism, Cytoplasm metabolism, Multiprotein Complexes metabolism, Nuclear Proteins metabolism, Transcription Factors metabolism
- Abstract
Iron-sulfur (Fe-S) clusters are essential cofactors of proteins with a wide range of biological functions. A dedicated cytosolic Fe-S cluster assembly (CIA) system is required to assemble Fe-S clusters into cytosolic and nuclear proteins. Here, we show that the mammalian nucleotide excision repair protein homolog MMS19 can simultaneously bind probable cytosolic iron-sulfur protein assembly protein CIAO1 and Fe-S proteins, confirming that MMS19 is a central protein of the CIA machinery that brings Fe-S cluster donor proteins and the receiving apoproteins into proximity. In addition, we show that mitotic spindle-associated MMXD complex subunit MIP18 also interacts with both CIAO1 and Fe-S proteins. Specifically, it binds the Fe-S cluster coordinating regions in Fe-S proteins. Furthermore, we show that ADP/ATP translocase 2 (ANT2) interacts with Fe-S apoproteins and MMS19 in the CIA complex but not with the individual proteins. Together, these results elucidate the composition and interactions within the late CIA complex.
- Published
- 2012
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27. Computational identification of transcriptionally co-regulated genes, validation with the four ANT isoform genes.
- Author
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Dupont PY, Guttin A, Issartel JP, and Stepien G
- Subjects
- Computational Biology, Oligonucleotide Array Sequence Analysis, Promoter Regions, Genetic genetics, Adenine Nucleotide Translocator 1 genetics, Adenine Nucleotide Translocator 2 genetics, Adenine Nucleotide Translocator 3 genetics, Gene Expression Regulation genetics, Mitochondrial ADP, ATP Translocases genetics, Transcription, Genetic genetics
- Abstract
Background: The analysis of gene promoters is essential to understand the mechanisms of transcriptional regulation required under the effects of physiological processes, nutritional intake or pathologies. In higher eukaryotes, transcriptional regulation implies the recruitment of a set of regulatory proteins that bind on combinations of nucleotide motifs. We developed a computational analysis of promoter nucleotide sequences, to identify co-regulated genes by combining several programs that allowed us to build regulatory models and perform a crossed analysis on several databases. This strategy was tested on a set of four human genes encoding isoforms 1 to 4 of the mitochondrial ADP/ATP carrier ANT. Each isoform has a specific tissue expression profile linked to its role in cellular bioenergetics., Results: From their promoter sequence and from the phylogenetic evolution of these ANT genes in mammals, we constructed combinations of specific regulatory elements. These models were screened using the full human genome and databases of promoter sequences from human and several other mammalian species. For each of transcriptionally regulated ANT1, 2 and 4 genes, a set of co-regulated genes was identified and their over-expression was verified in microarray databases., Conclusions: Most of the identified genes encode proteins with a cellular function and specificity in agreement with those of the corresponding ANT isoform. Our in silico study shows that the tissue specific gene expression is mainly driven by promoter regulatory sequences located up to about a thousand base pairs upstream the transcription start site. Moreover, this computational strategy on the study of regulatory pathways should provide, along with transcriptomics and metabolomics, data to construct cellular metabolic networks.
- Published
- 2012
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28. Adenovirus adenine nucleotide translocator-2 shRNA effectively induces apoptosis and enhances chemosensitivity by the down-regulation of ABCG2 in breast cancer stem-like cells.
- Author
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Jang JY, Kim MK, Jeon YK, Joung YK, Park KD, and Kim CW
- Subjects
- ATP Binding Cassette Transporter, Subfamily G, Member 2, ATP-Binding Cassette Transporters metabolism, Adenine Nucleotide Translocator 2 antagonists & inhibitors, Adenine Nucleotide Translocator 2 genetics, Antineoplastic Agents pharmacology, Apoptosis drug effects, Apoptosis genetics, Breast Neoplasms, Cadherins antagonists & inhibitors, Cadherins genetics, Cell Line, Tumor, Cell Survival drug effects, Cell Survival genetics, Cell Transdifferentiation drug effects, Doxorubicin pharmacology, Drug Resistance, Neoplasm drug effects, Epithelial-Mesenchymal Transition drug effects, Female, Gene Expression Regulation, Neoplastic drug effects, Gene Knockdown Techniques, Humans, Neoplasm Proteins metabolism, Neoplastic Stem Cells drug effects, Neoplastic Stem Cells pathology, Signal Transduction drug effects, ATP-Binding Cassette Transporters genetics, Adenoviridae genetics, Drug Resistance, Neoplasm genetics, Neoplasm Proteins genetics, Neoplastic Stem Cells metabolism, RNA, Small Interfering genetics
- Abstract
Cancer stem cells (CSCs) are resistant to chemo- and radio-therapy, and can survive to regenerate new tumors. This is an important reason why various anti- cancer therapies often fail to completely control tumors, although they kill and eliminate the bulk of cancer cells. In this study, we determined whether or not adenine nucleotide translocator-2 (ANT2) suppression could also be effective in inducing cell death of breast cancer stem-like cells. A sub-population (SP; CD44+/ CD24-) of breast cancer cells has been reported to have stem/progenitor cell properties. We utilized the adeno- ANT2 shRNA virus to inhibit ANT2 expression and then observed the treatment effect in a SP of breast cancer cell line. In this study, MCF7, MDA-MB-231 cells, and breast epithelial cells (MCF10A) mesenchymally-transdifferentiated through E-cadherin knockdown were used. ANT2 expression was high in both stem-like cells and non-stem-like cells of MCF7 and MDA-MB-231 cells, and was induced and up-regulated by mesenchymal transdifferentiation in MCF10A cells (MCF10A(EMT)). Knockdown of ANT2 by adeno-shRNA virus efficiently induced apoptotic cell death in the stem-like cells of MCF7 and MDA-MB-231 cells, and MCF10A(EMT). Stem-like cells of MCF7 and MDA-MB-231, and MCF10A(EMT) cells exhibited increased drug (doxorubicin) resistance, and expressed a multi-drug resistant related molecule, ABCG2, at a high level. Adeno-ANT2 shRNA virus markedly sensitized the stem-like cells of MCF7 and MDA-MB-231, and the MCF10A(EMT) cells to doxorubicin, which was accompanied by down-regulation of ABCG2. Our results suggest that ANT2 suppression by adeno-shRNA virus is an effective strategy to induce cell death and increase the chemosensitivity of stem-like cells in breast cancer.
- Published
- 2012
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29. The hepatoprotective effects of adenine nucleotide translocator-2 against aging and oxidative stress.
