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Jeyakanthan M, Tao K, Zou L, Meloncelli PJ, Lowary TL, Suzuki K, Boland D, Larsen I, Burch M, Shaw N, Beddows K, Addonizio L, Zuckerman W, Afzali B, Kim DH, Mengel M, Shapiro AMJ, West LJ. Chemical Basis for Qualitative and Quantitative Differences Between ABO Blood Groups and Subgroups: Implications for Organ Transplantation. Am J Transplant 2015; 15:2602-15. [PMID: 26014598 DOI: 10.1111/ajt.13328] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2015] [Revised: 03/01/2015] [Accepted: 03/20/2015] [Indexed: 01/25/2023]
Abstract
Blood group ABH(O) carbohydrate antigens are carried by precursor structures denoted type I-IV chains, creating unique antigen epitopes that may differ in expression between circulating erythrocytes and vascular endothelial cells. Characterization of such differences is invaluable in many clinical settings including transplantation. Monoclonal antibodies were generated and epitope specificities were characterized against chemically synthesized type I-IV ABH and related glycans. Antigen expression was detected on endomyocardial biopsies (n = 50) and spleen (n = 11) by immunohistochemical staining and on erythrocytes by flow cytometry. On vascular endothelial cells of heart and spleen, only type II-based ABH antigens were expressed; type III/IV structures were not detected. Type II-based ABH were expressed on erythrocytes of all blood groups. Group A1 and A2 erythrocytes additionally expressed type III/IV precursors, whereas group B and O erythrocytes did not. Intensity of A/B antigen expression differed among group A1 , A2 , A1 B, A2 B and B erythrocytes. On group A2 erythrocytes, type III H structures were largely un-glycosylated with the terminal "A" sugar α-GalNAc. Together, these studies define qualitative and quantitative differences in ABH antigen expression between erythrocytes and vascular tissues. These expression profiles have important implications that must be considered in clinical settings of ABO-incompatible transplantation when interpreting anti-ABO antibodies measured by hemagglutination assays with reagent erythrocytes.
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Affiliation(s)
- M Jeyakanthan
- Department of Pediatrics, University of Alberta, Edmonton, Alberta, Canada.,Department of Surgery, University of Alberta, Edmonton, Alberta, Canada.,Canadian National Transplant Research Program, Edmonton, Alberta, Canada.,Alberta Transplant Institute, Edmonton, Alberta, Canada
| | - K Tao
- Department of Pediatrics, University of Alberta, Edmonton, Alberta, Canada.,Alberta Transplant Institute, Edmonton, Alberta, Canada
| | - L Zou
- Alberta Glycomics Centre, Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada
| | - P J Meloncelli
- Alberta Glycomics Centre, Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada
| | - T L Lowary
- Alberta Glycomics Centre, Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada
| | - K Suzuki
- Alberta Diabetes Institute Molecular Biology Core, Faculty of Medicine, University of Alberta, Edmonton, Alberta, Canada
| | - D Boland
- Southern Alberta Cancer Research Institute Antibody Services, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada
| | - I Larsen
- Department of Pediatrics, University of Alberta, Edmonton, Alberta, Canada.,Alberta Transplant Institute, Edmonton, Alberta, Canada
| | - M Burch
- Pediatric Cardiology, Great Ormond Street Hospital, London, United Kingdom
| | - N Shaw
- Pediatric Cardiology, Great Ormond Street Hospital, London, United Kingdom
| | - K Beddows
- Division of Pediatric Cardiology, Columbia University, New York
| | - L Addonizio
- Division of Pediatric Cardiology, Columbia University, New York
| | - W Zuckerman
- Division of Pediatric Cardiology, Columbia University, New York
| | - B Afzali
- Department of Laboratory Medicine and Pathology, Edmonton, Alberta, Canada
| | - D H Kim
- Alberta Transplant Institute, Edmonton, Alberta, Canada.,Division of Medicine, Department of Cardiology, University of Alberta, University of Alberta, Edmonton, Alberta, Canada
| | - M Mengel
- Canadian National Transplant Research Program, Edmonton, Alberta, Canada.,Alberta Transplant Institute, Edmonton, Alberta, Canada.,Department of Laboratory Medicine and Pathology, Edmonton, Alberta, Canada
| | - A M J Shapiro
- Department of Surgery, University of Alberta, Edmonton, Alberta, Canada.,Canadian National Transplant Research Program, Edmonton, Alberta, Canada.,Alberta Transplant Institute, Edmonton, Alberta, Canada
| | - L J West
- Department of Pediatrics, University of Alberta, Edmonton, Alberta, Canada.,Department of Surgery, University of Alberta, Edmonton, Alberta, Canada.,Canadian National Transplant Research Program, Edmonton, Alberta, Canada.,Alberta Transplant Institute, Edmonton, Alberta, Canada.,Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada
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Nabbi A, Almami A, Thakur S, Suzuki K, Boland D, Bismar TA, Riabowol K. ING3 protein expression profiling in normal human tissues suggest its role in cellular growth and self-renewal. Eur J Cell Biol 2015; 94:214-22. [PMID: 25819753 DOI: 10.1016/j.ejcb.2015.03.002] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2015] [Revised: 03/03/2015] [Accepted: 03/03/2015] [Indexed: 12/17/2022] Open
Abstract
Members of the INhibitor of Growth (ING) family of proteins act as readers of the epigenetic code through specific recognition of the trimethylated form of lysine 4 of histone H3 (H3K4Me3) by their plant homeodomains. The founding member of the family, ING1, was initially identified as a tumor suppressor with altered regulation in a variety of cancer types. While alterations in ING1 and ING4 levels have been reported in a variety of cancer types, little is known regarding ING3 protein levels in normal or transformed cells due to a lack of reliable immunological tools. In this study we present the characterization of a new monoclonal antibody we have developed against ING3 that specifically recognizes human and mouse ING3. The antibody works in western blots, immunofluorescence, immunoprecipitation and immunohistochemistry. Using this antibody we show that ING3 is most highly expressed in small intestine, bone marrow and epidermis, tissues in which cells undergo rapid proliferation and renewal. Consistent with this observation, we show that ING3 is expressed at significantly higher levels in proliferating versus quiescent epithelial cells. These data suggest that ING3 levels may serve as a surrogate for growth rate, and suggest possible roles for ING3 in growth and self renewal and related diseases such as cancer.
