224 results on '"Marshall S. Horwitz"'
Search Results
2. Acute lymphoblastic leukemia in a child with Leri-Weill syndrome and complete SHOX gene deletion: A Case Report
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Jana Volejnikova, Jirina Zapletalova, Marie Jarosova, Helena Urbankova, Petr Vojta, Eva Klaskova, Marshall S. Horwitz, Marian Hajduch, and Vladimir Mihal
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acute lymphoblastic leukemia (all) ,childhood ,leri-weill syndrome (lws) ,pseudoautosomal region (par1) ,shox gene ,Medicine - Abstract
Background: Leri-Weill syndrome (LWS) ranks among conditions with short stature homeobox gene (SHOX) haploinsufficiency. Data on possible association of SHOX aberrations with malignant diseases are scarce. Methods and Results: We report a unique case of an 8-year-old girl who was successfully treated for acute lymphoblastic leukemia (pre-B ALL, intermediate risk) and was subsequently diagnosed with LWS due to characteristic clinical appearance (short disproportionate stature, Madelung deformity of the wrist) and molecular genetic examination (complete deletion of SHOX). An identical SHOX deletion was identified also in the patient's mother. Leukemic cells of the patient were retrospectively examined by array comparative genomic hybridization (aCGH), which revealed five regions of deletions at chromosome X, including the SHOX gene locus. Conclusion: Growth retardation in children with hemato-oncologic malignancies cannot always be attributed to cytotoxic treatment and should be carefully evaluated, especially with regards to growth hormone therapy.
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- 2018
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3. Use of Green Fluorescent Protein in Studies of Apoptosis of Transfected Cells
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Yongan Li and Marshall S. Horwitz
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Biology (General) ,QH301-705.5 - Published
- 1997
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4. Simultaneous brain cell type and lineage determined by scRNA-seq reveals stereotyped cortical development
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Donovan J. Anderson, Florian M. Pauler, Aaron McKenna, Jay Shendure, Simon Hippenmeyer, and Marshall S. Horwitz
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Mice ,Histology ,Neurogenesis ,Animals ,Brain ,Loss of Heterozygosity ,Cell Biology ,Single-Cell Analysis ,Article ,Pathology and Forensic Medicine ,Retrospective Studies - Abstract
Mutations are acquired frequently, such that each cell's genome inscribes its history of cell divisions. Common genomic alterations involve loss of heterozygosity (LOH). LOH accumulates throughout the genome, offering large encoding capacity for inferring cell lineage. Using only single-cell RNA sequencing (scRNA-seq) of mouse brain cells, we found that LOH events spanning multiple genes are revealed as tracts of monoallelically expressed, constitutionally heterozygous single-nucleotide variants (SNVs). We simultaneously inferred cell lineage and marked developmental time points based on X chromosome inactivation and the total number of LOH events while identifying cell types from gene expression patterns. Our results are consistent with progenitor cells giving rise to multiple cortical cell types through stereotyped expansion and distinct waves of neurogenesis. This type of retrospective analysis could be incorporated into scRNA-seq pipelines and, compared with experimental approaches for determining lineage in model organisms, is applicable where genetic engineering is prohibited, such as humans.
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- 2022
5. Restoring RUNX1 deficiency in RUNX1 familial platelet disorder by inhibiting its degradation
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Jin Dai, Sara Borst, Jean Ann Maguire, Pamela S. Becker, Sylvia Chien, Sioban Keel, Deborah L. French, Michelle C Krutein, Michaela R. DelPriore, Marshall S. Horwitz, Donovan J. Anderson, Matthew R. Hart, Jasmin Jeffery, Paul Gadue, Eirini P. Papapetrou, and Andriana G. Kotini
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Blood Platelets ,Myeloid Neoplasia ,Myeloid ,Chemistry ,Platelet disorder ,Myeloid leukemia ,Hematology ,Protein degradation ,medicine.disease ,Leukemia, Myeloid, Acute ,Leukemia ,chemistry.chemical_compound ,Blood Coagulation Disorders, Inherited ,medicine.anatomical_structure ,RUNX1 ,hemic and lymphatic diseases ,Core Binding Factor Alpha 2 Subunit ,embryonic structures ,medicine ,Cancer research ,Humans ,Blood Platelet Disorders ,Induced pluripotent stem cell - Abstract
RUNX1 familial platelet disorder (RUNX1-FPD) is an autosomal dominant disorder caused by a monoallelic mutation of RUNX1, initially resulting in approximately half-normal RUNX1 activity. Clinical features include thrombocytopenia, platelet functional defects, and a predisposition to leukemia. RUNX1 is rapidly degraded through the ubiquitin-proteasome pathway. Moreover, it may autoregulate its expression. A predicted kinetic property of autoregulatory circuits is that transient perturbations of steady-state levels result in continued maintenance of expression at adjusted levels, even after inhibitors of degradation or inducers of transcription are withdrawn, suggesting that transient inhibition of RUNX1 degradation may have prolonged effects. We hypothesized that pharmacological inhibition of RUNX1 protein degradation could normalize RUNX1 protein levels, restore the number of platelets and their function, and potentially delay or prevent malignant transformation. In this study, we evaluated cell lines, induced pluripotent stem cells derived from patients with RUNX1-FPD, RUNX1-FPD primary bone marrow cells, and acute myeloid leukemia blood cells from patients with RUNX1 mutations. The results showed that, in some circumstances, transient expression of exogenous RUNX1 or inhibition of steps leading to RUNX1 ubiquitylation and proteasomal degradation restored RUNX1 levels, thereby advancing megakaryocytic differentiation in vitro. Thus, drugs retarding RUNX1 proteolytic degradation may represent a therapeutic avenue for treating bleeding complications and preventing leukemia in RUNX1-FPD.
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- 2021
6. Normal peripheral blood neutrophil numbers accompanying ELANE whole gene deletion mutation
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Sioban Keel, Marshall S. Horwitz, and Mercy Y. Laurino
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Male ,0301 basic medicine ,Neutropenia ,Neutrophils ,Mutant ,Biology ,Leukocyte Count ,Young Adult ,03 medical and health sciences ,0302 clinical medicine ,Genome editing ,hemic and lymphatic diseases ,medicine ,Humans ,Genetic Predisposition to Disease ,Allele ,Genetic Association Studies ,Genetics ,Hematology ,Gene deletion ,Gene deletion mutation ,medicine.disease ,Peripheral blood ,Blood Cell Count ,030104 developmental biology ,030220 oncology & carcinogenesis ,Exceptional Case Report ,Chromosome Deletion ,Leukocyte Elastase ,Gene Deletion - Abstract
Key Points The patient reported here, along with collective observations in the literature, suggest that ELANE deletion does not cause neutropenia. Potential therapeutic genome editing involving knockout of the mutant ELANE allele is therefore not expected to produce neutropenia.
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- 2019
7. Simultaneous Identification of Brain Cell Type and Lineage via Single Cell RNA Sequencing
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Donovan J. Anderson, Marshall S. Horwitz, Jay Shendure, Aaron McKenna, Florian M. Pauler, and Simon Hippenmeyer
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Genetics ,Transcriptome ,Loss of heterozygosity ,Cell type ,Lineage (genetic) ,Somatic cell ,Progenitor cell ,Biology ,Gene ,Genome - Abstract
Acquired mutations are sufficiently frequent such that the genome of a single cell offers a record of its history of cell divisions. Among more common somatic genomic alterations are loss of heterozygosity (LOH). Large LOH events are potentially detectable in single cell RNA sequencing (scRNA-seq) datasets as tracts of monoallelic expression for constitutionally heterozygous single nucleotide variants (SNVs) located among contiguous genes. We identified runs of monoallelic expression, consistent with LOH, uniquely distributed throughout the genome in single cell brain cortex transcriptomes of F1 hybrids involving different inbred mouse strains. We then phylogenetically reconstructed single cell lineages and simultaneously identified cell types by corresponding gene expression patterns. Our results are consistent with progenitor cells giving rise to multiple cortical cell types through stereotyped expansion and distinct waves of neurogenesis. Compared to engineered recording systems, LOH events accumulate throughout the genome and across the lifetime of an organism, affording tremendous capacity for encoding lineage information and increasing resolution for later cell divisions. This approach can conceivably be computationally incorporated into scRNA-seq analysis and may be useful for organisms where genetic engineering is prohibitive, such as humans.
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- 2021
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8. Inducible expression of a disease-associated ELANE mutation impairs granulocytic differentiation, without eliciting an unfolded protein response
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Hrishikesh M Mehta, Seth J. Corey, Borwyn Wang, Marshall S. Horwitz, Bhavuk Garg, and Ralph Kamel
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0301 basic medicine ,Neutropenia ,Mutation, Missense ,Apoptosis ,Biology ,Granulocyte ,Biochemistry ,Gene Expression Regulation, Enzymologic ,03 medical and health sciences ,Mice ,Germline mutation ,Myeloblast ,Cell Line, Tumor ,medicine ,Animals ,Congenital Bone Marrow Failure Syndromes ,Humans ,Molecular Biology ,030102 biochemistry & molecular biology ,Endoplasmic reticulum ,Elastase ,Molecular Bases of Disease ,Cell Biology ,CEBPE ,Endoplasmic Reticulum Stress ,030104 developmental biology ,medicine.anatomical_structure ,Amino Acid Substitution ,Neutrophil elastase ,Cancer research ,biology.protein ,Unfolded protein response ,Unfolded Protein Response ,Leukocyte Elastase ,Granulocytes ,Transcription Factors - Abstract
Severe congenital neutropenia (SCN) is characterized by a near absence of neutrophils, rendering individuals with this disorder vulnerable to recurrent life-threatening infections. The majority of SCN cases arise because of germline mutations in the gene elastase, neutrophil-expressed (ELANE) encoding the neutrophil granule serine protease neutrophil elastase. Treatment with a high dose of granulocyte colony-stimulating factor increases neutrophil production and reduces infection risk. How ELANE mutations produce SCN remains unknown. The currently proposed mechanism is that ELANE mutations promote protein misfolding, resulting in endoplasmic reticulum stress and activation of the unfolded protein response (UPR), triggering death of neutrophil precursors and resulting in neutropenia. Here we studied the ELANE mutation p.G185R, often associated with greater clinical severity (e.g. decreased responsiveness to granulocyte colony-stimulating factor and increased leukemogenesis). Using an inducible expression system, we observed that this ELANE mutation diminishes enzymatic activity and granulocytic differentiation without significantly affecting cell proliferation, cell death, or UPR induction in murine myeloblast 32D and human promyelocytic NB4 cells. Impaired differentiation was associated with decreased expression of genes encoding critical hematopoietic transcription factors (Gfi1, Cebpd, Cebpe, and Spi1), cell surface proteins (Csf3r and Gr1), and neutrophil granule proteins (Mpo and Elane). Together, these findings challenge the currently prevailing model that SCN results from mutant ELANE, which triggers endoplasmic reticulum stress, UPR, and apoptosis.