- Author
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Kim HS, Je JH, Son TG, Park HR, Ji ST, Pokharel YR, Jeon HM, Kang KW, Kang HS, Chang SC, Kim HS, Chung HY, and Lee J
- Subjects
- Adenine Nucleotide Translocator 2 biosynthesis, Adenine Nucleotide Translocator 2 genetics, Animals, Disease Models, Animal, Hepatocytes metabolism, Hepatocytes pathology, Humans, Liver Cirrhosis metabolism, Male, Mice, Mice, Inbred ICR, Mitochondria metabolism, Rats, Rats, Sprague-Dawley, Reactive Oxygen Species metabolism, Specific Pathogen-Free Organisms, Transfection, Adenine Nucleotide Translocator 2 metabolism, Aging metabolism, Liver metabolism, Oxidative Stress physiology
- Abstract
Mitochondrial adenine nucleotide translocator (ANT) plays important roles in the regulation of mitochondrial permeability transition and cell bioenergetics. The mouse has three ANT isoforms (1, 2 and 4) showing tissue-specific expression patterns. Although ANT1 is known to have a pro-apoptotic property, the specific functions of ANT2 have not been well determined. In the present study, ANT2 expression was significantly lower in the aged rat liver and in a liver fibrosis model. To explore the protective role of ANT2 in the liver, we established a hepa1c1c7 cell line overexpressing ANT2. Overexpression of ANT2 caused hepa1c1c7 cells to be more resistant to oxidative stress, and mitochondrial membrane potential (MMP, ∆Ψm) was relatively intact in ANT2-overexpressing cells under oxidative stress. In addition, ANT2 was found to increase ATP production by influencing mitochondrial bioenergetics. These results imply that the hepatoprotective effect of ANT2 is due to the stabilization of MMP and enhanced ATP production, and thus, maintaining ANT2 levels in the liver might be important to enhance resistance to aging and oxidative stress., (© 2012 Informa UK, Ltd.)
- Published
- 2012
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30. TGF-β signals the formation of a unique NF1/Smad4-dependent transcription repressor-complex in human diploid fibroblasts.
- Author
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Luciakova K, Kollarovic G, Kretova M, Sabova L, and Nelson BD
- Subjects
- Animals, Diploidy, Humans, Mice, Transcription, Genetic, Transforming Growth Factor beta antagonists & inhibitors, Adenine Nucleotide Translocator 2 genetics, Fibroblasts metabolism, Gene Expression Regulation, Neurofibromin 1 metabolism, Repressor Proteins metabolism, Smad4 Protein metabolism, Transforming Growth Factor beta metabolism
- Abstract
We earlier reported the formation of a unique nuclear NF1/Smad complex in serum-restricted fibroblasts that acts as an NF1-dependent repressor of the human adenine nucleotide translocase-2 gene (ANT2) [K. Luciakova, G. Kollarovic, P. Barath, B.D. Nelson, Growth-dependent repression of human adenine nucleotide translocator-2 (ANT2) transcription: evidence for the participation of Smad and Sp family proteins in the NF1-dependent repressor complex, Biochem. J. 412 (2008) 123-130]. In the present study, we show that TGF-β, like serum-restriction: (a) induces the formation of NF1/Smad repressor complexes, (b) increases binding of the complexes to the repressor elements (Go elements) in the ANT2 promoter, and (c) inhibits ANT2 expression. Repression of ANT2 by TGF-β is eliminated by mutating the NF1 binding sites in the Go repressor elements. All of the above responses to TGF-β are prevented by inhibitors of TGF-β RI and MAPK p38. These inhibitors also prevent NF1/Smad4 repressor complex formation and repression of ANT2 expression in serum-restricted cells, suggesting that similar signaling pathways are initiated by TGF-β and serum-restriction. The present finding that NF1/Smad4 repressor complexes are formed through TGF-β signaling pathways suggests a new, but much broader, role for these complexes in the initiation or maintenance of the growth-inhibited state., (Copyright © 2011 Elsevier Inc. All rights reserved.)
- Published
- 2011
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31. Adenine nucleotide translocase 2 is a key mitochondrial protein in cancer metabolism.
- Author
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Chevrollier A, Loiseau D, Reynier P, and Stepien G
- Subjects
- Adenine Nucleotide Translocator 2 genetics, Adenine Nucleotide Translocator 2 metabolism, Animals, Energy Metabolism physiology, Humans, Isoenzymes genetics, Isoenzymes metabolism, Isoenzymes physiology, Mitochondria metabolism, Mitochondria pathology, Mitochondrial Proteins genetics, Mitochondrial Proteins metabolism, Mitochondrial Proteins physiology, Models, Biological, Neoplasms enzymology, Neoplasms genetics, Oxidative Phosphorylation, Adenine Nucleotide Translocator 2 physiology, Mitochondria enzymology, Neoplasms metabolism
- Abstract
Adenine nucleotide translocase (ANT), a mitochondrial protein that facilitates the exchange of ADP and ATP across the mitochondrial inner membrane, plays an essential role in cellular energy metabolism. Human ANT presents four isoforms (ANT1-4), each with a specific expression depending on the nature of the tissue, cell type, developmental stage and status of cell proliferation. Thus, ANT1 is specific to muscle and brain tissues; ANT2 occurs mainly in proliferative, undifferentiated cells; ANT3 is ubiquitous; and ANT4 is found in germ cells. ANT1 and ANT3 export the ATP produced by oxidative phosphorylation (OxPhos) from the mitochondria into the cytosol while importing ADP. In contrast, the expression of ANT2, which is linked to the rate of glycolytic metabolism, is an important indicator of carcinogenesis. In fact, cancers are characterized by major metabolic changes that switch cells from the normally dual oxidative and glycolytic metabolisms to an almost exclusively glycolytic metabolism. When OxPhos activity is impaired, ANT2 imports glycolytically produced ATP into the mitochondria. In the mitochondrial matrix, the F1F0-ATPase complex hydrolyzes the ATP, pumping out a proton into the intermembrane space. The reverse operations of ANT2 and F1F0-ATPase under glycolytic conditions contribute to maintaining the mitochondrial membrane potential, ensuring cell survival and proliferation. Unlike the ANT1 and ANT3 isoforms, ANT2 is not pro-apoptotic and may therefore contribute to carcinogenesis. Since the expression of ANT2 is closely linked to the mitochondrial bioenergetics of tumors, it should be taken into account for individualizing cancer treatments and for the development of anticancer strategies., (Copyright © 2010 Elsevier B.V. All rights reserved.)
- Published
- 2011
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32. Treatment with mANT2 shRNA enhances antitumor therapeutic effects induced by MUC1 DNA vaccination.
- Author
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Choi Y, Jeon YH, Jang JY, Chung JK, and Kim CW
- Subjects
- Adenofibroma immunology, Adenofibroma therapy, Animals, Apoptosis, CD8-Positive T-Lymphocytes immunology, Cancer Vaccines administration & dosage, Cancer Vaccines therapeutic use, Cell Line, Tumor, Combined Modality Therapy, DNA immunology, Female, Gene Expression, Immunotherapy, Melanoma immunology, Melanoma therapy, Mice, Mice, Inbred C57BL, Mucin-1 genetics, RNA Interference, T-Lymphocytes, Cytotoxic immunology, Vaccines, DNA administration & dosage, Vaccines, DNA therapeutic use, Adenine Nucleotide Translocator 2 genetics, Cancer Vaccines immunology, Mucin-1 immunology, RNA, Small Interfering, Vaccines, DNA immunology
- Abstract
In this study, we developed a combination therapy (pcDNA3/hMUC1+mANT2 shRNA) to enhance the efficiency of MUC1 DNA vaccination by combining it with mANT2 short hairpin RNA (shRNA) treatment in immunocompetent mice. mANT2 shRNA treatment alone increased the apoptosis of BMF cells (B16F1 murine melanoma cell line coexpressing an MUC1 and Fluc gene) and rendered BMF tumor cells more susceptible to lysis by MUC1-associated CD8(+) T cells. Furthermore, combined therapy enhanced MUC1 associated T-cell immune response and antitumor effects, and resulted in a higher cure rate than either treatment alone (pcDNA3/hMUC1 or mANT2 shRNA therapy alone). Human MUC1 (hMUC1)-loaded CD11c(+) cells in the draining lymph nodes of BMF-bearing mice treated with the combined treatment were found to be most effective at generating hMUC1-associated CD8(+)IFNγ(+) T cells. Furthermore, the in vitro killing activities of hMUC1-associated cytotoxic T cells (CTLs) in the combined therapy were greater than in the respective monotherapies. Cured animals treated with the combined treatment rejected a rechallenge by BMF cells, but not a rechallenge by B16F1-Fluc cells at 14 days after treatment, and showed MUC1 antigen-associated immune responses. These results suggest that combined therapy enhances antitumor activity, and that it offers an effective antitumor strategy for treating melanoma.