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Affiliation(s)
- Arash Nabbi
- Department of Biochemistry & Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada; Department of Oncology, Southern Alberta Cancer Research Institute, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada
| | - Amal Almami
- Department of Biochemistry & Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada; Department of Oncology, Southern Alberta Cancer Research Institute, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada
| | - Satbir Thakur
- Department of Biochemistry & Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada; Department of Oncology, Southern Alberta Cancer Research Institute, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada
| | - Keiko Suzuki
- Department of Biochemistry & Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada; Department of Oncology, Southern Alberta Cancer Research Institute, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada
| | - Donna Boland
- Department of Biochemistry & Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada; Department of Oncology, Southern Alberta Cancer Research Institute, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada
| | - Tarek A Bismar
- Department of Biochemistry & Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada; Department of Oncology, Southern Alberta Cancer Research Institute, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada; Department of Pathology & Laboratory Medicine, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada
| | - Karl Riabowol
- Department of Biochemistry & Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada; Department of Oncology, Southern Alberta Cancer Research Institute, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada.
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Bose P, Thakur S, Thalappilly S, Ahn BY, Satpathy S, Feng X, Suzuki K, Kim SW, Riabowol K. ING1 induces apoptosis through direct effects at the mitochondria. Cell Death Dis 2013; 4:e788. [PMID: 24008732 PMCID: PMC3789179 DOI: 10.1038/cddis.2013.321] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2013] [Revised: 07/23/2013] [Accepted: 07/29/2013] [Indexed: 12/29/2022]
Abstract
The ING family of tumor suppressors acts as readers and writers of the histone epigenetic code, affecting DNA repair, chromatin remodeling, cellular senescence, cell cycle regulation and apoptosis. The best characterized member of the ING family, ING1,interacts with the proliferating cell nuclear antigen (PCNA) in a UV-inducible manner. ING1 also interacts with members of the14-3-3 family leading to its cytoplasmic relocalization. Overexpression of ING1 enhances expression of the Bax gene and was reported to alter mitochondrial membrane potential in a p53-dependent manner. Here we show that ING1 translocates to the mitochondria of primary fibroblasts and established epithelial cell lines in response to apoptosis inducing stimuli, independent of the cellular p53 status. The ability of ING1 to induce apoptosis in various breast cancer cell lines correlates well with its degree of translocation to the mitochondria after UV treatment. Endogenous ING1 protein specifically interacts with the pro-apoptotic BCL2 family member BAX, and colocalizes with BAX in a UV-inducible manner. Ectopic expression of a mitochondria-targeted ING1 construct is more proficient in inducing apoptosis than the wild type ING1 protein. Bioinformatic analysis of the yeast interactome indicates that yeast ING proteins interact with 64 mitochondrial proteins. Also, sequence analysis of ING1 reveals the presence of a BH3-like domain. These data suggest a model in which stress-induced cytoplasmic relocalization of ING1 by14-3-3 induces ING1-BAX interaction to promote mitochondrial membrane permeability and represent a paradigm shift in our understanding of ING1 function in the cytoplasm and its contribution to apoptosis [corrected].
