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Nakamura A, Matsumoto M. Role of polyamines in intestinal mucosal barrier function. Semin Immunopathol 2025; 47:9. [PMID: 39836273 PMCID: PMC11750915 DOI: 10.1007/s00281-024-01035-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2024] [Accepted: 12/27/2024] [Indexed: 01/22/2025]
Abstract
The intestinal epithelium is a rapidly self-renewing tissue; the rapid turnover prevents the invasion of pathogens and harmful components from the intestinal lumen, preventing inflammation and infectious diseases. Intestinal epithelial barrier function depends on the epithelial cell proliferation and junctions, as well as the state of the immune system in the lamina propria. Polyamines, particularly putrescine, spermidine, and spermine, are essential for many cell functions and play a crucial role in mammalian cellular homeostasis, such as that of cell growth, proliferation, differentiation, and maintenance, through multiple biological processes, including translation, transcription, and autophagy. Although the vital role of polyamines in normal intestinal epithelial cell growth and barrier function has been known since the 1980s, recent studies have provided new insights into this topic at the molecular level, such as eukaryotic initiation factor-5A hypusination and autophagy, with rapid advances in polyamine biology in normal cells using biological technologies. This review summarizes recent advances in our understanding of the role of polyamines in regulating normal, non-cancerous, intestinal epithelial barrier function, with a particular focus on intestinal epithelial renewal, cell junctions, and immune cell differentiation in the lamina propria.
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Affiliation(s)
- Atsuo Nakamura
- Dairy Science and Technology Institute, Kyodo Milk Industry Co. Ltd, 20-1 Hirai, Hinode-Machi, Nishitama-Gun, Tokyo, 190-0182, Japan
| | - Mitsuharu Matsumoto
- Dairy Science and Technology Institute, Kyodo Milk Industry Co. Ltd, 20-1 Hirai, Hinode-Machi, Nishitama-Gun, Tokyo, 190-0182, Japan.
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2
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Bhagwat AC, Saroj SD. Polyamine as a microenvironment factor in resistance to antibiotics. Crit Rev Microbiol 2024; 50:504-513. [PMID: 37339480 DOI: 10.1080/1040841x.2023.2223277] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2023] [Revised: 04/30/2023] [Accepted: 06/05/2023] [Indexed: 06/22/2023]
Abstract
One of the main issues in modern medicine is the decrease in the efficacy of antibiotic therapy against resistant microorganisms. The advent of antimicrobial resistance has added significantly to the impact of infectious diseases, in number of infections, as well as added healthcare costs. The development of antibiotic tolerance and resistance is influenced by a variety of environmental variables, and it is important to identify these environmental factors as part of any strategy for combating antibiotic resistance. The review aims to emphasize that biogenic polyamines are one of such environmental cues that impacts the antibiotic resistance in bacteria. The biogenic polyamines can help bacteria acquire resistance to antibiotics either by regulating the level of number of porin channels in the outer membrane, by modifying the outer membrane liposaccharides or by protecting macromolecule from antibiotic stress. Thus, understanding the way polyamines function in bacteria can thus be beneficial while designing the drugs to combat diseases.
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Affiliation(s)
- Amrita C Bhagwat
- Symbiosis School of Biological Sciences, Symbiosis International (Deemed University), Pune, Maharashtra, India
| | - Sunil D Saroj
- Symbiosis School of Biological Sciences, Symbiosis International (Deemed University), Pune, Maharashtra, India
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3
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Guo Z, Zhang X, Lin C, Huang Y, Zhong Y, Guo H, Zheng Z, Weng S. METTL3-IGF2BP3-axis mediates the proliferation and migration of pancreatic cancer by regulating spermine synthase m6A modification. Front Oncol 2022; 12:962204. [PMID: 36276112 PMCID: PMC9582246 DOI: 10.3389/fonc.2022.962204] [Citation(s) in RCA: 15] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2022] [Accepted: 09/20/2022] [Indexed: 11/13/2022] Open
Abstract
Spermine synthase (SMS) is an enzyme participating in polyamine synthesis; however, its function and role in pancreatic cancer remains elusive. Here we report that SMS is upregulated in pancreatic cancer and predicts a worse overall survival and significantly promotes the proliferation and migration of pancreatic cancer cells. Excessive SMS reduces the accumulation of spermidine by converting spermidine into spermine, which activates the phosphorylation of serine/threonine kinase (AKT) and epithelial-mesenchymal transition (EMT) signaling pathway, thereby inhibiting pancreatic cancer cell proliferation and invasion. Moreover, SMS was identified as the direct target of both methyltransferase like 3 (METTL3) and insulin like growth factor 2 mRNA binding protein 3 (IGF2BP3), which directly bind to the m6A modification sites of SMS and inhibit mRNA degradation. Knockdown of METTL3 or IGF2BP3 significantly reduced the SMS protein expression and inhibited the migration of pancreatic cancer. We propose a novel regulatory mechanism in which the METTL3-IGF2BP3 axis mediates the mRNA degradation of SMS in an m6A-dependent manner to regulate spermine/spermidine conversion, which regulates AKT phosphorylation and EMT activation, thereby inducing tumor progression and migration in pancreatic cancer.
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Affiliation(s)
- Zhenyun Guo
- Department of Hepatobiliary Surgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China
- Fujian Abdominal Surgery Research Institute, The First Affiliated Hospital, Fujian Medical University, Fuzhou, China
| | - Xiang Zhang
- Department of Hepatobiliary Surgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China
- Fujian Abdominal Surgery Research Institute, The First Affiliated Hospital, Fujian Medical University, Fuzhou, China
| | - Chengjie Lin
- Department of Hepatobiliary Surgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China
- Fujian Abdominal Surgery Research Institute, The First Affiliated Hospital, Fujian Medical University, Fuzhou, China
| | - Yue Huang
- Department of Hepatobiliary Surgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China
- Fujian Abdominal Surgery Research Institute, The First Affiliated Hospital, Fujian Medical University, Fuzhou, China
| | - Yun Zhong
- Department of Hepatobiliary Surgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China
- Fujian Abdominal Surgery Research Institute, The First Affiliated Hospital, Fujian Medical University, Fuzhou, China
| | - Hailing Guo
- Department of Hepatobiliary Surgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China
- Fujian Abdominal Surgery Research Institute, The First Affiliated Hospital, Fujian Medical University, Fuzhou, China
| | - Zhou Zheng
- Department of Hepatobiliary Surgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China
- Fujian Abdominal Surgery Research Institute, The First Affiliated Hospital, Fujian Medical University, Fuzhou, China
| | - Shangeng Weng
- Department of Hepatobiliary Surgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China
- Fujian Abdominal Surgery Research Institute, The First Affiliated Hospital, Fujian Medical University, Fuzhou, China
- Key Laboratory of Gastrointestinal Cancer (Fujian Medical University), Ministry of Education, Fuzhou, China
- Fujian Key Laboratory of Tumor Microbiology, Department of Medical Microbiology, Fujian Medical University, Fuzhou, China
- *Correspondence: Shangeng Weng,
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Coradduzza D, Solinas T, Azara E, Culeddu N, Cruciani S, Zinellu A, Medici S, Maioli M, Madonia M, Carru C. Plasma Polyamine Biomarker Panels: Agmatine in Support of Prostate Cancer Diagnosis. Biomolecules 2022; 12:biom12040514. [PMID: 35454104 PMCID: PMC9024899 DOI: 10.3390/biom12040514] [Citation(s) in RCA: 12] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2022] [Revised: 03/10/2022] [Accepted: 03/26/2022] [Indexed: 01/03/2023] Open
Abstract
Prostate cancer is the most frequent malignant tumour among males (19%), often clinically silent and of difficult prognosis. Although several studies have highlighted the diagnostic and prognostic role of circulating biomarkers, such as PSA, their measurement does not necessarily allow the detection of the disease. Within this context, many authors suggest that the evaluation of circulating polyamines could represent a valuable tool, although several analytical problems still counteract their clinical practice. In particular, agmatine seems particularly intriguing, being a potential inhibitor of polyamines commonly derived from arginine. The aim of the present work was to evaluate the potential role of agmatine as a suitable biomarker for the identification of different classes of patients with prostate cancer (PC). For this reason, three groups of human patients—benign prostatic hyperplasia (BPH), precancerous lesion (PL), and prostate cancer (PC)—were recruited from a cohort of patients with suspected prostate cancer (n = 170), and obtained plasma was tested using the LC-HRMS method. Statistics on the receiver operating characteristics curve (ROC), and multivariate analysis were used to examine the predictive value of markers for discrimination among the three patient groups. Statistical analysis models revealed good discrimination using polyamine levels to distinguish the three classes of patients. AUC above 0.8, sensitivity ranging from 67% to 89%, specificity ranging from 74% to 89% and accuracy from 73% to 86%, considering the validation set, were achieved. Agmatine plasma levels were measured in PC (39.9 ± 12.06 ng/mL), BPH (77.62 ± 15.05 ng/mL), and PL (53.31 ± 15.27 ng/mL) patients. ROC analysis of the agmatine panel showed an AUC of 0.959 and p ≤ 0.001. These results could represent a future tool able to discriminate patients belonging to the three different clinical groups.
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Affiliation(s)
- Donatella Coradduzza
- Department of Biomedical Sciences, University of Sassari, 07100 Sassari, Italy; (D.C.); (S.C.); (A.Z.); (M.M.)
| | - Tatiana Solinas
- Department of Clinical and Experimental Medicine, Urologic Clinic, University of Sassari, 07100 Sassari, Italy; (T.S.); (M.M.)
| | - Emanuela Azara
- Institute of Biomolecular Chemistry, National Research Council, 07100 Sassari, Italy; (E.A.); (N.C.)
| | - Nicola Culeddu
- Institute of Biomolecular Chemistry, National Research Council, 07100 Sassari, Italy; (E.A.); (N.C.)
| | - Sara Cruciani
- Department of Biomedical Sciences, University of Sassari, 07100 Sassari, Italy; (D.C.); (S.C.); (A.Z.); (M.M.)
| | - Angelo Zinellu
- Department of Biomedical Sciences, University of Sassari, 07100 Sassari, Italy; (D.C.); (S.C.); (A.Z.); (M.M.)
| | - Serenella Medici
- Department of Chemistry and Pharmacy, University of Sassari, 07100 Sassari, Italy;
| | - Margherita Maioli
- Department of Biomedical Sciences, University of Sassari, 07100 Sassari, Italy; (D.C.); (S.C.); (A.Z.); (M.M.)
| | - Massimo Madonia
- Department of Clinical and Experimental Medicine, Urologic Clinic, University of Sassari, 07100 Sassari, Italy; (T.S.); (M.M.)
| | - Ciriaco Carru
- Department of Biomedical Sciences, University of Sassari, 07100 Sassari, Italy; (D.C.); (S.C.); (A.Z.); (M.M.)
- Department of Biomedical Sciences and University Hospital of Sassari (AOU), 07100 Sassari, Italy
- Correspondence:
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Hyperoxia Inhibits Proliferation of Retinal Endothelial Cells in a Myc-Dependent Manner. Life (Basel) 2021; 11:life11070614. [PMID: 34202240 PMCID: PMC8304924 DOI: 10.3390/life11070614] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2021] [Revised: 06/16/2021] [Accepted: 06/21/2021] [Indexed: 01/03/2023] Open
Abstract
Oxygen supplementation is necessary to prevent mortality in severely premature infants. However, the supraphysiological concentration of oxygen utilized in these infants simultaneously creates retinovascular growth attenuation and vasoobliteration that induces the retinopathy of prematurity. Here, we report that hyperoxia regulates the cell cycle and retinal endothelial cell proliferation in a previously unknown Myc-dependent manner, which contributes to oxygen-induced retinopathy.
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Wang J, Tan B, Li J, Kong X, Tan M, Wu G. Regulatory role of l-proline in fetal pig growth and intestinal epithelial cell proliferation. ACTA ACUST UNITED AC 2020; 6:438-446. [PMID: 33364460 PMCID: PMC7750805 DOI: 10.1016/j.aninu.2020.07.001] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2019] [Revised: 06/14/2020] [Accepted: 07/07/2020] [Indexed: 12/27/2022]
Abstract
l-proline (Pro) is a precursor of ornithine, which is converted into polyamines via ornithine decarboxylase (ODC). Polyamines plays a key role in the proliferation of intestinal epithelial cells. The study investigated the effect of Pro on polyamine metabolism and cell proliferation on porcine enterocytes in vivo and in vitro. Twenty-four Huanjiang mini-pigs were randomly assigned into 1 of 3 groups and fed a basal diet that contained 0.77% alanine (Ala, iso-nitrogenous control), 1% Pro or 1% Pro + 0.0167% α-difluoromethylornithine (DFMO) from d 15 to 70 of gestation. The fetal body weight and number of fetuses per litter were determined, and the small and large intestines were obtained on d 70 ± 1.78 of gestation. The in vitro study was performed in intestinal porcine epithelial (IPEC-J2) cells cultured in Dulbecco's modified Eagle medium-high glucose (DMEM-H) containing 0 μmol/L Pro, 400 μmol/L Pro, or 400 μmol/L Pro + 10 mmol/L DFMO for 4 d. The results showed that maternal dietary supplementation with 1% Pro increased fetal weight; the protein and DNA concentrations of the fetal small intestine; and mRNA levels for potassium voltage-gated channel, shaker-related subfamily, member 1 (Kv1.1) in the fetal small and large intestines (P < 0.05). Supplementing Pro to either gilts or IPEC-J2 cells increased ODC protein abundances and polyamine concentrations in the fetal intestines and IPEC-J2 cells (P < 0.05). In comparison with the Pro group, the combined administration of Pro and DFMO reduced the expression of ODC protein and spermine concentration in the fetal intestine, as well as the concentrations of putrescine, spermidine and spermine in IPEC-J2 cells (P < 0.05). Meanwhile, the percentage of cells in the S-phase and the mRNA levels of proto-oncogenes c-fos and c-myc were increased in response to Pro supplementation, whereas depletion of cellular polyamines with DFMO increased tumor protein p53 (p53) mRNA levels (P < 0.05). Taken together, dietary supplementation with Pro improved fetal pig growth and intestinal epithelial cell proliferation via enhancing polyamine synthesis.