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- 2020
9. SERF1 Is Required for G-CSF Resistance of Start-Codon Mutant ELANE Granulocytic Precursors
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Ramesh C. Nayak, Sana Emberesh, Abhishek Singh, Yarim Lee, Jose A. Cancelas, Marshall S. Horwitz, Theodosia A. Kalfa, Carolyn Lutzko, Lisa Trump, and Ashley M Wellendorf
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Start codon ,Immunology ,Mutant ,Cell Biology ,Hematology ,Biology ,Biochemistry ,Molecular biology - Abstract
Severe congenital neutropenia (SCN) is frequently associated with mutations in the ELANE gene encoding neutrophil elastase (NE), an azurophilic granule protein which represents a major fraction of all protein synthesized by neutrophilic promyelocytes and myelocytes. The reduction of mutant ELANE translation inversely correlates with neutrophil differentiation. Chronic administration of granulocyte colony-stimulating-factor (G-CSF) has been successfully used to treat some of these patients, especially those ones with mutations in C-terminus translating exons-4 and -5. Patients with proximal mutations of ELANE tend to be resistant to G-CSF therapy. The SCN pathobiology is poorly understood due to the lack of efficient disease animal models, and difficulty in obtaining relevant primary cell populations from patients. We have demonstrated that ELANE exon-3 mutations, sensitive to higher doses of G-CSF administration, cause maturation arrest and apoptosis of promyelocyte and myelocyte stages due to mutant NE inducing unfolded protein response (UPR) and endoplasmic reticulum (ER) stress. We have also shown that SCN patients with mutations in ELANE at its translation initiation codon (ELANE-ATG mut) result in synthesis of alternate truncated neutrophil elastase peptides from downstream in-frame ATG due to the presence of internal ribosomal entry sites. However, the mechanisms of SCN pathogenesis associated with mutations at the translation initiation codon of ELANE gene associated with G-CSF resistance remain unclear. We employed a disease-in-a-dish approach by directed hematopoietic and granulopoietic differentiation of gene edited (CRISPR/Cas9) isogenic patient-derived iPSC lines. We used four iPSC lines (healthy donor 28L and its start codon knock-in isogenic 28L-GTG; and patient-derived ATG mut SCN110 and its isogenic corrected line SCN110C) to evaluate if ELANE start codon mutations are both necessary and sufficient to cause disease pathogenesis, and to unravel the underlying molecular mechanisms. We found that myeloid precursors derived from 28-GTG and SCN110 iPSC lines generate low molecular weight truncated NE that is detected by a C-terminal region (C225-H238) specific anti-NE antibody (Ab). The survival, expansion and differentiation of ELANE start codon mutant hematopoietic progenitors were significantly reduced in cytokine supplemented in vitro myelopoiesis cultures, and the correction of the mutation (SCN110C) restored the granulocytic precursor expansion and differentiation. The reduced expansion in 28L-GTG and SCN110 lines was associated with both increased apoptosis and expression of proapoptotic BH3-only proteins in comparison to their isogenic 28L and SCN110C lines. However, this apoptosis was not associated with UPR/ER-stress in ELANE-ATG mut myeloid precursors. Granulopoietic differentiation of the 28L-GTG and SCN110 iPSC derived hematopoietic progenitors was significantly reduced, while it was rescued in corrected isogenic lines. Correlating with their clinical behavior, high-dose G-CSF in vitro did not restore the granulopoietic differentiation of ELANE ATG mutant hematopoietic progenitors. Mechanistically, 28L-GTG and SCN110 myeloid precursors contained high-molecular weight, sodium dodecyl sulfate (SDS) resistant NE bands that were detected by anti-NE Ab specific for the C-terminal (C225-238), but not the N-terminal region (C19-38), suggesting that truncated mutant NE generates insoluble NE aggregates. The association of truncated NE with classical autophagy marker LC3B strongly suggested the activation of an aggrephagy process in these cells. We found that SERF1 (small EDRK-rich factor), an RNA-chaperoning protein, known to localize in misfolded protein aggregates of neurodegenerative diseases, was highly upregulated in GTG knock-in and SCN patient myeloid precursors. SERF1 was distributed in both nucleus and cytoplasm, co-localized and physically interacted with mutant truncated NE in the cytoplasm of ELANE-ATG mut cells as shown by proximity ligation assays. Silencing of SERF1 made the survival and differentiation of 28L-GTG and SCN110 derived myeloid precursors sensitive to G-CSF. ELANE translation initiation mutant was associated with truncated NE aggregates and induced apoptosis mediated by SERF1. This data opens a path for therapeutic intervention of G-CSF resistant ELANEmut SCN. Disclosures Kalfa: Agios Pharmaceuticals, Inc.: Other: Steering Committee, Research Funding; FORMA Therapeutics, Inc: Research Funding. Lutzko: Aruvant Sciences: Patents & Royalties: preclinical vector development. Cancelas: Hemerus LLC: Research Funding; Vascular Solutions Inc.: Research Funding; Westat Inc: Research Funding; Fresenius-Kabi LLC: Research Funding; Cytosorbents Inc: Research Funding; Hemanext: Membership on an entity's Board of Directors or advisory committees, Research Funding; TerumoBCT: Research Funding; Cerus Co: Research Funding; University of South Florida/MEQU Inc: Research Funding.
- Published
- 2021
10. GATA factor mutations in hematologic disease
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John D. Crispino and Marshall S. Horwitz
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0301 basic medicine ,Immunology ,Biology ,medicine.disease_cause ,Biochemistry ,03 medical and health sciences ,Acute megakaryoblastic leukemia ,hemic and lymphatic diseases ,medicine ,Animals ,Humans ,GATA1 Transcription Factor ,Anemia, Diamond-Blackfan ,Mutation ,Review Series ,GATA2 ,Myeloid leukemia ,GATA1 ,Cell Biology ,Hematology ,medicine.disease ,Hematologic Diseases ,Hematopoiesis ,GATA2 Transcription Factor ,Haematopoiesis ,030104 developmental biology ,Hematologic disease ,Congenital dyserythropoietic anemia - Abstract
GATA family proteins play essential roles in development of many cell types, including hematopoietic, cardiac, and endodermal lineages. The first three factors, GATAs 1, 2, and 3, are essential for normal hematopoiesis, and their mutations are responsible for a variety of blood disorders. Acquired and inherited GATA1 mutations contribute to Diamond-Blackfan anemia, acute megakaryoblastic leukemia, transient myeloproliferative disorder, and a group of related congenital dyserythropoietic anemias with thrombocytopenia. Conversely, germ line mutations in GATA2 are associated with GATA2 deficiency syndrome, whereas acquired mutations are seen in myelodysplastic syndrome, acute myeloid leukemia, and in blast crisis transformation of chronic myeloid leukemia. The fact that mutations in these genes are commonly seen in blood disorders underscores their critical roles and highlights the need to develop targeted therapies for transcription factors. This review focuses on hematopoietic disorders that are associated with mutations in two prominent GATA family members, GATA1 and GATA2.
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- 2017
11. RUNX1-mutated families show phenotype heterogeneity and a somatic mutation profile unique to germline predisposed AML
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Andrew Dubowsky, Jinghua Feng, Amanda Wells, Stefan Fröhling, Meryl Altree, Andreas W. Schreiber, Sue Morgan, Lesley Rawlings, Richard J D'Andrea, Anna L. Brown, Chan-Eng Chong, Joëlle Michaud, Andrew H. Wei, Georges Natsoulis, Jeffrey Suttle, Rachel Susman, Cassandra Vakulin, Tilmann Bochtler, Uday R. Popat, Mark S. Currie, Paul Wang, Milena Babic, Ella J Wilkins, Christopher N. Hahn, Miriam Fine, Xiaochun Li, Jessica Burdett, Belinda Mercorella, Catherine Carmichael, Nigel Patton, Denae Henry, Marshall S. Horwitz, Peer Arts, Kerry Phillips, Julian Cooney, Sarah Moore, Sally Mapp, Nicola K. Poplawski, Thuong Ha, Sarah L King-Smith, Louise Jaensch, Shai Izraeli, Devendra K Hiwase, Julia Dobbins, Lucy A. Godley, Cecily Forsyth, Kenneth F. Bradstock, Carolyn M. Butcher, Helen Mar Fan, Grace McKavanagh, Hugh Y. Rienhoff, Hamish S. Scott, Mario Nicola, Elli Papaemmanuil, Ping Cannon, Ian D. Lewis, Claire C. Homan, Peter J. Brautigan, Alwin Krämer, Brown, Anna L, Arts, Peer, Babic, Milena, Dobbins, Julia, Feng, Jinghua, Ha, Thuong, Homan, Claire C, King-Smith, Sarah L, Li, Xiao-Chun, Brautigan, Peter, Butcher, Carolyn, D'Andrea, Richard J, Hahn, Christopher N, and Scott, Hamish S
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Genetics ,Mutation ,Myeloid Neoplasia ,Somatic cell ,Genetic heterogeneity ,Platelet disorder ,Hematology ,Biology ,medicine.disease_cause ,Phenotype ,Germline ,Epigenesis, Genetic ,Pedigree ,Leukemia, Myeloid, Acute ,Germline mutation ,Germ Cells ,hemic and lymphatic diseases ,embryonic structures ,Core Binding Factor Alpha 2 Subunit ,medicine ,Humans ,Allele - Abstract
First reported in 1999, germline runt-related transcription factor 1 (RUNX1) mutations are a well-established cause of familial platelet disorder with predisposition to myeloid malignancy (FPD-MM). We present the clinical phenotypes and genetic mutations detected in 10 novel RUNX1-mutated FPD-MM families. Genomic analyses on these families detected 2 partial gene deletions, 3 novel mutations, and 5 recurrent mutations as the germline RUNX1 alterations leading to FPD-MM. Combining genomic data from the families reported herein with aggregated published data sets resulted in 130 germline RUNX1 families, which allowed us to investigate whether specific germline mutation characteristics (type, location) could explain the large phenotypic heterogeneity between patients with familial platelet disorder and different HMs. Comparing the somatic mutational signatures between the available familial (n = 35) and published sporadic (n = 137) RUNX1-mutated AML patients showed enrichment for somatic mutations affecting the second RUNX1 allele and GATA2. Conversely, we observed a decreased number of somatic mutations affecting NRAS, SRSF2, and DNMT3A and the collective genes associated with CHIP and epigenetic regulation. This is the largest aggregation and analysis of germline RUNX1 mutations performed to date, providing a unique opportunity to examine the factors underlying phenotypic differences and disease progression from FPD to MM.
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- 2019
12. Activating PAX gene family paralogs to complement PAX5 leukemia driver mutations
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Marshall S. Horwitz, Tobias Neff, Matthew R. Hart, Christopher C. Porter, Donovan J. Anderson, and Michael Levin
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0301 basic medicine ,Male ,Cancer Research ,B Cells ,Cellular differentiation ,Paired Box ,Hypertonic Solutions ,Gene Expression ,medicine.disease_cause ,Kidney ,Biochemistry ,Hematologic Cancers and Related Disorders ,White Blood Cells ,Mice ,0302 clinical medicine ,Spectrum Analysis Techniques ,Osmoregulation ,NFAT5 ,immune system diseases ,Animal Cells ,hemic and lymphatic diseases ,Medicine and Health Sciences ,Small interfering RNAs ,RNA, Small Interfering ,Genetics (clinical) ,Mutation ,B-Lymphocytes ,Pax genes ,Cell Differentiation ,Hematology ,Precursor Cell Lymphoblastic Leukemia-Lymphoma ,Acute Lymphoblastic Leukemia ,Flow Cytometry ,3. Good health ,Cell biology ,Up-Regulation ,Nucleic acids ,Oncology ,Spectrophotometry ,030220 oncology & carcinogenesis ,Lymphoblastic Leukemia ,Female ,Cytophotometry ,Cellular Types ,Research Article ,lcsh:QH426-470 ,Immune Cells ,Immunology ,Primary Cell Culture ,Biology ,Research and Analysis Methods ,03 medical and health sciences ,Protein Domains ,Cell Line, Tumor ,Leukemias ,DNA-binding proteins ,medicine ,Genetics ,Animals ,Humans ,Antibody-Producing Cells ,Non-coding RNA ,Molecular Biology ,Ecology, Evolution, Behavior and Systematics ,Blood Cells ,HEK 293 cells ,PAX2 Transcription Factor ,Wild type ,PAX5 Transcription Factor ,Kidney metabolism ,Biology and Life Sciences ,Proteins ,Cancers and Neoplasms ,Cell Biology ,Xenograft Model Antitumor Assays ,Coculture Techniques ,Gene regulation ,Regulatory Proteins ,lcsh:Genetics ,030104 developmental biology ,HEK293 Cells ,RNA ,PAX5 ,Developmental Biology ,Transcription Factors - Abstract
PAX5, one of nine members of the mammalian paired box (PAX) family of transcription factors, plays an important role in B cell development. Approximately one-third of individuals with pre-B acute lymphoblastic leukemia (ALL) acquire heterozygous inactivating mutations of PAX5 in malignant cells, and heterozygous germline loss-of-function PAX5 mutations cause autosomal dominant predisposition to ALL. At least in mice, Pax5 is required for pre-B cell maturation, and leukemic remission occurs when Pax5 expression is restored in a Pax5-deficient mouse model of ALL. Together, these observations indicate that PAX5 deficiency reversibly drives leukemogenesis. PAX5 and its two most closely related paralogs, PAX2 and PAX8, which are not mutated in ALL, exhibit overlapping expression and function redundantly during embryonic development. However, PAX5 alone is expressed in lymphocytes, while PAX2 and PAX8 are predominantly specific to kidney and thyroid, respectively. We show that forced expression of PAX2 or PAX8 complements PAX5 loss-of-function mutation in ALL cells as determined by modulation of PAX5 target genes, restoration of immunophenotypic and morphological differentiation, and, ultimately, reduction of replicative potential. Activation of PAX5 paralogs, PAX2 or PAX8, ordinarily silenced in lymphocytes, may therefore represent a novel approach for treating PAX5-deficient ALL. In pursuit of this strategy, we took advantage of the fact that, in kidney, PAX2 is upregulated by extracellular hyperosmolarity. We found that hyperosmolarity, at potentially clinically achievable levels, transcriptionally activates endogenous PAX2 in ALL cells via a mechanism dependent on NFAT5, a transcription factor coordinating response to hyperosmolarity. We also found that hyperosmolarity upregulates residual wild type PAX5 expression in ALL cells and modulates gene expression, including in PAX5-mutant primary ALL cells. These findings specifically demonstrate that osmosensing pathways may represent a new therapeutic target for ALL and more broadly point toward the possibility of using gene paralogs to rescue mutations driving cancer and other diseases., Author summary Mutations inactivating PAX5 disrupt B cell differentiation and occur frequently in ALL. Others have previously shown that restoring PAX5 expression normalizes B cell differentiation and leads to disease remission in a mouse model of ALL. We found that exogenous expression of PAX5’s intact and closely related gene family members, PAX2 or PAX8, which are ordinarily silent in lymphocytes but expressed in kidney and other tissues, can substitute for PAX5 and restore differentiation in ALL cells. A new approach for treating ALL might therefore be to discover ways to activate expression of PAX2 or PAX8 in leukemic cells. In the kidney, PAX2 expression is activated by changes in extracellular osmolarity. We found that PAX2 retains the capacity for osmotic activation in ALL cells and that wild type PAX5 expression also increases when ALL cells are osmotically stressed. Adjustment of serum osmolarity—or treatment with drugs targeting pathways responding to osmotic stress—may offer a potential new avenue for ALL therapy by elevating expression of PAX gene family members. More generally, our studies point toward a novel strategy of recruiting paralogs to complement mutations in genes responsible for cancer and other diseases.