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Affiliation(s)
- P Bose
- Department of Biochemistry and Molecular Biology, Southern Alberta Cancer Research Institute, University of Calgary, Calgary, Alberta, Canada
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Abstract
The INhibitor of Growth 1 (ING1) is stoichiometric member of histone deacetylase (HDAC) complexes and functions as an epigenetic regulator and a type II tumor suppressor. It impacts cell growth, aging, apoptosis, and DNA repair, by affecting chromatin conformation and gene expression. Down regulation and mislocalization of ING1 have been reported in diverse tumor types and Ser/Thr phosphorylation has been implicated in both of these processes. Here we demonstrate that both in vitro and in vivo, the tyrosine kinase Src is able to physically associate with, and phosphorylate ING1, which results in a nuclear to cytoplasmic relocalization of ING1 in cells and a decrease of ING1 stability. Functionally, Src antagonizes the ability of ING1 to induce apoptosis, most likely through relocalization of ING1 and down regulation of ING1 levels. These effects were due to both kinase-dependent and kinase-independent properties of Src, and were most apparent at elevated levels of Src expression. These findings suggest that Src may play a major role in regulating ING1 levels during tumorigenesis in those cancers in which high levels of Src expression or activity are present. These data represent the first report of tyrosine kinase-mediated regulation of ING1 levels and suggest that kinase activation can impact chromatin structure through the ING1 epigenetic regulator.
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Helbing CC, Wagner MJ, Pettem K, Johnston J, Heimeier RA, Veldhoen N, Jirik FR, Shi YB, Browder LW. Modulation of thyroid hormone-dependent gene expression in Xenopus laevis by INhibitor of Growth (ING) proteins. PLoS One 2011; 6:e28658. [PMID: 22163049 PMCID: PMC3230625 DOI: 10.1371/journal.pone.0028658] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2011] [Accepted: 11/12/2011] [Indexed: 11/18/2022] Open
Abstract
BACKGROUND INhibitor of Growth (ING) proteins belong to a large family of plant homeodomain finger-containing proteins important in epigenetic regulation and carcinogenesis. We have previously shown that ING1 and ING2 expression is regulated by thyroid hormone (TH) during metamorphosis of the Xenopus laevis tadpole. The present study investigates the possibility that ING proteins modulate TH action. METHODOLOGY/PRINCIPAL FINDINGS Tadpoles expressing a Xenopus ING2 transgene (Trans(ING2)) were significantly smaller than tadpoles not expressing the transgene (Trans(GFP)). When exposed to 10 nM 3,5,3'-triiodothyronine (T(3)), premetamorphic Trans(ING2) tadpoles exhibited a greater reduction in tail, head, and brain areas, and a protrusion of the lower jaw than T(3)-treated Trans(GFP) tadpoles. Quantitative real time polymerase chain reaction (QPCR) demonstrated elevated TH receptor β (TRβ) and TH/bZIP transcript levels in Trans(ING2) tadpole tails compared to Trans(GFP) tadpoles while TRα mRNAs were unaffected. In contrast, no difference in TRα, TRβ or insulin-like growth factor (IGF2) mRNA abundance was observed in the brain between Trans(ING2) and Trans(GFP) tadpoles. All of these transcripts, except for TRα mRNA in the brain, were inducible by the hormone in both tissues. Oocyte transcription assays indicated that ING proteins enhanced TR-dependent, T(3)-induced TRβ gene promoter activity. Examination of endogenous T(3)-responsive promoters (TRβ and TH/bZIP) in the tail by chromatin immunoprecipitation assays showed that ING proteins were recruited to TRE-containing regions in T(3)-dependent and independent ways, respectively. Moreover, ING and TR proteins coimmunoprecipitated from tail protein homogenates derived from metamorphic climax animals. CONCLUSIONS/SIGNIFICANCE We show for the first time that ING proteins modulate TH-dependent responses, thus revealing a novel role for ING proteins in hormone signaling. This has important implications for understanding hormone influenced disease states and suggests that the induction of ING proteins may facilitate TR function during metamorphosis in a tissue-specific manner.
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Affiliation(s)
- Caren C. Helbing
- Department of Biochemistry & Microbiology, University of Victoria, Victoria, British Columbia, Canada
- * E-mail:
| | - Mary J. Wagner
- Department of Biochemistry & Microbiology, University of Victoria, Victoria, British Columbia, Canada
| | - Katherine Pettem
- Department of Biochemistry & Microbiology, University of Victoria, Victoria, British Columbia, Canada
| | - Jill Johnston
- Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada
| | - Rachel A. Heimeier
- Section on Molecular Morphogenesis, Laboratory of Gene Regulation and Development, Program on Cell Regulation and Metabolism, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Nik Veldhoen
- Department of Biochemistry & Microbiology, University of Victoria, Victoria, British Columbia, Canada
| | - Frank R. Jirik
- Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada
- The McCaig Institute for Bone and Joint Health, University of Calgary, Calgary, Alberta, Canada
| | - Yun-Bo Shi
- Section on Molecular Morphogenesis, Laboratory of Gene Regulation and Development, Program on Cell Regulation and Metabolism, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Leon W. Browder
- Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada
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Suzuki K, Boland D, Gong W, Riabowol K. Domain recognition of the ING1 tumor suppressor by a panel of monoclonal antibodies. Hybridoma (Larchmt) 2011; 30:239-45. [PMID: 21707358 DOI: 10.1089/hyb.2010.0124] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
The inhibitor of growth (ING) family of proteins play key roles in cell cycle arrest, apoptosis, cell aging, and the DNA damage response. To date, several domains including the plant homeodomain (PHD), lamin interacting domain (LID), and nuclear localization sequence (NLS) have been identified in the ING family of proteins that contribute to their function. To better understand the functional attributes of the ING proteins, we have developed and further characterized a panel of monoclonal IgGs that we call CAbs 1-9 based on their recognition sites, strength of binding affinity, and their specificity for ING1. All of the nine CAbs recognize the C-terminal half of the p33(ING1b) protein, which is fully conserved among all ING1 isoforms, being encoded by a common exon. Two of the nine CAbs bind a fragment that includes the PHD, which is the most conserved domain among ING family proteins (ING1-5), and one CAb cross-reacts with all ING family proteins that are encoded by different genes. Five of the nine CAbs recognized a fragment of ING1, which includes the NLS. Another two, CAb3 and CAb9, show affinity against an inter-domain sequence between the LID and the NLS. The sequence between the LID and NLS is less conserved among the ING proteins and, as expected, CAbs 3 and 9 were completely specific for ING1. Understanding the domains recognized by the different CAbs should further the functional analysis of the ING proteins that are known to participate in a wide variety of protein complexes, both in the cytoplasm and in the nucleus where they bind epigenetic histone marks via their PHD regions and lamin A via their LID domains.