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- 2011
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33. Evolutionary genomics implies a specific function of Ant4 in mammalian and anole lizard male germ cells.
- Author
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Lim CH, Hamazaki T, Braun EL, Wade J, and Terada N
- Subjects
- Adenine Nucleotide Translocator 1 genetics, Adenine Nucleotide Translocator 1 metabolism, Adenine Nucleotide Translocator 2 genetics, Adenine Nucleotide Translocator 2 metabolism, Adenine Nucleotide Translocator 3 genetics, Adenine Nucleotide Translocator 3 metabolism, Amino Acid Sequence, Animals, Avian Proteins genetics, Avian Proteins metabolism, Chickens, Chromosome Mapping, Evolution, Molecular, Female, Gene Expression Regulation, Enzymologic, Humans, Lizards metabolism, Male, Mammals metabolism, Mice, Mice, Inbred C57BL, Mitochondrial ADP, ATP Translocases classification, Mitochondrial ADP, ATP Translocases metabolism, Molecular Sequence Data, Phylogeny, Reptilian Proteins metabolism, Reverse Transcriptase Polymerase Chain Reaction, Sequence Homology, Amino Acid, Spermatozoa enzymology, Synteny, Testis cytology, Testis enzymology, Testis metabolism, Genomics methods, Lizards genetics, Mammals genetics, Mitochondrial ADP, ATP Translocases genetics, Reptilian Proteins genetics, Spermatozoa metabolism
- Abstract
Most vertebrates have three paralogous genes with identical intron-exon structures and a high degree of sequence identity that encode mitochondrial adenine nucleotide translocase (Ant) proteins, Ant1 (Slc25a4), Ant2 (Slc25a5) and Ant3 (Slc25a6). Recently, we and others identified a fourth mammalian Ant paralog, Ant4 (Slc25a31), with a distinct intron-exon structure and a lower degree of sequence identity. Ant4 was expressed selectively in testis and sperm in adult mammals and was indeed essential for mouse spermatogenesis, but it was absent in birds, fish and frogs. Since Ant2 is X-linked in mammalian genomes, we hypothesized that the autosomal Ant4 gene may compensate for the loss of Ant2 gene expression during male meiosis in mammals. Here we report that the Ant4 ortholog is conserved in green anole lizard (Anolis carolinensis) and demonstrate that it is expressed in the anole testis. Further, a degenerate DNA fragment of putative Ant4 gene was identified in syntenic regions of avian genomes, indicating that Ant4 was present in the common amniote ancestor. Phylogenetic analyses suggest an even more ancient origin of the Ant4 gene. Although anole lizards are presumed male (XY) heterogametic, like mammals, copy numbers of the Ant2 as well as its neighboring gene were similar between male and female anole genomes, indicating that the anole Ant2 gene is either autosomal or located in the pseudoautosomal region of the sex chromosomes, in contrast to the case to mammals. These results imply the conservation of Ant4 is not likely simply driven by the sex chromosomal localization of the Ant2 gene and its subsequent inactivation during male meiosis. Taken together with the fact that Ant4 protein has a uniquely conserved structure when compared to other somatic Ant1, 2 and 3, there may be a specific advantage for mammals and lizards to express Ant4 in their male germ cells.
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- 2011
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34. Short-hairpin RNA-induced suppression of adenine nucleotide translocase-2 in breast cancer cells restores their susceptibility to TRAIL-induced apoptosis by activating JNK and modulating TRAIL receptor expression.
- Author
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Jang JY, Jeon YK, Choi Y, and Kim CW
- Subjects
- Adenine Nucleotide Translocator 2 genetics, Animals, Apoptosis drug effects, Apoptosis genetics, Breast Neoplasms genetics, Cell Line, Tumor, Cell Survival drug effects, Cell Survival genetics, DNA (Cytosine-5-)-Methyltransferase 1, DNA (Cytosine-5-)-Methyltransferases genetics, DNA (Cytosine-5-)-Methyltransferases metabolism, Female, Genetic Vectors, Humans, Immunoblotting, JNK Mitogen-Activated Protein Kinases genetics, Mice, Mice, Nude, Polymerase Chain Reaction, Receptors, TNF-Related Apoptosis-Inducing Ligand genetics, Tumor Necrosis Factor Decoy Receptors genetics, Tumor Necrosis Factor Decoy Receptors metabolism, Adenine Nucleotide Translocator 2 metabolism, Breast Neoplasms drug therapy, Breast Neoplasms metabolism, JNK Mitogen-Activated Protein Kinases metabolism, Receptors, TNF-Related Apoptosis-Inducing Ligand metabolism, TNF-Related Apoptosis-Inducing Ligand therapeutic use
- Abstract
Background: Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL; apo2 ligand) induces apoptosis in cancer cells but has little effect on normal cells. However, many cancer cell types are resistant to TRAIL-induced apoptosis, limiting the clinical utility of TRAIL as an anti-cancer agent. We previously reported that the suppression of adenine nucleotide translocase-2 (ANT2) by short-hairpin RNA (shRNA) induces apoptosis of breast cancer cells, which frequently express high levels of ANT2. In the present study, we examined the effect of RNA shRNA-induced suppression of ANT2 on the resistance of breast cancer cells to TRAIL-induced apoptosis in vitro and in vivo., Results: ANT2 shRNA treatment sensitized MCF7, T47 D, and BT474 cells to TRAIL-induced apoptosis by up-regulating the expression of TRAIL death receptors 4 and 5 (DR4 and DR5) and down-regulating the TRAIL decoy receptor 2 (DcR2). In MCF7 cells, ANT2 knockdown activated the stress kinase c-Jun N-terminal kinase (JNK), subsequently stabilizing and increasing the transcriptional activity of p53 by phosphorylating it at Thr81; it also enhanced the expression and activity of DNA methyltransferase 1 (DNMT1). ANT2 shRNA-induced overexpression of DR4/DR5 and TRAIL sensitization were blocked by a p53 inhibitor, suggesting that p53 activation plays an important role in the transcriptional up-regulation of DR4/DR5. However, ANT2 knockdown also up-regulated DR4/DR5 in the p53-mutant cell lines BT474 and T47 D. In MCF7 cells, ANT2 shRNA treatment led to DcR2 promoter methylation and concomitant down-regulation of DcR2 expression, consistent with the observed activation of DNMT1. Treatment of the cells with a demethylating agent or JNK inhibitor prevented the ANT2 shRNA-induced down-regulation of DcR2 and activation of both p53 and DNMT1. In in vivo experiments using nude mice, ANT2 shRNA caused TRAIL-resistant MCF7 xenografts to undergo TRAIL-induced cell death, up-regulated DR4/DR5, and down-regulated DcR2. Co-treatment with ANT2 shRNA and TRAIL efficiently suppressed tumor growth in these mice., Conclusions: ANT2 suppression by shRNA might be exploited to overcome TRAIL-resistance in cancer.
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- 2010
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35. Degradation of HER2/neu by ANT2 shRNA suppresses migration and invasiveness of breast cancer cells.
