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Ding X, Zhu XL, Xu DH, Li S, Yang Q, Feng X, Wei YG, Li H, Yang L, Zhang YJ, Deng XL, Liu KC, Shi SL. NPM promotes hepatotoxin-induced fibrosis by inhibiting ROS-induced apoptosis of hepatic stellate cells and upregulating lncMIAT-induced TGF-β2. Cell Death Dis 2023; 14:575. [PMID: 37648688 PMCID: PMC10469196 DOI: 10.1038/s41419-023-06043-0] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2023] [Revised: 07/25/2023] [Accepted: 08/04/2023] [Indexed: 09/01/2023]
Abstract
Liver fibrosis is caused by a variety of chronic liver injuries and has caused significant morbidity and mortality in the world with increasing tendency. Elucidation of the molecular mechanism of liver fibrosis is the basis for intervention of this pathological process and drug development. Nucleophosmin (NPM) is a widely expressed nucleolar phosphorylated protein, which is particularly important for cell proliferation, differentiation and survival. The biological role of NPM in liver fibrosis remains unknown. Here we show that NPM promotes liver fibrosis through multiple pathways. Our study found that NPM was up-regulated in cirrhosis tissues and activated in hepatic stellate cells (HSCs). NPM inhibition reduced liver fibrosis markers expression in HSCs and inhibited the HSCs proliferation and migration. In mice model, NPM knockdown in HSCs or application of specific NPM inhibitor can remarkably attenuate hepatic fibrosis. Mechanistic analysis showed that NPM promotes hepatic fibrosis by inhibiting HSCs apoptosis through Akt/ROS pathway and by upregulating TGF-β2 through Akt-induced lncMIAT. LncMIAT up-regulated TGF-β2 mRNA by competitively sponging miR-16-5p. In response to liver injury, hepatocytes, Kupffer cells and HSCs up-regulated NPM to increase TGF-β2 secretion to activate HSCs in a paracrine or autocrine manner, leading to increased liver fibrosis. Our study demonstrated that NPM regulated hepatotoxin-induced fibrosis through Akt/ROS-induced apoptosis of HSCs and via the Akt/lncMIAT-up-regulated TGF-β2. Inhibition of NPM or application of NPM inhibitor CIGB300 remarkably attenuated liver fibrosis. NPM serves a potential new drug target for liver fibrosis.
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Affiliation(s)
- Xue Ding
- Cancer Research Center, School of Medicine, Xiamen University, Xiamen, China
- Department of Basic Medical Sciences, School of Medicine, Xiamen University, Xiamen, China
| | - Xin-Le Zhu
- Cancer Research Center, School of Medicine, Xiamen University, Xiamen, China
- Department of Basic Medical Sciences, School of Medicine, Xiamen University, Xiamen, China
| | - Dong-Hui Xu
- Cancer Research Center, School of Medicine, Xiamen University, Xiamen, China
- Department of Hepatic Biliary Pancreatic Vascular Surgery, the First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, China
| | - Shuang Li
- Cancer Research Center, School of Medicine, Xiamen University, Xiamen, China
| | - Qiong Yang
- Cancer Research Center, School of Medicine, Xiamen University, Xiamen, China
| | - Xian Feng
- Cancer Research Center, School of Medicine, Xiamen University, Xiamen, China
| | - Yong-Gui Wei
- Cancer Research Center, School of Medicine, Xiamen University, Xiamen, China
| | - Huan Li
- Cancer Research Center, School of Medicine, Xiamen University, Xiamen, China
| | - Ling Yang
- Cancer Research Center, School of Medicine, Xiamen University, Xiamen, China
| | - Yu-Jun Zhang
- Department of Basic Medical Sciences, School of Medicine, Xiamen University, Xiamen, China
| | - Xiao-Ling Deng
- Cancer Research Center, School of Medicine, Xiamen University, Xiamen, China
| | - Kuan-Can Liu
- Xiang'an Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, China.
| | - Song-Lin Shi
- Cancer Research Center, School of Medicine, Xiamen University, Xiamen, China.
- Department of Basic Medical Sciences, School of Medicine, Xiamen University, Xiamen, China.
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Rosengarten JF, Schatz S, Wolf T, Barbe S, Stitz J. Components of a HIV-1 vaccine mediate virus-like particle (VLP)-formation and display of envelope proteins exposing broadly neutralizing epitopes. Virology 2022; 568:41-48. [DOI: 10.1016/j.virol.2022.01.008] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2021] [Revised: 01/19/2022] [Accepted: 01/19/2022] [Indexed: 12/16/2022]
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Sun MJ, Cao ZQ, Leng P. The roles of galectins in hepatic diseases. J Mol Histol 2020; 51:473-484. [PMID: 32734557 DOI: 10.1007/s10735-020-09898-1] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2020] [Accepted: 07/14/2020] [Indexed: 12/24/2022]
Abstract
Hepatic diseases include all diseases that occur in the liver, including hepatitis, cirrhosis, hepatocellular carcinoma, etc. Hepatic diseases worldwide are characterized by high incidences of digestive system diseases, which present with subtle symptoms, are difficult to treat and have high mortality. Galectins are β-galactoside-binding proteins that have been found to be aberrantly expressed during hepatic disease progression. An increasing number of studies have shown that abnormal expression of galectins is extensively involved in hepatic diseases, such as hepatocellular carcinoma (HCC), liver cirrhosis, hepatitis and liver fibrosis. Galectins function as intracellular and extracellular hepatic disease regulators mainly through the binding of their carbohydrate recognition domain to glycoconjugates expressed in hepatocytes. In this review, we summarize current research on the various roles of galectins in cirrhosis, hepatitis, liver fibrosis and HCC, which may provide a preliminary theoretical basis for the exploration of new targets for the treatment of hepatic diseases.
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Affiliation(s)
- Mei-Juan Sun
- Department of Pharmacy, The Affiliated Hospital of Qingdao University, No. 16 Jiang Su Road, Qingdao, 266003, People's Republic of China
| | - Zhan-Qi Cao
- Department of Pharmacy, The Affiliated Hospital of Qingdao University, No. 16 Jiang Su Road, Qingdao, 266003, People's Republic of China
| | - Ping Leng
- Department of Pharmacy, The Affiliated Hospital of Qingdao University, No. 16 Jiang Su Road, Qingdao, 266003, People's Republic of China.
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Priyanka SH, Syam Das S, Nair SS, Rauf AA, Indira M. All trans retinoic acid modulates TNF-α and CYP2E1 pathways and enhances regression of ethanol-induced fibrosis markers in hepatocytes and HSCs in abstaining rodent model. Arch Physiol Biochem 2019; 125:302-310. [PMID: 29592769 DOI: 10.1080/13813455.2018.1455712] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Context: Our previous studies showed that all trans retinoic acid (ATRA) ameliorates alcohol-induced toxicity. Hence, we evaluated the efficacy of ATRA and abstention in the regression of alcohol-induced hepatotoxicity. Materials and methods: After ethanol administration to rats for 90 days, the regression of alcohol-induced toxicity was studied by supplementing ATRA at a dose of 100 μg/kg body weight for 30 days. It was also compared with animals in abstention. Results and discussion: Ethanol administration enhanced oxidative stress, activated HSCs and increased collagen deposition. All these alterations were reversed to a certain extent by ATRA supplementation. Conclusions: ATRA had better efficacy than just abstention in reducing ethanol-induced toxicity. The mechanism might be downregulation of CYP2E1, leading to reduced oxidative stress in the hepatocytes and thus impeding NFκB activation, cytokine production, activation of HSC and resulting in the reduction of inflammation and remodelling of fibrosis by modulating MMP and TIMP.