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Affiliation(s)
- Keiko Suzuki
- Department of Biochemistry & Molecular Biology, University of Calgary, Alberta, Canada
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Soliman MA, Berardi P, Pastyryeva S, Bonnefin P, Feng X, Colina A, Young D, Riabowol K. ING1a expression increases during replicative senescence and induces a senescent phenotype. Aging Cell 2008; 7:783-94. [PMID: 18691180 DOI: 10.1111/j.1474-9726.2008.00427.x] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
Abstract
The ING family of tumor suppressor proteins affects cell growth, apoptosis and response to DNA damage by modulating chromatin structure through association with different HAT and HDAC complexes. The major splicing isoforms of the ING1 locus are ING1a and INGlb. While INGlb plays a role in inducing apoptosis, the function of ING1a is currently unknown. Here we show that alternative splicing of the ING1 message alters the INGla:INGlb ratio by approximately 30-fold in senescent compared to low passage primary fibroblasts. INGla antagonizes INGlb function in apoptosis, induces the formation of structures resembling senescence-associated heterochromatic foci containing heterochromatin protein 1 gamma, the accumulation of senescence-associated beta-galactosidase activity and promotes senescent cell morphology and cell cycle arrest. Phenotypic effects may result from differential effects on gene expression since ING1a increases levels of both retinoblastoma and the p16 cyclin-dependent kinase inhibitor and ING1a and ING1b have opposite effects on the expression of proliferating nuclear cell antigen (PCNA), which is required for cell growth. Gene expression appears to be altered by targeting of HDAC complexes to gene promoters since INGla associates with several-fold higher levels of HDAC1 in senescent, compared to replication-competent cells and ING1 is found on the PCNA promoter by chromatin immunoprecipitation analysis. These data demonstrate a novel role for the ING1 proteins in differentially regulating senescence-associated chromatin remodeling vs. apoptosis and support the idea that altered ratios of the ING1 splicing isoforms may contribute to establishing the senescent phenotype through HDAC and HAT complex-mediated effects on chromatin structure.
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Affiliation(s)
- Mohamed A Soliman
- Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada
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Russell MW, Soliman MA, Schriemer D, Riabowol K. ING1 protein targeting to the nucleus by karyopherins is necessary for activation of p21. Biochem Biophys Res Commun 2008; 374:490-5. [PMID: 18655775 DOI: 10.1016/j.bbrc.2008.07.076] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2008] [Accepted: 07/11/2008] [Indexed: 12/21/2022]
Abstract
ING1 proteins affect apoptosis, growth, and DNA repair by binding histones and regulating chromatin structure and gene expression. ING1 is downregulated in cancers and cytoplasmic localization is associated with poor prognosis. Here, we report that ING1b interacts with karyopherins alpha2 and beta1 through several basic nuclear localization sequences (NLS) located adjacent to the ING1b PHD region. Deletion of NLS motifs resulted in failure of ING1b to completely localize to the nucleus and inhibited its ability to induce p21WAF1 expression. These observations support a general mechanism by which ING1b activity is regulated, in part, through dynamic subcellular partitioning between the nucleus and cytoplasm.