- Author
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Jang JY, Jeon YK, and Kim CW
- Subjects
- Adenine Nucleotide Translocator 2 antagonists & inhibitors, Adenine Nucleotide Translocator 2 genetics, Blotting, Western, Breast Neoplasms genetics, Breast Neoplasms pathology, Elafin genetics, Elafin metabolism, ErbB Receptors genetics, ErbB Receptors metabolism, Female, HSP90 Heat-Shock Proteins genetics, HSP90 Heat-Shock Proteins metabolism, Humans, Immunoprecipitation, Matrix Metalloproteinase 14 genetics, Matrix Metalloproteinase 14 metabolism, Matrix Metalloproteinases genetics, Matrix Metalloproteinases metabolism, Neoplasm Invasiveness, Proto-Oncogene Proteins c-akt genetics, Proto-Oncogene Proteins c-akt metabolism, RNA, Messenger genetics, RNA, Small Interfering pharmacology, Receptor, ErbB-2 genetics, Reverse Transcriptase Polymerase Chain Reaction, Tumor Cells, Cultured, Adenine Nucleotide Translocator 2 metabolism, Breast Neoplasms metabolism, Cell Movement, Receptor, ErbB-2 metabolism
- Abstract
Background: In breast cancer, the HER2/neu oncoprotein, which belongs to the epidermal growth factor receptor family, may trigger activation of the phosphoinositide-3 kinase (PI3K)/Akt pathway, which controls cell proliferation, survival, migration, and invasion. In this study, we examined the question of whether or not adenine nucleotide translocase 2 (ANT2) short hairpin RNA (shRNA)-mediated down-regulation of HER2/neu and inhibitory effects on the PI3K/Akt signaling pathway suppressed migration and invasiveness of breast cancer cells., Methods: We utilized an ANT2 vector-based RNA interference approach to inhibition of ANT2 expression, and the HER2/neu-overexpressing human breast cancer cell line, SK-BR3, was used throughout the study., Results: In this study, ANT2 shRNA decreased HER2/neu protein levels by promoting degradation of HER2/neu protein through dissociation from heat shock protein 90 (HSP90). As a result, ANT2 shRNA induced inhibitory effects on the PI3K/Akt signaling pathway. Inhibition of PI3K/Akt signaling by ANT2 shRNA caused down-regulation of membrane-type 1 matrix metalloproteinase (MT1-MMP) and vascular endothelial growth factor (VEGF) expression, decreased matrix metalloproteinase 2 (MMP2) and MMP9 activity, and suppressed migration and invasion of breast cancer cells., Conclusions: These results indicate that knock-down of ANT2 by shRNA down-regulates HER2/neu through suppression of HSP90's function and inhibits the PI3K/Akt signaling pathway, resulting ultimately in suppressed migration and invasion of breast cancer cells.
- Published
- 2010
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36. Isolation, nucleotide identification and tissue expression of three novel ovine genes-SLC25A4, SLC25A5 and SLC25A6.
- Author
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Yang L, He Y, Kong Q, Zhang W, Xi D, Mao H, and Deng W
- Subjects
- Adenine Nucleotide Translocator 1 chemistry, Adenine Nucleotide Translocator 1 metabolism, Adenine Nucleotide Translocator 2 chemistry, Adenine Nucleotide Translocator 2 metabolism, Adenine Nucleotide Translocator 3 chemistry, Adenine Nucleotide Translocator 3 metabolism, Animals, Base Sequence, Gene Expression Regulation, Molecular Sequence Data, Phylogeny, Protein Binding, Protein Structure, Secondary, Protein Structure, Tertiary, Reverse Transcriptase Polymerase Chain Reaction, Sequence Analysis, DNA, Adenine Nucleotide Translocator 1 genetics, Adenine Nucleotide Translocator 2 genetics, Adenine Nucleotide Translocator 3 genetics, Gene Expression Profiling, Sheep genetics
- Abstract
The complete coding sequences of three of sheep genes SLC25A4, SLC25A5 and SLC25A6 were firstly amplified using the reverse transcriptase polymerase chain reaction (RT-PCR) according to the conserved sequence information of the cattle or other mammals and known highly homologous sheep ESTs. Sheep SLC25A4, SLC25A5 and SLC25A6 genes encode three corresponding proteins of 298 amino acids which contain the identically conserved putative mitochondrial carrier protein domain. Sheep SLC25A4 protein has high homology with the SLC25A4 proteins of six species-cattle (99%), human (95%), rat (95%), mouse (94%), dog (94%) and chicken (89%). Sheep SLC25A5 protein has high identity with the SLC25A5 proteins of five species-cattle (100%), dog (99%), mouse (98%), rat (98%) and human (98%). Sheep SLC25A6 protein also has high homology with the SLC25A6 proteins of four species-cattle (99%), human (97%), pig (97%) and chicken (93%). The phylogenetic tree analysis demonstrated that sheep SLC25A4, SLC25A5 and SLC25A6 proteins share a common ancestor. Moreover, SLC25A4, SLC25A5 and SLC25A6 proteins present stronger interaction each other. The tissue expression analysis indicated that sheep SLC25A4, SLC25A5 and SLC25A6 genes were expressed in a range of tissues including leg muscle, kidney, skin, longissimus dorsi muscle, spleen, heart and liver. Our experiment is the first to provide the primary foundation for further insight into these three sheep genes.
- Published
- 2010
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37. Aromatase deficiency inhibits the permeability transition in mouse liver mitochondria.
- Author
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Moro L, Arbini AA, Hsieh JT, Ford J, Simpson ER, Hajibeigi A, and Oz OK
- Subjects
- Adenine Nucleotide Translocator 2 genetics, Adenine Nucleotide Translocator 2 metabolism, Adenosine Triphosphate metabolism, Analysis of Variance, Animals, Blotting, Western, Cardiolipins metabolism, Cell Fractionation, Estradiol pharmacology, Fatty Liver genetics, Fatty Liver metabolism, Fatty Liver pathology, Liver drug effects, Liver pathology, Male, Mice, Mice, Knockout, Mitochondria, Liver drug effects, Mitochondria, Liver genetics, Mitochondrial Membrane Transport Proteins genetics, Mitochondrial Membrane Transport Proteins metabolism, Mitochondrial Membranes drug effects, Organ Size genetics, Permeability drug effects, Proto-Oncogene Proteins c-bcl-2 genetics, Proto-Oncogene Proteins c-bcl-2 metabolism, RNA, Messenger genetics, RNA, Messenger metabolism, Reactive Oxygen Species metabolism, Reverse Transcriptase Polymerase Chain Reaction, Aromatase genetics, Electron Transport Complex IV metabolism, Liver metabolism, Mitochondria, Liver metabolism, Mitochondrial Membranes metabolism
- Abstract
Lack of estrogens affects male physiology in a number of ways, including severe changes in liver metabolism that result in lipid accumulation and massive hepatic steatosis. Here we investigated whether estrogen deficiency may alter the functionality and permeability properties of liver mitochondria using, as an experimental model, aromatase knockout (ArKO) male mice, which cannot synthesize endogenous estrogens due to a disruption of the Cyp19 gene. Liver mitochondria isolated from ArKO mice displayed increased activity of the mitochondrial respiratory complex IV compared with wild-type mice and were less prone to undergo cyclosporin A-sensitive mitochondrial permeability transition (MPT) induced by calcium loading. The altered permeability properties of the mitochondrial membranes were not due to changes in reactive oxygen species, ATP levels, or mitochondrial membrane potential but were associated with increased content of the phospholipid cardiolipin, structural component of the mitochondrial membranes and regulator of the MPT pore, and with increased mitochondrial protein levels of Bcl-2 and the adenine nucleotide translocator (ANT), regulator and component of the MPT pore, respectively. Real-time RT-PCR demonstrated increased mRNA levels for Bcl-2 and ANT2 but not for the ANT1 isoform in ArKO livers. Supplementation of 17beta-estradiol retrieved ArKO mice from massive hepatic steatosis and restored mitochondrial permeability properties, cardiolipin, Bcl-2, and ANT2 levels. Overall, our findings demonstrate an important role of estrogens in the modulation of hepatic mitochondrial function and permeability properties in males and suggest that estrogen deficiency may represent a novel positive regulator of Bcl-2 and ANT2 proteins, two inhibitors of MPT occurrence and powerful antiapoptotic molecules.