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Affiliation(s)
- Jing Wang
- Hunan International Joint Laboratory of Animal Intestinal Ecology and Health, Laboratory of Animal Nutrition and Human Health, College of Life Sciences, Hunan Normal University, Changsha 410081, China.,Laboratory of Animal Nutritional Physiology and Metabolic Process, Key Laboratory of Agro-ecological Processes in Subtropical Region, National Engineering Laboratory for Pollution Control and Waste Utilization in Livestock and Poultry Production, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha 410125, China
| | - Bi'e Tan
- College of Animal Science and Technology, Hunan Agricultural University, Changsha 410128, China.,Laboratory of Animal Nutritional Physiology and Metabolic Process, Key Laboratory of Agro-ecological Processes in Subtropical Region, National Engineering Laboratory for Pollution Control and Waste Utilization in Livestock and Poultry Production, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha 410125, China
| | - Jianjun Li
- Laboratory of Animal Nutritional Physiology and Metabolic Process, Key Laboratory of Agro-ecological Processes in Subtropical Region, National Engineering Laboratory for Pollution Control and Waste Utilization in Livestock and Poultry Production, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha 410125, China
| | - Xiangfeng Kong
- Laboratory of Animal Nutritional Physiology and Metabolic Process, Key Laboratory of Agro-ecological Processes in Subtropical Region, National Engineering Laboratory for Pollution Control and Waste Utilization in Livestock and Poultry Production, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha 410125, China
| | - Minjie Tan
- Laboratory of Animal Nutritional Physiology and Metabolic Process, Key Laboratory of Agro-ecological Processes in Subtropical Region, National Engineering Laboratory for Pollution Control and Waste Utilization in Livestock and Poultry Production, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha 410125, China
| | - Guoyao Wu
- Department of Animal Science, Texas A&M University, College Station, TX 77843, USA
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Capella Roca B, Doolan P, Barron N, O'Neill F, Clynes M. Altered gene expression in CHO cells following polyamine starvation. Biotechnol Lett 2020; 42:927-936. [PMID: 32078082 DOI: 10.1007/s10529-020-02841-0] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2019] [Accepted: 02/15/2020] [Indexed: 11/27/2022]
Abstract
AIM To investigate the impact of polyamine deprivation on the transcriptome of CHO cells RESULTS: Polyamines play a central but poorly-understood role in cell proliferation. Most studies to date have utilised chemical inhibitors to probe polyamine function. Here we exploit the fact that CHO cells grown in serum-free medium have an absolute requirement for putrescine supplementation due to their deficiency in activity of the enzyme arginase. A gene expression microarray (Affymetrix) analysis of CHO-K1 cells starved of polyamines for 3 days showed that cessation of growth, associated with increased G1/S transition and inhibition of M/G1 transition was accompanied by increased mRNA levels of mitotic complex checkpoint genes (Mad2l1, Tkk, Bub1b) and in the transition of G1- to S-phase (such as Skp2 and Tfdp1). mRNAs associated with DNA homologous recombination and repair (including Fanconi's anaemia-related genes) and with RNA splicing were consistently increased. Alterations in mRNA levels for genes related to protein processing in the ER, to ER stress, and to p53-related and apoptosis pathways were also observed. mRNAs showing highest levels of fold-change included several which code for membrane-localised proteins and receptors (Thbs1, Tfrc1, Ackr3, Extl1). CONCLUSIONS Growth-arrest induced by polyamine deprivation was associated with significant alterations in levels of mRNAs associated with cell cycle progression, DNA repair, RNA splicing, ER trafficking and membrane signalling as well as p53 and apoptosis-related pathways.
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Affiliation(s)
- Berta Capella Roca
- National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland
- SSPC-SFI Centre for Pharmaceuticals, Dublin City University, Dublin 9, Ireland
| | - Padraig Doolan
- National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland
| | - Niall Barron
- National Institute for Bioprocessing Research and Training, University College Dublin, Dublin, Ireland
| | - Fiona O'Neill
- National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland
| | - Martin Clynes
- National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland.
- SSPC-SFI Centre for Pharmaceuticals, Dublin City University, Dublin 9, Ireland.
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Arruabarrena-Aristorena A, Zabala-Letona A, Carracedo A. Oil for the cancer engine: The cross-talk between oncogenic signaling and polyamine metabolism. SCIENCE ADVANCES 2018; 4:eaar2606. [PMID: 29376126 PMCID: PMC5783676 DOI: 10.1126/sciadv.aar2606] [Citation(s) in RCA: 82] [Impact Index Per Article: 11.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/19/2017] [Accepted: 12/28/2017] [Indexed: 05/09/2023]
Abstract
The study of metabolism has provided remarkable information about the biological basis and therapeutic weaknesses of cancer cells. Classic biochemistry established the importance of metabolic alterations in tumor biology and revealed the importance of various metabolite families to the tumorigenic process. We have evidence of the central role of polyamines, small polycatonic metabolites, in cell proliferation and cancer growth from these studies. However, how cancer cells activate this metabolic pathway and the molecular cues behind the oncogenic action of polyamines has remained largely obscure. In contrast to the view of metabolites as fuel (anabolic intermediates) for cancer cells, polyamines are better defined as the oil that lubricates the cancer engine because they affect the activity of biological processes. Modern research has brought back to the limelight this metabolic pathway, providing a strong link between genetic, metabolic, and signaling events in cancer. In this review, we enumerate and discuss current views of the regulation and activity of polyamine metabolism in tumor cell biology.
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Affiliation(s)
| | - Amaia Zabala-Letona
- CIC bioGUNE, Bizkaia Technology Park, 801A Building, 48160 Derio, Bizkaia, Spain
- CIBERONC Centro de Investigación Biomédica en Red de Cáncer, Avenida Monforte de Lemos, Madrid, Spain
| | - Arkaitz Carracedo
- CIC bioGUNE, Bizkaia Technology Park, 801A Building, 48160 Derio, Bizkaia, Spain
- CIBERONC Centro de Investigación Biomédica en Red de Cáncer, Avenida Monforte de Lemos, Madrid, Spain
- Ikerbasque, Basque Foundation for Science, 48011 Bilbao, Spain
- Biochemistry and Molecular Biology Department, University of the Basque Country (UPV/EHU), P.O. Box 644, E-48080 Bilbao, Spain
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Fecal Microbiota and Metabolome in a Mouse Model of Spontaneous Chronic Colitis: Relevance to Human Inflammatory Bowel Disease. Inflamm Bowel Dis 2016; 22:2767-2787. [PMID: 27824648 DOI: 10.1097/mib.0000000000000970] [Citation(s) in RCA: 42] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
BACKGROUND Dysbiosis of the gut microbiota may be involved in the pathogenesis of inflammatory bowel disease (IBD). However, the mechanisms underlying the role of the intestinal microbiome and metabolome in IBD onset and its alteration during active treatment and recovery remain unknown. Animal models of chronic intestinal inflammation with similar microbial and metabolomic profiles would enable investigation of these mechanisms and development of more effective treatments. Recently, the Winnie mouse model of colitis closely representing the clinical symptoms and characteristics of human IBD has been developed. In this study, we have analyzed fecal microbial and metabolomic profiles in Winnie mice and discussed their relevance to human IBD. METHODS The 16S rRNA gene was sequenced from fecal DNA of Winnie and C57BL/6 mice to define operational taxonomic units at ≥97% similarity threshold. Metabolomic profiling of the same fecal samples was performed by gas chromatography-mass spectrometry. RESULTS Composition of the dominant microbiota was disturbed, and prominent differences were evident at all levels of the intestinal microbiome in fecal samples from Winnie mice, similar to observations in patients with IBD. Metabolomic profiling revealed that chronic colitis in Winnie mice upregulated production of metabolites and altered several metabolic pathways, mostly affecting amino acid synthesis and breakdown of monosaccharides to short chain fatty acids. CONCLUSIONS Significant dysbiosis in the Winnie mouse gut replicates many changes observed in patients with IBD. These results provide justification for the suitability of this model to investigate mechanisms underlying the role of intestinal microbiota and metabolome in the pathophysiology of IBD.
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Zhou X, Merchak K, Lee W, Grande JP, Cascalho M, Platt JL. Cell Fusion Connects Oncogenesis with Tumor Evolution. THE AMERICAN JOURNAL OF PATHOLOGY 2015; 185:2049-60. [PMID: 26066710 DOI: 10.1016/j.ajpath.2015.03.014] [Citation(s) in RCA: 45] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/21/2014] [Revised: 02/08/2015] [Accepted: 03/02/2015] [Indexed: 12/30/2022]
Abstract
Cell fusion likely drives tumor evolution by undermining chromosomal and DNA stability and/or by generating phenotypic diversity; however, whether a cell fusion event can initiate malignancy and direct tumor evolution is unknown. We report that a fusion event involving normal, nontransformed, cytogenetically stable epithelial cells can initiate chromosomal instability, DNA damage, cell transformation, and malignancy. Clonal analysis of fused cells reveals that the karyotypic and phenotypic potential of tumors formed by cell fusion is established immediately or within a few cell divisions after the fusion event, without further ongoing genetic and phenotypic plasticity, and that subsequent evolution of such tumors reflects selection from the initial diverse population rather than ongoing plasticity of the progeny. Thus, one cell fusion event can both initiate malignancy and fuel evolution of the tumor that ensues.
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Affiliation(s)
- Xiaofeng Zhou
- Departments of Microbiology and Immunology and Surgery, University of Michigan, Ann Arbor, Michigan; Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan
| | - Kevin Merchak
- Departments of Microbiology and Immunology and Surgery, University of Michigan, Ann Arbor, Michigan
| | - Woojin Lee
- Departments of Microbiology and Immunology and Surgery, University of Michigan, Ann Arbor, Michigan
| | - Joseph P Grande
- Division of Anatomic Pathology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota
| | - Marilia Cascalho
- Departments of Microbiology and Immunology and Surgery, University of Michigan, Ann Arbor, Michigan
| | - Jeffrey L Platt
- Departments of Microbiology and Immunology and Surgery, University of Michigan, Ann Arbor, Michigan.
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11
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Landau G, Ran A, Bercovich Z, Feldmesser E, Horn-Saban S, Korkotian E, Jacob-Hirsh J, Rechavi G, Ron D, Kahana C. Expression profiling and biochemical analysis suggest stress response as a potential mechanism inhibiting proliferation of polyamine-depleted cells. J Biol Chem 2012; 287:35825-37. [PMID: 22942278 DOI: 10.1074/jbc.m112.381335] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Polyamines are small organic polycations that are absolutely required for cell growth and proliferation; yet the basis for this requirement is mostly unknown. Here, we combined a genome-wide expression profiling with biochemical analysis to reveal the molecular basis for inhibited proliferation of polyamine-depleted cells. Transcriptional responses accompanying growth arrest establishment in polyamine-depleted cells or growth resumption following polyamine replenishment were monitored and compared. Changes in the expression of genes related to various fundamental cellular processes were established. Analysis of mirror-symmetric expression patterns around the G(1)-arrest point identified a set of genes representing a stress-response signature. Indeed, complementary biochemical analysis demonstrated activation of the PKR-like endoplasmic reticulum kinase arm of the unfolded protein response and of the stress-induced p38 MAPK. These changes were accompanied by induction of key growth-inhibitory factors such as p21 and Gadd45a and reduced expression of various cyclins, most profoundly cyclin D1, setting the basis for the halted proliferation. However, although the induced stress response could arrest growth, polyamine depletion also inhibited proliferation of PKR-like endoplasmic reticulum kinase and p38α-deficient cells and of cells harboring a nonphosphorylatable mutant eIF2α (S51A), suggesting that additional yet unidentified mechanisms might inhibit proliferation of polyamine-depleted cells. Despite lengthy persistence of the stress and activation of apoptotic signaling, polyamine-depleted cells remained viable, apparently due to induced expression of protective genes and development of autophagy.