- Published
- 2018
13. Inducible Expression of Mutant ELANE Correlates with Disruption of Differentiation Program, but Not the Unfolded Protein Response
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Hrishikesh M Mehta, Seth J. Corey, Marshall S. Horwitz, Ralph Kamel, and Bhavuk Garg
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Cell growth ,Immunology ,Mutant ,Wild type ,Cell Biology ,Hematology ,CEBPE ,Biology ,Biochemistry ,Molecular biology ,Gene expression profiling ,Gene expression ,CEBPA ,CEBPB - Abstract
Background. Severe congenital neutropenia (SCN) is characterized by a critical reduction in absolute neutrophil count (ANC < 500/μl), which renders the affected individual vulnerable to recurrent, life-threatening infections. A majority of SCN cases arise due to germline mutations in the neutrophil elastase gene (ELANE). Treatment with high dose granulocyte colony stimulating factor (GCSF) increases the number of neutrophils and eliminates the risk of infection. SCN patients are at a significantly high risk of developing myelodysplastic syndrome (MDS) or acute myelogenous leukemia (AML). About 70% of the MDS/AML cases report somatic mutations in the GCSF receptor gene (CSF3R), suggesting a correlation between GCSF treatment and development of MDS/AML. The current favored mechanism for ELANE mutations promoting SCN is induction of an unfolded protein response (UPR), triggering death of neutrophil precursors resulting in neutropenia. However multiple studies of UPR activation reported a requirement of treatment with chemical UPR inducers such as tunicamycin or bortezomib to observe significant changes. The mechanism of mutant ELANE-mediated neutrophil deficit thus remains elusive. In addition, the ability of G-CSF to alleviate neutropenia and its role in progression from SCN to MDS/AML is still a confounding factor in G-CSF therapy for SCN cases. Methods.We created a doxycycline inducible expression system of ELANE, consisting of wild type (wt) and point mutation (G185R) variant associated with SCN, in the murine myeloblast cell line 32D and the human promyelocytic leukemia cell line NB4. We determined the effect of mutant ELANE expression on cell proliferation (Trypan blue exclusion), survival (Trypan blue exclusion, AnnexinV-PI staining) and differentiation (Wright-Giemsa staining, CD11b expression). Underlying molecular changes were investigated using immunoblotting and qPCR based gene expression analysis. Results . Induction of wt or mutant ELANEexpression with doxycycline did not have any detrimental effects on cell proliferation and survival. However, a partial block in differentiation with an accumulation of myeloid progenitors was observed in cells expression mutant ELANE. Profiling of gene expression for markers of differentiation identified reduced expression of key transcriptional factors (Cebpa, Cebpe, Cebpb, Cebpd, Gfi1, Spi1) coupled with reduced levels of other markers of differentiation (e.g., Mpo, Ltf). Increased transcript levels were also observed in some cell cycle genes (Pola1, Ccnd1). We did not observe an increase in expression of UPR genes upon mutant ELANEinduction. Conclusion.Our data demonstrate that expression of mutant ELANE promotes an aberrant differentiation gene expression profile that leads to a partial block in differentiation. In addition, our data also suggest that mutant ELANE expression does not promote UPR. We also show a concomitant upregulation of cell proliferation genes upon mutant ELANE expression. This detour from a differentiation profile may indicate pre-leukemic underpinnings for G-CSF treatment in SCN. Disclosures No relevant conflicts of interest to declare.
- Published
- 2019
14. Pathogenesis of ELANE-mutant severe neutropenia revealed by induced pluripotent stem cells
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Jose A. Cancelas, Patrick J. Paddison, H. Leighton Grimes, C. Alexander Valencia, Lisa Trump, Theodosia A. Kalfa, Carolyn Lutzko, Marshall S. Horwitz, Kasiani C. Myers, Bruce J. Aronow, Ashley M Wellendorf, Parinda A. Mehta, and Ramesh C. Nayak
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Male ,Neutropenia ,Neutrophils ,Induced Pluripotent Stem Cells ,Biology ,Granulopoiesis ,Pathogenesis ,Granulocyte Colony-Stimulating Factor ,CEBPA ,CEBPB ,Humans ,Point Mutation ,Granulocyte Precursor Cells ,Induced pluripotent stem cell ,Protein kinase B ,Cells, Cultured ,Myelopoiesis ,CCAAT-Enhancer-Binding Protein-beta ,Genetic Diseases, Inborn ,General Medicine ,Endoplasmic Reticulum Stress ,3. Good health ,Immunology ,CCAAT-Enhancer-Binding Proteins ,Unfolded Protein Response ,Unfolded protein response ,Cancer research ,Female ,Leukocyte Elastase ,Research Article ,Promyelocyte - Abstract
Severe congenital neutropenia (SCN) is often associated with inherited heterozygous point mutations in ELANE, which encodes neutrophil elastase (NE). However, a lack of appropriate models to recapitulate SCN has substantially hampered the understanding of the genetic etiology and pathobiology of this disease. To this end, we generated both normal and SCN patient-derived induced pluripotent stem cells (iPSCs), and performed genome editing and differentiation protocols that recapitulate the major features of granulopoiesis. Pathogenesis of ELANE point mutations was the result of promyelocyte death and differentiation arrest, and was associated with NE mislocalization and activation of the unfolded protein response/ER stress (UPR/ER stress). Similarly, high-dose G-CSF (or downstream signaling through AKT/BCL2) rescues the dysgranulopoietic defect in SCN patient-derived iPSCs through C/EBPβ-dependent emergency granulopoiesis. In contrast, sivelestat, an NE-specific small-molecule inhibitor, corrected dysgranulopoiesis by restoring normal intracellular NE localization in primary granules; ameliorating UPR/ER stress; increasing expression of CEBPA, but not CEBPB; and promoting promyelocyte survival and differentiation. Together, these data suggest that SCN disease pathogenesis includes NE mislocalization, which in turn triggers dysfunctional survival signaling and UPR/ER stress. This paradigm has the potential to be clinically exploited to achieve therapeutic responses using lower doses of G-CSF combined with targeting to correct NE mislocalization.
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- 2015
15. Mechanisms and clinical applications of chromosomal instability in lymphoid malignancy
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Marshall S. Horwitz, Oliver W. Press, Timothy Tidwell, and Maxwell M. Krem
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DNA Replication ,Recombination, Genetic ,DNA Breaks ,Aneuploidy ,Chromosomal translocation ,DNA, Neoplasm ,Hematology ,Biology ,medicine.disease ,medicine.disease_cause ,Telomere ,Lymphoma ,Malignant transformation ,Chromosomal Instability ,Hematologic Neoplasms ,Chromosome instability ,Immunology ,medicine ,Humans ,Carcinogenesis ,Multiple myeloma - Abstract
Lymphocytes are unique among cells in that they undergo programmed DNA breaks and translocations, but that special property predisposes them to chromosomal instability (CIN), a cardinal feature of neoplastic lymphoid cells that manifests as whole chromosome- or translocation-based aneuploidy. In several lymphoid malignancies translocations may be the defining or diagnostic markers of the diseases. CIN is a cornerstone of the mutational architecture supporting lymphoid neoplasia, though it is perhaps one of the least understood components of malignant transformation in terms of its molecular mechanisms. CIN is associated with prognosis and response to treatment, making it a key area for impacting treatment outcomes and predicting prognoses. Here we will review the types and mechanisms of CIN found in Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma and the lymphoid leukaemias, with emphasis placed on pathogenic mutations affecting DNA recombination, replication and repair; telomere function; and mitotic regulation of spindle attachment, centrosome function, and chromosomal segregation. We will discuss the means by which chromosome-level genetic aberrations may give rise to multiple pathogenic mutations required for carcinogenesis and conclude with a discussion of the clinical applications of CIN and aneuploidy to diagnosis, prognosis and therapy.
- Published
- 2015
16. Germline ETV6 mutations in familial thrombocytopenia and hematologic malignancy
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R. Coleman Lindsley, Inga Hofmann, Sioban Keel, Colin C. Pritchard, Scott A. Coats, Tom Walsh, Rafael Márquez, Boglarka Gyurkocza, Mary Claire King, David A. Williams, Barbara Neistadt, Marshall S. Horwitz, Michael Y. Zhang, Melissa Forouhar, Christopher J. Mariani, Keith R. Loeb, Ryan Basom, Jeffrey J. Delrow, Ming K. Lee, Suleyman Gulsuner, Akiko Shimamura, Bradford S Schwartz, Janis L. Abkowitz, Marilyn Sanchez-Bonilla, Lucy A. Godley, and Jane E. Churpek
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Male ,Models, Molecular ,Molecular Sequence Data ,Mutation, Missense ,Biology ,medicine.disease_cause ,Article ,Germline ,Malignant transformation ,Germline mutation ,Genes, Reporter ,Genetics ,medicine ,Humans ,Missense mutation ,Gene ,Germ-Line Mutation ,Cell Proliferation ,Mutation ,Proto-Oncogene Proteins c-ets ,Sequence Analysis, RNA ,Cancer ,Exons ,medicine.disease ,Thrombocytopenia ,Recombinant Proteins ,Pedigree ,Protein Structure, Tertiary ,Repressor Proteins ,ETV6 ,Hematologic Neoplasms ,Cancer research ,Female ,HeLa Cells - Abstract
We report germline missense mutations in ETV6 segregating with the dominant transmission of thrombocytopenia and hematologic malignancy in three unrelated kindreds, defining a new hereditary syndrome featuring thrombocytopenia with susceptibility to diverse hematologic neoplasms. Two variants, p.Arg369Gln and p.Arg399Cys, reside in the highly conserved ETS DNA-binding domain. The third variant, p.Pro214Leu, lies within the internal linker domain, which regulates DNA binding. These three amino acid sites correspond to hotspots for recurrent somatic mutation in malignancies. Functional studies show that the mutations abrogate DNA binding, alter subcellular localization, decrease transcriptional repression in a dominant-negative fashion and impair hematopoiesis. These familial genetic studies identify a central role for ETV6 in hematopoiesis and malignant transformation. The identification of germline predisposition to cytopenias and cancer informs the diagnosis and medical management of at-risk individuals.