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Affiliation(s)
- Michael W Russell
- Department of Biochemistry & Molecular Biology, Faculty of Medicine, University of Calgary, 311 HMRB, 3330 Hospital Dr. NW, Calgary, Alta., Canada T2N 4N1
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Tallen UG, Truss M, Kunitz F, Wellmann S, Unryn B, Sinn B, Lass U, Krabbe S, Holtkamp N, Hagemeier C, Wurm R, Henze G, Riabowol KT, von Deimling A. Down-regulation of the inhibitor of growth 1 (ING1) tumor suppressor sensitizes p53-deficient glioblastoma cells to cisplatin-induced cell death. J Neurooncol 2007; 86:23-30. [PMID: 17763999 DOI: 10.1007/s11060-007-9436-x] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2007] [Accepted: 06/11/2007] [Indexed: 12/21/2022]
Abstract
Impaired tumor suppressor functions, such as deficient p53, are characteristic for glioblastoma multiforme (GBM) and can cause resistance to DNA-damaging agents like cisplatin. We have recently shown that the INhibitor of Growth 1 (ING1) tumor suppressor is down-regulated in malignant gliomas and that the decrease of ING1 expression correlates with histological grade of malignancy, suggesting a role for ING1 in the pathogenesis and progression of malignant gliomas. Based on this background, the purpose of our current study was to examine the potential impact of ING1 protein levels on DNA-damage response in GBM. Using LN229 GBM cells, which express ING1 proteins and harbor mutant TP53, we are the first to show that DNA damage by cisplatin or ionizing radiation differentially induced the two major ING1 splicing isoforms. The p47 ING1a isoform, that promotes deacetylation of histones, thus formation of heterochromatic regions of DNA, which are less susceptible to DNA damage, was preferentially induced by >50-fold. This might represent a response to protect DNA from damage. Also, ING1 knockdown by siRNA accelerated transit of cells through G1 phase, consistent with ING1 serving a tumor suppressor function, and caused cells to enter apoptosis more rapidly in response to cisplatin. Our results indicate that malignant gliomas may down-regulate ING1 to allow more efficient tumor growth and progression. Also, ING1 down-regulation may sensitize GBM cells with deficient p53 to treatment with cisplatin.
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Affiliation(s)
- Ute Gesche Tallen
- Department of Pediatric Oncology and Hematology, Children's Hospital, Charité, Universitätsmedizin-Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, 13353, Berlin, Germany.
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Gong W, Russell M, Suzuki K, Riabowol K. Subcellular targeting of p33ING1b by phosphorylation-dependent 14-3-3 binding regulates p21WAF1 expression. Mol Cell Biol 2006; 26:2947-54. [PMID: 16581770 PMCID: PMC1446971 DOI: 10.1128/mcb.26.8.2947-2954.2006] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2005] [Revised: 10/05/2005] [Accepted: 12/30/2005] [Indexed: 02/05/2023] Open
Abstract
ING1 is a type II tumor suppressor that affects cell growth, stress signaling, apoptosis, and DNA repair by altering chromatin structure and regulating transcription. Decreased ING1 expression is seen in several human cancers, and mislocalization has been noted in diverse types of cancer cells. Aberrant targeting may, therefore, functionally inactivate ING1. Bioinformatics analysis identified a sequence between the nuclear localization sequence and plant homeodomain domains of ING1 that closely matched the binding motif of 14-3-3 proteins that target cargo proteins to specific subcellular locales. We find that the widely expressed p33(ING1b) splicing isoform of ING1 interacts with members of the 14-3-3 family of proteins and that this interaction is regulated by the phosphorylation status of ING1. 14-3-3 binding resulted in significant amounts of p33(ING1b) protein being tethered in the cytoplasm. As shown previously, ectopic expression of p33(ING1b) increased levels of the p21(Waf1) cyclin-dependent kinase inhibitor upon UV-induced DNA damage. Overexpression of 14-3-3 inhibited the up-regulation of p21(Waf1) by p33(ING1b), consistent with the idea that mislocalization blocks at least one of ING1's biological activities. These data support the idea that the 14-3-3 proteins play a crucial role in regulating the activity of p33(ING1b) by directing its subcellular localization.
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Affiliation(s)
- Wei Gong
- Southern Alberta Cancer Research Institute, Dept. of Biochemistry, University of Calgary, #370 Heritage Medical Research Building, 3330 Hospital Drive NW, Calgary, Alberta T2N 4N1, Canada
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Zhu JJ, Li FB, Zhu XF, Liao WM. The p33ING1b tumor suppressor cooperates with p53 to induce apoptosis in response to etoposide in human osteosarcoma cells. Life Sci 2006; 78:1469-77. [PMID: 16325212 DOI: 10.1016/j.lfs.2005.07.044] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2005] [Accepted: 07/12/2005] [Indexed: 02/03/2023]
Abstract
p33ING1b induces cell cycle arrest and stimulates DNA repair, apoptosis and chemosensitivity. The magnitude of some p33ING1b effects may be due to activation of the tumor suppressor p53. To investigate if the p33ING1b protein affected chemosensitivity of osteosarcoma cells, we overexpressed p33ING1b in p53+/+ U2OS cells or in p53-mutant MG63 cells, and then assessed for growth arrest and apoptosis after treatment with etoposide. p33ING1b increased etoposide-induced growth inhibition and apoptosis to a much greater degree in p53+/+ U2OS cells than in p53-mutant MG63 cells. Moreover, ectopic expression of p33ING1b markedly upregulated p53, p21WAF1 and bax protein levels and activated caspase-3 protein kinase in etoposide-treated U2OS cells. Together, our data indicate that p33ING1b prominently enhances etoposide-induced apoptosis through p53-dependent pathways in human osteosarcoma cells. p33ING1b may be an important marker and/or therapeutic target in the prevention and treatment of metastatic osteosarcoma.