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Affiliation(s)
- Guy Landau
- Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel
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12
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Zou T, Rao JN, Liu L, Xiao L, Cui YH, Jiang Z, Ouyang M, Donahue JM, Wang JY. Polyamines inhibit the assembly of stress granules in normal intestinal epithelial cells regulating apoptosis. Am J Physiol Cell Physiol 2012; 303:C102-11. [PMID: 22555848 DOI: 10.1152/ajpcell.00009.2012] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Polyamines regulate multiple signaling pathways and are implicated in many aspects of cellular functions, but the exact molecular processes governed by polyamines remain largely unknown. In response to environmental stress, repression of translation is associated with the assembly of stress granules (SGs) that contain a fraction of arrested mRNAs and are thought to function as mRNA storage. Here we show that polyamines modulate the assembly of SGs in normal intestinal epithelial cells (IECs) and that induced SGs following polyamine depletion are implicated in the protection of IECs against apoptosis. Increasing the levels of cellular polyamines by ectopic overexpression of the ornithine decarboxylase gene decreased cytoplasmic levels of SG-signature constituent proteins eukaryotic initiation factor 3b and T-cell intracellular antigen-1 (TIA-1)-related protein and repressed the assembly of SGs induced by exposure to arsenite-induced oxidative stress. In contrast, depletion of cellular polyamines by inhibiting ornithine decarboxylase with α-difluoromethylornithine increased cytoplasmic eukaryotic initiation factor 3b and TIA-1 related protein abundance and enhanced arsenite-induced SG assembly. Polyamine-deficient cells also exhibited an increase in resistance to tumor necrosis factor-α/cycloheximide-induced apoptosis, which was prevented by inhibiting SG formation with silencing SG resident proteins Sort1 and TIA-1. These results indicate that the elevation of cellular polyamines represses the assembly of SGs in normal IECs and that increased SGs in polyamine-deficient cells are crucial for increased resistance to apoptosis.
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Affiliation(s)
- Tongtong Zou
- Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore, USA
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13
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Cui YH, Xiao L, Rao JN, Zou T, Liu L, Chen Y, Turner DJ, Gorospe M, Wang JY. miR-503 represses CUG-binding protein 1 translation by recruiting CUGBP1 mRNA to processing bodies. Mol Biol Cell 2011; 23:151-62. [PMID: 22072795 PMCID: PMC3248894 DOI: 10.1091/mbc.e11-05-0456] [Citation(s) in RCA: 61] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
This study shows that microRNA-503 interacts with the CUG-binding protein 1 (CUGBP1) mRNA and represses its translation by recruiting the CUGBP1 mRNA to processing bodies. microRNAs (miRNAs) and RNA-binding proteins (RBPs) jointly regulate gene expression at the posttranscriptional level and are involved in many aspects of cellular functions. The RBP CUG-binding protein 1 (CUGBP1) destabilizes and represses the translation of several target mRNAs, but the exact mechanism that regulates CUGBP1 abundance remains elusive. In this paper, we show that miR-503, computationally predicted to associate with three sites of the CUGBP1 mRNA, represses CUGBP1 expression. Overexpression of an miR-503 precursor (pre-miR-503) reduced the de novo synthesis of CUGBP1 protein, whereas inhibiting miR-503 by using an antisense RNA (antagomir) enhanced CUGBP1 biosynthesis and elevated its abundance; neither intervention changed total CUGBP1 mRNA levels. Studies using heterologous reporter constructs revealed a greater repressive effect of miR-503 through the CUGBP1 coding region sites than through the single CUGBP1 3′-untranslated region target site. CUGBP1 mRNA levels in processing bodies (P-bodies) increased in cells transfected with pre-miR-503, while silencing P-body resident proteins Ago2, RCK, or LSm4 decreased miR-503–mediated repression of CUGBP1 expression. Decreasing the levels of cellular polyamines reduced endogenous miR-503 levels and promoted CUGBP1 expression, an effect that was prevented by ectopic miR-503 overexpression. Repression of CUGBP1 by miR-503 in turn altered the expression of CUGBP1 target mRNAs and thus increased the sensitivity of intestinal epithelial cells to apoptosis. These findings identify miR-503 as both a novel regulator of CUGBP1 expression and a modulator of intestinal epithelial homoeostasis.
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Affiliation(s)
- Yu-Hong Cui
- Department of Surgery, University of Maryland School of Medicine, Baltimore, MD 21201, USA
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14
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Carvajal-Gamez BI, Arroyo R, Camacho-Nuez M, Lira R, Martínez-Benitez M, Alvarez-Sánchez ME. Putrescine is required for the expression of eif-5a in Trichomonas vaginalis. Mol Biochem Parasitol 2011; 180:8-16. [PMID: 21801756 DOI: 10.1016/j.molbiopara.2011.07.003] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2010] [Revised: 07/04/2011] [Accepted: 07/09/2011] [Indexed: 11/21/2022]
Abstract
Recently, we found that Trichomonas vaginalis contains a eukaryotic translation initiation factor 5A (TveIF-5A) with unknown function in this parasite. eIF-5A is the only cellular protein dependent of polyamines to form a hypusine residue, an unusual basic amino acid that is post-translationally formed by modification of a single specific lysine residue in an eIF-5A precursor protein. The purpose of this study was to determine the effect of a putrescine analogue, 1,4-diamino-2-butanone (DAB), on tveif-5a mRNA and TveIF-5A protein expression. TveIF-5A protein expression was reduced by inhibition of putrescine biosynthesis, and tveif-5a mRNA levels were reduced ∼90%, as shown by western blot and immunofluorescence assays. Cycloheximide treatment reduced the amount of mature TveIF-5A protein at 4h and decreased the tveif-5a transcript level at 2h, according to western blot, RT-PCR and qRT-PCR analyses. Actinomycin D treatment showed that the tveif-5a mRNA had half-life of ∼2.5h in DAB-treated parasites. The half-life of tveif-5a mRNA was ∼4.5h under exogenous putrescine conditions. These results suggest that putrescine is required for tveif-5a mRNA stability, and it is necessary for the expression, stability and maturation of TveIF-5A protein.
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15
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Affiliation(s)
- Rao N. Jaladanki
- University of Maryland School of Medicine and Baltimore Veterans Affairs Medical Center
| | - Jian-Ying Wang
- University of Maryland School of Medicine and Baltimore Veterans Affairs Medical Center
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16
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Aksenova GE, Logvinovich OS, Fialkovskaya LA, Afanasyev VN, Ignat'ev DA, Kolomiytseva IK, Fesenko E E. The effect of hypothermia on the ornithine decarboxylase activity in tissues of rats. DOKL BIOCHEM BIOPHYS 2010; 428:249-51. [PMID: 20848911 DOI: 10.1134/s160767290905007x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Affiliation(s)
- G E Aksenova
- Institute of Cell Biophysics, Russian Academy of Sciences, ul. Institutskaya 3, Pushchino, Moscow oblast 142292, Russia
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17
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Polyamines regulate the stability of JunD mRNA by modulating the competitive binding of its 3' untranslated region to HuR and AUF1. Mol Cell Biol 2010; 30:5021-32. [PMID: 20805360 DOI: 10.1128/mcb.00807-10] [Citation(s) in RCA: 51] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
Polyamines critically regulate all mammalian cell growth and proliferation by mechanisms such as the repression of growth-inhibitory proteins, including JunD. Decreasing the levels of cellular polyamines stabilizes JunD mRNA without affecting its transcription, but the exact mechanism whereby polyamines regulate JunD mRNA degradation has not been elucidated. RNA-binding proteins HuR and AUF1 associate with labile mRNAs bearing AU-rich elements located in the 3' untranslated regions (3'-UTRs) and modulate their stability. Here, we show that JunD mRNA is a target of HuR and AUF1 and that polyamines modulate JunD mRNA degradation by altering the competitive binding of HuR and AUF1 to the JunD 3'-UTR. The depletion of cellular polyamines enhanced HuR binding to JunD mRNA and decreased the levels of JunD transcript associated with AUF1, thus stabilizing JunD mRNA. The silencing of HuR increased AUF1 binding to the JunD mRNA, decreased the abundance of HuR-JunD mRNA complexes, rendered the JunD mRNA unstable, and prevented increases in JunD mRNA and protein in polyamine-deficient cells. Conversely, increasing the cellular polyamines repressed JunD mRNA interaction with HuR and enhanced its association with AUF1, resulting in an inhibition of JunD expression. These results indicate that polyamines modulate the stability of JunD mRNA in intestinal epithelial cells through HuR and AUF1 and provide new insight into the molecular functions of cellular polyamines.
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18
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Liu L, Rao JN, Zou T, Xiao L, Wang PY, Turner DJ, Gorospe M, Wang JY. Polyamines regulate c-Myc translation through Chk2-dependent HuR phosphorylation. Mol Biol Cell 2009; 20:4885-98. [PMID: 19812253 DOI: 10.1091/mbc.e09-07-0550] [Citation(s) in RCA: 82] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022] Open
Abstract
All mammalian cells depend on polyamines for normal growth and proliferation, but the exact roles of polyamines at the molecular level remain largely unknown. The RNA-binding protein HuR modulates the stability and translation of many target mRNAs. Here, we show that in rat intestinal epithelial cells (IECs), polyamines enhanced HuR association with the 3'-untranslated region of the c-Myc mRNA by increasing HuR phosphorylation by Chk2, in turn promoting c-Myc translation. Depletion of cellular polyamines inhibited Chk2 and reduced the affinity of HuR for c-Myc mRNA; these effects were completely reversed by addition of the polyamine putrescine or by Chk2 overexpression. In cells with high content of cellular polyamines, HuR silencing or Chk2 silencing reduced c-Myc translation and c-Myc expression levels. Our findings demonstrate that polyamines regulate c-Myc translation in IECs through HuR phosphorylation by Chk2 and provide new insight into the molecular functions of cellular polyamines.
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Affiliation(s)
- Lan Liu
- Cell Biology Group, Department of Surgery, and Department of Pathology, University of Maryland School of Medicine and Baltimore Veterans Affairs Medical Center, Baltimore, MD 21201, USA
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19
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Wei G, DeFeo K, Hayes CS, Woster PM, Mandik-Nayak L, Gilmour SK. Elevated ornithine decarboxylase levels activate ataxia telangiectasia mutated-DNA damage signaling in normal keratinocytes. Cancer Res 2008; 68:2214-22. [PMID: 18381427 DOI: 10.1158/0008-5472.can-07-5030] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
We examined the effect of increased expression of ornithine decarboxylase (ODC), a key rate-limiting enzyme in polyamine biosynthesis, on cell survival in primary cultures of keratinocytes isolated from the skin of K6/ODC transgenic mice (Ker/ODC) and their normal littermates (Ker/Norm). Although elevated levels of ODC and polyamines stimulate proliferation of keratinocytes, Ker/ODC undergo apoptotic cell death within days of primary culture unlike Ker/Norm that continue to proliferate. Phosphorylation of ataxia telangiectasia mutated (ATM) and its substrate p53 are significantly induced both in Ker/ODC and in K6/ODC transgenic skin. Chromatin immunoprecipitation analyses show that the increased level of p53 in Ker/ODC is accompanied by increased recruitment of p53 to the Bax proximal promoter. ATM activation is polyamine dependent because alpha-difluoromethylornithine, a specific inhibitor of ODC activity, blocks its phosphorylation. Ker/ODC also displays increased generation of H(2)O(2), acrolein-lysine conjugates, and protein oxidation products as well as polyamine-dependent DNA damage, as measured by the comet assay and the expression of the phosphorylated form of the histone variant gamma H2AX. Both reactive oxygen species generation and apoptotic cell death of Ker/ODC may, at least in part, be due to induction of a polyamine catabolic pathway that generates both H(2)O(2) and cytotoxic aldehydes, because spermine oxidase (SMO) levels are induced in Ker/ODC. In addition, treatment with MDL 72,527, an inhibitor of SMO, blocks the production of H(2)O(2) and increases the survival of Ker/ODC. These results show a novel activation of the ATM-DNA damage signaling pathway in response to increased ODC activity in nontumorigenic keratinocytes.
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Affiliation(s)
- Gang Wei
- Lankenau Institute for Medical Research, Wynnewood, PA 19096, USA
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20
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Lescoat G, Chantrel-Groussard K, Pasdeloup N, Nick H, Brissot P, Gaboriau F. Antiproliferative and apoptotic effects in rat and human hepatoma cell cultures of the orally active iron chelator ICL670 compared to CP20: a possible relationship with polyamine metabolism. Cell Prolif 2007; 40:755-67. [PMID: 17877614 PMCID: PMC6495977 DOI: 10.1111/j.1365-2184.2007.00468.x] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023] Open
Abstract
OBJECTIVE Iron loading has been observed to have a hyperproliferative effect on hepatocytes in vitro and on tumour cells in vivo; removal of this iron being required to induce antitumour activity. MATERIAL AND METHODS Antiproliferative effects of orally active tridentate iron chelator ICL670 (deferasirox) and bidentate iron chelator CP20 (deferiprone), mediated through the chelation of intracellular iron, were compared in rat hepatoma cell line FAO and human hepatoma cell line HUH7. RESULTS In FAO cell cultures, we have shown that ICL670 decreased cell viability and DNA replication and induced apoptosis more efficiently than an iron-binding equivalent concentration of CP20. Moreover, ICL670 decreased significantly the number of the cells in G(2)-M phase. In the HUH7 cell cultures, ICL670 and a four-time higher iron-binding equivalent concentration of CP20, decreased cell viability and DNA replication in the same range. CP20 increased the number of the cells in G(2)-M phase. However, ICL670 inhibited polyamine biosynthesis by decreasing ornithine decarboxylase mRNA level; in contrast, CP20 increased polyamine biosynthesis, particularly putrescine level, by stimulating spermidine-spermine N(1)-acetyl transferase activity that could activate the polyamine retro-conversion pathway. By mass spectrometry, we observed that ICL670 cellular uptake was six times higher than CP20. CONCLUSIONS These results suggest that ICL670 has a powerful antitumoural effect and blocks cell proliferation in neoplastic cells by a pathway different from that of CP20 and may constitute a potential adjuvant drug for anticancer therapy.