- Published
- 2015
17. Case Report of an Adolescent Male With Unexplained Pancytopenia
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Michelle van Hee, Sioban Keel, Rofida Nofal, Hadi Sawaf, Allison Jay, Marshall S. Horwitz, Roshini S. Abraham, Adonis Lorenzana, and Lauren Azevedo
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0301 basic medicine ,Pediatrics ,medicine.medical_specialty ,medicine.diagnostic_test ,business.industry ,Brief Report ,GATA2 ,MEDLINE ,Bone marrow failure ,medicine.disease ,Pancytopenia ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Pediatrics, Perinatology and Child Health ,Medicine ,business ,030215 immunology ,Genetic testing - Published
- 2017
18. Prolonged pharmacological inhibition of cathepsin C results in elimination of neutrophil serine proteases
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Erhan Firatli, Cécile Croix, Sandrine Dallet-Choisy, Niels Borregaard, Marshall S. Horwitz, John Pedersen, Brice Korkmaz, Marie-Claude Viaud-Massuard, Nathalie Heuzé-Vourc'h, Francis Gauthier, Conni Lauritzen, Sylvain Marchand-Adam, Anne Sophie Lamort, Yveline Hamon, Thomas Baranek, Carla Guarino, Mustapha Si-Tahar, Adam Lesner, and Dieter E. Jenne
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0301 basic medicine ,Proteases ,Neutrophils ,medicine.medical_treatment ,Biology ,Cysteine Proteinase Inhibitors ,Biochemistry ,Cathepsin C ,Serine ,03 medical and health sciences ,0302 clinical medicine ,Papillon-Lefevre Disease ,Proteinase 3 ,medicine ,Animals ,Humans ,Pharmacology ,Serine protease ,Protease ,Elastase ,Cysteine protease ,3. Good health ,Macaca fascicularis ,030104 developmental biology ,030220 oncology & carcinogenesis ,Case-Control Studies ,biology.protein ,Female ,Serine Proteases ,Leukocyte Elastase ,Bronchoalveolar Lavage Fluid - Abstract
Cathepsin C (CatC) is a tetrameric cysteine dipeptidyl aminopeptidase that plays a key role in activation of pro-inflammatory serine protease zymogens by removal of a N-terminal pro-dipeptide sequence. Loss of function mutations in the CatC gene is associated with lack of immune cell serine protease activities and cause Papillon-Lefevre syndrome (PLS). Also, only very low levels of elastase-like protease zymogens are detected by proteome analysis of neutrophils from PLS patients. Thus, CatC inhibitors represent new alternatives for the treatment of neutrophil protease-driven inflammatory or autoimmune diseases. We aimed to experimentally inactivate and lower neutrophil elastase-like proteases by pharmacological blocking of CatC-dependent maturation in cell-based assays and in vivo. Isolated, immature bone marrow cells from healthy donors pulse-chased in the presence of a new cell permeable cyclopropyl nitrile CatC inhibitor almost totally lack elastase. We confirmed the elimination of neutrophil elastase-like proteases by prolonged inhibition of CatC in a non-human primate. We also showed that neutrophils lacking elastase-like protease activities were still recruited to inflammatory sites. These preclinical results demonstrate that the disappearance of neutrophil elastase-like proteases as observed in PLS patients can be achieved by pharmacological inhibition of bone marrow CatC. Such a transitory inhibition of CatC might thus help to rebalance the protease load during chronic inflammatory diseases, which opens new perspectives for therapeutic applications in humans.
- Published
- 2017
19. Targeted correction of RUNX1 mutation in FPD patient-specific induced pluripotent stem cells rescues megakaryopoietic defects
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Abdel G. Elkahloun, Marshall S. Horwitz, Jon P Connelly, Niraj S. Trivedi, Linzhao Cheng, Erika M. Kwon, Yongxing Gao, and Pengfei Liu
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Myeloid ,Platelet disorder ,Induced Pluripotent Stem Cells ,Immunology ,Mutation, Missense ,Biology ,Biochemistry ,Thrombopoiesis ,Mice ,chemistry.chemical_compound ,Blood Coagulation Disorders, Inherited ,hemic and lymphatic diseases ,medicine ,Animals ,Humans ,Induced pluripotent stem cell ,Gene Expression Profiling ,Autosomal dominant trait ,Myeloid leukemia ,Cell Biology ,Hematology ,Platelets and Thrombopoiesis ,medicine.disease ,Leukemia, Myeloid, Acute ,Leukemia ,Haematopoiesis ,medicine.anatomical_structure ,RUNX1 ,chemistry ,embryonic structures ,Core Binding Factor Alpha 2 Subunit ,Blood Platelet Disorders ,Targeted Gene Repair - Abstract
Familial platelet disorder with predisposition to acute myeloid leukemia (FPD/AML) is an autosomal dominant disease of the hematopoietic system that is caused by heterozygous mutations in RUNX1. FPD/AML patients have a bleeding disorder characterized by thrombocytopenia with reduced platelet numbers and functions, and a tendency to develop AML. No suitable animal models exist for FPD/AML, as Runx11/2 mice and zebra fish do not develop bleeding disorders or leukemia. Here we derived induced pluripotent stem cells (iPSCs) from 2 patients in a family with FPD/AML, and found that the FPD iPSCs display defects in megakaryocytic differentiation in vitro. We corrected the RUNX1 mutation in 1 FPD iPSC line through gene targeting, which led to normalization of megakaryopoiesis of the iPSCs in culture. Our results demonstrate successful in vitro modeling of FPD with patient-specific iPSCs and confirm that RUNX1 mutations are responsible for megakaryopoietic defects in FPD patients.
- Published
- 2014
20. The Role Of FIP-2 (optineurin) In Regulation Of The Chemokines And Kinases
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Leonid Tarassishin and Marshall S. Horwitz
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Chemokine ,Microarray ,biology ,Kinase ,Chemistry ,Gene expression ,biology.protein ,Interleukin 8 ,Cell biology ,Optineurin - Published
- 2014
21. Clonal Expansions and Short Telomeres Are Associated with Neoplasia in Early-onset, but not Late-onset, Ulcerative Colitis
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Jesse J. Salk, Peter S. Rabinovitch, David A. Crispin, Cigdem Himmetoglu Ussakli, Lisa A. Lai, Lawrence A. Loeb, Rosa Ana Risques, Aasthaa Bansal, Teresa A. Brentnall, Marshall S. Horwitz, and Mary P. Bronner
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Adult ,Male ,medicine.medical_specialty ,Adolescent ,Colorectal cancer ,Biology ,Polymerase Chain Reaction ,Inflammatory bowel disease ,Gastroenterology ,Article ,Young Adult ,Internal medicine ,medicine ,Humans ,Immunology and Allergy ,Age of Onset ,Child ,Aged ,Aged, 80 and over ,Case-control study ,Cancer ,Middle Aged ,Telomere ,Prognosis ,medicine.disease ,Ulcerative colitis ,Clone Cells ,Dysplasia ,Case-Control Studies ,Colonic Neoplasms ,Mutation ,Poly G ,Immunology ,Disease Progression ,Colitis, Ulcerative ,Female ,Age of onset ,Precancerous Conditions ,Biomarkers ,Follow-Up Studies - Abstract
Background: Patients with ulcerative colitis (UC) are at risk of developing colorectal cancer. We have previously reported that cancer progression is associated with the presence of clonal expansions and shorter telomeres in nondysplastic mucosa. We sought to validate these findings in an independent case–control study. Methods: This study included 33 patients with UC: 14 progressors (patients with high-grade dysplasia or cancer) and 19 nonprogressors. For each patient, a mean of 5 nondysplastic biopsies from proximal, mid, and distal colon were assessed for clonal expansions, as determined by clonal length altering mutations in polyguanine tracts, and telomere length, as measured by quantitative PCR. Both parameters were compared with individual clinicopathological characteristics. Results: Clonal expansions and shorter telomeres were more frequent in nondysplastic biopsies from UC progressors than nonprogressors, but only for patients with early-onset of UC (diagnosis at younger than 50 years of age). Late-onset progressor patients had very few or no clonal expansions and longer telomeres. A few nonprogressors exhibited clonal expansions, which were associated with older age and shorter telomeres. In progressors, clonal expansions were associated with proximity to dysplasia. The mean percentage of clonally expanded mutations distinguished early-onset progressors from nonprogressors with 100% sensitivity and 80% specificity. Conclusions: Early-onset progressors develop cancer in a field of clonally expanded epithelium with shorter telomeres. The detection of these clones in a few random nondysplastic colon biopsies is a promising cancer biomarker in early-onset UC. Curiously, patients with late-onset UC seem to develop cancer without the involvement of such fields. (Inflamm Bowel Dis 2013;19:2593–2602)
- Published
- 2013
22. ELANE Mutations in Cyclic and Severe Congenital Neutropenia
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Seth J. Corey, Timothy Tidwell, Marshall S. Horwitz, and H. Leighton Grimes
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Genetics ,Mutation ,biology ,Genetic heterogeneity ,Hematology ,Neutropenia ,medicine.disease_cause ,medicine.disease ,Pathogenesis ,Cyclic neutropenia ,nervous system ,Oncology ,ELANE Gene ,Neutrophil elastase ,Immunology ,medicine ,biology.protein ,sense organs ,Congenital Neutropenia - Abstract
The 2 main forms of hereditary neutropenia are cyclic (CN) and severe congenital (SCN) neutropenia. CN is an autosomal dominant disorder in which neutrophil counts fluctuate with 21-day periodicity. SCN consists of static neutropenia, with promyelocytic maturation arrest in the bone marrow. Unlike CN, SCN displays frequent acquisition of somatic mutations in the gene CSF3R. CN is caused by heterozygous mutations in the gene ELANE, encoding neutrophil elastase. SCN is genetically heterogeneous but is most frequently associated with ELANE mutations. We discuss how the mutations provide clues into the pathogenesis of neutropenia and describe current hypotheses for its molecular mechanisms.
- Published
- 2013
23. Whole-organism lineage tracing by combinatorial and cumulative genome editing
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Jay Shendure, Alexander F. Schier, James A. Gagnon, Aaron McKenna, Gregory M. Findlay, and Marshall S. Horwitz
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0301 basic medicine ,Lineage (genetic) ,Zygote ,Computational biology ,medicine.disease_cause ,03 medical and health sciences ,0302 clinical medicine ,Bacterial Proteins ,Genome editing ,Single-cell analysis ,CRISPR-Associated Protein 9 ,medicine ,Animals ,DNA Barcoding, Taxonomic ,CRISPR ,Cell Lineage ,Zebrafish ,Genetics ,Mutation ,Multidisciplinary ,biology ,Cas9 ,Stem Cells ,Endonucleases ,biology.organism_classification ,Multicellular organism ,030104 developmental biology ,Cell Tracking ,CRISPR-Cas Systems ,Single-Cell Analysis ,Genetic Engineering ,Cell Division ,030217 neurology & neurosurgery - Abstract
INTRODUCTION The developmental path by which a fertilized egg gives rise to the cells of a multicellular organism is termed the cell lineage. In 1983, John Sulston and colleagues documented the invariant cell lineage of the roundworm Caenorhabditis elegans as determined by visual observation. However, tracing cell lineage in nearly all other multicellular organisms is vastly more challenging. Contemporary methods rely on genetic markers or somatic mutations, but these approaches have limitations that preclude their application at the level of a whole, complex organism. RATIONALE For a technology to comprehensively trace cell lineages in a complex multicellular system, it must uniquely and incrementally mark cells and their descendants over many divisions and in a way that does not interfere with normal development. These unique marks must also accumulate irreversibly over time, allowing the reconstruction of lineage trees. Finally, the full set of marks must be read out from each of many single cells. We hypothesized that genome editing, which introduces diverse, irreversible edits in a highly programmable fashion, could be repurposed for cell lineage tracing in a way that realizes these characteristics. To this end, we developed a method termed genome editing of synthetic target arrays for lineage tracing (GESTALT). This method uses genome editing to generate a combinatorial diversity of mutations that accumulate over many cell divisions within a compact DNA barcode consisting of multiple clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 target sites. Lineage relationships can be readily queried by sequencing the edited barcodes and relating the patterns of edits observed. RESULTS We first developed this approach in cell culture, editing synthetic arrays of 9 to 12 CRISPR/Cas9 target sites to generate thousands of unique derivative barcodes. We show that edited barcodes can be read by targeted sequencing of either DNA or RNA. In addition, the rates and patterns of barcode editing are tunable and the diverse edits accumulate over successive divisions in a way that is informative of cell lineage. We then applied GESTALT to the zebrafish Danio rerio by injecting fertilized eggs with editing reagents that target a genomic barcode bearing 10 target sites. Across dozens of embryos, we demonstrate the accumulation of hundreds to thousands of uniquely edited barcodes per animal, from which lineage relationships can be inferred on the basis of shared mutations. In adult zebrafish, we evaluated the edited barcodes from ~200,000 cells and observed that the majority of cells in each organ are derived from a small number of progenitor cells. Furthermore, ancestral progenitors, inferred on the basis of shared mutations among subsets of cells, can contribute to different germ layers and organ systems. CONCLUSION Our proof-of-principle experiments show that combinatorial, cumulative genome editing of a compact barcode can be used to record lineage information in multicellular systems. Further optimization of GESTALT will enable mapping of the complete cell lineage in diverse organisms. This method could also be adapted to link cell lineage information to molecular profiles of the same cells. In the long term, we envision that rich, systematically generated maps of organismal development—wherein lineage, epigenetic, transcriptional, and positional information are concurrently captured at single-cell resolution—will advance our understanding of development in both healthy and disease states. More broadly, cumulative and combinatorial genome editing could stably record other types of biological information and history in living cells. GESTALT. ( Left ) A barcode of CRISPR/Cas9 target sites is progressively edited over many cell divisions. ( Right ) Edited barcode sequences are related to one another on the basis of shared mutations in order to reconstruct lineage trees.