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Affiliation(s)
- Jin-Jun Zhu
- Department of Orthopedic Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510080, China
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12
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Zhu Z, Lin J, Qu JH, Feitelson MA, Ni CR, Li FM, Zhu MH. Inhibitory effect of tumor suppressor p33 ING1b and its synergy with p53 gene in hepatocellular carcinoma. World J Gastroenterol 2005; 11:1903-9. [PMID: 15800978 PMCID: PMC4305709 DOI: 10.3748/wjg.v11.i13.1903] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the inhibitory effect of tumor suppressor p33ING1b and its synergy with p53 gene in hepatocellular carcinoma (HCC).
METHODS: Recombinant sense and antisense p33ING1b plasmids were transfected into hepatoma cell line HepG2 with lipofectamine. Apoptosis, G0/G1 arrest, cell growth rate and cloning efficiency in soft agar of HepG2 were analyzed after transfection. In three hepatoma cell lines with different endogenous p53 gene expressions, the synergistic effect of p33ING1b with p53 was analyzed by flow cytometry and luciferase assay was performed to detect the activation of p53 downstream gene p21WAF1/CIP1. In addition, the expression and mutation rates of p33ING1b in HCC tissues were measured by immunohistochemistry and polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP).
RESULTS: Overexpression of p33ING1b inhibited cell growth of HepG2, induced more apoptosis and protected cells from growth in soft agar. Combined transfer of p33ING1b and p53 gene promoted hepatoma cell apoptosis, G0/G1 arrest and elevated expression of p21WAF1/CIP1. Immunostaining results showed co-localized p33ING1b with P53 protein in HCC tissues and there was a significant relation between protein expression rates of these two genes (P<0.01). Among 28 HCC samples, p33ING1b presented a low gene mutation rate (7.1%).
CONCLUSION: p33ING1b collaborates with p53 in cell growth inhibition, cell cycle arrest and apoptosis in HCC. Loss or inactivation of p33ING1b normal function may be an important mechanism for the development of HCC retaining wild-type p53.
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Affiliation(s)
- Zhi Zhu
- Department of Pathology, Changhai Hospital, Second Military Medical University, 174 Changhai Road, Shanghai 200433, China.
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Shen DH, Chan KYK, Khoo US, Ngan HYS, Xue WC, Chiu PM, Ip P, Cheung ANY. Epigenetic and genetic alterations of p33 ING1b in ovarian cancer. Carcinogenesis 2005; 26:855-63. [PMID: 15677627 DOI: 10.1093/carcin/bgi011] [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: 11/12/2022] Open
Abstract
p33ING1b is a candidate tumor suppressor gene and a nuclear protein. We investigated whether genetic and epigenetic mechanisms affect p33ING1b expression in ovarian cancer thus contributing toward its pathogenesis. A total of 111 ovarian cancers collected from Beijing and Hong Kong were used for this study. Weak or negative p33ING1b protein expression was demonstrated by immunohistochemistry on tissue microarray in 28/111 cases. Real-time quantitative RT-PCR also showed overall significant reduction of p33ING1b mRNA expression (P = 0.0137), with 53.1% (17/32) cases showing 2- to 5-fold reduction and absence of expression. The reduction of mRNA expression in cancer correlated with decreased p33ING1b protein expression (P < 0.0001). While no p33ING1b mutation was found, allelic loss at the p33ING1b locus was demonstrated in 25% (8/32) cases. The allelic loss profiles also showed statistical significant correlation with reduction of p33ING1b protein and mRNA expression (P = 0.031 and 0.030). Promoter methylation as assessed by methylation specific PCR was found in 23.9% (21/88) cases analyzed. Bisulfite sequencing results confirmed the p33ING1b promoter methylation status of these methylation positive cases. Statistical significant correlation between methylation and mRNA expression (P = 0.006) was demonstrated. Treatment with demethylating drug, 5'-aza-2'-deoxycytidine, resulted in dosage-dependent elevated mRNA expression of p33ING1b in ovarian cancer cell lines. This is the first study reporting epigenetic mechanism regulating the p33ING1b expression. Our findings support that genetic and epigenetic alteration of p33ING1b are likely to contribute towards the pathogenesis of ovarian cancers.