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MESH Headings
- Animals
- Apoptosis/drug effects
- Base Sequence
- Benzoates/pharmacokinetics
- Benzoates/pharmacology
- Carcinoma, Hepatocellular/drug therapy
- Carcinoma, Hepatocellular/genetics
- Carcinoma, Hepatocellular/metabolism
- Carcinoma, Hepatocellular/pathology
- Cell Cycle/drug effects
- Cell Line, Tumor
- Cell Proliferation/drug effects
- Cell Survival/drug effects
- Chemotherapy, Adjuvant
- DNA Primers/genetics
- DNA Replication/drug effects
- Deferasirox
- Deferiprone
- Humans
- Iron Chelating Agents/pharmacokinetics
- Iron Chelating Agents/pharmacology
- Liver Neoplasms/drug therapy
- Liver Neoplasms/genetics
- Liver Neoplasms/metabolism
- Liver Neoplasms/pathology
- Liver Neoplasms, Experimental/drug therapy
- Liver Neoplasms, Experimental/genetics
- Liver Neoplasms, Experimental/metabolism
- Liver Neoplasms, Experimental/pathology
- Polyamines/metabolism
- Pyridones/pharmacokinetics
- Pyridones/pharmacology
- Rats
- Triazoles/pharmacokinetics
- Triazoles/pharmacology
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Affiliation(s)
- G Lescoat
- Inserm, U522, Rennes, F-35000, Rennes cedex, France.
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21
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Xiao L, Rao JN, Zou T, Liu L, Marasa BS, Chen J, Turner DJ, Zhou H, Gorospe M, Wang JY. Polyamines regulate the stability of activating transcription factor-2 mRNA through RNA-binding protein HuR in intestinal epithelial cells. Mol Biol Cell 2007; 18:4579-90. [PMID: 17804813 PMCID: PMC2043536 DOI: 10.1091/mbc.e07-07-0675] [Citation(s) in RCA: 63] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Maintenance of intestinal mucosal epithelial integrity requires polyamines that modulate the expression of various genes involved in cell proliferation and apoptosis. Recently, polyamines were shown to regulate the subcellular localization of the RNA-binding protein HuR, which stabilizes its target transcripts such as nucleophosmin and p53 mRNAs. The activating transcription factor-2 (ATF-2) mRNA encodes a member of the ATF/CRE-binding protein family of transcription factors and was computationally predicted to be a target of HuR. Here, we show that polyamines negatively regulate ATF-2 expression posttranscriptionally and that polyamine depletion stabilizes ATF-2 mRNA by enhancing the interaction of the 3'-untranslated region (UTR) of ATF-2 with cytoplasmic HuR. Decreasing cellular polyamines by inhibiting ornithine decarboxylase (ODC) with alpha-difluoromethylornithine increased the levels of ATF-2 mRNA and protein, whereas increasing polyamines by ectopic ODC overexpression repressed ATF-2 expression. Polyamine depletion did not alter transcription via the ATF-2 gene promoter but increased the stability of ATF-2 mRNA. Increased cytoplasmic HuR in polyamine-deficient cells formed ribonucleoprotein complexes with the endogenous ATF-2 mRNA and specifically bound to 3'-UTR of ATF-2 mRNA on multiple nonoverlapping 3'-UTR segments. Adenovirus-mediated HuR overexpression elevated ATF-2 mRNA and protein levels, whereas HuR silencing rendered the ATF-2 mRNA unstable and prevented increases in ATF-2 mRNA and protein. Furthermore, inhibition of ATF-2 expression prevented the increased resistance of polyamine-deficient cells to apoptosis induced by treatment with tumor necrosis factor-alpha and cycloheximide. These results indicate that polyamines modulate the stability of ATF-2 mRNA by altering cytoplasmic HuR levels and that polyamine-modulated ATF-2 expression plays a critical role in regulating epithelial apoptosis.
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Affiliation(s)
- Lan Xiao
- *Cell Biology Group, Department of Surgery, and
- Baltimore Veterans Affairs Medical Center, Baltimore, MD 21201
| | - Jaladanki N. Rao
- *Cell Biology Group, Department of Surgery, and
- Baltimore Veterans Affairs Medical Center, Baltimore, MD 21201
| | - Tongtong Zou
- *Cell Biology Group, Department of Surgery, and
- Baltimore Veterans Affairs Medical Center, Baltimore, MD 21201
| | - Lan Liu
- *Cell Biology Group, Department of Surgery, and
- Baltimore Veterans Affairs Medical Center, Baltimore, MD 21201
| | - Bernard S. Marasa
- *Cell Biology Group, Department of Surgery, and
- Department of Pathology, University of Maryland School of Medicine, Baltimore, MD 21201
| | - Jie Chen
- *Cell Biology Group, Department of Surgery, and
- Baltimore Veterans Affairs Medical Center, Baltimore, MD 21201
| | - Douglas J. Turner
- *Cell Biology Group, Department of Surgery, and
- Baltimore Veterans Affairs Medical Center, Baltimore, MD 21201
| | - Huiping Zhou
- Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond, VA 23298; and
| | - Myriam Gorospe
- Laboratory of Cellular and Molecular Biology, National Institute on Aging-Intramural Research Program, National Institutes of Health, Baltimore, MD 21224
| | - Jian-Ying Wang
- *Cell Biology Group, Department of Surgery, and
- Department of Pathology, University of Maryland School of Medicine, Baltimore, MD 21201
- Baltimore Veterans Affairs Medical Center, Baltimore, MD 21201
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22
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Chen J, Rao JN, Zou T, Liu L, Marasa BS, Xiao L, Zeng X, Turner DJ, Wang JY. Polyamines are required for expression of Toll-like receptor 2 modulating intestinal epithelial barrier integrity. Am J Physiol Gastrointest Liver Physiol 2007; 293:G568-76. [PMID: 17600044 DOI: 10.1152/ajpgi.00201.2007] [Citation(s) in RCA: 77] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
The Toll-like receptors (TLRs) allow mammalian intestinal epithelium to detect various microbes and activate innate immunity after infection. TLR2 and TLR4 have been identified in intestinal epithelial cells (IECs) as fundamental components of the innate immune response to bacterial pathogens, but the exact mechanism involved in control of TLR expression remains unclear. Polyamines are implicated in a wide variety of biological functions, and regulation of cellular polyamines is a central convergence point for the multiple signaling pathways driving different epithelial cell functions. The current study determined whether polyamines regulate TLR expression, thereby modulating intestinal epithelial barrier function. Depletion of cellular polyamines by inhibiting ornithine decarboxylase (ODC) with alpha-difluoromethylornithine decreased levels of TLR2 mRNA and protein, whereas increased polyamines by ectopic overexpression of the ODC gene enhanced TLR2 expression. Neither intervention changed basal levels of TLR4. Exposure of normal IECs to low-dose (5 microg/ml) LPS increased ODC enzyme activity and stimulated expression of TLR2 but not TLR4, while polyamine depletion prevented this LPS-induced TLR2 expression. Decreased TLR2 in polyamine-deficient cells was associated with epithelial barrier dysfunction. In contrast, increased TLR2 by the low dose of LPS enhanced epithelial barrier function, which was abolished by inhibition of TLR2 expression with specific, small interfering RNA. These results indicate that polyamines are necessary for TLR2 expression and that polyamine-induced TLR2 activation plays an important role in regulating epithelial barrier function.
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Affiliation(s)
- Jie Chen
- Department of Surgery, Baltimore Veterans Affairs Medical Center, 10 North Greene Street, Baltimore, MD 21201, USA.
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23
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Zhang AH, Rao JN, Zou T, Liu L, Marasa BS, Xiao L, Chen J, Turner DJ, Wang JY. p53-Dependent NDRG1 expression induces inhibition of intestinal epithelial cell proliferation but not apoptosis after polyamine depletion. Am J Physiol Cell Physiol 2007; 293:C379-89. [PMID: 17442733 DOI: 10.1152/ajpcell.00547.2006] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023]
Abstract
Normal intestinal mucosal growth requires polyamines that regulate expression of various genes involved in cell proliferation, growth arrest, and apoptosis. Our previous studies have shown that polyamine depletion stabilizes p53, resulting in inhibition of intestinal epithelial cell (IEC) proliferation, but the exact downstream targets of induced p53 are still unclear. The NDRG1 (N- myc downregulated gene-1) gene encodes a growth-related protein, and its transcription can be induced in response to stress. The current study tests the hypothesis that induced p53 inhibits IEC proliferation by upregulating NDRG1 expression following polyamine depletion. Depletion of cellular polyamines by inhibiting ornithine decarboxylase (ODC) with α-difluoromethylornithine not only induced p53 but also increased NDRG1 transcription as indicated by induction of the NDRG1 promoter activity and increased levels of NDRG1 mRNA and protein, all of which were prevented by using specific p53 siRNA and in cells with a targeted deletion of p53. In contrast, increased levels of cellular polyamines by ectopic expression of the ODC gene decreased p53 and repressed expression of NDRG1. Consistently, polyamine depletion-induced activation of the NDRG1-promoter was decreased when p53-binding sites within the NDRG1 proximal promoter region were deleted. Ectopic expression of the wild-type NDRG1 gene inhibited DNA synthesis and decreased final cell numbers regardless of the presence or absence of endogenous p53, whereas silencing NDRG1 promoted cell growth. However, overexpression of NDRG1 failed to directly induce cell death and to alter susceptibility to apoptosis induced by tumor necrosis factor-α/cycloheximide. These results indicate that NDRG1 is one of the direct mediators of induced p53 following polyamine depletion and that p53-dependent NDRG1 expression plays a critical role in the negative control of IEC proliferation.
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Affiliation(s)
- Ai-Hong Zhang
- Dept. of Surgery, University of Maryland School of Medicine, Baltimore, MD 21201, USA
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24
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Turner DJ, Alaish SM, Zou T, Rao JN, Wang JY, Strauch ED. Bile salts induce resistance to apoptosis through NF-kappaB-mediated XIAP expression. Ann Surg 2007; 245:415-25. [PMID: 17435549 PMCID: PMC1877019 DOI: 10.1097/01.sla.0000236631.72698.99] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
Apoptosis plays a critical role in intestinal mucosal homeostasis. We previously showed that the bile salt taurodeoxycholate has a beneficial effect on the intestinal mucosa through an increase in resistance to apoptosis mediated by nuclear factor (NF)-kappaB. The current study further characterizes the effect of bile salts on intestinal epithelial cell susceptibility to apoptosis and determines if the X-linked inhibitor of apoptosis protein (XIAP) regulates bile salt-induced resistance to apoptosis. Exposure of normal intestinal epithelial cells (IEC-6) to the conjugated bile salts taurodeoxycholate (TDCA) and taurochenodeoxycholate (TCDCA) resulted in an increase in resistance to tumor necrosis factor (TNF)-alpha and cycloheximide (CHX)-induced apoptosis, and NF-kappaB activation. Treatment with TDCA and TCDCA resulted in an increase in XIAP expression. Specific inhibition of NF-kappaB by infection with an adenoviral vector that expresses the IkappaBalpha super-repressor (IkappaBSR) prevented the induction of XIAP expression and the bile salt-mediated resistance to apoptosis. Treatment with the specific XIAP inhibitor Smac also overcame this increase in enterocyte resistance to apoptosis. Bile salts inhibited formation of the active caspase-3 from its precursor procaspase-3. Smac prevented the inhibitory effect of bile salts on caspase-3 activation. These results indicate that bile salts increase intestinal epithelial cell resistance to apoptosis through NF-kappaB-mediated XIAP expression. Bile salt-induced XIAP mediates resistance to TNF-alpha/CHX-induced apoptosis, at least partially, through inhibition of caspase-3 activity. These data support an important beneficial role of bile salts in regulation of mucosal integrity. Decreased enterocyte exposure to luminal bile salts, as occurs during starvation and parenteral nutrition, may have a detrimental effect on mucosal integrity.
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Affiliation(s)
- Douglas J Turner
- Departments of Surgery, University of Maryland School of Medicine, Baltimore, MD, USA.
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25
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Xiao L, Rao J, Zou T, Liu L, Marasa B, Chen J, Turner D, Passaniti A, Wang JY. Induced JunD in intestinal epithelial cells represses CDK4 transcription through its proximal promoter region following polyamine depletion. Biochem J 2007; 403:573-81. [PMID: 17253961 PMCID: PMC1876376 DOI: 10.1042/bj20061436] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2006] [Revised: 01/17/2007] [Accepted: 01/25/2007] [Indexed: 02/06/2023]
Abstract
Maintenance of intestinal epithelial integrity requires cellular polyamines that regulate expression of various genes involved in cell proliferation, growth arrest and apoptosis. In prior studies, depletion of cellular polyamines has been shown to stabilize JunD, a member of the AP-1 (activator protein-1) family of transcription factors, leading to inhibition of intestinal epithelial cell proliferation, but the exact downstream targets of induced JunD remain elusive. CDK4 (cyclin-dependent kinase 4) is essential for the G1- to S-phase transition during the cell cycle and its expression is primarily controlled at the transcriptional level. In the present study, we show that induced JunD in IECs (intestinal epithelial cells) is a transcriptional repressor of the CDK4 gene following polyamine depletion. Increased JunD in polyamine-deficient cells was associated with a significant inhibition of CDK4 transcription, as indicated by repression of CDK4-promoter activity and decreased levels of CDK4 mRNA and protein, all of which were prevented by using specific antisense JunD oligomers. Ectopic expression of the wild-type junD also repressed CDK4-promoter activity and decreased levels of CDK4 mRNA and protein without any effect on CDK2 expression. Gel shift and chromatin immunoprecipitation assays revealed that JunD bound to the proximal region of the CDK4-promoter in vitro as well as in vivo, while experiments using different CDK4-promoter mutants showed that transcriptional repression of CDK4 by JunD was mediated through an AP-1 binding site within this proximal sequence of the CDK4-promoter. These results indicate that induced JunD in IECs represses CDK4 transcription through its proximal promoter region following polyamine depletion.