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Affiliation(s)
- S H Priyanka
- a Department of Biochemistry, University of Kerala , Thiruvananthapuram , India
| | - S Syam Das
- a Department of Biochemistry, University of Kerala , Thiruvananthapuram , India
| | - Saritha S Nair
- a Department of Biochemistry, University of Kerala , Thiruvananthapuram , India
| | - Arun A Rauf
- a Department of Biochemistry, University of Kerala , Thiruvananthapuram , India
| | - M Indira
- a Department of Biochemistry, University of Kerala , Thiruvananthapuram , India
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Wang Y, Guan M, Zhao X, Li X. Effects of garlic polysaccharide on alcoholic liver fibrosis and intestinal microflora in mice. PHARMACEUTICAL BIOLOGY 2018; 56:325-332. [PMID: 29969576 PMCID: PMC6130653 DOI: 10.1080/13880209.2018.1479868] [Citation(s) in RCA: 78] [Impact Index Per Article: 11.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/08/2023]
Abstract
CONTEXT Alcoholic liver fibrosis (ALF) is treatable and reversible consequence of liver disease. Intestinal microflora plays an important role in the progression of liver disease. Garlic (Allium sativum L. [Amaryllidaceae]) has been consumed as a traditional medicine to treat liver injury. OBJECTIVE To investigate the effects of garlic polysaccharide (GP) on ALF and intestinal microflora in mice. MATERIALS AND METHODS KM mice were orally administered with alcohol (56%, 6 mL/kg) for 30 d to establish ALF model, and divided into four groups together with control group (water only). Hugan tablet (60 mg/kg) or GP (250 and 150 mg/kg) were given 5 h after each dose of alcohol. Biochemical markers in serum and liver homogenate were determined with kits. Alteration of intestinal microflora, and protein expressions of TGF-β1, TNF-α and decorin were detected. RESULTS In GP-H group, ALT and AST decreased to 18.85 ± 4.71 U/L and 40.84 ± 7.89 U/L. MDA, TC, TG and LDL-C decreased to 2.32 ± 0.86 mmol/mg, 0.21 ± 0.12 mmol/L, 0.96 ± 0.31 mmol/L and 0.084 ± 0.027 mmol/L. SOD, GSH-Px and GSH increased to 118.32 ± 16.32 U/mg, 523.72 ± 64.20 U/mg and 0.56 ± 0.05 mg/g. Ratios of TGF-β1 and TNF-α decreased to 0.608 ± 0.170 and 1.057 ± 0.058, decorin increased to 2.182 ± 0.129. Lachnospiraceae and Lactobacillus increased, Facklamia and Firmicutes decreased with GP pretreatment. DISCUSSION AND CONCLUSIONS Intestinal microflora provides novel insight into the mechanisms of GP that may be used to treat ALF and intestinal microflora dysbiosis.
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Affiliation(s)
- Yuchuan Wang
- Department of Pediatrics, The Second Hospital of Dalian Medical University, Dalian, PR China
| | - Min Guan
- Department of Pediatrics, The Second Hospital of Dalian Medical University, Dalian, PR China
| | - Xin Zhao
- Department of Biotechonolgy, Dalian Medical University, Dalian, PR China
| | - Xinli Li
- Department of Biotechonolgy, Dalian Medical University, Dalian, PR China
- CONTACT Xinli Li Department of Biotechnology, Dalian Medical University, No. 9, West-Middle Section of Lvshun South Road, Dalian116044, Liaoning, PR China
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Huang Y, Huang D, Weng J, Zhang S, Zhang Q, Mai Z, Gu W. Effect of reversine on cell cycle, apoptosis, and activation of hepatic stellate cells. Mol Cell Biochem 2016; 423:9-20. [DOI: 10.1007/s11010-016-2815-x] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2016] [Accepted: 08/29/2016] [Indexed: 12/21/2022]
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Mazidi M, Karimi E, Meydani M, Ghayour-Mobarhan M, Ferns GA. Potential effects of curcumin on peroxisome proliferator-activated receptor-γ in vitro and in vivo. World J Methodol 2016; 6:112-117. [PMID: 27019802 PMCID: PMC4804246 DOI: 10.5662/wjm.v6.i1.112] [Citation(s) in RCA: 37] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/18/2015] [Revised: 02/01/2016] [Accepted: 03/07/2016] [Indexed: 02/06/2023] Open
Abstract
Natural peroxisome proliferator-activated receptor-γ (PPAR-γ) agonists are found in food and may be important for health through their anti-inflammatory properties. Curcumin (Cur) is a bright yellow spice, derived from the rhizome of Curcuma longa Linn. It has been shown to have many biological properties that appear to operate through diverse mechanisms. Some of these potentially beneficial effects of Cur are due to activation of the nuclear transcription factor PPAR-γ. It is reported (using in vitro and in vivo models) that Cur plays a potential role against several diseases. In this review article, we present the current literature on the effects of Cur on the modulation of inflammatory processes that are mediated through PPAR-γ.
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Cuesta-Domínguez Á, León-Rico D, Álvarez L, Díez B, Bodega-Mayor I, Baños R, Martín-Rey MÁ, Santos-Roncero M, Gaspar ML, Martín-Acosta P, Almarza E, Bueren JA, Río P, Fernández-Ruiz E. BCR-JAK2 drives a myeloproliferative neoplasm in transplanted mice. J Pathol 2015; 236:219-28. [DOI: 10.1002/path.4513] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2014] [Revised: 01/28/2015] [Accepted: 02/05/2015] [Indexed: 01/13/2023]
Affiliation(s)
- Álvaro Cuesta-Domínguez
- Molecular Biology Unit; Instituto de Investigación Sanitaria Princesa (IIS-P, UAM), Hospital Universitario de La Princesa; Madrid Spain
| | - Diego León-Rico
- Division of Haematopoietic Innovative Therapies; CIEMAT/CIBERER; Madrid Spain
- Instituto de Investigaciones Sanitarias Fundación Jiménez Díaz (IIS-FJD, UAM); Madrid Spain
| | - Lara Álvarez
- Division of Haematopoietic Innovative Therapies; CIEMAT/CIBERER; Madrid Spain
- Instituto de Investigaciones Sanitarias Fundación Jiménez Díaz (IIS-FJD, UAM); Madrid Spain
| | - Begoña Díez
- Division of Haematopoietic Innovative Therapies; CIEMAT/CIBERER; Madrid Spain
- Instituto de Investigaciones Sanitarias Fundación Jiménez Díaz (IIS-FJD, UAM); Madrid Spain
| | - Irene Bodega-Mayor
- Molecular Biology Unit; Instituto de Investigación Sanitaria Princesa (IIS-P, UAM), Hospital Universitario de La Princesa; Madrid Spain
| | - Rocío Baños
- Division of Haematopoietic Innovative Therapies; CIEMAT/CIBERER; Madrid Spain
- Instituto de Investigaciones Sanitarias Fundación Jiménez Díaz (IIS-FJD, UAM); Madrid Spain
| | - Miguel Ángel Martín-Rey
- Division of Haematopoietic Innovative Therapies; CIEMAT/CIBERER; Madrid Spain
- Instituto de Investigaciones Sanitarias Fundación Jiménez Díaz (IIS-FJD, UAM); Madrid Spain
| | - Matilde Santos-Roncero
- Molecular Biology Unit; Instituto de Investigación Sanitaria Princesa (IIS-P, UAM), Hospital Universitario de La Princesa; Madrid Spain
| | - María Luisa Gaspar
- Centro Nacional de Microbiología; Instituto de Salud Carlos III (ISCIII); Majadahonda Spain
| | - Paloma Martín-Acosta
- Servicio de Anatomía Patológica; Hospital Universitario Puerta de Hierro; Majadahonda Spain
| | - Elena Almarza
- Division of Haematopoietic Innovative Therapies; CIEMAT/CIBERER; Madrid Spain
- Instituto de Investigaciones Sanitarias Fundación Jiménez Díaz (IIS-FJD, UAM); Madrid Spain
| | - Juan A. Bueren
- Division of Haematopoietic Innovative Therapies; CIEMAT/CIBERER; Madrid Spain
- Instituto de Investigaciones Sanitarias Fundación Jiménez Díaz (IIS-FJD, UAM); Madrid Spain
| | - Paula Río
- Division of Haematopoietic Innovative Therapies; CIEMAT/CIBERER; Madrid Spain
- Instituto de Investigaciones Sanitarias Fundación Jiménez Díaz (IIS-FJD, UAM); Madrid Spain
| | - Elena Fernández-Ruiz
- Molecular Biology Unit; Instituto de Investigación Sanitaria Princesa (IIS-P, UAM), Hospital Universitario de La Princesa; Madrid Spain
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Duval F, Moreno-Cuevas JE, González-Garza MT, Rodríguez-Montalvo C, Cruz-Vega DE. Protective mechanisms of medicinal plants targeting hepatic stellate cell activation and extracellular matrix deposition in liver fibrosis. Chin Med 2014; 9:27. [PMID: 25606051 PMCID: PMC4299307 DOI: 10.1186/s13020-014-0027-4] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2014] [Accepted: 11/26/2014] [Indexed: 01/18/2023] Open
Abstract
During chronic liver injury, hepatic stellate cells (HSC) are activated and proliferate, which causes excessive extracellular matrix (ECM) deposition, leading to scar formation and fibrosis. Medicinal plants are gaining popularity as antifibrotic agents, and are often safe, cost-effective, and versatile. This review aims to describe the protective role and mechanisms of medicinal plants in the inhibition of HSC activation and ECM deposition during the pathogenesis of liver fibrosis. A systematic literature review on the anti-fibrotic mechanisms of hepatoprotective plants was performed in PubMed, which yielded articles about twelve relevant plants. Many of these plants act via disruption of the transforming growth factor beta 1 signaling pathway, possibly through reduction in oxidative stress. This reduction could explain the inhibition of HSC activation and reduction in ECM deposition. Medicinal plants could be a source of anti-liver fibrosis compounds.