- Published
- 2010
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38. Growth-dependent repression of human adenine nucleotide translocator-2 (ANT2) transcription: evidence for the participation of Smad and Sp family proteins in the NF1-dependent repressor complex.
- Author
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Luciakova K, Kollarovic G, Barath P, and Nelson BD
- Subjects
- Cells, Cultured, Culture Media, Serum-Free pharmacology, Humans, Models, Biological, Multigene Family physiology, Multiprotein Complexes metabolism, Multiprotein Complexes physiology, Neurofibromin 1 metabolism, Promoter Regions, Genetic, Protein Binding drug effects, Repressor Proteins metabolism, Repressor Proteins physiology, Smad Proteins metabolism, Sp Transcription Factors metabolism, Transcription, Genetic, Adenine Nucleotide Translocator 2 genetics, Cell Proliferation drug effects, Down-Regulation drug effects, Neurofibromin 1 physiology, Smad Proteins physiology, Sp Transcription Factors physiology
- Abstract
NF1 (nuclear factor 1) binds to two upstream elements of the human ANT2 (adenine nucleotide translocator-2) promoter and actively represses expression of the gene in growth-arrested diploid skin fibroblasts [Luciakova, Barath, Poliakova, Persson and Nelson (2003) J. Biol. Chem. 278, 30624-30633]. ChIP (chromatin immunoprecipitation) and co-immunoprecipitation analyses of nuclear extracts from growth-arrested and growth-activated diploid cells demonstrate that NF1, when acting as a repressor, is part of a multimeric complex that also includes Smad and Sp-family proteins. This complex appears to be anchored to both the upstream NF1-repressor elements and the proximal promoter, Sp1-dependent activation elements in growth-arrested cells. In growth-activated cells, the repressor complex dissociates and NF1 leaves the promoter. As revealed by co-immunoprecipitation experiments, NF1-Smad4-Sp3 complexes are present in nuclear extracts only from growth-inhibited cells, suggesting that the growth-state-dependent formation of these complexes is not an ANT2 promoter-specific event. Consistent with the role of Smad proteins in the repression complex, TGF-beta (transforming growth factor-beta) can fully repress ANT2 transcription in normally growing fibroblasts. Finally, pull-down experiments of in vitro transcribed/translated NF1 isoforms by GST (glutathione transferase)-Smad and GST-Smad MH fusion proteins indicate direct physical interactions between members of the two families. These findings suggest a possible functional relationship between the NF1 and Smad proteins that has not been previously observed.
- Published
- 2008
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39. Carboxyatractyloside effects on brown-fat mitochondria imply that the adenine nucleotide translocator isoforms ANT1 and ANT2 may be responsible for basal and fatty-acid-induced uncoupling respectively.
- Author
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Shabalina IG, Kramarova TV, Nedergaard J, and Cannon B
- Subjects
- Adenine Nucleotide Translocator 1 antagonists & inhibitors, Adenine Nucleotide Translocator 1 biosynthesis, Adenine Nucleotide Translocator 1 genetics, Adenine Nucleotide Translocator 2 antagonists & inhibitors, Adenine Nucleotide Translocator 2 biosynthesis, Adenine Nucleotide Translocator 2 genetics, Adipose Tissue, Brown metabolism, Animals, Atractyloside pharmacology, Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone pharmacology, Cell Respiration drug effects, Crosses, Genetic, Fatty Acids metabolism, Guanosine Diphosphate pharmacology, Ion Channels deficiency, Ion Channels genetics, Mice, Mice, Inbred C57BL, Mice, Knockout, Mitochondria metabolism, Mitochondria, Liver drug effects, Mitochondria, Liver metabolism, Mitochondrial Proteins deficiency, Mitochondrial Proteins genetics, Models, Biological, Oleic Acid pharmacology, Organ Specificity, Oxygen Consumption drug effects, Palmitates pharmacology, Protons, Pyruvic Acid pharmacology, RNA, Messenger biosynthesis, RNA, Messenger genetics, Thermogenesis drug effects, Thermogenesis physiology, Uncoupling Protein 1, Adenine Nucleotide Translocator 1 physiology, Adenine Nucleotide Translocator 2 physiology, Adipose Tissue, Brown drug effects, Atractyloside analogs & derivatives, Fatty Acids pharmacology, Mitochondria drug effects, Uncoupling Agents pharmacology
- Abstract
In brown-fat mitochondria, fatty acids induce thermogenic uncoupling through activation of UCP1 (uncoupling protein 1). However, even in brown-fat mitochondria from UCP1-/- mice, fatty-acid-induced uncoupling exists. In the present investigation, we used the inhibitor CAtr (carboxyatractyloside) to examine the involvement of the ANT (adenine nucleotide translocator) in the mediation of this UCP1-independent fatty-acid-induced uncoupling in brown-fat mitochondria. We found that the contribution of ANT to fatty-acid-induced uncoupling in UCP1-/- brown-fat mitochondria was minimal (whereas it was responsible for nearly half the fatty-acid-induced uncoupling in liver mitochondria). As compared with liver mitochondria, brown-fat mitochondria exhibit a relatively high (UCP1-independent) basal respiration ('proton leak'). Unexpectedly, a large fraction of this high basal respiration was sensitive to CAtr, whereas in liver mitochondria, basal respiration was CAtr-insensitive. Total ANT protein levels were similar in brown-fat mitochondria from wild-type mice and in liver mitochondria, but the level was increased in brown-fat mitochondria from UCP1-/- mice. However, in liver, only Ant2 mRNA was found, whereas in brown adipose tissue, Ant1 and Ant2 mRNA levels were equal. The data are therefore compatible with a tentative model in which the ANT2 isoform mediates fatty-acid-induced uncoupling, whereas the ANT1 isoform may mediate a significant part of the high basal proton leak in brown-fat mitochondria.
- Published
- 2006
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40. Chemosensitization by knockdown of adenine nucleotide translocase-2.