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Key Words
- activator protein-1 (ap-1)
- α-difluoromethylornithine
- growth arrest
- intestinal epithelium
- ornithine decarboxylase
- transcriptional regulation
- ap-1, activator protein-1
- cdk, cyclin-dependent kinase
- chip, chromatin immunoprecipitation
- dfmo, α-difluoromethylornithine
- emsa, electrophoretic mobility-shift assay
- fbs, fetal bovine serum
- gapdh, glyceraldehyde-3-phosphate dehydrogenase
- iec, intestinal epithelial cell
- luc, luciferase
- pbs-t, pbs containing tween 20
- q-pcr, quantitative pcr
- rb, retinoblastoma tumour suppressor protein
- rt, reverse transcriptase
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Affiliation(s)
- Lan Xiao
- *Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore, MD 21201, U.S.A
- ‡Baltimore Veterans Affairs Medical Center, Baltimore, MD 21201, U.S.A
| | - Jaladanki N. Rao
- *Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore, MD 21201, U.S.A
- ‡Baltimore Veterans Affairs Medical Center, Baltimore, MD 21201, U.S.A
| | - Tongtong Zou
- *Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore, MD 21201, U.S.A
- ‡Baltimore Veterans Affairs Medical Center, Baltimore, MD 21201, U.S.A
| | - Lan Liu
- *Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore, MD 21201, U.S.A
- ‡Baltimore Veterans Affairs Medical Center, Baltimore, MD 21201, U.S.A
| | - Bernard S. Marasa
- *Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore, MD 21201, U.S.A
- †Department of Pathology, University of Maryland School of Medicine, Baltimore, MD 21201, U.S.A
- ‡Baltimore Veterans Affairs Medical Center, Baltimore, MD 21201, U.S.A
| | - Jie Chen
- *Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore, MD 21201, U.S.A
- ‡Baltimore Veterans Affairs Medical Center, Baltimore, MD 21201, U.S.A
| | - Douglas J. Turner
- *Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore, MD 21201, U.S.A
- ‡Baltimore Veterans Affairs Medical Center, Baltimore, MD 21201, U.S.A
| | - Antonino Passaniti
- †Department of Pathology, University of Maryland School of Medicine, Baltimore, MD 21201, U.S.A
- ‡Baltimore Veterans Affairs Medical Center, Baltimore, MD 21201, U.S.A
| | - Jian-Ying Wang
- *Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore, MD 21201, U.S.A
- †Department of Pathology, University of Maryland School of Medicine, Baltimore, MD 21201, U.S.A
- ‡Baltimore Veterans Affairs Medical Center, Baltimore, MD 21201, U.S.A
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26
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Shantz LM, Levin VA. Regulation of ornithine decarboxylase during oncogenic transformation: mechanisms and therapeutic potential. Amino Acids 2007; 33:213-23. [PMID: 17443268 DOI: 10.1007/s00726-007-0531-2] [Citation(s) in RCA: 82] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2006] [Accepted: 02/01/2007] [Indexed: 01/10/2023]
Abstract
The activity of ornithine decarboxylase (ODC(1)), the first enzyme in polyamine biosynthesis, is induced during carcinogenesis by a variety of oncogenic stimuli. Intracellular levels of ODC and the polyamines are tightly controlled during normal cell growth, and regulation occurs at the levels of transcription, translation and protein degradation. Several known proto-oncogenic pathways appear to control ODC transcription and translation, and dysregulation of pathways downstream of ras and myc result in the constitutive elevation of ODC activity that occurs with oncogenesis. Inhibition of ODC activity reverts the transformation of cells in vitro and reduces tumor growth in several animal models, suggesting high levels of ODC are necessary for the maintenance of the transformed phenotype. The ODC irreversible inactivator DFMO has proven to be not only a valuable tool in the study of ODC in cancer, but also shows promise as a chemopreventive and chemotherapeutic agent in certain types of malignancies.
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Affiliation(s)
- L M Shantz
- Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, PA 17033, USA.
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27
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Wang JY. Polyamines and mRNA stability in regulation of intestinal mucosal growth. Amino Acids 2007; 33:241-52. [PMID: 17404803 DOI: 10.1007/s00726-007-0518-z] [Citation(s) in RCA: 51] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2006] [Accepted: 02/01/2007] [Indexed: 10/23/2022]
Abstract
The mammalian intestinal epithelium is a rapidly self-renewing tissue in the body, and its homeostasis is preserved through strict regulation of epithelial cell proliferation, growth arrest, and apoptosis. Polyamines are necessary for normal intestinal mucosal growth and decreasing cellular polyamines inhibits cell proliferation and disrupts epithelial integrity. An increasing body of evidence indicates that polyamines regulate intestinal epithelial cell renewal by virtue of their ability to modulate expression of various genes and that growth inhibition following polyamine depletion results primarily from the activation of growth-inhibiting genes rather than a simple decrease in expression of growth-promoting genes. In this review article, we will focus on changes in expression of growth-inhibiting genes following polyamine depletion and further analyze in some detail the mechanisms through which mRNA stability is regulated by RNA-binding proteins.
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Affiliation(s)
- J-Y Wang
- Cell Biology Group, Departments of Surgery and Pathology, University of Maryland School of Medicine and Baltimore Veterans Affairs Medical Center, Baltimore, MD 21201, USA.
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28
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Liu L, Guo X, Rao J, Zou T, Marasa B, Chen J, Greenspon J, Casero R, Wang JY. Polyamine-modulated c-Myc expression in normal intestinal epithelial cells regulates p21Cip1 transcription through a proximal promoter region. Biochem J 2006; 398:257-67. [PMID: 16706751 PMCID: PMC1550304 DOI: 10.1042/bj20060217] [Citation(s) in RCA: 42] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
Abstract
Maintenance of intestinal mucosal epithelial integrity requires cellular polyamines that regulate expression of various genes involved in cell proliferation, growth arrest and apoptosis. Our previous studies have shown that polyamines are essential for expression of the c-myc gene and that polyamine-induced c-Myc plays a critical role in stimulation of normal IEC (intestinal epithelial cell) proliferation, but the exact downstream targets of induced c-Myc are still unclear. The p21Cip1 protein is a major player in cell cycle control, which is primarily regulated at the transcriptional level. The current study was designed to determine whether induced c-Myc stimulates normal IEC proliferation by repressing p21Cip1 transcription following up-regulation of polyamines. Overexpression of the ODC (ornithine decarboxylase) gene increased levels of cellular polyamines, induced c-Myc expression and inhibited p21Cip1 transcription, as indicated by repression of p21Cip1 promoter activity and a decrease in p21Cip1 protein levels. In contrast, depletion of cellular polyamines by inhibiting ODC enzyme activity with alpha-difluoromethylornithine decreased c-Myc, but increased p21Cip1 transcription. Ectopic expression of wild-type c-myc not only inhibited basal levels of p21Cip1 transcription in control cells, but also prevented increased p21Cip1 in polyamine-deficient cells. Experiments using different p21Cip1 promoter mutants showed that transcriptional repression of p21Cip1 by c-Myc was mediated through Miz-1- and Sp1-binding sites within the proximal region of the p21Cip1 promoter in normal IECs. These findings confirm that p21Cip1 is one of the direct mediators of induced c-Myc following increased polyamines and that p21Cip1 repression by c-Myc is implicated in stimulation of normal IEC proliferation.
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Affiliation(s)
- Lan Liu
- *Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore, MD 21201, U.S.A
- †Baltimore Veterans Affairs Medical Center, Baltimore, MD 21201, U.S.A
| | - Xin Guo
- *Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore, MD 21201, U.S.A
- †Baltimore Veterans Affairs Medical Center, Baltimore, MD 21201, U.S.A
| | - Jaladanki N. Rao
- *Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore, MD 21201, U.S.A
- †Baltimore Veterans Affairs Medical Center, Baltimore, MD 21201, U.S.A
| | - Tongtong Zou
- *Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore, MD 21201, U.S.A
- †Baltimore Veterans Affairs Medical Center, Baltimore, MD 21201, U.S.A
| | - Bernard S. Marasa
- *Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore, MD 21201, U.S.A
- ‡Department of Pathology, University of Maryland School of Medicine, Baltimore, MD 21201, U.S.A
| | - Jie Chen
- *Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore, MD 21201, U.S.A
- †Baltimore Veterans Affairs Medical Center, Baltimore, MD 21201, U.S.A
| | - Jose Greenspon
- *Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore, MD 21201, U.S.A
| | - Robert A. Casero
- §Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD 21231, U.S.A
| | - Jian-Ying Wang
- *Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore, MD 21201, U.S.A
- †Baltimore Veterans Affairs Medical Center, Baltimore, MD 21201, U.S.A
- ‡Department of Pathology, University of Maryland School of Medicine, Baltimore, MD 21201, U.S.A
- To whom correspondence should be addressed (email )
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29
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Zou T, Mazan-Mamczarz K, Rao JN, Liu L, Marasa BS, Zhang AH, Xiao L, Pullmann R, Gorospe M, Wang JY. Polyamine depletion increases cytoplasmic levels of RNA-binding protein HuR leading to stabilization of nucleophosmin and p53 mRNAs. J Biol Chem 2006; 281:19387-94. [PMID: 16690610 DOI: 10.1074/jbc.m602344200] [Citation(s) in RCA: 103] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Polyamines are essential for maintaining normal intestinal epithelial integrity, an effect that relies, at least in part, on their ability to keep low levels of nucleophosmin (NPM) and p53 mRNAs. The RNA-binding protein HuR associates with the p53 mRNA, as reported previously, and with the NPM mRNA, computationally predicted to be a target of HuR. Here, we show that HuR binds the NPM and p53 3'-untranslated regions and stabilizes these mRNAs in polyamine-depleted intestinal epithelial cells. Depletion of cellular polyamines by inhibiting ornithine decarboxylase with alpha-difluoromethylornithine dramatically enhanced the cytoplasmic abundance of HuR, whereas ectopic ornithine decarboxylase overexpression decreased cytoplasmic HuR; neither intervention changed whole-cell HuR levels. HuR was found to specifically bind the 3'-untranslated regions of NPN and p53 mRNAs. HuR silencing rendered the NPM and p53 mRNAs unstable and prevented increases in NPM and p53 mRNA and protein in polyamine-deficient cells. These results indicate that polyamines modulate cytoplasmic HuR levels in intestinal epithelial cells, in turn controlling the stability of the NPM and p53 mRNAs and influencing NPM and p53 protein levels.
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Affiliation(s)
- Tongtong Zou
- Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore 21201, USA
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30
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Sánchez-Moreiras AM, Reigosa MJ. Whole plant response of lettuce after root exposure to BOA (2(3H)-benzoxazolinone). J Chem Ecol 2005; 31:2689-703. [PMID: 16273435 DOI: 10.1007/s10886-005-7620-z] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2004] [Revised: 03/22/2005] [Accepted: 07/16/2005] [Indexed: 10/25/2022]
Abstract
The goal of our work was to expand the knowledge about plant stress response to the allelochemical 2(3H)-benzoxazolinone (BOA). We focused on physiological processes that are affected by this secondary metabolite. Physiological and biochemical characteristics of plants exposed to BOA help us to better understand its mode of action and open the gate to the use of allelochemicals as "natural" herbicides. Measurements on photosynthesis, fluorescence, water relations, antioxidant enzymes (superoxide dismutase, peroxidase), ATPases, and lipid peroxidation indicated that a phytotoxic effect follows BOA exposition. This effect was intense enough to interfere with plant growth and development and to produce "induced senescence." Based on this, we propose a multifaceted mode of action for BOA with effects at different levels and in different parts of the plant.