- Published
- 2016
24. Whole organism lineage tracing by combinatorial and cumulative genome editing
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Marshall S. Horwitz, Alexander F. Schier, Aaron McKenna, Jay Shendure, James A. Gagnon, and Gregory M. Findlay
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0303 health sciences ,Lineage (genetic) ,Cell division ,biology ,Cas9 ,Computational biology ,biology.organism_classification ,03 medical and health sciences ,Multicellular organism ,0302 clinical medicine ,Genome editing ,CRISPR ,Developmental biology ,Zebrafish ,030217 neurology & neurosurgery ,030304 developmental biology - Abstract
Multicellular systems develop from single cells through a lineage, but current lineage tracing approaches scale poorly to whole organisms. Here we use genome editing to progressively introduce and accumulate diverse mutations in a DNA barcode over multiple rounds of cell division. The barcode, an array of CRISPR/Cas9 target sites, records lineage relationships in the patterns of mutations shared between cells. In cell culture and zebrafish, we show that rates and patterns of editing are tunable, and that thousands of lineage-informative barcode alleles can be generated. By sampling hundreds of thousands of cells from individual zebrafish, we find that most cells in adult zebrafish organs derive from relatively few embryonic progenitors. Genome editing of synthetic target arrays for lineage tracing (GESTALT) will help generate large-scale maps of cell lineage in multicellular systems.
- Published
- 2016
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25. HIF1α induced switch from bivalent to exclusively glycolytic metabolism during ESC-to-EpiSC/hESC transition
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Wenyu Zhou, Lilyana Margaretha, C. Anthony Blau, Michael Choi, Daciana Margineantu, Christopher Cavanaugh, Carol B. Ware, Marshall S. Horwitz, Jennifer Hesson, Hannele Ruohola-Baker, and David M. Hockenbery
- Subjects
General Immunology and Microbiology ,General Neuroscience ,Cellular differentiation ,Biology ,Embryonic stem cell ,Warburg effect ,Phenotype ,General Biochemistry, Genetics and Molecular Biology ,Cell biology ,Biochemistry ,Epiblast ,embryonic structures ,Inner cell mass ,Glycolysis ,biological phenomena, cell phenomena, and immunity ,Stem cell ,Molecular Biology ,reproductive and urinary physiology - Abstract
The function of metabolic state in stemness is poorly understood. Mouse embryonic stem cells (ESC) and epiblast stem cells (EpiSC) are at distinct pluripotent states representing the inner cell mass (ICM) and epiblast embryos. Human embryonic stem cells (hESC) are similar to EpiSC stage. We now show a dramatic metabolic difference between these two stages. EpiSC/hESC are highly glycolytic, while ESC are bivalent in their energy production, dynamically switching from glycolysis to mitochondrial respiration on demand. Despite having a more developed and expanding mitochondrial content, EpiSC/hESC have low mitochondrial respiratory capacity due to low cytochrome c oxidase (COX) expression. Similarly, in vivo epiblasts suppress COX levels. These data reveal EpiSC/hESC functional similarity to the glycolytic phenotype in cancer (Warburg effect). We further show that hypoxia-inducible factor 1α (HIF1α) is sufficient to drive ESC to a glycolytic Activin/Nodal-dependent EpiSC-like stage. This metabolic switch during early stem-cell development may be deterministic.
- Published
- 2012
26. Loss-of-function germline GATA2 mutations in patients with MDS/AML or MonoMAC syndrome and primary lymphedema reveal a key role for GATA2 in the lymphatic vasculature
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Hamish S. Scott, Milena Babic, Tom Walsh, Michael Y. Zhang, Peter G Bardy, Christopher N. Hahn, Amy P. Hsu, Robert S. Wildin, Sarah Dyack, Genevieve A. Secker, Marshall S. Horwitz, Conrad V. Fernandez, Natasha L. Harvey, Jan Kazenwadel, Chan Eng Chong, Akiko Shimamura, Dennis D. Hickstein, Jill A. Rosenfeld, Steven M. Holland, Jennifer Cuellar-Rodriguez, Yajuan J. Liu, Kazenwadel, Jan, Secker, Genevieve A, Liu, Yajuan J, Rosenfeld, Jill A, Chong, Chan-Eng, Scott, Hamish S, and Harvey, Natasha L
- Subjects
Adult ,Male ,Adolescent ,Immunology ,Mice, Transgenic ,Biology ,Biochemistry ,Monocytes ,Frameshift mutation ,Mice ,Young Adult ,sporadic monocytopenia ,hemic and lymphatic diseases ,medicine ,Animals ,Humans ,Missense mutation ,Primary lymphedema ,Lymphedema ,Lymphangiogenesis ,Child ,Cells, Cultured ,Germ-Line Mutation ,transcription factor ,Lymphatic Vessels ,Myeloid Neoplasia ,hematopoietic defect ,Splice site mutation ,GATA2 Deficiency ,Infant, Newborn ,Syndrome ,Cell Biology ,Hematology ,medicine.disease ,MonoMAC ,GATA2 Transcription Factor ,Mice, Inbred C57BL ,Leukemia, Myeloid, Acute ,Myelodysplastic Syndromes ,Cancer research ,Female ,acute myeloid-leukemia ,Haploinsufficiency - Abstract
Recent work has established that heterozygous germline GATA2 mutations predispose carriers to familial myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML), “MonoMAC” syndrome, and DCML deficiency. Here, we describe a previously unreported MDS family carrying a missense GATA2 mutation (p.Thr354Met), one patient with MDS/AML carrying a frameshift GATA2 mutation (p.Leu332Thrfs*53), another with MDS harboring a GATA2 splice site mutation, and 3 patients exhibiting MDS or MDS/AML who have large deletions encompassing the GATA2 locus. Intriguingly, 2 MDS/AML or “MonoMAC” syndrome patients with GATA2 deletions and one with a frameshift mutation also have primary lymphedema. Primary lymphedema occurs as a result of aberrations in the development and/or function of lymphatic vessels, spurring us to investigate whether GATA2 plays a role in the lymphatic vasculature. We demonstrate here that GATA2 protein is present at high levels in lymphatic vessel valves and that GATA2 controls the expression of genes important for programming lymphatic valve development. Our data expand the phenotypes associated with germline GATA2 mutations to include predisposition to primary lymphedema and suggest that complete haploinsufficiency or loss of function of GATA2, rather than missense mutations, is the key predisposing factor for lymphedema onset. Moreover, we reveal a crucial role for GATA2 in lymphatic vascular development.
- Published
- 2012
27. Target protein interactions of indole-3-carbinol and the highly potent derivative 1-benzyl-I3C with the C-terminal domain of human elastase uncouples cell cycle arrest from apoptotic signaling
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Maria N. Preobrazhenskaya, Ida Aronchik, Marshall S. Horwitz, Leonard F. Bjeldanes, Tony J Chen, Gary L. Firestone, and Kathleen A. Durkin
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Cancer Research ,Cell cycle checkpoint ,Allosteric regulation ,Elastase ,Biology ,Cell cycle ,chemistry.chemical_compound ,Biochemistry ,chemistry ,Cancer cell ,Indole-3-carbinol ,Target protein ,Binding site ,Molecular Biology - Abstract
Elastase is the only currently identified target protein for indole-3-carbinol (I3C), a naturally occurring hydrolysis product of glucobrassicin in cruciferous vegetables such as broccoli, cabbage, and Brussels sprouts that induces a cell cycle arrest and apoptosis of human breast cancer cells. In vitro elastase enzymatic assays demonstrated that I3C and at lower concentrations its more potent derivative 1-benzyl-indole-3-carbinol (1-benzyl-I3C) act as non-competitive allosteric inhibitors of elastase activity. Consistent with these results, in silico computational simulations have revealed the first predicted interactions of I3C and 1-benzyl-I3C with the crystal structure of human neutrophil elastase, and identified a potential binding cluster on an external surface of the protease outside of the catalytic site that implicates elastase as a target protein for both indolecarbinol compounds. The Δ205 carboxyterminal truncation of elastase, which disrupts the predicted indolecarbinol binding site, is enzymatically active and generates a novel I3C resistant enzyme. Expression of the wild type and Δ205 elastase in MDA-MB-231 human breast cancer cells demonstrated that the carboxyterminal domain of elastase is required for the I3C and 1-benzyl-I3C inhibition of enzymatic activity, accumulation of the unprocessed form of the CD40 elastase substrate (a tumor necrosis factor receptor family member), disruption of NFκB nuclear localization and transcriptional activity, and induction of a G1 cell cycle arrest. Surprisingly, expression of the Δ205 elastase molecule failed to reverse indolecarbinol stimulated apoptosis, establishing an elastase-dependent bifurcation point in anti-proliferative signaling that uncouples the cell cycle and apoptotic responses in human breast cancer cells. © 2011 Wiley Periodicals, Inc.
- Published
- 2011
28. Heritable GATA2 Mutations Associated with Familial Myelodysplastic Syndrome and Acute Myeloid Leukemia
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Ella J Wilkins, Richard J D'Andrea, Xiaochun Li, Anna L. Brown, Hamish S. Scott, Robert Escher, Chung H. Kok, Graeme Suthers, Marshall S. Horwitz, Milena Babic, Catherine Carmichael, Ian D. Lewis, Sarah Moore, Christopher N. Hahn, Chan-Eng Chong, Ming-Chih Lin, Kathryn Friend, Paul G Ekert, Peter J. Brautigan, Andrew E. Timms, Amandine Carmagnac, Peter Bardy, L. Bik To, Jan Storek, Meryl Altree, Lucia Gagliardi, Carolyn M. Butcher, Young Koung Lee, Hahn, Christopher N, Chong, Chan-Eng, Carmichael, Catherine L, Wilkins, Ella J, Brautigan, Peter J, Brown, Anna L, D'Andrea, Richard J, and Scott, Hamish S
- Subjects
Myeloid ,Biology ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,hemic and lymphatic diseases ,Genetics ,medicine ,GATA2 ,Missense mutation ,acute myeloid leukaemia ,030304 developmental biology ,genetic predisposition to disease ,0303 health sciences ,Myelodysplastic syndromes ,Myeloid leukemia ,medicine.disease ,3. Good health ,MonoMAC ,myelodysplastic syndrome ,Leukemia ,medicine.anatomical_structure ,RUNX1 ,chemistry ,030220 oncology & carcinogenesis ,Cancer research ,mutation - Abstract
We report the discovery of GATA2 as a new myelodysplastic syndrome (MDS)-acute myeloid leukemia (AML) predisposition gene. We found the same, previously unidentified heterozygous c.1061C>T (p.Thr354Met) missense mutation in the GATA2 transcription factor gene segregating with the multigenerational transmission of MDS-AML in three families and a GATA2 c.1063_1065delACA (p.Thr355del) mutation at an adjacent codon in a fourth MDS family. The resulting alterations reside within the second zinc finger of GATA2, which mediates DNA-binding and protein-protein interactions. We show differential effects of the mutations on the transactivation of target genes, cellular differentiation, apoptosis and global gene expression. Identification of such predisposing genes to familial forms of MDS and AML is critical for more effective diagnosis and prognosis, counseling, selection of related bone marrow transplant donors and development of therapies. Refereed/Peer-reviewed
- Published
- 2011
29. Poor prognosis in familial acute myeloid leukaemia with combined biallelic CEBPA mutations and downstream events affecting the ATM, FLT3 and CDX2 genes
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Ella J Wilkins, Hamish S. Scott, Paul C. Vincent, Christopher N. Hahn, Henrik Bengtsson, Graham P Young, Robert Escher, Catherine Carmichael, Marshall S. Horwitz, and Terence P. Speed
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Male ,Myeloid ,Biology ,medicine.disease_cause ,Myeloid Neoplasm ,CEBPA ,medicine ,Humans ,Genetic Predisposition to Disease ,CDX2 ,Mutation ,Base Sequence ,Cancer ,Hematology ,Middle Aged ,Prognosis ,medicine.disease ,Neoplasm Proteins ,Pedigree ,Leukemia, Myeloid, Acute ,Leukemia ,medicine.anatomical_structure ,CCAAT-Enhancer-Binding Proteins ,Cancer research ,Female ,Genes, Neoplasm ,Chronic myelogenous leukemia - Published
- 2010
30. KLHDC8Bin Hodgkin lymphoma and possibly twinning
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Marshall S. Horwitz and Andrew E. Timms
- Subjects
Genetics ,Binucleated cells ,Balanced Chromosomal Translocation ,Chromosomal translocation ,Single-nucleotide polymorphism ,Biology ,Article Addendum ,Midbody ,Polar body ,immune system diseases ,hemic and lymphatic diseases ,General Agricultural and Biological Sciences ,Mitosis ,Cytokinesis - Abstract
A key feature of Hodgkin lymphoma is that the malignant cells are binucleated, as a consequence of failed cytokinesis. We recently ascertained a family in which multiple cases of Hodgkin lymphoma had occurred among individuals who inherited a balanced chromosomal translocation. We cloned the translocation breakpoints and found that it disrupted a previously uncharacterized gene, KLHDC8B, encoding a Kelch family protein whose deficiency impairs cytokinesis and leads to binucleated cells. In other families we found a rare single nucleotide polymorphism affecting mitotic translation of KLHDC8B that was associated with and linked to Hodgkin lymphoma. Interestingly, the index family demonstrated an unusual frequency of twins, and there is a previously reported association between Hodgkin lymphoma and twins. Here we review the unusual genetic features of Hodgkin lymphoma, including gender concordance among siblings, and genetically test the hypothesis that KLHDC8B may participate in twinning by disrupting cytokinesis through impediment of polar body separation from oocytes.