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Affiliation(s)
- Florent Duval
- Cell Therapy Department, School of Medicine, Tecnológico de Monterrey, Monterrey, NL CP 63710 Mexico
| | - Jorge E Moreno-Cuevas
- Cell Therapy Department, School of Medicine, Tecnológico de Monterrey, Monterrey, NL CP 63710 Mexico
| | | | | | - Delia Elva Cruz-Vega
- Cell Therapy Department, School of Medicine, Tecnológico de Monterrey, Monterrey, NL CP 63710 Mexico
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Inhibition of acid-sensing ion channel 1a in hepatic stellate cells attenuates PDGF-induced activation of HSCs through MAPK pathway. Mol Cell Biochem 2014; 395:199-209. [PMID: 24939363 DOI: 10.1007/s11010-014-2125-0] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2014] [Accepted: 06/02/2014] [Indexed: 12/11/2022]
Abstract
Acid-sensing ion channels (ASICs), a group of Na(+)-selective and Ca(2+)-permeant ligand-gated cation channels, can be transiently activated by extracellular acid. Among seven subunits of ASICs, acid-sensing ion channel 1a (ASIC1a), which is responsible for Ca(2+) transportation, is elevated in response to inflammation, tumor, and ischemic injury in central nervous system and non-neuronal tissues. In this study, we demonstrated for the first time the presence of ASIC1a in rat liver and hepatic stellate cells (HSCs). Furthermore, the expression of ASIC1a was increased in primary HSCs and liver tissues of CCl4-treated rats, suggesting that ASIC1a may play certain role in liver fibrosis. Interestingly, we identified that the level of ASIC1a was significantly elevated in response to platelet-derived growth factor (PDGF) induction in a time- and dose-dependent manner. It was also established that Ca(2+)-transporting ASIC1a was involved in acid-induced injury of different cell types. Moreover, inhibition or silencing of ASIC1a was able to inhibit PDGF-induced pro-fibrogenic effects of activated rat HSCs, including cell activation, de novo synthesis of extracellular matrix components through mitogen-activated protein kinase signaling pathway. Collectively, our studies identified that ASIC1a was expressed in rat liver and HSCs and provided a strong evidence for the involvement of the ASIC1a in the progression of hepatic fibrosis.
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Watanabe R, Fujii H, Shirai T, Saito S, Ishii T, Harigae H. Autophagy plays a protective role as an anti-oxidant system in human T cells and represents a novel strategy for induction of T-cell apoptosis. Eur J Immunol 2014; 44:2508-20. [DOI: 10.1002/eji.201344248] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2013] [Revised: 03/24/2014] [Accepted: 04/30/2014] [Indexed: 12/14/2022]
Affiliation(s)
- Ryu Watanabe
- Department of Hematology and Rheumatology; Tohoku University Graduate School of Medicine; Sendai Japan
| | - Hiroshi Fujii
- Department of Hematology and Rheumatology; Tohoku University Graduate School of Medicine; Sendai Japan
| | - Tsuyoshi Shirai
- Department of Hematology and Rheumatology; Tohoku University Graduate School of Medicine; Sendai Japan
| | - Shinichiro Saito
- Department of Hematology and Rheumatology; Tohoku University Graduate School of Medicine; Sendai Japan
| | - Tomonori Ishii
- Department of Hematology and Rheumatology; Tohoku University Graduate School of Medicine; Sendai Japan
| | - Hideo Harigae
- Department of Hematology and Rheumatology; Tohoku University Graduate School of Medicine; Sendai Japan
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12
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Correction of glycogen storage disease type III with rapamycin in a canine model. J Mol Med (Berl) 2014; 92:641-50. [PMID: 24509886 DOI: 10.1007/s00109-014-1127-4] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2013] [Revised: 12/27/2013] [Accepted: 01/23/2014] [Indexed: 01/09/2023]
Abstract
UNLABELLED Recently, we reported that progression of liver fibrosis and skeletal myopathy caused by extensive accumulation of cytoplasmic glycogen at advanced age is the major feature of a canine model of glycogen storage disease (GSD) IIIa. Here, we aim to investigate whether rapamycin, a specific inhibitor of mTOR, is an effective therapy for GSD III. Our data show that rapamycin significantly reduced glycogen content in primary muscle cells from human patients with GSD IIIa by suppressing the expression of glycogen synthase and glucose transporter 1. To test the treatment efficacy in vivo, rapamycin was daily administered to GSD IIIa dogs starting from age 2 (early-treatment group) or 8 months (late-treatment group), and liver and skeletal muscle biopsies were performed at age 12 and 16 months. In both treatment groups, muscle glycogen accumulation was not affected at age 12 months but significantly inhibited at 16 months. Liver glycogen content was reduced in the early-treatment group but not in the late-treatment group at age 12 months. Both treatments effectively reduced liver fibrosis at age 16 months, consistent with markedly inhibited transition of hepatic stellate cells into myofibroblasts, the central event in the process of liver fibrosis. Our results suggest a potential useful therapy for GSD III. KEY MESSAGES Rapamycin inhibited glycogen accumulation in GSD IIIa patient muscle cells. Rapamycin reduced muscle glycogen content in GSD IIIa dogs at advanced age. Rapamycin effectively prevented progression of liver fibrosis in GSD IIIa dogs. Our results suggest rapamycin as potential useful therapy for patients with GSD III.
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Fernández-Morales B, Pavón L, Calés C. CDC6 expression is regulated by lineage-specific transcription factor GATA1. Cell Cycle 2012; 11:3055-66. [PMID: 22871742 DOI: 10.4161/cc.21471] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023] Open
Abstract
GATA1 is a hematopoietic transcription factor essential for expression of most genes encoding erythro-megakaryocytic proteins, i.e., globins and platelet glycoproteins. A role for GATA1 as a cell proliferation regulator has been proposed, as some of its bona fide targets comprise global regulators, such as c-KIT or c-MYC, or cell cycle factors, i.e., CYCLIN D or p21CIP1. In this study, we describe that GATA1 directly regulates the expression of replication licensing factor CDC6. Using reporter transactivation, electrophoretic mobility shift and chromatin immunoprecipitation assays, we show that GATA1 stimulates CDC6 transcription by binding to a canonical binding site located within a 166bp enhancer region upstream CDC6 promoter. This evolutionary conserved GATA binding site conforms to recently described chromatin occupancy rules, i.e., preferred bases within core WGATAR (TGATAA), 5' and 3' flanking bases (GGTGATAAGG) and distance to the transcription initiation site. We also found adjacent conserved binding sites for ubiquitously expressed transcription factor CP2, needed for GATA activity on CDC6 enhancer. Our results add to the growing evidence for GATA1 acting as a direct transcriptional regulator of the cell cycle machinery, thus linking cell proliferation control and specific gene expression programs during lineage differentiation.