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Affiliation(s)
- A M Sánchez-Moreiras
- Laboratory of Plant Ecophysiology, Faculty of Biology, University of Vigo, Campus Lagoas-Marcosende s/n, 36310 Vigo, Spain
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31
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Zou T, Rao JN, Liu L, Marasa BS, Keledjian KM, Zhang AH, Xiao L, Bass BL, Wang JY. Polyamine depletion induces nucleophosmin modulating stability and transcriptional activity of p53 in intestinal epithelial cells. Am J Physiol Cell Physiol 2005; 289:C686-96. [PMID: 15872011 DOI: 10.1152/ajpcell.00085.2005] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Our previous studies have shown that polyamines are required for normal intestinal mucosal growth and that decreased levels of polyamines inhibit intestinal epithelial cell (IEC) proliferation by stabilizing p53 and other growth-inhibiting proteins. Nucleophosmin (NPM) is a multifunctional protein that recently has been shown to regulate p53 activity. In the present study, we sought to determine whether polyamine depletion increases NPM modulating the stability and transcriptional activity of p53 in a normal IEC-6 intestinal epithelial cell line. Depletion of cellular polyamines by alpha-difluoromethylornithine, the specific inhibitor of polyamine biosynthesis, stimulated expression of the NPM gene and induced nuclear translocation of NPM protein. Polyamine depletion stimulated NPM expression primarily by increasing NPM gene transcription and its mRNA stability, and it induced NPM nuclear translocation through activation of phosphorylation of mitogen-activated protein kinase kinase. Increased NPM interacted with p53 and formed a NPM/p53 complex in polyamine-deficient cells. Inhibition of NPM expression by small interfering RNA targeting NPM (siNPM) not only destabilized p53 as indicated by a decrease in its protein half-life but also prevented the increased p53-dependent transactivation as shown by suppression of the p21 promoter activity. Decreased expression of NPM by siNPM also promoted cell growth in polyamine-deficient cells. These results indicate that 1) polyamine depletion increases expression of the NPM gene and enhances NPM nuclear translocation and 2) increased NPM interacts with and stabilizes p53, leading to inhibition of IEC-6 cell proliferation.
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Affiliation(s)
- Tongtong Zou
- Dept. of Surgery, University of Maryland School of Medicine, Baltimore, MD, USA
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32
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Gugliucci A. Polyamines as clinical laboratory tools. Clin Chim Acta 2005; 344:23-35. [PMID: 15149868 DOI: 10.1016/j.cccn.2004.02.022] [Citation(s) in RCA: 95] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2004] [Revised: 02/25/2004] [Accepted: 02/25/2004] [Indexed: 12/12/2022]
Abstract
Since their discovery by Antoni van Leeuwenhoek in 1678 until the recent development of transgenic mice expressing proteins altering polyamine levels in a tissue-specific manner, polyamines have been the object of intense research efforts which have shed light on several biological and pathological processes. From the discovery of a particular form of proteasome regulation of the catabolism of the key regulatory enzyme in their synthetic pathway, to the experimental cancer treatment or prevention with polyamine antagonists or inhibitors of the latter enzyme, a whole spectrum of interests can be revealed. Still, many aspects of their functions remain elusive and difficulties inherent in their analysis, which relies on sophisticated high-performance liquid chromatographic (HPLC) methods, and the lack of standardization; have hampered the transit from the research realm to the standard clinical laboratory domain. Their assay in biological fluids has been used for cancer diagnosis and for monitoring anticancer treatment. In this article, we attempt to provide an overview of polyamine structure, nutritional value, metabolism, and physiological roles. Next, we will summarize the main analytical methods on which we count, and finally we will address their role in diagnosis of cancer as well their proposed role as antioxidant and antiglycation agents.
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Affiliation(s)
- A Gugliucci
- Laboratory of Biochemistry, Division of Basic Medical Sciences, Touro University College of Osteopathic Medicine, 1310 Johnson Lane, Mare Island, Vallejo, CA 94592, USA.
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33
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Liu L, Han L, Wong DYL, Yue PYK, Ha WY, Hu YH, Wang PX, Wong RNS. Effects of Si-Jun-Zi decoction polysaccharides on cell migration and gene expression in wounded rat intestinal epithelial cells. Br J Nutr 2005; 93:21-9. [PMID: 15705221 DOI: 10.1079/bjn20041295] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Si-Jun-Zi decoction (SJZD), a traditional Chinese herbal prescription, has been used clinically for treating patients with disorders of the digestive system. Previous studies indicated that the polysaccharides of SJZD (SJZPS) are the active components contributing towards its pharmacological effects in improving gastrointestinal function and immunity. However, the protective and restitutive effects on intestinal epithelial cells remain unknown. In the present study, SJZPS were first extracted and chemically characterized. Then their stimulatory and restitutive effects on intestinal epithelial cells (IEC-6 cells) were elicited by different in vitro models including migration of wounded IEC-6 cells and cell proliferation. Results indicated that SJZPS not only protects the cells against the harmful impairment of indomethacin but also enhances re-epithelialization of a wounded monolayer at an optimal dose of 100 mug/ml at 24 h incubation. To elucidate the modulatory effect of SJZPS on wounded IEC-6 cells at the molecular level, an oligonucleotide microarray was employed to study differential gene expression of SJZPS-treated IEC-6 cells and the candidate genes were validated by RT-PCR. There was increased expression of genes coding for ion channels and transporters, which are critical to cell migration and restoration of wounded intestinal cells, suggesting a possible mechanism for re-epithelialization. In conclusion, our data show for the first time that SJZPS can enhance intestinal restitution and protect against indomethacin-induced damage of intestinal epithelial cells. These findings provide new insight into the mechanism of action of a traditional Chinese herbal prescription, SJZD, in intestinal wound restitution.
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Affiliation(s)
- L Liu
- Research and Development Division, School of Chinese Medicine, Hong Kong Baptist University, Hong Kong
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34
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Liu L, Li L, Rao JN, Zou T, Zhang HM, Boneva D, Bernard MS, Wang JY. Polyamine-modulated expression of c-myc plays a critical role in stimulation of normal intestinal epithelial cell proliferation. Am J Physiol Cell Physiol 2004; 288:C89-99. [PMID: 15355849 DOI: 10.1152/ajpcell.00326.2004] [Citation(s) in RCA: 48] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
The nuclear protein c-Myc is a transcription factor involved in the control of cell cycle. Our previous studies indicated that cellular polyamines are absolutely required for cell proliferation in crypts of small intestinal mucosa and that polyamines have the ability to stimulate expression of the c-myc gene. The current study went further to determine whether induced nuclear c-Myc plays a role in stimulation of cell proliferation by polyamines in intestinal crypt cells (IEC-6 line). Exposure of normal quiescent cells after 24-h serum deprivation to 5% dialyzed fetal bovine serum (dFBS) increased both cellular polyamines and expression of the c-myc gene. Increased c-Myc protein formed heterodimers with its binding partner, Max, and specifically bound to the Myc/Max binding site, which was associated with an increase in DNA synthesis. Depletion of cellular polyamines by pretreatment with alpha-difluoromethylornithine (DFMO) prevented increases in c-myc expression and DNA synthesis induced by 5% dFBS. c-Myc gene transcription and cell proliferation decreased in polyamine-deficient cells, whereas the natural polyamine spermidine given together with DFMO maintained c-myc gene expression and cell growth at normal levels. Disruption of c-myc expression using specific c-myc antisense oligomers not only inhibited normal cell growth (without DFMO) but also prevented the restoration of cell proliferation by spermidine in polyamine-deficient cells. Ectopic expression of wild-type c-myc by recombinant adenoviral vector containing c-myc cDNA increased cell growth. These results indicate that polyamine-induced nuclear c-Myc interacts with Max, binds to the specific DNA sequence, and plays an important role in stimulation of normal intestinal epithelial cell proliferation.
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Affiliation(s)
- Lan Liu
- Department of Surgery, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA
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35
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Zhang HM, Rao JN, Guo X, Liu L, Zou T, Turner DJ, Wang JY. Akt kinase activation blocks apoptosis in intestinal epithelial cells by inhibiting caspase-3 after polyamine depletion. J Biol Chem 2004; 279:22539-47. [PMID: 15024023 DOI: 10.1074/jbc.m314337200] [Citation(s) in RCA: 83] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Apoptosis plays a critical role in the maintenance of gut mucosal homeostasis and is regulated by numerous factors including polyamines. Although the exact roles of polyamines in apoptotic pathway are still unclear, inhibition of polyamine synthesis promotes the resistance of intestinal epithelial cells to apoptosis. Akt is a serine-threonine kinase that has been established as an important intracellular signaling in regulating cell survival. The current studies test the hypothesis that polyamines are involved in the control of Akt activity in normal intestinal epithelial cells (IEC-6 line) and that activated Akt mediates suppression of apoptosis following polyamine depletion. Depletion of cellular polyamines by alpha-difluoromethylornithine induced levels of phosphorylated Akt and increased Akt kinase activity, although it had no effect on expression of total Akt, pERK, p38, and Bcl-2 proteins. This activated Akt was associated with both decreased levels of active caspase-3 and increased resistance to tumor necrosis factor-alpha/cycloheximide-induced apoptosis. Inactivation of Akt by either treatment with LY294002 or ectopic expression of a dominant negative Akt mutant (DNMAkt) not only enhanced the caspase-3 activation in polyamine-deficient cells but also prevented the increased resistance to tumor necrosis factor-alpha/cycloheximide-induced apoptosis. Phosphorylation of glycogen synthase kinase-3, a downstream target of Akt, was also increased in alpha-difluoromethylornithine-treated cells, which was prevented by inactivation of Akt by LY294002 or DNMAkt overexpression. These results indicate that polyamine depletion induces the Akt activation mediating suppression of apoptosis via inhibition of caspase-3 in normal intestinal epithelial cells.
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Affiliation(s)
- Huifang M Zhang
- Departments of Surgery and Pathology, University of Maryland School of Medicine and Baltimore Veterans Affairs Medical Center, Baltimore, Maryland 21201, USA
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36
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Zou T, Rao JN, Guo X, Liu L, Zhang HM, Strauch ED, Bass BL, Wang JY. NF-kappaB-mediated IAP expression induces resistance of intestinal epithelial cells to apoptosis after polyamine depletion. Am J Physiol Cell Physiol 2003; 286:C1009-18. [PMID: 15075199 DOI: 10.1152/ajpcell.00480.2003] [Citation(s) in RCA: 77] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Apoptosis plays a crucial role in maintenance of intestinal epithelial integrity and is highly regulated by numerous factors, including cellular polyamines. We recently showed that polyamines regulate nuclear factor (NF)-kappaB activity in normal intestinal epithelial (IEC-6) cells and that polyamine depletion activates NF-kappaB and promotes resistance to apoptosis. The current study went further to determine whether the inhibitors of apoptosis (IAP) family of proteins, c-IAP2 and XIAP, are downstream targets of activated NF-kappaB and play a role in antiapoptotic activity of polyamine depletion in IEC-6 cells. Depletion of cellular polyamines by alpha-difluoromethylornithine not only activated NF-kappaB activity but also increased expression of c-IAP2 and XIAP. Specific inhibition of NF-kappaB by the recombinant adenoviral vector containing IkappaBalpha superrepressor (AdIkappaBSR) prevented the induction of c-IAP2 and XIAP in polyamine-deficient cells. Decreased levels of c-IAP2 and XIAP proteins by inactivation of NF-kappaB through AdIkappaBSR infection or treatment with the specific inhibitor Smac also overcame the resistance of polyamine-depleted cells to apoptosis induced by the combination of tumor necrosis factor (TNF)-alpha and cycloheximide (CHX). Although polyamine depletion did not alter levels of procaspase-3 protein, it inhibited formation of the active caspase-3. Decreased levels of c-IAP2 and XIAP by Smac prevented the inhibitory effect of polyamine depletion on the cleavage of procaspase-3 to the active caspase-3. These results indicate that polyamine depletion increases expression of c-IAP2 and XIAP by activating NF-kappaB in intestinal epithelial cells. Increased c-IAP2 and XIAP after polyamine depletion induce the resistance to TNF-alpha/CHX-induced apoptosis, at least partially, through inhibition of the caspase-3 activity.
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Affiliation(s)
- Tongtong Zou
- Dept. of Surgery, Baltimore Veterans Affairs Medical Center, 10 North Greene St., Baltimore, MD 21201, USA.
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37
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Liu L, Santora R, Rao JN, Guo X, Zou T, Zhang HM, Turner DJ, Wang JY. Activation of TGF-beta-Smad signaling pathway following polyamine depletion in intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol 2003; 285:G1056-67. [PMID: 12855402 DOI: 10.1152/ajpgi.00151.2003] [Citation(s) in RCA: 51] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Smad proteins are transcription activators that are critical for transmitting transforming growth factor-beta (TGF-beta) superfamily signals from the cell surface receptors to the nucleus. Our previous studies have shown that cellular polyamines are essential for normal intestinal mucosal growth and that a decreased level of polyamines inhibits intestinal epithelial cell proliferation, at least partially, by increasing expression of TGF-beta/TGF-beta receptors. The current study went further to determine the possibility that Smads are the downstream intracellular effectors of activated TGF-beta/TGF-beta receptor signaling following polyamine depletion. Studies were conducted in IEC-6 cells derived from rat small intestinal crypts. Depletion of cellular polyamines by alpha-difluoromethylornithine (DFMO) increased basal levels of Smad3 and Smad4 proteins, induced their nuclear translocation, and stimulated Smad sequence-specific DNA-binding activity. Polyamine depletion-induced Smads were also associated with a significant increase in transcription activation as measured by luciferase reporter gene activity of Smad-dependent promoters. Inhibition of Smads by a dominant-negative mutant Smad4 in the DFMO-treated cells prevented the increased Smad transcription activation. Polyamine-deficient cells highly expressed TGF-beta and were growth-arrested at the G1 phase. Inhibition of TGF-beta by treatment with either immunoneutralizing anti-TGF-beta antibody or TGF-beta antisense oligodeoxyribonucleotides not only blocked the induction of Smad activity but also decreased the Smad-mediated transcriptional activation in polyamine-depleted cells. These findings suggest that Smads are involved in the downstream cellular processes mediated by cellular polyamines and that increased TGF-beta/TGF-beta receptor signaling following polyamine depletion activates Smads, thus resulting in the stimulation of Smad target gene expression.