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MESH Headings
- Adenocarcinoma, Clear Cell/genetics
- Adenocarcinoma, Clear Cell/metabolism
- Adenocarcinoma, Clear Cell/pathology
- Antimetabolites, Antineoplastic/pharmacology
- Azacitidine/analogs & derivatives
- Azacitidine/pharmacology
- Carcinoma, Endometrioid/genetics
- Carcinoma, Endometrioid/metabolism
- Carcinoma, Endometrioid/pathology
- Case-Control Studies
- Cell Cycle Proteins
- CpG Islands
- Cystadenocarcinoma, Mucinous/genetics
- Cystadenocarcinoma, Mucinous/metabolism
- Cystadenocarcinoma, Mucinous/pathology
- Cystadenocarcinoma, Serous/genetics
- Cystadenocarcinoma, Serous/metabolism
- Cystadenocarcinoma, Serous/pathology
- DNA Methylation
- DNA-Binding Proteins
- Decitabine
- Female
- Gene Expression Regulation, Neoplastic
- Genes, Tumor Suppressor
- Humans
- Immunoenzyme Techniques
- Inhibitor of Growth Protein 1
- Intracellular Signaling Peptides and Proteins
- Loss of Heterozygosity
- Mutation/genetics
- Nuclear Proteins
- Ovarian Neoplasms/genetics
- Ovarian Neoplasms/metabolism
- Ovarian Neoplasms/pathology
- Ovary/metabolism
- Ovary/pathology
- Promoter Regions, Genetic/genetics
- Proteins/genetics
- RNA, Messenger/genetics
- RNA, Messenger/metabolism
- Reverse Transcriptase Polymerase Chain Reaction
- Tumor Cells, Cultured
- Tumor Suppressor Proteins
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Affiliation(s)
- Dan-Hua Shen
- Department of Pathology, People's Hospital, Peking University, Beijing, Republic of China
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14
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Tallen G, Kaiser I, Krabbe S, Lass U, Hartmann C, Henze G, Riabowol K, von Deimling A. NoING1 mutations in human brain tumours but reduced expression in high malignancy grades of astrocytoma. Int J Cancer 2004; 109:476-9. [PMID: 14961591 DOI: 10.1002/ijc.11715] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022]
Abstract
The ING1 family of proteins has been shown to have regulatory functions in oncogenesis, apoptosis, DNA repair and cell cycle regulation. Here we present the first report on LOH analysis of the ING1 locus, mutation analysis of the complete coding sequence including intron-exon boundaries and expression analysis of the different ING1 splice products and protein isoforms in primary brain tumours. No somatic ING1 mutations were detected. Semi-quantitative analysis revealed higher levels of p33ING1b RNA in benign than in malignant lesions. This correlation was significant in a subset of 37 astrocytic tumours WHO grades I to IV. ING1 protein isoforms p47ING1a, p33ING1b and p24ING1c were found to be expressed variably in this series. Our findings support a regulatory contribution of ING1 to the development or progression of brain tumours.
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Affiliation(s)
- Gesche Tallen
- Department of Pediatric Oncology, Charité, Universitätsmedizin Berlin, Berlin, Germany.
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15
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Nouman GS, Anderson JJ, Lunec J, Angus B. The role of the tumour suppressor p33 ING1b in human neoplasia. J Clin Pathol 2003; 56:491-6. [PMID: 12835293 PMCID: PMC1769994 DOI: 10.1136/jcp.56.7.491] [Citation(s) in RCA: 39] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 01/26/2003] [Indexed: 12/25/2022]
Abstract
The inhibitor of growth (ING) genes (ING1-4) probably descend from tumour suppressor genes. ING1 was the first to be identified and later isolated using an approach to detect genes whose expression is suppressed in cancer. The others were isolated through homology and similarity searches in human and mouse databases. All members contain a plant homeodomain involved in macromolecule recognition. Apart from the extensively studied ING1, little is known about the number of transcripts encoded by the other members or their gene structure. ING1 encodes several differentially spliced mRNAs, which may produce a family of proteins. The most widely expressed protein isoform is p33(INGb1), which is involved in restriction of cell growth and proliferation, apoptosis, tumour anchorage independent growth, cellular senescence, maintenance of genomic stability, and modulation of cell cycle checkpoints. ING1 gene mutation is uncommon in cancer, although the subcellular localisation of p33(INGb1) may have an effect on its function. The p33(INGb1) cellular compartmental shift from the nucleus to the cytoplasm may cause loss of normal cellular function, and may play a central role in the pathogenesis of several cancers.
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Affiliation(s)
- G S Nouman
- Pathology Department, Royal Victoria Infirmary, Newcastle upon Tyne NE1 4PH, UK.
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16
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Abstract
The ING family of proteins are involved in chromatin remodelling, and bind to and affect the activity of histone acetyltransferase, histone deacetylase, and factor acetyltransferase protein complexes. Some family members affect transcription, including the expression of p53-inducible genes such as p21 and Bax, and ING2 induces p53 acetylation on a site implicated in the regulation of p53 activity. ING1 promotes DNA repair and interacts with proliferating cell nuclear antigen, thus linking DNA repair, apoptosis and chromatin remodelling. Here, we summarize what is known about the molecular interactions of ING1 family proteins and, based on these interactions, develop a model to better understand the impact of ING proteins on multiple biological processes.