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Affiliation(s)
- Bárbara Fernández-Morales
- Department of Cancer Biology, Instituto de Investigaciones Biomédicas Alberto Sols, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid-IdiPAZ, Madrid, Spain
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Ping A, Yihao T, Jingxing D, Minkai C, Hesheng L. Ca²⁺/calmodulin-dependent protein kinase II mediates platelet-derived growth factor-induced human hepatic stellate cell proliferation. Dig Dis Sci 2012; 57:935-942. [PMID: 22215519 DOI: 10.1007/s10620-011-2014-2] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/26/2011] [Accepted: 12/01/2011] [Indexed: 01/26/2023]
Abstract
BACKGROUND AND AIM Proliferation and activation of myofibroblastic hepatic stellate cells (HSCs) in response to growth factors is essential for the development of liver fibrosis. As one of the most potent factors, platelet-derived growth factor (PDGF) activates intracellular signals and contributes to sustained HSCs activation. Growing evidence has suggested that the Ca(2+) signal is involved in PDGF pathways. We showed previously for the first time that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is essential for human HSC proliferation. The inhibition of CaMKII by its specific inhibitor, KN-93, significantly decreased the HSC growth and increased expression of cell cycle suppressive regulators P53 and P21. METHODS In the present study, we investigated the role of CaMKII in PDGF-induced HSC proliferation and underlying mechanisms. RESULTS We confirmed that in human HSCs, PDGF significantly increased CaMKII mRNA levels, protein expression, and phosphorylation. The interruption of CaMKII by KN-93, specific inhibitory peptide (AIP), or specific CaMKII knockdown by its siRNA not only attenuated PDGF-induced HSC proliferation but also ERK1/2 phosphorylation. However, CaMKII had no effect on JNK phosphorylation. In addition, inhibitors of ERK1/2 (PD98059) and JNK (SP600125) did not affect CaMKII expression. Interruption of CaMKII-ERK cascade, not JNK signal, inhibited PDGF-induced HSC proliferation. CONCLUSION We confirmed that CaMKII mediated PDGF-induced human HSC proliferation through ERK1/2 but not the JNK mechanism. Our study shed light on CaMKII as a crucial signal in PDGF-activated HSCs and a potential therapeutic point in hepatic fibrosis.
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Affiliation(s)
- An Ping
- Division of Gastroenterology and Hepatology, Renmin Hospital of Wuhan University, 238 Jiefang Road, Wuhan 430060, China
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Lu P, Liu H, Yin H, Yang L. Expression of angiotensinogen during hepatic fibrogenesis and its effect on hepatic stellate cells. Med Sci Monit 2011; 17:BR248-56. [PMID: 21873937 PMCID: PMC3560510 DOI: 10.12659/msm.881928] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
BACKGROUND The liver renin-angiotensin system (RAS) plays an important role in promoting the development of hepatic fibrogenesis. Angiotensinogen (AGT) is an important precursor in tissue RAS. This study aimed to investigate the expression and cellular source of AGT in hepatic fibrogenesis and its effect on proliferation and collagen metabolism of hepatic stellate cells. MATERIAL/METHODS In a rat carbon tetrachloride (CCl4)-induced liver fibrosis model the mRNA expression of AGT was determined by real-time PCR and the cellular source of AGT was determined by immunohistochemical staining. In vitro HSC-T6 cells were transfected with AGT, and the expression plasmid, AGT shRNA plasmid and negative shRNA plasmid were constructed. Real-time PCR and ELISA were applied to determine the mRNA expressions and contents of TIMP-1, TGF-β1, type I collagen and type III collagen of the cells or in the supernatants. RESULTS Compared to normal liver, the AGT and α-SMA mRNA expressions increased at the early stage of hepatic fibrosis and decreased in hepatic cirrhosis. The expressions of AGT and α-SMA mRNA were correlated with the hepatic fibrosis (r=0.915, P=0.03). Immunohistochemistry demonstrated the activated HSCs were the main source of AGT due to colocalization of AGT and α-SMA expressions. The mRNA and protein of TGF-β1, TIMP-1, type I collagen and type III collagen were markedly up-regulated. CONCLUSIONS ACEI and angiotensin II type 1 receptor antagonist (AT1RA) could attenuate the progression of hepatic fibrosis in the early stage. Direct inhibition of AGT from aHSCs may become an effective antifibrotic anti-liver fibrosis strategy.
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Affiliation(s)
- Ping Lu
- Department of Geriatrics, 9th People's Hospital Affiliated to School of Medicine, Shanghai Jiaotong University, Shanghai, China
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16
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Doty RT, Sabo KM, Chen J, Miller AD, Abkowitz JL. An all-feline retroviral packaging system for transduction of human cells. Hum Gene Ther 2011; 21:1019-27. [PMID: 20222826 DOI: 10.1089/hum.2010.032] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Abstract The subgroup C feline leukemia virus (FeLV-C) receptor FLVCR is a widely expressed 12-transmembrane domain transporter that exports cytoplasmic heme and is a promising target for retrovirus-mediated gene delivery. Previous studies demonstrated that FeLV-C pseudotype vectors were more efficient at targeting human hematopoietic stem cells than those pseudotyped with gibbon ape leukemia virus (GALV), and thus we developed an all FeLV-C-based packaging system, termed CatPac. CatPac is helper-virus free and can produce higher titer vectors than existing gammaretroviral packaging systems, including systems mixing Moloney murine leukemia virus (MoMLV) Gag-Pol and FeLV-C Env proteins. The vectors can be readily concentrated (>30-fold), refrozen (three to five times), and held on ice (>2 days) with little loss of titer. Furthermore, we demonstrate that CatPac pseudotype vectors efficiently target early CD34(+)CD38(-) stem/progenitor cells, monocytic and erythroid progenitors, activated T cells, mature macrophages, and cancer cell lines, suggesting utility for human cell and cell line transduction and possibly gene therapy.
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Affiliation(s)
- Raymond T Doty
- Department of Medicine, University of Washington, Seattle, WA 98195, USA
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Zaldívar I, Muñoz-Fernández MA, Alarcón B, San José E. Expression of a modified form of CD4 results in the release of an anti-HIV factor derived from the Env sequence. THE JOURNAL OF IMMUNOLOGY 2009; 183:1188-96. [PMID: 19553524 DOI: 10.4049/jimmunol.0802944] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
We have studied the inhibitory effect of a CD4 chimera (CD4epsilon15) on HIV replication. This chimera is retained in the endoplasmic reticulum and traps the HIV envelope precursor gp160, preventing its maturation. Retroviral expression of the chimera strongly inhibited HIV replication even when it is expressed by only a minority of the T cell population. This protective effect on bystander nontransduced cells is mediated by a soluble factor that we identified as a fragment of HIV gp120 envelope protein and accordingly, we named this factor Env-derived antiviral factor (EDAF). Biochemical and immunoreactivity data show that EDAF is comprised of the gp120 C3-C5 regions and indeed, a recombinant protein bearing this sequence reproduces the anti-HIV properties of EDAF. Surprisingly, three tryptic peptides derived from EDAF are homologous but not identical with the corresponding sequences of the HIV isolate used to generate EDAF. We propose that EDAF results from an alternative intracellular processing of the Env protein provoked by its association to CD4epsilon15 and the selection of the best fitted Env protein sequences contained within the HIV isolate. The presence of EDAF improves the therapeutic potential of the CD4epsilon15 gene and it opens new possibilities for antiviral treatment and vaccine development.
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Affiliation(s)
- Irene Zaldívar
- Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Spain
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Bassi AM, Casu A, Canepa C, Maloberti G, Nanni G. Chronic High Doses of Thioacetamide Followed by Vitamin A Modify Dolichol, Dolichol Isoprenoids, and Retinol Content in Rat Liver Cells. Drug Chem Toxicol 2008; 28:91-104. [PMID: 15720038 DOI: 10.1081/dct-39721] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/03/2022]
Abstract
Our line of researches follows the hypothesis that dolichol and retinol metabolism might be interrelated and involved in liver fibrosis. To this end, in this study rats were subjected to chronic treatment with thioacetamide (TAA) (300 mg/L liquid diet) for 1 and 2 months and, after liver damage had occurred, supplemented with vitamin A before sacrifice. Dolichol, dolichol isoprene units, and retinol content were determined in isolated parenchymal and sinusoidal liver cells (hepatic stellate cells; Kupffer cells; sinusoidal endothelial cells). Dolichol increased in hepatocytes after TAA treatment, with or without vitamin A. Dolichol decreased in the other cells. Retinol in general decreased. In hepatocytes, retinol decreased only on normal nutrition, while the vitamin A load was taken up normally. The percentages of dolichol isoprene units (Dol-16 to Dol-20, in rats) confirm that Dol-18, which was not modified in percentage by TAA on normal nutrition, did not increase after vitamin A, as it did in control cells (7-12%). The behavior of Dol-18 was similar in all the cells studied. Vitamin A might reveal a latent damage produced by TAA on dolichol homologues. These data support previous hypotheses that the action of TAA depends on the administration modality, the dosage, and the diet, and that Dol-18 might have different functions and compartmentalization in the cells. Furthermore, the results support the hypothesis that dolichol chain length might be interrelated with retinol metabolism, perhaps through their metabolites.