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Affiliation(s)
- Lan Liu
- Dept. of Surgery, Baltimore Veterans Affairs Medical Center, 10 North Greene St., Baltimore, MD 21201, USA
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38
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Gardini G, Cravanzola C, Autelli R, Testore G, Cesa R, Morando L, Solinas SP, Muzio G, Grillo MA, Colombatto S. Agmatine inhibits the proliferation of rat hepatoma cells by modulation of polyamine metabolism. J Hepatol 2003; 39:793-9. [PMID: 14568263 DOI: 10.1016/s0168-8278(03)00386-6] [Citation(s) in RCA: 20] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/02/2023]
Abstract
BACKGROUND/AIMS Previous experiments have shown that agmatine, the product of arginine decarboxylase, is transported in competition with putrescine into quiescent rat hepatocytes, where it promotes several effects, including marked decrease of intracellular polyamines and induction of apoptosis. The primary aim of the present study was to assess the action of agmatine on transformed and proliferating hepatic rat cells. METHODS To assess the effect of agmatine on hepatoma cells, analysis by flow cytometry, Western blotting, reverse transcription-polymerase chain reaction, scanning and transmission electron microscopy, immunofluorescence detection of beta-actin and alpha-tubulin were performed. RESULTS The results showed that agmatine has antiproliferative effects on the cell lines studied (HTC, JM2, HepG2). Further experiments were performed on HTC cells. The effect was proportional to agmatine concentration (in a range between 50 and 500 microM). It was not correlated with induction of necrosis or apoptosis and was accompanied by accumulation in G(2)/M cell cycle phase and by dramatic modification of cell morphology. Spermidine reversed these effects, suggesting that the marked decrease of the polyamine pool is the main target of agmatine . CONCLUSIONS The results obtained show a relationship between the decrease of intracellular polyamine content, the rate of cell growth and the cytoskeleton organization.
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Affiliation(s)
- Giulia Gardini
- Dipartimento di Medicina e Oncologia Sperimentale, Sezione di Biochimica, Università di Torino, Via Michelangelo 27, 10126 Turin, Italy
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39
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Eklou-Kalonji E, Andriamihaja M, Reinaud P, Mayeur C, Camous S, Robert V, Charpigny G, Blachier F. Prostaglandin-independent effects of aspirin on cell cycle and putrescine synthesis in human colon carcinoma cells. Can J Physiol Pharmacol 2003; 81:443-50. [PMID: 12774850 DOI: 10.1139/y03-058] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Aspirin consumption has been reported to be able to reduce colorectal cancer risk in humans and in animal models of colon carcinogenesis. Although the mechanism involved in such an effect is not yet clear, both prostaglandin-dependent and -independent effects have been proposed. Using HT-29 Glc(-/+)cells, which originate from a human colon adenocarcinoma, we demonstrated in this study a dose-dependent effect of millimolar concentration of aspirin on cell growth that was concomitant with a rapid accumulation of the cells in the G0/G1 phase, followed by an accumulation in the G2/M phase and by a minor increase in the proportion of cells undergoing nuclear condensation. Cell membrane integrity and cell release into the culture medium were not affected by this treatment. The aspirin effects were apparently unrelated to prostaglandin biosynthesis inhibition, since although these cells were found to express high levels of cyclooxygenase 1 (COX-1) and low levels of COX-2 proteins, they did not produce any measurable net amounts of prostaglandins, based on both utilization of radiolabelled arachidonic acid and the radioimmunoassay of prostaglandins E2 and F2 alpha. In contrast, we identified polyamine biosynthesis as a cellular target of aspirin, since the treatment of HT-29 Glc(-/+) cells with aspirin reduced the flux of L-ornithine through ornithine decarboxylase, an effect that could not be explained by an acute action of the drug on the ornithine decarboxylase catalytic activity. Since polyamine biosynthesis is strictly necessary for HT-29 cell growth, our data suggest that reduced flux through ornithine decarboxylase may participate in the antiproliferative activity of aspirin towards colonic tumoral cells. It is concluded that in HT-29 Glc(-/+) cells that are not functional for prostaglandin production, aspirin can affect cell growth, cell cycle, and polyamine biosynthesis without affecting cell membrane integrity.
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Affiliation(s)
- E Eklou-Kalonji
- Laboratoire de Nutrition et Sécurité Alimentaire, Institut National de la Recherche Agronomique, 78350 Jouy-en-Josas, France
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Strauch ED, Bass BL, Rao JN, Vann JA, Wang JY. NF-kappaB regulates intestinal epithelial cell and bile salt-induced migration after injury. Ann Surg 2003; 237:494-501. [PMID: 12677145 PMCID: PMC1514469 DOI: 10.1097/01.sla.0000060459.03270.e7] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
OBJECTIVE To determine if NF-kappa B regulates intestinal epithelial cell migration and if it has a role during bile salt-induced migration. SUMMARY BACKGROUND DATA Mucosal restitution is an important repair modality in the gastrointestinal tract. The authors have shown that taurodeoxycholate (TDCA) increases intestinal epithelial cell migration. NF-kappa B regulates activation of a number of genes involved in inflammatory responses. METHODS Studies were conducted in IEC-6 cells. I kappa B protein expression was determined by Western blot analysis. Sequence-specific NF-kappa B binding activity was measured by EMSA shift assays and nuclear localization by immunohistochemistry. Cell migration was examined by using an in vitro model that mimics the early cell division-independent stages of epithelial restitution. RESULTS The process of cell migration over the wounded area was associated with a significant increase in NF-kappa B binding activity in IEC-6 cells. Immunohistochemistry revealed translocation of NF-kappa B into the nucleus. Western blot analysis showed that injury decreased I kappa B protein expression. Inhibition of the binding activity by treatment with a specific NF-kappa B inhibitor, MG-132, inhibited cell migration during restitution. Further, exposure to TDCA at the physiologic concentration that induces intestinal epithelial cell migration increased NF-kappa B binding activity, induced NF-kappa B translocation into the nucleus, and decreased I kappa B protein expression. MG-132 also inhibits bile salt-induced cell migration. CONCLUSIONS NF-kappa B regulates intestinal epithelial cell migration. Bile salts at physiologic concentrations increase cell migration by activation of NF-kappa B. These data show that bile salts may have a role in the maintenance of intestinal mucosal integrity.
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Affiliation(s)
- Eric D Strauch
- Department of Surgery, University of Maryland School of Medicine, 22 South Greene Street, Baltimore, MD 21201, USA.
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Li L, Liu L, Rao JN, Esmaili A, Strauch ED, Bass BL, Wang JY. JunD stabilization results in inhibition of normal intestinal epithelial cell growth through P21 after polyamine depletion. Gastroenterology 2002; 123:764-79. [PMID: 12198703 DOI: 10.1053/gast.2002.35386] [Citation(s) in RCA: 73] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/02/2022]
Abstract
BACKGROUND & AIMS Normal intestinal mucosal growth requires cellular polyamines that regulate expression of various genes involved in cell proliferation, growth arrest, and apoptosis. We have recently shown that growth inhibition after polyamine depletion is associated with an increase in JunD/AP-1 activity in normal intestinal epithelial cells (IEC-6 line). The current study tests the hypothesis that polyamine depletion-induced JunD/activator protein 1 (AP-1) activity results from the activation of junD gene expression and plays a critical role in regulation of intestinal epithelial cell growth. METHODS The junD gene transcription was examined by nuclear run-on assays, and messenger RNA (mRNA) stability was measured by determination of JunD mRNA half-life. Functions of JunD were investigated by using JunD antisense oligodeoxyribonucleotides and transient transfection with the junD-expressing vector. RESULTS Depletion of cellular polyamines by DL-alpha-difluoromethylornithine (DFMO) induced levels of JunD mRNA and protein, which was associated with an increase in G(1) phase growth arrest. Polyamine depletion did not increase the rate of junD gene transcription but significantly increased the stability of JunD mRNA. Decreasing JunD protein by using JunD antisense oligomers promoted cell growth in polyamine-deficient cells. Growth arrest following polyamine depletion also was accompanied by increases in both p21 expression and its promoter activity. Treatment with JunD antisense oligomers inhibited the p21 promoter and prevented the increase in p21 expression in the presence of DFMO. Ectopic expression of the wild-type junD increased p21-promoter activity and inhibited epithelial cell growth. CONCLUSIONS Polyamines negatively regulate junD gene expression posttranscriptionally, and increased JunD/AP-1 inhibits intestinal epithelial cell proliferation at least partially through the activation of p21 promoter.
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Affiliation(s)
- Li Li
- Department of Surgery, University of Maryland School of Medicine, Baltimore, USA
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Zou C, Vlastos AT, Yang L, Wang J, Nishioka K, Follen M. Effects of difluoromethylornithine on growth inhibition and apoptosis in human cervical epithelial and cancerous cell lines. Gynecol Oncol 2002; 85:266-73. [PMID: 11972386 DOI: 10.1006/gyno.2002.6599] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/24/2023]
Abstract
OBJECTIVE Difluoromethylornithine(DFMO), an irreversible inhibitor of ornithine decarboxylase and an angiogenesis inhibitor, has been used in phase I cervical intraepithelial neoplasia (CIN) trials, producing a 50% regression of CIN 3 lesions. DFMO is currently in phase II trials. In the experiments reported here, DFMO's growth inhibition and apoptosis induction were explored in an in vitro model to elucidate mechanisms of action. METHODS Four immortalized cervical epithelial cell lines, serving as in vitro models of precancerous CIN lesions, and nine cervical carcinoma cell lines were studied. DFMO's growth inhibitory effect was tested in monolayer culture and in semisolid medium, and concentrations required for a 50% growth inhibition (IC(50)) with a 5-day treatment were determined. Apoptosis induction was analyzed using the terminal deoxynucleotidyl transferase assay of DNA fragmentation. RESULTS DFMO inhibited growth of immortalized cervical epithelial cell lines and cervical cancer cell lines in monolayer culture and in semisolid medium. The immortalized cervical epithelial cell lines were more sensitive than the cervical cancer cell lines to DFMO's growth inhibitory effect. Concentrations required for 50% growth inhibition after a 5-day treatment ranged from 100 microM to >5 mM for cervical carcinoma cell lines and from 100 microM to 1 mM for immortalized cervical epithelial cell lines. DFMO induced apoptosis in precancerous and cancerous cell lines at a concentration of 5 mM, regardless of the cells' human papillomavirus status. CONCLUSION DFMO inhibits the growth of cervical precancerous and cancerous cells in vitro in a dose-dependent and time-dependent manner, partially through inducing apoptosis.
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Affiliation(s)
- Changping Zou
- Department of Obstetrics, Gynecology, and Reproductive Sciences, University of Texas Medical School at Houston, 1515 Holcombe Boulevard, Houston, TX 77030, USA
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Abstract
AIM: To determine whether the gastrin stimulated intestinal crypt cell (IEC-6) proliferation by induction of ornithine decarboxylase (ODC).
METHODS: IEC-6 cells were grown in DMEM containing 50 mL·L-1 dialyzed fetal bovine serum for 24 h and then were treated with gastrin. The proliferative capability of the cells was monitored subsequently on d 1, 2, 3, and 4 after treatment with MTT assay at aborbance 570 nm. The cellular ODC mRNA expression, ODC activity, and putrescine content were examined by RT-PCR method, radiometric technique and high-performance liquid chromatography(HPLC) analysis respectively after 12 h of treatment.
RESULTS: On d1 after exposure of IEC-6 cells to pentagastrin, the proliferation increased initially and reached a peak on d3 at 250 μg·L-1 concentration. Pentagastrin 500 μg·L-1 increased cell proliferation on day 1 and day 2, and then decreased. Compared with control group, pentagastrin 250 μg·L-1 increased ODC mRNA level by 1.09-fold (P < 0.05), ODC activity by 1.71-fold(P < 0.01), and putrescine content 5.30-fold (P < 0.01), respectively. Similarly, pentagastrin of 500 μg·L-1 also increased ODC mRNA level by 1.16-fold (P < 0.05), ODC activity 1.63-fold(P < 0.05), and putrescine content 4.41-fold (P < 0.01), respectively. But there was not significant difference between them.
CONCLUSION: Gastrin is an agent which promotes IEC-6 cell proliferation involved in regulating ODC activity mechanism.