- Author
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Le Bras M, Borgne-Sanchez A, Touat Z, El Dein OS, Deniaud A, Maillier E, Lecellier G, Rebouillat D, Lemaire C, Kroemer G, Jacotot E, and Brenner C
- Subjects
- Adenine Nucleotide Translocator 1 biosynthesis, Adenine Nucleotide Translocator 1 genetics, Adenine Nucleotide Translocator 1 metabolism, Adenine Nucleotide Translocator 2 biosynthesis, Adenine Nucleotide Translocator 2 deficiency, Adenosine Triphosphate metabolism, Apoptosis physiology, Cell Line, Tumor, Drug Resistance, Neoplasm, Gene Silencing, HeLa Cells, Humans, Mitochondrial Membranes drug effects, Mitochondrial Membranes metabolism, Mitochondrial Membranes physiology, Permeability, Proto-Oncogene Proteins c-bcl-2 metabolism, RNA, Small Interfering genetics, Adenine Nucleotide Translocator 2 genetics, Adenine Nucleotide Translocator 2 metabolism, Antineoplastic Agents pharmacology, Apoptosis drug effects, Indazoles pharmacology
- Abstract
Mitochondrial membrane permeabilization (MMP) is a rate-limiting step of apoptosis, including in anticancer chemotherapy. Adenine nucleotide translocase (ANT) mediates the exchange of ADP and ATP on the inner mitochondrial membrane in healthy cells. In addition, ANT can cooperate with Bax to form a lethal pore during apoptosis. Humans possess four distinct ANT isoforms, encoded by four genes, whose transcription depends on the cell type, developmental stage, cell proliferation, and hormone status. Here, we show that the ANT2 gene is up-regulated in several hormone-dependent cancers. Knockdown of ANT2 by RNA interference induced no major changes in the aspect of the mitochondrial network or cell cycle but provoked minor increase in mitochondrial transmembrane potential and reactive oxygen species level and reduced intracellular ATP concentration without affecting glycolysis. At expression and functional levels, ANT2 depletion was not compensated by other ANT isoforms. Most importantly, ANT2, but not ANT1, silencing facilitated MMP induction by lonidamine, a mitochondrion-targeted antitumor compound already used in clinical studies for breast, ovarian, glioma, and lung cancer as well as prostate adenoma. The combination of ANT2 knockdown with lonidamine induced apoptosis irrespective of the Bcl-2 status. These data identify ANT2 as an endogenous inhibitor of MMP and suggest that its selective inhibition could constitute a promising strategy of chemosensitization.
- Published
- 2006
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41. An isoform shift in the cardiac adenine nucleotide translocase expression alters the kinetic properties of the carrier in dilated cardiomyopathy.
- Author
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Dörner A, Giessen S, Gaub R, Grosse Siestrup H, Schwimmbeck PL, Hetzer R, Poller W, and Schultheiss HP
- Subjects
- Adenine Nucleotide Translocator 1 blood, Adenine Nucleotide Translocator 2 blood, Blotting, Western, Cardiomyopathy, Dilated genetics, Cardiomyopathy, Dilated pathology, Chromatography, Gel, Energy Metabolism physiology, Female, Humans, Isoenzymes genetics, Isoenzymes metabolism, Male, Middle Aged, Mitochondria, Heart pathology, Mutation, Adenine Nucleotide Translocator 1 genetics, Adenine Nucleotide Translocator 2 genetics, Cardiomyopathy, Dilated enzymology, DNA genetics, Gene Expression, Mitochondria, Heart enzymology, Protein Transport physiology
- Abstract
Background: Impaired mitochondrial ADP/ATP transport and altered adenine nucleotide translocase (ANT) isoform expression characterized by enhanced ANT1 and decreased ANT2 expression have been implicated in the pathophysiology of dilated cardiomyopathy (DCM). It is still unknown whether restricted ANT function results from exogenous factors, or mutations in the ANT genes, or whether the imbalance in the isoform composition causes the reduced ADP/ATP transport. We performed DNA mutation screening of ANT genes and analyzed the kinetic properties of ANT protein isolated from DCM hearts and controls in a reconstituted system excluding natural environmental influences., Results: A G1409T polymorphism in ANT2 leads to an exchange from Arg111 to Leu111 in healthy blood donors (n = 60) with allele frequencies of 76% and 24%. This polymorphism was neither associated with DCM (74%, 26%; n = 93) nor with altered myocardial ANT isoform expression or restricted ANT function (89%, 11%; n = 8). However, there was a remarkable reduction in the maximum transport activity (v(max)) of reconstituted ANT from DCM hearts with altered ANT isoform expression (498 +/- 113 micromol min(-1) g(-1) incorporated protein vs. 1112 +/- 178 micromol min(-1) g(-1) incorporated protein, p < 0.01). Moreover, the substrate affinity of DCM myocardial ANT to ATP was slightly reduced with an increased K(m) value of 104.3 +/- 2.4 microM vs. 90.4 +/- 2.9 microM in controls (p < 0.03)., Conclusion: The altered isoform expression in DCM hearts entails changes in the kinetic properties of total ANT protein restricting ANT function and contributing to disturbed energy metabolism in DCM.
- Published
- 2006
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42. Mitochondrial permeability transition in apoptosis and necrosis.
- Author
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Zamzami N, Larochette N, and Kroemer G
- Subjects
- Adenine Nucleotide Translocator 1 genetics, Adenine Nucleotide Translocator 2 genetics, Animals, Peptidyl-Prolyl Isomerase F, Cyclophilins genetics, Cyclophilins physiology, Humans, Intracellular Membranes physiology, Ion Channels physiology, Mice, Mice, Knockout, Mitochondrial Membrane Transport Proteins, Mitochondrial Permeability Transition Pore, Permeability, Apoptosis physiology, Mitochondria physiology, Necrosis
- Published
- 2005
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43. Silencing of the tumor suppressor gene SLC5A8 is associated with BRAF mutations in classical papillary thyroid carcinomas.
- Author
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Porra V, Ferraro-Peyret C, Durand C, Selmi-Ruby S, Giroud H, Berger-Dutrieux N, Decaussin M, Peix JL, Bournaud C, Orgiazzi J, Borson-Chazot F, Dante R, and Rousset B
- Subjects
- Adenine Nucleotide Translocator 2 genetics, Cells, Cultured, DNA Methylation, Female, Humans, MAP Kinase Signaling System, Male, Membrane Transport Proteins genetics, Monocarboxylic Acid Transporters, Mutation, Sulfate Transporters, Thyroid Gland metabolism, Carcinoma, Papillary genetics, Cation Transport Proteins genetics, Gene Silencing, Proto-Oncogene Proteins B-raf genetics, Thyroid Neoplasms genetics
- Abstract
SLC5A8, proposed as a thyroid apical iodide transporter, was recently defined as a Na+-coupled transporter of short-chain fatty acid. To document the expression pattern of SLC5A8 in the thyroid, we analyzed the regulation of its expression in normal human thyrocytes in culture and in tissues with distinct functional activity. To determine whether SLC5A8 expression is altered in all thyroid carcinomas or only in particular subtypes, we investigated the level of its expression in a series of 50 hypofunctioning tumors. SLC5A8 expression was studied at the transcript level and compared with that of SLC26A4 or Pendrin and SLC5A5 or Na+/iodide symporter. SLC5A8 expression, unlike that of SLC5A5 and SLC26A4, was not regulated by TSH in normal human thyrocytes in culture and was not related to the functional state of thyroid tissue; toxic adenomas and adjacent resting tissues exhibited the same SLC5A8 transcript content. SLC5A8 expression was selectively down-regulated (40-fold) in papillary thyroid carcinomas of classical form (PTC-cf.). Methylation-specific PCR analyses showed that SLC5A8 was methylated in 90% of PTC-cf. and in about 20% of other papillary thyroid carcinomas. In a series of 52 PTC-cf., a low SLC5A8 expression was highly significantly associated with the presence of BRAF T1796A mutation. These data identify a relationship between the methylation-associated silencing of the tumor-suppressor gene SLC5A8 and the T1796A point mutation of the BRAF gene in the PTC-cf. subtype of thyroid carcinomas.
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- 2005
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44. Two-step differential expression analysis reveals a new set of genes involved in thyroid oncocytic tumors.