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Affiliation(s)
- Anna Maria Bassi
- Section of General Pathology, Department of Experimental Medicine, University of Genoa, Genoa, Italy.
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Targeted inhibition of platelet-derived growth factor receptor-beta subunit in hepatic stellate cells ameliorates hepatic fibrosis in rats. Gene Ther 2008; 15:1424-35. [PMID: 18509379 DOI: 10.1038/gt.2008.93] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
The activation of hepatic stellate cells (HSCs) is the key event of the pathogenesis of hepatic fibrosis. Platelet-derived growth factor (PDGF) is the most potent mitogen for HSCs, and PDGF receptor-beta subunit (PDGFR-beta) is required for the proliferation of HSCs induced by PDGF. In this study, a high gene-silencing-efficacy PDGFR-beta small interference RNA (siRNA) was synthesized that could suppress the PDGFR-beta expression and inhibit the activation and proliferation but could not induce the apoptosis of HSCs in vitro. To avoid the side effect of nonspecific interference of PDGFR-beta, we constructed an HSCs-specific short hairpin RNA (shRNA) expression plasmid in which PDGFR-beta shRNA was driven by a glial fibrillary acidic protein (GFAP) promoter. The double-staining immunofluorescence examination indicated that GFAP promoter could target the transgene expression into HSCs in carbon tetrachloride induced acute injured rat's liver and bile duct ligation (BDL)-induced chronic injured rat's liver. Furthermore, HSCs-specific PDGFR-beta shRNA could relieve liver injury and hepatic fibrosis in the rat's model induced by BDL. This study demonstrates that PDGFR-beta siRNA may be presented as an antifibrogenic agent. The application of HSCs-specific RNA interference induced by the GFAP promoter might supply a new powerful tool for cell-specific gene therapy of hepatic fibrogenesis.
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20
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Salas AL, Montezuma TD, Fariña GG, Reyes-Esparza J, Rodríguez-Fragoso L. Genistein modifies liver fibrosis and improves liver function by inducing uPA expression and proteolytic activity in CCl4-treated rats. Pharmacology 2007; 81:41-9. [PMID: 17823541 DOI: 10.1159/000107968] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2007] [Accepted: 05/14/2007] [Indexed: 01/18/2023]
Abstract
AIM To evaluate the effect of genistein on the fibrosis and matrix degradation caused by experimentally induced fibrosis in rats. METHODS Hepatic fibrosis was brought about by chronic administration of carbon tetrachloride to rats. To evaluate the effect of genistein on liver fibrosis and function, total collagen content and proteolytic activity in the liver were quantified. Urokinase-type plasminogen activator (uPA) expression during experimental fibrosis was localized by immunohistochemistry. Histopathological changes were evaluated using light and electron microscopy. RESULTS Animals with fibrosis and treated with genistein showed an important reduction (73%) in hepatic collagen content as well as an improvement in liver function (p < 0.001). Genistein increased the capacity of the liver to degrade type I collagen and Matrigel (3.1- and 3.7-fold, respectively; p < 0.001) in animals with liver fibrosis. Genistein increased the number of uPA-immunoreactive cells. The increase in the uPA expression correlated with an increase in proteolytic activity. Histological analysis revealed a reduction in the number of fiber septa in pericentral and perisinusoidal areas. Transmission electron micrographs of livers from animals with fibrosis and treated with genistein showed a reduction in the number of hepatic stellate cells activated and a smaller number of collagen fibers. CONCLUSION Genistein is able to improve the liver after injury and fibrosis induced by chronic administration of carbon tetrachloride. This finding suggests that genistein has antifibrogenic potential and could therefore be useful for treating chronic liver disease.
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Affiliation(s)
- Alfonso Leija Salas
- Facultad de Farmacia, Universidad Autónoma del Estado de Morelos, Cuernavaca, Morelos, México
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21
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Zheng SM, Jiang MD, Zeng WZ, Xu H, Wang YX, Ma HD, Xie FW, Zhang Y, Qin JP, Wu XL. Effects of extracellular signal-regulated kinase on rat cultured hepatic stellate cells stimulated by acetaldehyde. J Dig Dis 2007; 8:148-53. [PMID: 17650227 DOI: 10.1111/j.1443-9573.2007.00302.x] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
OBJECTIVE To investigate the effects of PD98059 on the cell cycle, cell proliferation, the secretion of type I collagen and expression of transforming growth factor-beta-1 mRNA in rat hepatic stellate cells stimulated by acetaldehyde. METHODS Rat hepatic stellate cells stimulated by acetaldehyde were incubated with different concentrations of PD98059. The cell cycle was analyzed by flow cytometry. Cell proliferation was assessed by methyl thiazolyl tetrazolium colorimetric assay. The mRNA expression of transforming growth factor-beta-1 was examined by reverse transcriptase polymerase chain reaction. Type I collagen of the culture medium was detected by enzyme-linked immunoadsorbent assay. RESULTS Twenty, 50 and 100 micromol/L PD98059 could significantly inhibit the proliferation and provoke a G0/G1-phase arrest of hepatic stellate cells stimulated by acetaldehyde in a dose-dependent manner. The secretion of type I collagen and transforming growth factor-beta-1 mRNA expression of acetaldehyde-induced hepatic stellate cells were markedly inhibited by 50 and 100 micromol/L PD98059, respectively. CONCLUSION Extracellular signal-regulated kinase signal transduction pathway could regulate cell proliferation, the secretion of type I collagen and transforming growth factor-beta-1 mRNA expression of rat hepatic stellate cells stimulated by acetaldehyde. This is most likely related to its regulative effect on the cell cycle.
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Affiliation(s)
- Shu Mei Zheng
- Department of Gastroenterology, General Hospital of Chengdu Military Command, Chengdu, China.
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Gómez-Benito M, Balsas P, Carvajal-Vergara X, Pandiella A, Anel A, Marzo I, Naval J. Mechanism of apoptosis induced by IFN-alpha in human myeloma cells: role of Jak1 and Bim and potentiation by rapamycin. Cell Signal 2006; 19:844-54. [PMID: 17158029 DOI: 10.1016/j.cellsig.2006.10.009] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2006] [Revised: 10/16/2006] [Accepted: 10/22/2006] [Indexed: 11/21/2022]
Abstract
Interferon-alpha (IFN-alpha) has been used for the last 20 years in the maintenance therapy of multiple myeloma (MM), though it is only effective in some patients. Congruent with this, IFN-alpha induces apoptosis in some MM cell lines. Understanding the mechanism of IFN-alpha-induced apoptosis could be useful in establishing criteria of eligibility for therapy. Here we show that IFN-alpha-induced apoptosis in the MM cell lines U266 and H929 was completely blocked by a specific inhibitor of Jak1. The mTOR inhibitor rapamycin mitigated apoptosis in U266 but potentiated it in H929 cells. IFN-alpha induced PS exposure, DeltaPsi(m) loss and pro-apoptotic conformational changes of Bak, but not of Bax, and was fully prevented by Mcl-1 overexpression in U266 cells. IFN-alpha treatment caused the release of cytochrome c from mitochondria to cytosol and consequently, a limited proteolytic processing of caspases. Apoptosis induced by IFN-alpha was only slightly prevented by caspase inhibitors. Levels of the BH3-only proteins PUMA and Bim increased during IFN-alpha treatment. Bim increase and apoptosis was prevented by transfection with the siRNA for Bim. PUMA-siRNA transfection reduced electroporation-induced apoptosis but had no effect on apoptosis triggered by IFN-alpha. The potentiating effect of rapamycin on apoptosis in H929 cells was associated to an increase in basal and IFN-alpha-induced Bim levels. Our results indicate that IFN-alpha causes apoptosis in myeloma cells through a moderate triggering of the mitochondrial route initiated by Bim and that mTOR inhibitors may be useful in IFN-alpha maintenance therapy of certain MM patients.