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Affiliation(s)
- Zi-Li Zhang
- Piei Institute,Guangzhou University of Traditional Chinese Medicine, Guangzhou 510405, China
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Li L, Rao JN, Guo X, Liu L, Santora R, Bass BL, Wang JY. Polyamine depletion stabilizes p53 resulting in inhibition of normal intestinal epithelial cell proliferation. Am J Physiol Cell Physiol 2001; 281:C941-53. [PMID: 11502571 DOI: 10.1152/ajpcell.2001.281.3.c941] [Citation(s) in RCA: 66] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The p53 nuclear phosphoprotein plays a critical role in transcriptional regulation of target genes involved in growth arrest and apoptosis. The natural polyamines, including spermidine, spermine, and their precursor putrescine, are required for cell proliferation, and decreasing cellular polyamines inhibits growth of the small intestinal mucosa. In the current study, we investigated the mechanisms of regulation of p53 gene expression by cellular polyamines and further determined the role of the gene product in the process of growth inhibition after polyamine depletion. Studies were conducted both in vivo and in vitro using rats and the IEC-6 cell line, derived from rat small intestinal crypt cells. Levels for p53 mRNA and protein, transcription and posttranscription of the p53 gene, and cell growth were examined. Depletion of cellular polyamines by treatment with alpha-difluoromethylornithine (DFMO) increased p53 gene expression and caused growth inhibition in the intact small intestinal mucosa and the cultured cells. Polyamine depletion dramatically increased the stability of p53 mRNA as measured by the mRNA half-life but had no effect on p53 gene transcription in IEC-6 cells. Induction of p53 mRNA levels in DFMO-treated cells was paralleled by an increase in the rate of newly synthesized p53 protein. The stability of p53 protein was also increased after polyamine depletion, which was associated with a decrease in Mdm2 expression. When polyamine-deficient cells were exposed to exogenous spermidine, a decrease in p53 gene expression preceded an increase in cellular DNA synthesis. Inhibition of the p53 gene expression by using p53 antisense oligodeoxyribonucleotides significantly promoted cell growth in the presence of DFMO. These findings indicate that polyamines downregulate p53 gene expression posttranscriptionally and that growth inhibition of small intestinal mucosa after polyamine depletion is mediated, at least partially, through the activation of p53 gene.
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Affiliation(s)
- L Li
- Department of Surgery, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA
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Maurici D, Monti P, Campomenosi P, North S, Frebourg T, Fronza G, Hainaut P. Amifostine (WR2721) restores transcriptional activity of specific p53 mutant proteins in a yeast functional assay. Oncogene 2001; 20:3533-40. [PMID: 11429700 DOI: 10.1038/sj.onc.1204428] [Citation(s) in RCA: 42] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2001] [Revised: 02/07/2001] [Accepted: 02/26/2001] [Indexed: 01/01/2023]
Abstract
Many p53 mutants found in human cancer have an altered ability to bind DNA and transactivate gene expression. Re-expression of functional p53 in cells in which the endogenous TP53 gene is inactivated has been demonstrated to restore a non-tumorigenic phenotype. Pharmacological modulation of p53 mutant conformation may therefore represent a mechanism to reactivate p53 function and consequently improve response to radio- and chemotherapy. We have recently reported that the radio- and chemoprotector Amifostine (WR2721, Ethyol) activates wild-type p53 in cultured mammalian cells. In the present study, we have used a yeast functional assay to investigate the effect of WR2721 on the transcriptional activity of p53. WR2721 restored this activity in a temperature-sensitive mutant V272M (valine to methionine at codon 272) expressed at the non-permissive temperature and it also partially restored the transcriptional activity of several other conformationally flexible p53 mutants. The results indicate that the yeast functional assay may be used to identify compounds that modulate p53 activity, with potential therapeutic implications.
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Affiliation(s)
- D Maurici
- Group of Molecular Carcinogenesis, International Agency for Research on Cancer, 150 Cours Albert Thomas, 69372 Lyon, France
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Favre C, Carnovale CE, Monti JA, Carrillo MC. Inhibition by interferon α-2b of rat liver regeneration: effect on ornithine decarboxylase and total protein synthesis ☆ ☆This work was presented, in part, at the 1999 Meeting of the Pan-American Association for Biochemistry and Molecular Biology (PABMB) and the American Society for Biochemistry and Molecular Biology (ASBMB). 1 1Abbreviations: IL, interleukin; TNFα, tumor necrosis factor-α; TGFβ, transforming growth factor-β; IFNα, interferon-α and ODC, ornithine decarboxylase. Biochem Pharmacol 2001; 61:1587-93. [PMID: 11377389 DOI: 10.1016/s0006-2952(01)00595-0] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
Polyamines are key factors in macromolecule synthesis during liver regeneration. It has been postulated that interferon-alpha (IFNalpha) decreases putrescine levels in regenerating liver by inhibiting ornithine decarboxylase (ODC) activity, the main enzyme in polyamine biosynthesis. In the present study, we analysed the effects of a pharmacological dose of IFNalpha on polyamine and ODC levels during the regenerative process following partial hepatectomy in rats. Synthesis of ODC by isolated hepatocytes from IFN-treated rats with regenerating livers was also assessed. Furthermore, we investigated the effect of IFNalpha-2b on DNA and total protein synthesis in 24-hr regenerating livers. No effect on DNA synthesis was observed at the dose of IFNalpha-2b used, but total protein synthesis decreased significantly in IFNalpha-2b-treated rats undergoing liver regeneration (7.0 +/- 2.0 and 12.1 +/- 1.7%. min(-1) in hepatectomized rats treated with IFNalpha-2b and saline, respectively). ODC levels were also reduced significantly (by 50%) in hepatectomized rats treated with IFNalpha-2b versus saline. In parallel with the ODC decrease, the concentrations of putrescine and spermidine (63 +/- 25 vs 101 +/- 15 nmol/g liver and 1.08 +/- 0.35 vs 2.14 +/- 0.22 micromol/g liver, respectively, in IFNalpha-2b- and saline-treated hepatectomized rats) showed similar, significant diminutions. Moreover, the incorporation of [35S]methionine into ODC was decreased dramatically in isolated hepatocytes from IFNalpha-2b-treated hepatectomized rats 12 hr after surgery. In conclusion, the protein synthesis rate in regenerating liver was impaired by therapeutic doses of IFNalpha-2b. In addition, the results presented in this study suggest that IFNalpha-2b negatively regulates ODC synthesis, causing a reduction in polyamine levels during liver regeneration.
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Affiliation(s)
- C Favre
- Institute of Experimental Physiology, National Council of Scientific and Technical Research, Faculty of Biochemical and Pharmaceutical Sciences, National University of Rosario, Suipacha 570, Rosario 2000, Argentina
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Li L, Rao JN, Bass BL, Wang JY. NF-kappaB activation and susceptibility to apoptosis after polyamine depletion in intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol 2001; 280:G992-G1004. [PMID: 11292609 DOI: 10.1152/ajpgi.2001.280.5.g992] [Citation(s) in RCA: 78] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
The maintenance of intestinal mucosal integrity depends on a balance between cell renewal and cell death, including apoptosis. The natural polyamines, putrescine, spermidine, and spermine, are essential for mucosal growth, and decreasing polyamine levels cause G(1) phase growth arrest in intestinal epithelial (IEC-6) cells. The present study was done to determine changes in susceptibility of IEC-6 cells to apoptosis after depletion of cellular polyamines and to further elucidate the role of nuclear factor-kappaB (NF-kappaB) in this process. Although depletion of polyamines by alpha-difluoromethylornithine (DFMO) did not directly induce apoptosis, the susceptibility of polyamine-deficient cells to staurosporine (STS)-induced apoptosis increased significantly as measured by changes in morphological features and internucleosomal DNA fragmentation. In contrast, polyamine depletion by DFMO promoted resistance to apoptotic cell death induced by the combination of tumor necrosis factor-alpha (TNF-alpha) and cycloheximide. Depletion of cellular polyamines also increased the basal level of NF-kappaB proteins, induced NF-kappaB nuclear translocation, and activated the sequence-specific DNA binding activity. Inhibition of NF-kappaB binding activity by sulfasalazine or MG-132 not only prevented the increased susceptibility to STS-induced apoptosis but also blocked the resistance to cell death induced by TNF-alpha in combination with cycloheximide in polyamine-deficient cells. These results indicate that 1) polyamine depletion sensitizes intestinal epithelial cells to STS-induced apoptosis but promotes the resistance to TNF-alpha-induced cell death, 2) polyamine depletion induces NF-kappaB activation, and 3) disruption of NF-kappaB function is associated with altered susceptibility to apoptosis induced by STS or TNF-alpha. These findings suggest that increased NF-kappaB activity after polyamine depletion has a proapoptotic or antiapoptotic effect on intestinal epithelial cells determined by the nature of the death stimulus.
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Affiliation(s)
- L Li
- Department of Surgery, University of Maryland School of Medicine, 10 North Greene St., Baltimore, MD 21201, USA
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Scorcioni F, Corti A, Davalli P, Astancolle S, Bettuzzi S. Manipulation of the expression of regulatory genes of polyamine metabolism results in specific alterations of the cell-cycle progression. Biochem J 2001; 354:217-23. [PMID: 11171097 PMCID: PMC1221646 DOI: 10.1042/0264-6021:3540217] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
We have previously reported that cyclical phases of accumulation and depletion of polyamines occur during cell-cycle progression. Regulatory ornithine decarboxylase (ODC) catalyses the first step of polyamine biosynthesis. Ornithine decarboxylase antizyme (OAZ), induced by high polyamine levels, inhibits ODC activity and prevents extracellular polyamine uptake. Spermidine/spermine N1-acetyltransferase (SSAT) regulates the polyamine degradation/excretion pathway. Here we show that 24 h transient transfection of immortalized human prostatic epithelial cells (PNT1A and PNT2) with antisense ODC RNA or OAZ cDNA, or both, while effectively causing marked decreases of ODC activity and polyamine (especially putrescine) concentrations, resulted in accumulation of cells in the S phase of the cell cycle. Transfection with SSAT cDNA led to more pronounced decreases in spermidine and spermine levels and resulted in accumulation of cells in the G2/M phases. Transfection with all three constructs together produced maximal depletion of all polyamines, accompanied by accumulation of PNT1A cells in the S phase and PNT2 cells in the G0/G1 and G2/M phases. Accumulation of PNT1A cells in the S phase progressively increased at 15, 18 and 24 h of transfection with antisense ODC and/or OAZ cDNA. At 24 h, the DNA content was always reduced, as a possible outcome of altered chromosome condensation. A direct link between polyamine metabolism, cell proliferation and chromatin structure is thus proposed.
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Affiliation(s)
- F Scorcioni
- Dipartimento di Scienze Biomediche, Università di Modena e Reggio Emilia, Via G. Campi 287, 41100 Modena, Italy
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Nemoto T, Kamei S, Seyama Y, Kubota S. p53 independent G(1) arrest induced by DL-alpha-difluoromethylornithine. Biochem Biophys Res Commun 2001; 280:848-54. [PMID: 11162600 DOI: 10.1006/bbrc.2000.4227] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Ornithine decarboxylase (ODC), which catalyzes polyamine biosynthesis, plays an essential role in cell growth. DL-alpha-Difluoromethylornithine (DFMO), a synthetic inhibitor of ODC, inhibits cell growth. However, the exact mechanism by which polyamine depletion by DFMO results in growth inhibition remains to be elucidated. We clarified the mechanisms by which DFMO inhibits human gastric cancer cell (MKN45) growth. DFMO induced MKN45 cell G(1) phase arrest after 48 h, and the percentage of G(1) arrest cells continued to increase until 72 h. Expression of p21 and phosphorylation of Stat1 were significantly induced by DFMO at 24 h. Luciferase assay and gel shift assay showed specific binding of Stat1 to the p21 promoter, and promoter activity was activated at 24 h. In dominant negative p53 expressing cells, DFMO significantly induced p21 expression, arrested cells at G(1) phase, and suppressed cell growth effectively. These results suggest that DFMO induced MKN45 cell arrest at G(1) phase in a p53 independent manner, and Stat1 is, at least in part, involved in G(1) arrest.
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Affiliation(s)
- T Nemoto
- Department of Physiological Chemistry and Metabolism, Graduate School of Medicine, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
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Rao JN, Li L, Bass BL, Wang JY. Expression of the TGF-beta receptor gene and sensitivity to growth inhibition following polyamine depletion. Am J Physiol Cell Physiol 2000; 279:C1034-44. [PMID: 11003584 DOI: 10.1152/ajpcell.2000.279.4.c1034] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Our previous studies have shown that inhibition of polyamine biosynthesis increases the sensitivity of intestinal epithelial cells to growth inhibition induced by exogenous transforming growth factor-beta (TGF-beta). This study went further to determine whether expression of the TGF-beta receptor genes is involved in this process. Studies were conducted in the IEC-6 cell line, derived from rat small intestinal crypt cells. Administration of alpha-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase (the rate-limiting enzyme for polyamine synthesis), for 4 and 6 days depleted cellular polyamines putrescine, spermidine, and spermine in IEC-6 cells. Polyamine depletion by DFMO increased levels of the TGF-beta type I receptor (TGF-betaRI) mRNA and protein but had no effect on the TGF-beta type II receptor expression. The induced TGF-betaRI expression after polyamine depletion was associated with an increased sensitivity to growth inhibition induced by exogenous TGF-beta but not by somatostatin. Extracellular matrix laminin inhibited IEC-6 cell growth without affecting the TGF-beta receptor expression. Laminin consistently failed to induce the sensitivity of TGF-beta-mediated growth inhibition. In addition, decreasing TGF-betaRI expression by treatment with retinoic acid not only decreased TGF-beta-mediated growth inhibition in normal cells but also prevented the increased sensitivity to exogenous TGF-beta in polyamine-deficient cells. These results indicate that 1) depletion of cellular polyamines by DFMO increases expression of the TGF-betaRI gene and 2) increased TGF-betaRI expression plays an important role in the process through which polyamine depletion sensitizes intestinal epithelial cells to growth inhibition induced by TGF-beta.
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Affiliation(s)
- J N Rao
- Department of Surgery, University of Maryland School of Medicine and Baltimore Veterans Affairs Medical Center, Baltimore, Maryland 21201, USA
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