- Published
- 2010
31. Mutations in a gene encoding a midbody protein in binucleated Reed-Sternberg cells of Hodgkin lymphoma
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Marshall S. Horwitz, Maxwell M. Krem, and Stephen J. Salipante
- Subjects
Ubiquitin-Protein Ligases ,Binucleated cells ,Cell Cycle Proteins ,Biology ,medicine.disease_cause ,Article ,immune system diseases ,hemic and lymphatic diseases ,BARD1 ,medicine ,Humans ,Reed-Sternberg Cells ,Kelch protein ,Molecular Biology ,Cytokinesis ,BRCA2 Protein ,Mutation ,BRCA1 Protein ,Tumor Suppressor Proteins ,Cell Biology ,medicine.disease ,Hodgkin Disease ,Midbody ,Reed–Sternberg cell ,Cancer research ,HeLa Cells ,Developmental Biology - Abstract
Classical Hodgkin lymphoma (cHL) is a cancer in which malignant "Reed-Sternberg" cells comprise just a fraction of the bulk of the tumor and are characteristically binucleated. We recently identified a novel gene, KLHDC8B, which appears responsible for some familial cases of cHL. KLHDC8B encodes a midbody kelch protein expressed during cytokinesis. Deficiency of KLHDC8B leads to binucleated cells, implicating its involvement in Reed-Sternberg cell formation. Interestingly, other cancer genes, such as BRG41 and BRCA2, also encode proteins locating to the midbody during cytokinesis, even though their participation in other pathways has received greater attention. Midbody components may be an over-looked source of tumor suppressor genes.
- Published
- 2010
32. Phylogenetic analysis of developmental and postnatal mouse cell lineages
- Author
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Arnold Kas, Stephen J. Salipante, Marshall S. Horwitz, and Eva McMonagle
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Genetics ,Germline mutation ,Phylogenetic tree ,Fate mapping ,Somatic cell ,Phylogenetics ,Lineage (evolution) ,Embryology ,Gene regulatory network ,Biology ,Ecology, Evolution, Behavior and Systematics ,Developmental Biology - Abstract
Fate maps depict how cells relate together through past lineage relationships, and are useful tools for studying developmental and somatic processes. However, with existing technologies, it has not been possible to generate detailed fate maps of complex organisms such as the mouse. We and others have therefore proposed a novel approach, "phylogenetic fate mapping," where patterns of somatic mutation carried by the individual cells of an animal are used to retrospectively deduce lineage relationships through phylogenetic inference. Here, we have cataloged genomic polymorphisms at 324 mutation-prone polyguanine tracts for nearly 300 cells isolated from a single mouse, and have explored the cells' lineage relationships both phylogenetically and through a network-based approach. We present a model of mouse embryogenesis, where an early period of substantial cell mixing is followed by more coherent growth of clones later. We find that cells from certain tissues have greater numbers of close relatives in other specific tissues than expected from chance, suggesting that those populations arise from a similar pool of ancestral lineages. Finally, we have investigated the dynamics of cell turnover (the frequency of cell loss and replacement) in postnatal tissues. This work offers a longitudinal study of developmental lineages, from conception to adulthood, and provides insight into basic questions of mouse embryology as well as the somatic processes that occur after birth.
- Published
- 2010
33. Clonal expansions in ulcerative colitis identify patients with neoplasia
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Marshall S. Horwitz, Peter S. Rabinovitch, Lin Li, Jesse J. Salk, Teresa A. Brentnall, Mary P. Bronner, Rosa Ana Risques, Lawrence A. Loeb, Stephen J. Salipante, and David A. Crispin
- Subjects
Guanine ,Genotype ,Colorectal cancer ,Biology ,medicine.disease_cause ,Models, Biological ,Somatic evolution in cancer ,Inflammatory bowel disease ,medicine ,Humans ,Colitis ,Cell Proliferation ,Electrophoresis, Agar Gel ,Multidisciplinary ,Cancer ,Biological Sciences ,medicine.disease ,Ulcerative colitis ,Clone Cells ,Colonic Neoplasms ,Mutation ,Immunology ,Cancer research ,Colitis, Ulcerative ,Carcinogenesis - Abstract
Chronic inflammation predisposes to a variety of human cancers. Affected tissues slowly accumulate mutations, some of which affect growth regulation and drive successive waves of clonal evolution, whereas a far greater number are functionally neutral and serve only to passively mark expanding clones. Ulcerative colitis (UC) is an inflammatory bowel disease, in which up to 10% of patients eventually develop colon cancer. Here we have mapped mutations in hypermutable intergenic and intronic polyguanine tracts in patients with UC to delineate the extent of clonal expansions associated with carcinogenesis. We genotyped colon biopsies for length altering mutations at 28 different polyguanine markers. In eight patients without neoplasia, we detected only two mutations in a single individual from among 37 total biopsies. In contrast, for 11 UC patients with neoplasia elsewhere in the colon, we identified 63 mutations in 51 nondysplastic biopsies, and every patient possessed at least one mutant clone. A subset of clones were large and extended over many square centimeters of colon. Of these, some occurred as isolated populations in nondysplastic tissue, considerably distant from neoplastic lesions. Other large clones included regions of cancer, suggesting that the tumor arose within a preexisting clonal field. Our results demonstrate that neutral mutations in polyguanine tracts serve as a unique tool for identifying fields of clonal expansions, which may prove clinically useful for distinguishing a subset of UC patients who are at risk for developing cancer.
- Published
- 2009
34. Mutations in a gene encoding a midbody kelch protein in familial and sporadic classical Hodgkin lymphoma lead to binucleated cells
- Author
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Peter H. Wiernik, Mary L. McMaster, Stephen J. Salipante, Margaret A. Tucker, Govind Bhagat, Mikhail Roshal, Jeremy Wechsler, Marshall S. Horwitz, Kenneth Offit, Shinae Namkoong, Yajuan Liu, Maxwell M. Krem, Henry T. Lynch, Lynn R. Goldin, Tomas Kirchhoff, Jonathan R. Fromm, and Matthew E. Mealiffe
- Subjects
Adult ,Male ,Adolescent ,Binucleated cells ,Chromosomal translocation ,macromolecular substances ,Biology ,Loss of heterozygosity ,Young Adult ,Antigens, Neoplasm ,Sequence Homology, Nucleic Acid ,polycyclic compounds ,medicine ,Animals ,Humans ,Genetic Predisposition to Disease ,Reed-Sternberg Cells ,Kelch protein ,Mitosis ,Aged ,Aged, 80 and over ,Cell Nucleus ,Multidisciplinary ,Base Sequence ,Middle Aged ,Biological Sciences ,medicine.disease ,Hodgkin Disease ,Molecular biology ,Pedigree ,Midbody ,Reed–Sternberg cell ,Chromosomes, Human, Pair 2 ,Mutation ,Female ,Chromosomes, Human, Pair 3 ,5' Untranslated Regions ,Sequence Alignment ,Cytokinesis - Abstract
Classical Hodgkin lymphoma (cHL) is a malignancy of B-cell origin in which the neoplastic cells, known as “Reed-Sternberg” (RS) cells, are characteristically binucleated. Here we describe a family where multiple individuals developing cHL have inherited a reciprocal translocation between chromosomes 2 and 3. The translocation disrupts KLHDC8B , an uncharacterized gene from a region (3p21.31) previously implicated in lymphoma and related malignancies, resulting in its loss of expression. We tested KLHDC8B as a candidate gene for cHL and found that a 5′-UTR polymorphism responsible for decreasing its translational expression is associated with cHL in probands from other families with cHL and segregates with disease in those pedigrees. In one of three informative sporadic cases of cHL, we detected loss of heterozygosity (LOH) for KLHDC8B in RS cells, but not reactive T lymphocytes, purified from a malignant lymph node. KLHDC8B encodes a protein predicted to contain seven kelch repeat domains. KLHDC8B is expressed during mitosis, where it localizes to the midbody structure connecting cells about to separate during cytokinesis, and it is degraded after cell division. Depletion of KLHDC8B through RNA interference leads to an increase in binucleated cells, implicating its reduced expression in the formation of cHL's signature RS cell.
- Published
- 2009
35. Contributions to Neutropenia from PFAAP5 (N4BP2L2), a Novel Protein Mediating Transcriptional Repressor Cooperation between Gfi1 and Neutrophil Elastase
- Author
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Kathleen F. Benson, Stephen J. Salipante, Marshall S. Horwitz, Brice Korkmaz, Richard E. Person, H. Leighton Grimes, Zhijun Duan, Jeremy Wechsler, and Meghan E. B. Rojas
- Subjects
Chromatin Immunoprecipitation ,Neutropenia ,Transcription, Genetic ,HL-60 Cells ,Cyclic neutropenia ,Genes, Reporter ,Neutrophil differentiation ,RNA interference ,Two-Hybrid System Techniques ,White blood cell ,medicine ,Animals ,Humans ,RNA, Small Interfering ,Congenital Neutropenia ,Molecular Biology ,Cell Proliferation ,biology ,Tumor Suppressor Proteins ,Cell Differentiation ,Articles ,Cell Biology ,Hematopoietic Stem Cells ,medicine.disease ,Cell biology ,DNA-Binding Proteins ,Repressor Proteins ,medicine.anatomical_structure ,Gene Expression Regulation ,Neutrophil elastase ,Immunology ,biology.protein ,Carrier Proteins ,Leukocyte Elastase ,Chromatin immunoprecipitation ,Transcription Factors - Abstract
"Neutropenia" refers to deficient numbers of neutrophils, the most abundant type of white blood cell. Two main forms of inherited neutropenia are cyclic neutropenia, in which neutrophil counts oscillate with a 21-day frequency, and severe congenital neutropenia, in which static neutropenia may evolve at times into leukemia. Mutations of ELA2, encoding the protease neutrophil elastase, can cause both disorders. Among other genes, severe congenital neutropenia can also result from mutations affecting the transcriptional repressor Gfi1, one of whose genetic targets is ELA2, suggesting that the two act through similar mechanisms. In order to identify components of a common pathway regulating neutrophil production, we conducted yeast two-hybrid screens with Gfi1 and neutrophil elastase and detected a novel protein, PFAAP5 (also known as N4BP2L2), interacting with both. Expression of PFAAP5 allows neutrophil elastase to potentiate the repression of Gfi1 target genes, as determined by reporter assays, RNA interference, chromatin immunoprecipitation, and impairment of neutrophil differentiation in HSCs with PFAAP5 depletion, thus delineating a mechanism through which neutrophil elastase could regulate its own synthesis. Our findings are consistent with theoretical models of cyclic neutropenia proposing that its periodicity can be explained through disturbance of a feedback circuit in which mature neutrophils inhibit cell proliferation, thereby homeostatically regulating progenitor populations.
- Published
- 2009
36. Lentivectors encoding immunosuppressive proteins genetically engineer pancreatic β-cells to correct diabetes in allogeneic mice
- Author
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Teresa P. DiLorenzo, Tsoline Kojaoghlanian, Aviva Joseph, Harris Goldstein, Antonia Follenzi, Margarita Leiser, Jian Hua Zheng, Norman Fleischer, and Marshall S. Horwitz
- Subjects
Graft Rejection ,Genetic enhancement ,Genetic Vectors ,Islets of Langerhans Transplantation ,Mice, Inbred Strains ,Gene delivery ,Biology ,Major histocompatibility complex ,Article ,Diabetes Mellitus, Experimental ,Immune tolerance ,Mice ,Downregulation and upregulation ,Transduction, Genetic ,Insulin-Secreting Cells ,Adenovirus E3 Proteins ,Immune Tolerance ,Genetics ,medicine ,Animals ,Molecular Biology ,Mice, Inbred C3H ,Reverse Transcriptase Polymerase Chain Reaction ,Adenovirus Early Proteins ,Lentivirus ,Transplantation ,Disease Models, Animal ,Diabetes Mellitus, Type 1 ,medicine.anatomical_structure ,Cell culture ,Immunology ,biology.protein ,Molecular Medicine ,Female ,Genetic Engineering ,Pancreas - Abstract
The effectiveness of genetic engineering with lentivectors to protect transplanted cells from allogeneic rejection was examined using, as a model, type 1 diabetes treatment with beta-cell transplantation, whose widespread use has been limited by the requirement for sustained immunosuppressive treatment to prevent graft rejection. We examined whether lentivectors expressing select immunosuppressive proteins encoded by the adenoviral genome early region 3 (AdE3) would protect transplanted beta-cells from an alloimmune attack. The insulin-producing beta-cell line beta TC-tet (C3HeB/FeJ-derived) was transduced with lentiviruses encoding the AdE3 proteins gp19K and RID alpha/beta. The efficiency of lentiviral transduction of beta TC-tet cells exceeded 85%. Lentivector expression of gp19K decreased surface class I major histocompatibility complex expression by over 90%, whereas RID alpha/beta expression inhibited cytokine-induced Fas upregulation by over 75%. beta TC-tet cells transduced with gp19K and RID alpha/beta lentivectors, but not with a control lentivector, provided prolonged correction of hyperglycemia after transplantation into diabetic BALB/c severe combined immunodeficient mice reconstituted with allogeneic immune effector cells or into diabetic allogeneic BALB/c mice. Thus, genetic engineering of beta-cells using gp19K- and RID alpha/beta-expressing lentiviral vectors may provide an alternative that has the potential to eliminate or reduce treatment with the potent immunosuppressive agents necessary at present for prolonged engraftment with transplanted islets.