- Author
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Jacques C, Baris O, Prunier-Mirebeau D, Savagner F, Rodien P, Rohmer V, Franc B, Guyetant S, Malthiery Y, and Reynier P
- Subjects
- Adenine Nucleotide Translocator 2 genetics, Adenoma metabolism, DNA, Mitochondrial genetics, Humans, Ion Channels, Membrane Transport Proteins genetics, Mitochondrial Proteins genetics, Neoplasm Proteins metabolism, Oligonucleotide Array Sequence Analysis, Thyroid Neoplasms metabolism, Uncoupling Protein 2, Adenoma genetics, Gene Expression Profiling, Thyroid Neoplasms genetics
- Abstract
Thyroid oncocytic adenomas are a class of tumors characterized by the presence of abundant mitochondria. We performed a differential display RT-PCR analysis on two oncocytic adenomas and their paired controls. We then carried out a microarray analysis using the 460 selected, differentially expressed clones on four other oncocytomas and their paired controls. Thirty genes, 12 encoded by mitochondrial DNA and 18 nuclear-encoded, were overexpressed by a factor of at least 2 in the tumors compared with the controls. Seven of the 18 nuclear-encoded genes are involved in protein metabolism: DKFZP434I116, B3GTL, SNX19, RP42, SENP1, UBE2D3, and the CTSB gene, which is known to be particularly deregulated in most thyroid tumors. Other genes are implicated in signal transduction (ITGAV) or tumorigenesis (AF1q). Immunohistochemistry allowed us to confirm overexpression of the ITGAV and CTSB genes at the protein level and showed a marked relocation of the CTSB protein. We confirmed the overexpression of the AF1q oncogene in 56% of 18 oncocytic tumors by quantitative RT-PCR analysis, which attested to the heterogeneity of these tumors. Our results show an increased expression of genes involved in protein metabolism in oncocytoma, the significance of which requires investigation.
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- 2005
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- View/download PDF
45. Valine 181 is critical for the nucleotide exchange activity of human mitochondrial ADP/ATP carriers in yeast.
- Author
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De Marcos Lousa C, Trézéguet V, David C, Postis V, Arnou B, Pebay-Peyroula E, Brandolin G, and Lauquin GJ
- Subjects
- Adenine Nucleotide Translocator 1 genetics, Adenine Nucleotide Translocator 2 genetics, Adenine Nucleotide Translocator 3 genetics, Amino Acid Substitution genetics, Animals, Cattle, Genetic Complementation Test, Humans, Methionine genetics, Mitochondria enzymology, Mitochondria genetics, Mitochondria metabolism, Mutagenesis, Site-Directed, Saccharomyces cerevisiae enzymology, Saccharomyces cerevisiae genetics, Ultraviolet Rays, Adenine Nucleotide Translocator 1 metabolism, Adenine Nucleotide Translocator 2 metabolism, Adenine Nucleotide Translocator 3 metabolism, Guanine Nucleotide Exchange Factors metabolism, Mitochondrial ADP, ATP Translocases metabolism, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism, Valine genetics
- Abstract
We isolated yeast Saccharomyces cerevisiae cells transformed with one of the three human adenine nucleotide carrier genes (HANC) that exhibited higher growth capacity than previously observed. The HANC genes were isolated from these clones, and we identified two independent mutations of HANC that led to replacement of valine 181 located in the fourth transmembrane segment by methionine or phenylalanine. Tolerance of this position toward substitution with various amino acids was systematically investigated, and since HANC/V181M was among the most efficient in growth complementation, it was more extensively studied. Here we show that increased growth capacities were associated with higher ADP/ATP exchange activities and not with higher human carrier amount in yeast mitochondria. These results are discussed in the light of the bovine Ancp structure, that shares more than 90% amino acid identity with Hancps, and its interaction with the lipid environment.
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- 2005
- Full Text
- View/download PDF
46. ANT2 expression under hypoxic conditions produces opposite cell-cycle behavior in 143B and HepG2 cancer cells.
- Author
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Chevrollier A, Loiseau D, Gautier F, Malthièry Y, and Stepien G
- Subjects
- Adenine Nucleotide Translocator 2 metabolism, Adenine Nucleotide Translocator 3 genetics, Adenine Nucleotide Translocator 3 metabolism, Glucose metabolism, Hexokinase genetics, Hexokinase metabolism, Humans, Lactic Acid metabolism, RNA, Messenger metabolism, Vascular Endothelial Growth Factor A genetics, Vascular Endothelial Growth Factor A metabolism, Adenine Nucleotide Translocator 2 genetics, Gene Expression Regulation, Neoplastic physiology, Hypoxia metabolism, Neoplasms metabolism
- Abstract
Under hypoxic conditions, mitochondrial ATP production ceases, leaving cells entirely dependent on their glycolytic metabolism. The cytoplasmic and intramitochondrial ATP/ADP ratios, partly controlled by the adenine nucleotide translocator (ANT), are drastically modified. In dividing and growing cells that have a predominantly glycolytic metabolism, the ANT isoform 2, which has kinetic properties allowing ATP import into mitochondria, is over-expressed in comparison to control cells. We studied the cellular metabolic and proliferative response to hypoxia in two transformed human cell lines with different metabolic backgrounds: HepG2 and 143B, and in their rho(o) derivatives, i.e., cells with no mitochondrial DNA. Transformed 143B and rho(o) cells continued their proliferation whereas HepG2 cells, with a more differentiated phenotype, arrested their cell-cycle at the G(1)/S checkpoint. Hypoxia induced an increase in glycolytic activity, correlated to an induction of VEGF and hexokinase II (HK II) expression. Thus, according to their tumorigenicity, transformed cells may adopt one of two distinct behaviors to support hypoxic stress, i.e., proliferation or quiescence. Our study links the constitutive glycolytic activity and ANT2 expression levels of transformed cells with the loss of cell-cycle control after oxygen deprivation. ATP import by ANT2 allows cells to maintain their mitochondrial integrity while acquiring insensitivity to any alterations in the proteins involved in oxidative phosphorylation. This loss of cell dependence on oxidative metabolism is an important factor in the development of tumors.
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- 2005
- Full Text
- View/download PDF
47. Identification of NF1 as a silencer protein of the human adenine nucleotide translocase-2 gene.
- Author
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Barath P, Poliakova D, Luciakova K, and Nelson BD
- Subjects
- Amino Acid Sequence, Base Sequence, DNA metabolism, Gene Silencing, Humans, Molecular Sequence Data, NFI Transcription Factors, Promoter Regions, Genetic, Adenine Nucleotide Translocator 2 genetics, CCAAT-Enhancer-Binding Proteins physiology, Repressor Proteins physiology, Transcription Factors physiology
- Abstract
The human adenine nucleotide translocase-2 (ANT2) promoter contains a silencer region that confers partial repression on the heterologous herpes simplex virus thymidine kinase (HSVtk) promoter [Barath, P., Albert-Fournier, B., Luciakova, K., Nelson, B.D. (1999) J. Biol. Chem.274, 3378-3384]. Two sequences in the silencer (Site-2 and Site-3) are protected in the DNase I assay in vitro, and one of these is a repeated GTCCTG element previously shown to act as the active repressor element. We have now purified the DNA binding protein, and identified it using MALDI-TOF MS as a 33-kDa member of the nuclear factor 1 (NF1) family of transcription factors. NF1 purified from rat liver and HeLa cell nuclei bind to both silencer Site-2 and Site-3, resulting in a DNase I footprint identical to that obtained with purified recombinant NF1. Furthermore, transient transfection experiments with reporter constructs containing mutated silencer Site-2 and/or Site-3 show that both sites contribute to repression of the HSVtk promoter. Finally, chromatin immunoprecipitation analysis reveals that NF1 is bound to both elements on the endogenous HeLa cell ANT2 promoter. Our data support the belief that NF1 acts as a repressor when bound to silencing Site-2 and Site-3 of the ANT2 gene.