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Affiliation(s)
- Maria Gómez-Benito
- Departamento de Bioquimica, Biologia Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, 50009 Zaragoza, Spain
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23
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IJzer J, Roskams T, Molenbeek RF, Ultee T, Penning LC, Rothuizen J, van den Ingh TSGAM. Morphological characterisation of portal myofibroblasts and hepatic stellate cells in the normal dog liver. COMPARATIVE HEPATOLOGY 2006; 5:7. [PMID: 17109742 PMCID: PMC1660578 DOI: 10.1186/1476-5926-5-7] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/04/2005] [Accepted: 11/16/2006] [Indexed: 11/21/2022]
Abstract
Background Hepatic fibrosis is a common outcome of hepatic injury in both man and dog. Activated fibroblasts which develop myofibroblastic characteristics play an essential role in hepatic fibrogenesis, and are comprised of three subpopulations: 1) portal or septal myofibroblasts, 2) interface myofibroblasts and 3) the perisinusoidally located hepatic stellate cells (HSC). The present study was performed to investigate the immunohistochemical characteristics of canine portal myofibroblasts (MF) and HSC in the normal unaffected liver as a basis for further studies on fibrogenesis in canine liver disease. Results In the formalin-fixed and paraffin embedded normal canine liver vimentin showed staining of hepatic fibroblasts, probably including MF in portal areas and around hepatic veins; however, HSC were in general negative. Desmin proved to react with both portal MF and HSC. A unique feature of these HSC was the positive immunostaining for alpha-smooth muscle actin (α-SMA) and muscle-specific actin clone HHF35 (HHF35), also portal MF stained positive with these antibodies. Synaptophysin and glial fibrillary acidic protein (GFAP) were consistently negative in the normal canine liver. In a frozen chronic hepatitis case (with expected activated hepatic MF and HSC), HSC were negative to synaptophysin, GFAP and NCAM. Transmission electron microscopy (TEM) immunogold labelling for α-SMA and HHF35 recognized the positive cells as HSC situated in the space of Disse. Conclusion In the normal formalin-fixed and paraffin embedded canine liver hepatic portal MF and HSC can be identified by α-SMA, HHF35 and to a lesser extent desmin immunostaining. These antibodies can thus be used in further studies on hepatic fibrosis. Synaptophysin, GFAP and NCAM do not seem suitable for marking of canine HSC. The positivity of HSC for α-SMA and HHF35 in the normal canine liver may eventually reflect a more active regulation of hepatic sinusoidal flow by these HSC compared to other species.
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Affiliation(s)
- Jooske IJzer
- Department of Pathobiology, Faculty of Veterinary Medicine, Utrecht University, The Netherlands
| | - Tania Roskams
- Laboratory of Morphology and Molecular Pathology, University of Leuven (K.U. Leuven), Leuven, Belgium
| | - Ronald F Molenbeek
- Department of Pathobiology, Faculty of Veterinary Medicine, Utrecht University, The Netherlands
| | - Ton Ultee
- Department of Pathobiology, Faculty of Veterinary Medicine, Utrecht University, The Netherlands
| | - Louis C Penning
- Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, The Netherlands
| | - Jan Rothuizen
- Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, The Netherlands
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Muñoz AL, Prieto C, Tabarés E. A comparison of enhanced green fluorescent protein expression induced by immediate-early cytomegalovirus (IE-CMV) and gG pseudorabies virus (gG-PRV) promoters, using pseudorabies virus amplicons as vectors. J Virol Methods 2006; 136:257-60. [PMID: 16712964 DOI: 10.1016/j.jviromet.2006.04.010] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2006] [Revised: 04/03/2006] [Accepted: 04/06/2006] [Indexed: 11/19/2022]
Abstract
This study compares the expression efficiencies of the IE-CMV and gG-PRV promoters following their transfection into cultured human and monkey cells, using pseudorabies virus amplicons as vectors and enhanced green fluorescence protein (EGFP) as an expression marker. EGFP expression was similarly strong with both promoters. Pseudorabies virus amplicons appear to be useful vectors in gene expression studies due to their replication in the presence of helpers and their wide range of cellular hosts.
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Affiliation(s)
- A L Muñoz
- Departamento de Medicina Preventiva, Salud Pública y Microbiología, Facultad de Medicina, Universidad Autónoma de Madrid, Arzobispo Morcillo 4, E-28029 Madrid, Spain
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25
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Demetris AJ, Lunz JG. Early HCV-associated stellate cell activation in aggressive recurrent HCV: what can liver allografts teach about HCV pathogenesis? Liver Transpl 2005; 11:1172-6. [PMID: 16184566 DOI: 10.1002/lt.20506] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
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Valenzuela-Fernández A, Alvarez S, Gordon-Alonso M, Barrero M, Ursa A, Cabrero JR, Fernández G, Naranjo-Suárez S, Yáñez-Mo M, Serrador JM, Muñoz-Fernández MA, Sánchez-Madrid F. Histone deacetylase 6 regulates human immunodeficiency virus type 1 infection. Mol Biol Cell 2005; 16:5445-54. [PMID: 16148047 PMCID: PMC1266439 DOI: 10.1091/mbc.e05-04-0354] [Citation(s) in RCA: 108] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022] Open
Abstract
Efficient human immunodeficiency virus (HIV)-1 infection depends on multiple interactions between the viral gp41/gp120 envelope (Env) proteins and cell surface receptors. However, cytoskeleton-associated proteins that modify membrane dynamics may also regulate the formation of the HIV-mediated fusion pore and hence viral infection. Because the effects of HDAC6-tubulin deacetylase on cortical alpha-tubulin regulate cell migration and immune synapse organization, we explored the possible role of HDAC6 in HIV-1-envelope-mediated cell fusion and infection. The binding of the gp120 protein to CD4+-permissive cells increased the level of acetylated alpha-tubulin in a CD4-dependent manner. Furthermore, overexpression of active HDAC6 inhibited the acetylation of alpha-tubulin, and remarkably, prevented HIV-1 envelope-dependent cell fusion and infection without affecting the expression and codistribution of HIV-1 receptors. In contrast, knockdown of HDAC6 expression or inhibition of its tubulin deacetylase activity strongly enhanced HIV-1 infection and syncytia formation. These results demonstrate that HDAC6 plays a significant role in regulating HIV-1 infection and Env-mediated syncytia formation.
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Svegliati-Baroni G, Inagaki Y, Rincon-Sanchez AR, Else C, Saccomanno S, Benedetti A, Ramirez F, Rojkind M. Early response of alpha2(I) collagen to acetaldehyde in human hepatic stellate cells is TGF-beta independent. Hepatology 2005; 42:343-52. [PMID: 16025520 PMCID: PMC1314984 DOI: 10.1002/hep.20798] [Citation(s) in RCA: 69] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Acetaldehyde is fibrogenic and induces the expression of type I collagen genes in hepatic stellate cells. Some of these acetaldehyde-dependent events are mediated by H(2)O(2) and thus establish a direct connection between oxidative stress and collagen upregulation. We localized to the -378 to -183 region of the alpha2(I) collagen (COL1A2) promoter an acetaldehyde-responsive element (AcRE) functional in human hepatic stellate cells (HHSCs) and investigated molecular mechanisms whereby acetaldehyde stimulates and modulates its transcriptional activity. Because the AcRE co-localized with a previously described transforming growth factor beta (TGF-beta)1-responsive element, and both acetaldehyde and this cytokine induce their effects through H(2)O(2), we investigated whether all fibrogenic actions of acetaldehyde were mediated by this cytokine. Here we show that acetaldehyde-induced COL1A2 upregulation in HHSCs recognizes two distinct but overlapping early and late stages that last from 1 to 6 hours and from 6 to 24 hours, respectively. We present several lines of evidence to show that early acetaldehyde-mediated events are independent of TGF-beta1. These include significant time-course differences in the expression of COL1A2 and TGF-beta1 mRNAs and inability of neutralizing antibodies to TGF-beta1 to inhibit acetaldehyde-dependent collagen gene transcription and Smad 3 phosphorylation. We also show that although acetaldehyde-dependent upregulation of collagen was PI3K dependent, that of TGF-beta1 was PI3K independent. In conclusion, acetaldehyde-dependent mechanisms involved in COL1A2 upregulation are similar, but not identical, to those of TGF-beta1. We suggest that early acetaldehyde-dependent events induce the late expression of TGF-beta1 and create an H(2)O(2)-dependent autocrine loop that may sustain and amplify the fibrogenic response of this alcohol metabolite.