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Affiliation(s)
- Xiaolan Feng
- Department of Biochemistry and Molecular Biology, Faculty of Medicine, The University of Calgary, 3330 Hospital Drive, NW, Calgary, Alberta, Canada T2N 4N1
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17
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Vieyra D, Loewith R, Scott M, Bonnefin P, Boisvert FM, Cheema P, Pastyryeva S, Meijer M, Johnston RN, Bazett-Jones DP, McMahon S, Cole MD, Young D, Riabowol K. Human ING1 proteins differentially regulate histone acetylation. J Biol Chem 2002; 277:29832-9. [PMID: 12015309 DOI: 10.1074/jbc.m200197200] [Citation(s) in RCA: 85] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023] Open
Abstract
ING1 proteins are nuclear, growth inhibitory, and regulate apoptosis in different experimental systems. Here we show that similar to their yeast homologs, human ING1 proteins interact with proteins associated with histone acetyltransferase (HAT) activity, such as TRRAP, PCAF, CBP, and p300. Human ING1 immunocomplexes contain HAT activity, and overexpression of p33(ING1b), but not of p47(ING1a), induces hyperacetylation of histones H3 and H4, in vitro and in vivo at the single cell level. p47(ING1a) inhibits histone acetylation in vitro and in vivo and binds the histone deacetylase HDAC1. Finally, we present evidence indicating that p33(ING1b) affects the degree of physical association between proliferating cell nuclear antigen (PCNA) and p300, an association that has been proposed to link DNA repair to chromatin remodeling. Together with the finding that human ING1 proteins bind PCNA in a DNA damage-dependent manner, these data suggest that ING1 proteins provide a direct linkage between DNA repair, apoptosis, and chromatin remodeling via multiple HAT.ING1.PCNA protein complexes.
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Affiliation(s)
- Diego Vieyra
- Department of Biochemistry, University of Calgary, Calgary, Alberta T2N 4N1, Canada
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18
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Scott M, Bonnefin P, Vieyra D, Boisvert FM, Young D, Bazett-Jones DP, Riabowol K. UV-induced binding of ING1 to PCNA regulates the induction of apoptosis. J Cell Sci 2001; 114:3455-62. [PMID: 11682605 DOI: 10.1242/jcs.114.19.3455] [Citation(s) in RCA: 106] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Previous studies have shown that UV-induced binding of p21WAF1 to PCNA through the PCNA-interacting protein (PIP) domain in p21WAF1 promotes a switch from DNA replication to DNA repair by altering the PCNA protein complex. Here we show that the p33ING1b isoform of the ING1 candidate tumour suppressor contains a PIP domain. UV rapidly induces p33ING1b to bind PCNA competitively through this domain, a motif also found in DNA ligase, the DNA repair-associated FEN1 and XPG exo/endonucleases, and DNA methyltransferase. Interaction of p33ING1b with PCNA occurs between a significant proportion of ING1 and PCNA, increases more than tenfold in response to UV and is specifically inhibited by overexpression of p21WAF1, but not by p16MTS1, which has no PIP sequence. In contrast to wild-type p33ING1b, ING1 PIP mutants that do not bind PCNA do not induce apoptosis, but protect cells from UV-induced apoptosis, suggesting a role for this PCNA-p33ING1b interaction in eliminating UV-damaged cells through programmed cell death. These data indicate that ING1 competitively binds PCNA through a site used by growth regulatory and DNA damage proteins, and may contribute to regulating the switch from DNA replication to DNA repair by altering the composition of the PCNA protein complex.
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Affiliation(s)
- M Scott
- Department of Biochemistry, Faculty of Medicine, The University of Calgary, 3330 Hospital Drive, NW, Calgary, Alberta T2N 4N1, Canada
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Scott M, Boisvert FM, Vieyra D, Johnston RN, Bazett-Jones DP, Riabowol K. UV induces nucleolar translocation of ING1 through two distinct nucleolar targeting sequences. Nucleic Acids Res 2001; 29:2052-8. [PMID: 11353074 PMCID: PMC55466 DOI: 10.1093/nar/29.10.2052] [Citation(s) in RCA: 83] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
The ING1 candidate tumor suppressor is downregulated in a variety of primary tumors and established cancer cell lines. Blocking its expression experimentally promotes unregulated growth in vitro and in vivo, using cell and animal models. Alternative splicing products encode proteins that localize to the nucleus, inhibit cell cycle progression and affect apoptosis in different model systems. Here we show that ING1 proteins translocate to the nucleolus 12-48 h after UV-induced DNA damage. When a small 50 amino acid portion of ING1 was fused to green fluorescent protein, the fusion protein was efficiently targeted to the nucleolus, indicating that ING1 possesses an intrinsic nucleolar targeting sequence (NTS). We mapped this activity to two distinct 4 amino acid regions, which individually direct fused heterologous proteins to the nucleolus. Overexpression of ING1 induced apoptosis of primary fibroblasts in the presence and absence of UV exposure. In contrast, NTS mutants of ING1 that were not targeted to the nucleolus did not efficiently induce apoptosis when overexpressed and instead protected cells from UV-induced apoptosis. Taken together, these results indicate that UV induces ING1 to translocate to the nucleolus and that this translocation may facilitate apoptosis.
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Affiliation(s)
- M Scott
- Department of Biochemistry and Molecular Biology, Faculty of Medicine, The University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta T2N 4N1, Canada
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