- Published
- 2004
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48. The ADP/ATP translocator is not essential for the mitochondrial permeability transition pore.
- Author
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Kokoszka JE, Waymire KG, Levy SE, Sligh JE, Cai J, Jones DP, MacGregor GR, and Wallace DC
- Subjects
- Adenine Nucleotide Translocator 1 genetics, Adenine Nucleotide Translocator 2 genetics, Animals, Cell Death, Gene Deletion, Hepatocytes cytology, Hepatocytes metabolism, Isoenzymes deficiency, Isoenzymes genetics, Isoenzymes metabolism, Mice, Mice, Knockout, Mitochondria, Liver metabolism, Mitochondrial Membrane Transport Proteins, Mitochondrial Permeability Transition Pore, Adenine Nucleotide Translocator 1 deficiency, Adenine Nucleotide Translocator 1 metabolism, Adenine Nucleotide Translocator 2 deficiency, Adenine Nucleotide Translocator 2 metabolism, Ion Channels metabolism
- Abstract
A sudden increase in permeability of the inner mitochondrial membrane, the so-called mitochondrial permeability transition, is a common feature of apoptosis and is mediated by the mitochondrial permeability transition pore (mtPTP). It is thought that the mtPTP is a protein complex formed by the voltage-dependent anion channel, members of the pro- and anti-apoptotic BAX-BCL2 protein family, cyclophilin D, and the adenine nucleotide (ADP/ATP) translocators (ANTs). The latter exchange mitochondrial ATP for cytosolic ADP and have been implicated in cell death. To investigate the role of the ANTs in the mtPTP, we genetically inactivated the two isoforms of ANT in mouse liver and analysed mtPTP activation in isolated mitochondria and the induction of cell death in hepatocytes. Mitochondria lacking ANT could still be induced to undergo permeability transition, resulting in release of cytochrome c. However, more Ca2+ than usual was required to activate the mtPTP, and the pore could no longer be regulated by ANT ligands. Moreover, hepatocytes without ANT remained competent to respond to various initiators of cell death. Therefore, ANTs are non-essential structural components of the mtPTP, although they do contribute to its regulation.
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- 2004
- Full Text
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49. Repression of the human adenine nucleotide translocase-2 gene in growth-arrested human diploid cells: the role of nuclear factor-1.
- Author
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Luciakova K, Barath P, Poliakova D, Persson A, and Nelson BD
- Subjects
- 3T3 Cells, Animals, Base Sequence, CCAAT-Enhancer-Binding Proteins metabolism, Cell Division physiology, Diploidy, Down-Regulation physiology, Humans, Mice, Molecular Sequence Data, Mutagenesis physiology, NFI Transcription Factors, Nuclear Proteins, Promoter Regions, Genetic physiology, Skin cytology, Suppression, Genetic, Transcription, Genetic physiology, Y-Box-Binding Protein 1, Adenine Nucleotide Translocator 2 genetics, DNA-Binding Proteins, Resting Phase, Cell Cycle physiology, Transcription Factors
- Abstract
Adenine nucleotide translocase-2 (ANT2) catalyzes the exchange of ATP for ADP across the mitochondrial membrane, thus playing an important role in maintaining the cytosolic phosphorylation potential required for cell growth. Expression of ANT2 is activated by growth stimulation of quiescent cells and is down-regulated when cells become growth-arrested. In this study, we address the mechanism of growth arrest repression. Using a combination of transfection, in vivo dimethyl sulfate mapping, and in vitro DNase I mapping experiments, we identified two protein-binding elements (Go-1 and Go-2) that are responsible for growth arrest of ANT2 expression in human diploid fibroblasts. Proteins that bound the Go elements were purified and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry as members of the NF1 family of transcription factors. Chromatin immunoprecipitation analysis showed that NF1 was bound to both Go-1 and Go-2 in quiescent human diploid cells in vivo, but not in the same cells stimulated to growth by serum. NF1 binding correlated with the disappearance of ANT2 transcripts in quiescent cells. Furthermore, overexpression of NF1-A, -C, and -X in NIH3T3 cells repressed expression of an ANT2-driven reporter gene construct. Two additional putative repressor elements in the ANT2 promoter, an Sp1 element juxtaposed to the transcription start site and a silencer centered at nucleotide -332, did not appear to contribute to growth arrest repression. Thus, enhanced binding of NF1 is a key step in the growth arrest repression of ANT2 transcription. To our knowledge, this is the first report showing a role for NF1 in growth arrest.
- Published
- 2003
- Full Text
- View/download PDF
50. Expression of mitochondrial uncoupling protein 3 and adenine nucleotide translocase 1 genes in developing rat heart: putative involvement in control of mitochondrial membrane potential.
- Author
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Skárka L, Bardová K, Brauner P, Flachs P, Jarkovská D, Kopecký J, and Ostádal B
- Subjects
- Adenine Nucleotide Translocator 1 genetics, Adenine Nucleotide Translocator 2 genetics, Animals, Animals, Newborn, Carrier Proteins genetics, Carrier Proteins physiology, Ion Channels, Male, Membrane Potentials, Mitochondria metabolism, Mitochondrial Proteins, Myocardium metabolism, Rats, Rats, Wistar, Receptors, Cytoplasmic and Nuclear genetics, Transcription Factors genetics, Uncoupling Protein 3, Adenine Nucleotide Translocator 1 metabolism, Carrier Proteins metabolism, Heart growth & development, Heart physiology, Mitochondria physiology
- Abstract
Postnatal maturation of the heart depends on the switch from glycolytic to oxidative metabolism and it is associated with decreasing tolerance to oxygen deprivation. Therefore, changes in composition and function of cardiac mitochondria during postnatal development require detailed characterization. Left-ventricular myocardium of prenatal, and 1-, 2-, 5-, 10-, 20-, 28-, 50-, 60-, and 90-d-old male Wistar rats was studied. The expression of uncoupling proteins (UCPs), adenine nucleotide translocase (ANT), and peroxisome proliferator-activated receptor alpha (PPARalpha) genes was characterized by northern blotting (UCP2), real-time quantitative RT-PCR (UCP2, UCP3, ANT1, ANT2, and PPARalpha), and by immunoblotting (UCP3). In isolated mitochondria, cytochromes a + a(3) were quantified by a spectrophotometry, and mitochondrial membrane potential (MMP) was measured using Rhodamine 123 (by spectrofluorimetry and flow cytometry). The specific content of cytochromes in mitochondria increased two-fold between birth and day 30, similarly, as the expression of ANT1 and PPARalpha genes. Postnatal activation in the expression of UCP2, UCP3, ANT1 and PPARalpha genes resulted in the expression maxima between days 20 and 30. The content/expression declined following day 20 (UCP2, UCP3, and PPARalpha) or 30 (cytochromes and ANT1), while expression of ANT2 declined continuously during the first month of life. In 1-d-old animals a single population of mitochondria with a relatively high MMP was observed; with increasing age, a second population of mitochondria with a significantly lower MMP appeared. The results support the view that mitochondrial energy conversion in heart changes during ontogeny and suggest the involvement of UCP3 and/or ANT1 in the control mechanism.
- Published
- 2003
- Full Text
- View/download PDF
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