- Published
- 2008
37. Phylogenetic Fate Mapping: Theoretical and Experimental Studies Applied to the Development of Mouse Fibroblasts
- Author
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Marshall S. Horwitz, James M. Thompson, and Stephen J. Salipante
- Subjects
Genetics ,Mutation rate ,Mutation ,Models, Genetic ,Cell division ,Phylogenetic tree ,Lineage (evolution) ,Bayes Theorem ,DNA ,Investigations ,Fibroblasts ,Biology ,medicine.disease_cause ,Mice ,Multicellular organism ,Phylogenetics ,Fate mapping ,medicine ,Animals ,Computer Simulation ,Caenorhabditis elegans ,Cell Division ,Phylogeny - Abstract
Mutations are an inevitable consequence of cell division. Similarly to how DNA sequence differences allow inferring evolutionary relationships between organisms, we and others have recently demonstrated how somatic mutations may be exploited for phylogenetically reconstructing lineages of individual cells during development in multicellular organisms. However, a problem with such “phylogenetic fate maps” is that they cannot be verified experimentally; distinguishing actual lineages within clonal populations requires direct observation of cell growth, as was used to construct the fate map of Caenorhabditis elegans, but is not possible in higher organisms. Here we employ computer simulation of mitotic cell division to determine how factors such as the quantity of cells, mutation rate, and the number of examined marker sequences contribute to fidelity of phylogenetic fate maps and to explore statistical methods for assessing accuracy. To experimentally evaluate these factors, as well as for the purpose of investigating the developmental origins of connective tissue, we have produced a lineage map of fibroblasts harvested from various organs of an adult mouse. Statistical analysis demonstrates that the inferred relationships between cells in the phylogenetic fate map reflect biological information regarding the origin of fibroblasts and is suggestive of cell migration during mesenchymal development.
- Published
- 2008
38. Lymphadenopathy as the primary manifestation of malignant transformation in two patients with severe congenital neutropenia
- Author
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Clifford M. Takemoto, Jeffrey Schowinsky, Christopher Gamper, John J. Strouse, Marshall S. Horwitz, and Michael J. Borowitz
- Subjects
medicine.medical_specialty ,Pathology ,Neutropenia ,Myeloid ,Malignant transformation ,Bone Marrow ,hemic and lymphatic diseases ,medicine ,Humans ,Child ,Congenital Neutropenia ,Lymphatic Diseases ,Chromosome 7 (human) ,Extramural ,business.industry ,Infant, Newborn ,Infant ,Myeloid leukemia ,Hematology ,Flow Cytometry ,medicine.disease ,Dermatology ,Leukemia, Myeloid, Acute ,Leukemia ,Cell Transformation, Neoplastic ,medicine.anatomical_structure ,nervous system ,Oncology ,Mutation ,Pediatrics, Perinatology and Child Health ,Etiology ,Leukocyte Elastase ,business - Abstract
We present the cases of two patients with severe congenital neutropenia (SCN) who both developed generalized adenopathy. Although both had recent histories that placed infection high on the differential of causes for the adenopathy, biopsies demonstrated acute myeloid leukemia (AML) as the etiology. The risk of malignant transformation in SCN is known to be significantly elevated, and these cases illustrate the need for physicians of such patients to keep myelodysplastic syndrome (MDS) and AML high on the differential when patients manifest atypical symptoms.
- Published
- 2008
39. IKKγ (NEMO) is involved in the coordination of the AP-1 and NF-κB pathways
- Author
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Amde Selassie Shifera, Joshua M. Friedman, and Marshall S. Horwitz
- Subjects
Programmed cell death ,Leucine zipper ,Activator (genetics) ,Kinase ,Clinical Biochemistry ,Cell ,c-jun ,NF-κB ,Cell Biology ,General Medicine ,Biology ,Cell biology ,chemistry.chemical_compound ,medicine.anatomical_structure ,chemistry ,medicine ,Cancer research ,Phosphorylation ,Molecular Biology - Abstract
Tumor necrosis factor alpha (TNFα) activates the nuclear factor-kappaB (NF-κB) pathway in various cell types, leading to expression of cell survival and inflammatory proteins. One mechanism of cell survival brought about by NF-κB is the inhibition of Activator Protein-1 (AP-1), which when activated, could lead to cell death. However, TNFα can also induce the AP-1 pathway, and the mechanisms by which these two pathways are regulated in response to TNFα are poorly understood. We proposed that Inhibitor of κB Kinase gamma (IKKγ) (which is also known as NF-κB essential modulator, NEMO) plays a key role in integrating and coordinating these two pathways. Our results showed that IKKγ activates the AP-1 pathway, via a mechanism that is dependent on the first leucine zipper (LZ) domain of IKKγ, by interacting with two proteins of the AP-1 complex, c-Jun and c-Fos, and changing the phosphorylation status of c-Jun. Even though IKKγ is required for the activation of NF-κB, we found that it reduced the activity of NF-κB when it was overexpressed. In summary, we demonstrated that transfected IKKγ, while inhibiting the NF-κB pathway, directly interacts with the AP-1 proteins and activates the AP-1 pathway independent of its effects on NF-κB. Our results indicate that IKKγ regulates TNFα signaling by coordinating cell responses mediated by the AP-1 and NF-κB pathways.
- Published
- 2007
40. The Clinical, Immunohematological, and Molecular Study of Iranian Patients with Severe Congenital Neutropenia
- Author
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Zahra Pourpak, Mostafa Moin, Maryam Mahmoudi, Magda Grudzien, Marshall S. Horwitz, Roya Sherkat, Fatemeh Mahjoub, Asghar Aghamohammadi, Manuela Germeshausen, Mina Izadyar, Mehdi Yeganeh, Abolhassan Farhoudi, Zahra Chavoshzadeh, Christoph Klein, Asghar Ramyar, and Nima Rezaei
- Subjects
Adult ,Male ,medicine.medical_specialty ,Neutropenia ,Adolescent ,Immunology ,Mucocutaneous zone ,Iran ,Mucocutaneous Candidiasis ,Leukocyte Count ,Sepsis ,medicine ,Humans ,Immunology and Allergy ,Child ,Congenital Neutropenia ,business.industry ,Infant ,medicine.disease ,Dermatology ,Immunoglobulin A ,Pedigree ,Surgery ,Pneumonia ,Otitis ,Immunoglobulin M ,Child, Preschool ,Immunoglobulin G ,Chronic Disease ,Primary immunodeficiency ,Absolute neutrophil count ,Female ,medicine.symptom ,business - Abstract
Severe congenital neutropenia (SCN) is a rareE primary immunodeficiency disorder characterized by early onset recurrent infections in association with persistent severe agranulocytosis. To identify the clinical, immunohematological, and molecular characteristics of patients with SCN, 18 Iranian patients with the mean age of 8.8 +/- 5.8 years were investigated in this study. All of these patients experienced severe neutropenia; the mean of absolute neutrophil count was 281.4 +/- 137.7 cells/mm3. Bone marrow findings were typified by a myeloid maturation arrest at the promyelocyte-myelocyte stage in these patients. Molecular analysis revealed different mutations in the ELA-2 gene of one patient and in the HAX-1 gene of another three patients. The most common presenting complaints in these patients were superficial abscesses, oral ulcers, cutaneous infections, omphalitis, and pneumonia. During the course of illness, all patients developed mucocutaneous manifestations, and 16 cases had respiratory infections. The most commonly manifestations were abscesses, oral ulcers, pneumonia, periodontitis, otitis media, cutaneous infections, mucocutaneous candidiasis, and acute diarrhea. Three patients died because of a severe infection. Although SCN is a rare disorder, early onset of severe and recurrent infections should always raise a suspicion, which deserves further evaluation for detecting such disorder.
- Published
- 2007
41. Neutrophil elastase in cyclic and severe congenital neutropenia
- Author
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Zhijun Duan, Hu-Hui Lee, Marshall S. Horwitz, Brice Korkmaz, Matthew E. Mealiffe, and Stephen J. Salipante
- Subjects
Neutropenia ,Genotype ,Immunology ,G6PC3 ,Review Article ,Models, Biological ,Biochemistry ,AP3B1 ,Cyclic neutropenia ,medicine ,Animals ,Humans ,Congenital Neutropenia ,biology ,Cell Differentiation ,Cell Biology ,Hematology ,medicine.disease ,HAX1 ,Neutrophil elastase ,Mutation ,biology.protein ,Leukocyte Elastase ,Kostmann syndrome - Abstract
Mutations in ELA2 encoding the neutrophil granule protease, neutrophil elastase (NE), are the major cause of the 2 main forms of hereditary neutropenia, cyclic neutropenia and severe congenital neutropenia (SCN). Genetic evaluation of other forms of neutropenia in humans and model organisms has helped to illuminate the role of NE. A canine form of cyclic neutropenia corresponds to human Hermansky-Pudlak syndrome type 2 (HPS2) and results from mutations in AP3B1 encoding a subunit of a complex involved in the subcellular trafficking of vesicular cargo proteins (among which NE appears to be one). Rare cases of SCN are attributable to mutations in the transcriptional repressor Gfi1 (among whose regulatory targets also include ELA2). The ultimate biochemical consequences of the mutations are not yet known, however. Gene targeting of ELA2 has thus far failed to recapitulate neutropenia in mice. The cycling phenomenon and origins of leukemic transformation in SCN remain puzzling. Nevertheless, mutations in all 3 genes are capable of causing the mislocalization of NE and may also induce the unfolded protein response, suggesting that there might a convergent pathogenic mechanism focusing on NE.
- Published
- 2006
42. Adenovirus RID complex enhances degradation of internalized tumour necrosis factor receptor 1 without affecting its rate of endocytosis
- Author
-
Y. Rebecca Chin and Marshall S. Horwitz
- Subjects
Endosome ,medicine.medical_treatment ,education ,Cell ,Biology ,Endocytosis ,medicine.disease_cause ,Receptors, Tumor Necrosis Factor ,Adenoviridae ,Cell Line ,Viral Proteins ,Virology ,Adenovirus E3 Proteins ,medicine ,Humans ,Receptor ,technology, industry, and agriculture ,respiratory system ,Up-Regulation ,Tumor Necrosis Factor Decoy Receptors ,medicine.anatomical_structure ,Cytokine ,Receptors, Tumor Necrosis Factor, Type I ,Multiprotein Complexes ,Biotinylation ,Tumor necrosis factor alpha ,human activities - Abstract
The receptor internalization and degradation (RID) complex of adenovirus plays an important role in modulating the immune response by downregulating the surface levels of tumour necrosis factor receptor 1 (TNFR1), thereby inhibiting NF-κB activation. Total cellular content of TNFR1 is also reduced in the presence of RID, which can be inhibited by treatment with lysosomotropic agents. In this report, surface biotinylation experiments revealed that, although RID and TNFR1 were able to form a complex on the cell surface, the rate of TNFR1 endocytosis was not affected by RID. However, the degradation of internalized TNFR1 was enhanced significantly in the presence of RID. Therefore, these data suggest that RID downregulates TNFR1 levels by altering the fate of internalized TNFR1 that becomes associated with RID at the plasma membrane, probably by promoting its sorting into endosomal/lysosomal degradation compartments.