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Affiliation(s)
| | - Yutaka Inagaki
- Department of Community Health, Tokai University School of Medicine, Bohseidai, Isehara, Japan
| | - Ana-Rosa Rincon-Sanchez
- Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine, New York, NY
| | - Cindy Else
- Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine, New York, NY
- Laboratory of Genetics and Organogenesis, Hospital for Special Surgery at the Weill Medical College of Cornell University, New York, NY; and
| | - Stefania Saccomanno
- From the Clinica di Gastroenterologia, Universita Politecnica delle Marche, Ancona, Italy
| | - Antonio Benedetti
- From the Clinica di Gastroenterologia, Universita Politecnica delle Marche, Ancona, Italy
| | - Francesco Ramirez
- Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine, New York, NY
- Laboratory of Genetics and Organogenesis, Hospital for Special Surgery at the Weill Medical College of Cornell University, New York, NY; and
| | - Marcos Rojkind
- Departments of Biochemistry and Molecular Biology and of Pathology, The George Washington University Medical Center, Washington, DC
- Address reprint requests to: Marcos Rojkind, M.D., Ph.D., Departments of Biochemistry and Molecular Biology, The George Washington University Medical Center, 2300 I St., NW, Washington DC, 20037. E-mail:
; fax: 202-994-8974
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Hellerbrand C, Bataille F, Schlegel J, Hartmann A, Mühlbauer M, Schölmerich J, Büttner R, Hofstädter F, Bosserhoff AK. In situ expression patterns of melanoma inhibitory activity 2 in healthy and diseased livers. Liver Int 2005; 25:357-66. [PMID: 15780062 DOI: 10.1111/j.1478-3231.2005.01099.x] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/21/2023]
Abstract
UNLABELLED Recently we identified a novel gene of the MIA gene family, melanoma inhibitory activity 2 (MIA2) and found that MIA2 mRNA is selectively expressed in hepatocytes. Here, we analyzed the in situ expression of MIA2 protein and mRNA in healthy and diseased livers to get first insights into the function of MIA2. METHODS We analyzed liver tissue of patients with chronic hepatitis C (HepC) infection and hepatocellular carcinoma (HCC) as well as a human multi-tissue array, primary human hepatocytes and the hepatoma cell-lines HepG2, Hep3B and PLC by immunohistochemical staining and quantitative RT-PCR. In addition to MIA2, the expression of alpha-smooth muscle actin (alpha-sma), a marker for activated hepatic stellate cells (HSCs)/myofibroblast, was analyzed. RESULTS Hepatocytes were confirmed as the exclusive cellular source of MIA2 expression, with a granular, cytoplasmatic staining pattern without enhancement at the cell membrane. In contrast, only low MIA2 expression levels were detected in most HCC and hepatoma cell lines. Only in HCC that contained fibrous stroma or thick hyalinized bundles, adjacent atypical hepatocytes revealed strong staining. In accordance, MIA2 expression was also upregulated in non-tumorous livers of patients with HepC and correlated with the staging of fibrosis. Interestingly, both in HCC and liver tissues of patients with HepC we found a correlation of MIA2 and alpha-sma expression. DISCUSSION We define for the first time in situ expression patterns of MIA2 in healthy and diseased livers. Our data raise the hypothesis that activation of HSCs/myofibroblasts has influence on MIA2 expression in vivo, consistent with our previous in vitro findings. Since the staining pattern and the protein structure highly suggests that MIA2 is a secreted protein, it may possibly serve as a marker of hepatic fibrosis.
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Affiliation(s)
- Claus Hellerbrand
- Department of Internal Medicine I, University of Regensburg, Regensburg, Germany
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Dupré L, Trifari S, Follenzi A, Marangoni F, Lain de Lera T, Bernad A, Martino S, Tsuchiya S, Bordignon C, Naldini L, Aiuti A, Roncarolo MG. Lentiviral Vector-Mediated Gene Transfer in T Cells from Wiskott–Aldrich Syndrome Patients Leads to Functional Correction. Mol Ther 2004; 10:903-15. [PMID: 15509508 DOI: 10.1016/j.ymthe.2004.08.008] [Citation(s) in RCA: 80] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2004] [Accepted: 08/12/2004] [Indexed: 10/26/2022] Open
Abstract
Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency with a median survival below the age of 20 due to infections, severe hemorrhage, and lymphomas. Transplantation of hematopoietic stem cells from HLA-identical sibling donors is a resolutive treatment, but is available for a minority of patients. Transplantation of genetically corrected autologous hematopoietic stem cells or T cells could represent an alternative treatment applicable to all patients. We investigated whether WAS gene transfer with MMLV-based oncoretroviral and HIV-based lentiviral vectors could restore normal functions of patients' T cells. T cells transduced either with lentiviral vectors expressing the WAS protein (WASP) from the ubiquitous PGK promoter or the tissue-specific WASP promoter or with an oncoretroviral vector expressing WASP from the LTR, reached normal levels of WASP with correction of functional defects, including proliferation, IL-2 production, and lipid raft upregulation. Lentiviral vectors transduced T cells from WAS patients at higher rates, compared to oncoretroviral vectors, and efficiently transduced both activated and naive WAS T cells. Furthermore, a selective growth advantage of T cells corrected with the lentiviral vectors was demonstrated. The observation that lentiviral vector-mediated gene transfer results in correction of T cell defects in vitro supports their application for gene therapy in WAS patients.
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Affiliation(s)
- Loïc Dupré
- San Raffaele Telethon Institute for Gene Therapy, 20132 Milan, Italy
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Monjas A, Alcover A, Alarcón B. Engaged and bystander T cell receptors are down-modulated by different endocytotic pathways. J Biol Chem 2004; 279:55376-84. [PMID: 15516342 DOI: 10.1074/jbc.m409342200] [Citation(s) in RCA: 54] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
T cell antigen receptor (TCR) engagement by stimulatory antibodies or its major histocompatibility complex-antigen ligand results in its down-modulation from the cell surface, a phenomenon that is thought to play a role in T cell desensitization. However, TCR engagement results in the down-modulation not only of the engaged receptors but also of non-engaged bystander TCRs. We have investigated the mechanisms that mediate the down-modulation of engaged and bystander receptors and show that co-modulation of the bystander TCRs requires protein-tyrosine kinase activity and is mediated by clathrin-coated pits. In contrast, the down-modulation of engaged TCRs is independent of protein-tyrosine kinases and clathrin pits, suggesting that this process is mediated by an alternate mechanism. Indeed, down-modulation of engaged TCRs appears to depend upon lipid rafts, because cholesterol depletion with methyl-beta-cyclodextrin completely blocks this process. Thus, two independent pathways of internalization are involved in TCR down-modulation and act differentially on directly engaged and bystander receptors. Finally, we propose that although both mechanisms coexist, the predominance of one or the other mechanisms will depend on the dose of ligand.
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Affiliation(s)
- Alicia Monjas
- Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Cantoblanco, Madrid 28049, Spain
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Gómez-Sebastián S, Tabarés E. Negative regulation of herpes simplex virus type 1 ICP4 promoter by IE180 protein of pseudorabies virus. J Gen Virol 2004; 85:2125-2130. [PMID: 15269350 DOI: 10.1099/vir.0.80119-0] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Recombinant pseudorabies viruses (PRVs) gIS8 and N1aHTK were constructed by the insertion of a chimeric gene (alpha4-TK) from herpes simplex virus type 1 (HSV-1) into wild-type PRV. HSV-1 TK expression by these recombinant viruses resulted in enhanced sensitivity to ganciclovir, compared to that of the wild-type PRV, and was similar to the sensitivity shown by HSV-1. Infection with gIS8 or N1aHTK recombinant viruses led to expression of HSV-1 TK mRNA as an immediate-early (IE) gene, observed by downregulation of the HSV-1 alpha4 promoter. This negative regulation was due to a PRV IE protein, IE180. IE180, however, does not have all the regulatory functions of the infected-cell protein ICP4, as it does not restore the growth of ICP4-deficient HSV-1 mutants.
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Affiliation(s)
- S Gómez-Sebastián
- Departamento de Medicina Preventiva, Salud Pública y Microbiología, Facultad de Medicina, Universidad Autónoma de Madrid, Arzobispo Morcillo 4, E-28029 Madrid, Spain
| | - E Tabarés
- Departamento de Medicina Preventiva, Salud Pública y Microbiología, Facultad de Medicina, Universidad Autónoma de Madrid, Arzobispo Morcillo 4, E-28029 Madrid, Spain
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de la Fuente R, Abad JL, García-Castro J, Fernández-Miguel G, Petriz J, Rubio D, Vicario-Abejón C, Guillén P, González MA, Bernad A. Dedifferentiated adult articular chondrocytes: a population of human multipotent primitive cells. Exp Cell Res 2004; 297:313-28. [PMID: 15212937 DOI: 10.1016/j.yexcr.2004.02.026] [Citation(s) in RCA: 68] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2003] [Revised: 02/10/2004] [Indexed: 01/13/2023]
Abstract
OBJECTIVE To test the hypothesis that dedifferentiated adult human cartilage chondrocytes (HAC) are a true multipotent primitive population. METHODS Studies to characterize dedifferentiated HAC included cell cycle and quiescence analysis, cell fusion, flow-FISH telomere length assays, and ABC transporter analysis. Dedifferentiated HAC were characterized by flow cytometry, in parallel with bone marrow mesenchymal stem cells (MSC) and processed lipoaspirate (PLA) cells. The in vitro differentiation potential of dedifferentiated HAC was studied by cell culture under several inducing conditions, in multiclonal and clonal cell populations. RESULTS Long-term HAC cultures were chromosomically stable and maintained cell cycle dynamics while showing telomere shortening. The phenotype of dedifferentiated HAC was quite similar to that of human bone marrow MSC. In addition, this population expressed human embryonic stem cell markers. Multiclonal populations of dedifferentiated HAC differentiated to chondrogenic, osteogenic, adipogenic, myogenic, and neurogenic lineages. Following VEGF induction, dedifferentiated HAC expressed characteristics of endothelial cells, including AcLDL uptake. A total of 53 clonal populations of dedifferentiated HAC were efficiently expanded; 17 were able to differentiate to chondrogenic, osteogenic, and adipogenic lineages. No correlation was observed between telomere length or quiescent population and differentiation potential in the clones assayed. CONCLUSION Dedifferentiated HAC should be considered a human multipotent primitive population.
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Affiliation(s)
- Ricardo de la Fuente
- Department of Immunology and Oncology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Madrid, Spain
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Nieto M, Samper E, Fraga MF, González de Buitrago G, Esteller M, Serrano M. The absence of p53 is critical for the induction of apoptosis by 5-aza-2'-deoxycytidine. Oncogene 2004; 23:735-43. [PMID: 14737108 DOI: 10.1038/sj.onc.1207175] [Citation(s) in RCA: 65] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
The absence of functional p53 has complex consequences on the cellular responses to cytotoxic drugs. Here, we have examined the role of p53 in the response to 5-aza-2'-deoxycytidine (5-aza-dC or decitabine). Primary mouse embryonic fibroblasts deficient for p53 undergo apoptosis after treatment with 5-aza-dC. When compared with other demethylating drugs or chemotherapeutic treatments, 5-aza-dC showed the highest selectivity ratio for triggering apoptosis in p53-deficient cells relative to wild-type cells. Moreover, the apoptotic efficacy of 5-aza-dC is proprietary of p53-deficient cells, not being observed in cells lacking other cell-cycle regulators, such as p19ARF, p16INK4a, p21(CIP1/WAF1), E2F-1, or E2F-2. Interestingly, treatment with 5-aza-dC results in the same degree of global genomic hypomethylation in wild-type and p53-null cells. However, wild-type cells activate p53 and arrest at G2/M, whereas p53-null cells accumulate severe chromosomal aberrations and undergo apoptosis. Significantly, the impact of p53-deficiency on the response to 5-aza-dC is not exclusive of primary non-neoplastic cells, but it is also present in neoplastically transformed cells. Finally, treatment of mice bearing genetically defined tumors with nontoxic doses of 5-aza-dC results in therapeutical responses only on tumors lacking p53, but not on tumors lacking p19ARF. Together, our results put forward the hypothesis that the absence of p53 may determine a higher chemotherapeutic index for 5-aza-dC.
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Affiliation(s)
- María Nieto
- Department of Immunology and Oncology, Spanish National Center of Biotechnology (CNB), Madrid, Spain
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Kusser KL, Randall TD. Simultaneous detection of EGFP and cell surface markers by fluorescence microscopy in lymphoid tissues. J Histochem Cytochem 2003; 51:5-14. [PMID: 12502749 DOI: 10.1177/002215540305100102] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022] Open
Abstract
Enhanced GFP (EGFP) is a powerful tool for the visualization of tagged proteins and transfected cells and is easily detected by fluorescence microscopy or flow cytometry in living cells. However, soluble EGFP molecules can be lost if cell integrity is disrupted by freezing, sectioning, or permeablization. Furthermore, the fluorescence of EGFP is dependent on its conformation. Therefore, fixation protocols that immobilize EGFP may also destroy its usefulness as a fluorescent reporter. Here we determined which methods of preparing murine lymphoid tissues immobilized soluble EGFP protein and retained its fluorescence while simultaneously maintaining the antigenicity of various immunologically important molecules and best preserving the overall morphology of the tissues. We found that EGFP could not be visualized in frozen sections of spleen that had not been fixed before freezing. However, robust EGFP fluorescence could be observed in frozen sections of tissues fixed under various conditions. Fixation was important to immobilize EGFP rather than to maintain conformation, because only minimal EGFP could be detected by immunofluorescence in unfixed frozen sections. Although it had little effect on EGFP fluorescence, the inclusion of sucrose during fixation better preserved the morphology of fixed tissues. These methods also preserved the antigenicity of a wide variety of molecules used to identify cell types in lymphoid tissues.
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Affiliation(s)
- Kim L Kusser
- Trudeau Institute, Saranac Lake, New York 12983, USA.
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Bermejo R, Vilaboa N, Calés C. Regulation of CDC6, geminin, and CDT1 in human cells that undergo polyploidization. Mol Biol Cell 2002; 13:3989-4000. [PMID: 12429841 PMCID: PMC133609 DOI: 10.1091/mbc.e02-04-0217] [Citation(s) in RCA: 44] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2002] [Revised: 07/23/2002] [Accepted: 08/08/2002] [Indexed: 01/10/2023] Open
Abstract
Endomitosis is the process by which mammalian megakaryocytes become polyploid during terminal differentiation. As in other endoreplicating cells, cyclin-cdk complexes are distinctly regulated, probably to overcome the strict mechanisms that prevent rereplication in most somatic cells. We have asked whether key factors involved in the assembly and licensing of replication origins are equally regulated during endomitosis. Cdc6, cdt1, and geminin expression was analyzed during differentiation of two human megakaryoblastic cell lines, HEL and K562, which respectively do and do not establish endoreplication cycles. Geminin was downregulated, whereas cdt1 levels were maintained upon differentiation of both cell lines, independently of whether cells entered extra S-phases. In contrast, cdc6 was present and remained nuclear only in differentiated endoreplicating cells. Interestingly, cdc6 protein expression was reestablished in K562 cells that underwent endomitosis after transient or stable cyclin E overexpression. The high levels of cyclin E reached in these cells appeared to influence the stabilization of cdc6 protein rather than its RNA transcription rate. Finally, cdc6 overexpression drove HEL cells into endoreplication cycles in the absence of differentiation stimuli. Our results show that both cdt1 and cdc6 are differentially regulated during megakaryocytic differentiation and suggest an active role of cdc6 in endomitosis.
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Affiliation(s)
- Rodrigo Bermejo
- Department of Biochemistry, Instituto de Investigaciones Biomédicas Alberto Sols, Universidad Autónoma de Madrid, CSIC, Arturo Duperier, 4.28029 Madrid, Spain
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