- Published
- 2006
43. Adenovirus RIDαβ Complex Inhibits Lipopolysaccharide Signaling without Altering TLR4 Cell Surface Expression
- Author
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Fernando Delgado-López and Marshall S. Horwitz
- Subjects
Lipopolysaccharides ,Chemokine ,education ,Immunology ,Cell ,Microbiology ,Cell Line, Tumor ,Virology ,Adenovirus E3 Proteins ,medicine ,Humans ,Receptor ,Inflammation ,Regulation of gene expression ,biology ,Tumor Necrosis Factor-alpha ,technology, industry, and agriculture ,Virus-Cell Interactions ,Cell biology ,Toll-Like Receptor 4 ,medicine.anatomical_structure ,Gene Expression Regulation ,Receptors, Tumor Necrosis Factor, Type I ,Multiprotein Complexes ,Insect Science ,biology.protein ,TLR4 ,Tumor necrosis factor alpha ,Chemokines ,Signal transduction ,human activities ,Intracellular ,Interleukin-1 ,Signal Transduction - Abstract
The transmembrane heterotrimer complex 10.4K/14.5K, also known as RID (for “receptor internalization and degradation”), is encoded by the adenovirus E3 region, and it down-regulates the cell surface expression of several unrelated receptors. We recently showed that RID expression correlates with down-regulation of the cell surface expression of the tumor necrosis factor (TNF) receptor 1 in several human cells. This observation provided the first mechanistic explanation for the inhibition of TNF alpha-induced chemokines by RID. Here we analyze the immunoregulatory activities of RID on lipopolysaccharide (LPS) and interleukin-1 beta (IL-1β)-mediated responses. Although both signaling pathways are strongly inhibited by RID, the chemokines up-regulated by IL-1β stimulation are only marginally inhibited. In addition, RID inhibits signaling induced by LPS without affecting the expression of the LPS receptor Toll-like receptor 4, demonstrating that RID need not target degradation of the receptor to alter signal transduction. Taken together, our data demonstrate the inhibitory effect of RID on two additional cell surface receptor-mediated signaling pathways involved in inflammatory processes. The data suggest that RID has intracellular targets that impair signal transduction and chemokine expression without evidence of receptor down-regulation.
- Published
- 2006
44. Mechanisms of dominant congenital neutropenias
- Author
-
Stephen J. Salipante and Marshall S. Horwitz
- Subjects
Protease ,biology ,medicine.medical_treatment ,Neutropenia ,medicine.disease ,AP3B1 ,Cyclic neutropenia ,Membrane protein ,hemic and lymphatic diseases ,Neutrophil elastase ,Drug Discovery ,Immunology ,medicine ,biology.protein ,Molecular Medicine ,Congenital Neutropenia ,Intracellular - Abstract
The two major inherited forms of human neutropenia are cyclic neutropenia, in which neutrophil numbers oscillate in three-week cycles, and severe congenital neutropenia (SCN), where the neutropenia is static. Mutations in the gene ELA2, which encodes the protease neutrophil elastase, are the major cause of both. Recent findings that mutations in the genes for the Gfi1 transcriptional repressor and the intracellular transporter AP3B1 also cause neutropenia suggest a mechanistic model involving mistargeting of neutrophil elastase to membranes. The hypothesis that neutrophil elastase can be a membrane protein has implications for its catalytic mechanism.
- Published
- 2005
45. Targeted transcriptional repression of Gfi1 by GFI1 and GFI1B in lymphoid cells
- Author
-
Loretta L. Doan, Marshall S. Horwitz, Philip N. Tsichlis, Marcella M. Flubacher, Susan D. Porter, Zhijun Duan, H. Leighton Grimes, C. Blake Gilks, and Diego Montoya
- Subjects
Transcription, Genetic ,T-Lymphocytes ,Molecular Sequence Data ,Repressor ,Thymus Gland ,Biology ,Response Elements ,Jurkat cells ,Cell Line ,Conserved sequence ,Jurkat Cells ,Mice ,Proto-Oncogene Proteins ,Sequence Homology, Nucleic Acid ,Genetics ,Animals ,Humans ,RNA, Messenger ,Lymphopoiesis ,Promoter Regions, Genetic ,Transcription factor ,Psychological repression ,Cells, Cultured ,Conserved Sequence ,Regulation of gene expression ,Base Sequence ,DNA ,Articles ,Molecular biology ,Rats ,DNA-Binding Proteins ,Repressor Proteins ,Thymocyte ,Gene Expression Regulation ,Protein Binding ,Transcription Factors - Abstract
Growth factor independence-1 (GFI1) and GFI1B are closely related, yet differentially expressed transcriptional repressors with nearly identical DNA binding domains. GFI1 is upregulated in the earliest thymocyte precursors, while GFI1B expression is restricted to T lymphopoiesis stages coincident with activation. Transgenic expression of GFI1 potentiates T-cell activation, while forced GFI1B expression decreases activation. Both mice and humans with mutant Gfi1 display lymphoid abnormalities. Here we describe autoregulation of Gfi1 in primary mouse thymocytes and a human T-cell line. GFI1 binding to cis-element sequences conserved between rat, mouse and human Gfi1 mediates direct and potent transcriptional repression. In addition, dramatic regulation of Gfi1 can also be mediated by GFI1B. These data provide the first example of a gene directly targeted by GFI1 and GFI1B. Moreover, they support a role for auto- and trans-regulation of Gfi1 by GFI1 and GFI1B in maintaining the normal expression patterns of Gfi1, and suggest that GFI1B may indirectly affect T-cell activation through repression of Gfi1.
- Published
- 2004
46. Hereditary neutropenia: dogs explain human neutrophil elastase mutations
- Author
-
Marshall S. Horwitz, Zhijun Duan, Richard E. Person, Kathleen F. Benson, and Feng-Qian Li
- Subjects
Models, Molecular ,Neutropenia ,Time Factors ,Adaptor Protein Complex 3 ,Protein Conformation ,medicine.disease_cause ,Models, Biological ,AP3B1 ,Cyclic neutropenia ,Dogs ,medicine ,Animals ,Humans ,Adaptor Protein Complex beta Subunits ,Molecular Biology ,Genetics ,Mutation ,biology ,Membrane transport protein ,Cell Membrane ,Elastase ,Membrane Transport Proteins ,medicine.disease ,Molecular biology ,Transmembrane protein ,Protein Structure, Tertiary ,Disease Models, Animal ,Protein Transport ,Transmembrane domain ,Neutrophil elastase ,biology.protein ,Molecular Medicine ,Leukocyte Elastase - Abstract
Mutations in ELA2, the gene encoding neutrophil elastase (NE), cause the human diseases cyclic neutropenia (CN) and severe congenital neutropenia (SCN). Numerous mutations are known, but their lack of consistent biochemical effect has proven puzzling. The recent finding that mutation of AP3B1, which encodes the beta subunit of adaptor protein complex 3 (AP3), is the cause of canine CN suggests a model for the molecular basis of hereditary neutropenias, involving the mistrafficking of NE: AP3 recognizes NE as a cargo protein, and their interaction implies that NE is a transmembrane protein. Computerized algorithms predict two NE transmembrane domains. Most CN mutations fall within predicted transmembrane domains and lead to excessive deposition of NE in granules, whereas SCN mutations usually disrupt the AP3 recognition sequence, resulting in excessive transport to the plasma membrane.
- Published
- 2004
47. Function of adenovirus E3 proteins and their interactions with immunoregulatory cell proteins
- Author
-
Marshall S. Horwitz
- Subjects
Allogeneic transplantation ,Adenoviridae Infections ,Genetic enhancement ,Cell ,Computational biology ,Biology ,Adenoviridae ,Adjuvants, Immunologic ,Drug Discovery ,Adenovirus E3 Proteins ,Genetics ,medicine ,Animals ,Humans ,Molecular Biology ,Gene ,Genetics (clinical) ,Early region ,NF-kappa B ,Proteins ,medicine.anatomical_structure ,Autoimmune diabetes ,Immunology ,Molecular Medicine ,Chemokines ,Signal transduction ,Function (biology) ,Signal Transduction - Abstract
This manuscript will contain a brief review of adenovirus (Ad) biology including its structure and the spectrum of clinical diseases that it causes. Newer findings about the interactions of Ad early region 3 (E3) proteins with host proteins and signal transduction processes, which control the inflammatory response, will be described. Some of these processes affect the strategies for using Ads as vectors for gene therapy. There are many excellent reviews of some of these aspects 1. The history of the discoveries that led to the use of Ads as vectors as well as key experiments that resulted in improvements to various aspects of these systems have also been reviewed recently 2. The use of Ad E3 immunoregulatory genes to facilitate allogeneic transplantation and prevent autoimmune diabetes will be described.
- Published
- 2004
48. A Novel Notch Protein, N2N, Targeted by Neutrophil Elastase and Implicated in Hereditary Neutropenia
- Author
-
Kayleen Williams, Feng-Qian Li, Kimberly Meade-White, Jeremy Wechsler, Marshall S. Horwitz, Kathleen F. Benson, and Zhijun Duan
- Subjects
Neutropenia ,EGF-like domain ,Proteolysis ,medicine.disease_cause ,medicine ,Humans ,Cloning, Molecular ,Cell Growth and Development ,Molecular Biology ,Serine protease ,Mutation ,Receptors, Notch ,biology ,medicine.diagnostic_test ,Membrane Proteins ,Sequence Analysis, DNA ,Cell Biology ,Neutrophil extracellular traps ,Molecular biology ,Notch proteins ,Neutrophil elastase ,biology.protein ,Signal transduction ,Leukocyte Elastase ,Signal Transduction - Abstract
Mutations in ELA2, encoding the human serine protease neutrophil elastase, cause cyclic and severe congenital neutropenia, and recent evidence indicates that the mutations alter the membrane trafficking of neutrophil elastase. These disorders feature impaired bone marrow production of neutrophils along with excess monocytes—terminally differentiated lineages corresponding to the two alternative fates of myeloid progenitor cells. We utilized a modified yeast two-hybrid system and identified a new, widely expressed gene, N2N, whose product is homologous to Notch2, that interacts with neutrophil elastase. N2N is a 36-kDa protein distributed throughout the cell and secreted. Its amino-terminal sequence consists of several EGF repeats identical to those of the extracellular region of Notch2, and its carboxyl terminus contains a unique 24-residue domain required for interaction with neutrophil elastase. Neutrophil elastase cleaves N2N within EGF repeats in vitro and in living cells, but the C-terminal domain retards proteolysis. In vitro, N2N represses transcriptional activities of Notch proteins. Disease-causing mutations of neutrophil elastase disrupt the interaction with N2N, impair proteolysis of N2N and Notch2, and interfere with Notch2 signaling, suggesting defective proteolysis of an inhibitory form of Notch as an explanation for the alternate switching of cell fates characteristic of hereditary neutropenia.
- Published
- 2004
49. Congenital and Acquired Neutropenia
- Author
-
Marshall S. Horwitz, Nancy Berliner, and Thomas P. Loughran
- Subjects
Leukemia, T-Cell ,Neutropenia ,Antigen-Antibody Complex ,Granulocyte ,Pathogenesis ,Cyclic neutropenia ,hemic and lymphatic diseases ,medicine ,Humans ,Congenital Neutropenia ,Autoantibodies ,biology ,business.industry ,Molecular pathology ,Hematology ,medicine.disease ,Leukemia ,medicine.anatomical_structure ,Gene Expression Regulation ,Neutrophil elastase ,Mutation ,Immunology ,Felty Syndrome ,biology.protein ,Leukocyte Elastase ,business - Abstract
Our understanding of the pathogenesis of congenital and acquired neutropenia is rapidly evolving. New ground-breaking observations have identified the genes responsible for many of the congenital neutropenia syndromes and are also providing new insights into normal neutrophil commitment and differentiation. Acquired neutropenia remains a poorly understood syndrome, although new insights into its pathogenesis are also emerging, especially with regard to subsets of immune neutropenia.In Section I, Dr. Marshall Horwitz reviews the current understanding of the genetic basis, molecular pathology, and approaches to treatment of congenital neutropenia and cyclic hematopoiesis. Mutations in the ELA2 gene, which encodes for neutrophil elastase, cause cyclic hematopoiesis. ELA2 mutations are also the most common cause of congenital neutropenia, where their presence may equate with a more severe clinical course and higher frequency of leukemic progression. Emerging evidence indicates interrelatedness with Hermansky Pudlak syndrome and other disorders of neutrophil and platelet granules.In Section II, Dr. Nancy Berliner presents an overview of the clinical approach to the evaluation and treatment of acquired neutropenia. This includes a review of the pathogenesis of primary and secondary immune neutropenia, drug-induced neutropenia, and non-immune chronic idiopathic neutropenia of adults. Studies used to evaluate patients for potential immune neutropenia are reviewed. Management issues, especially the use of granulocyte colony-stimulating factor (G-CSF), are discussed.In Section III, Dr. Thomas Loughran, Jr., reviews the pathogenesis and clinical manifestations of large granular lymphocyte (LGL) leukemia. Possible mechanisms of neutropenia are discussed. In particular, discussion focuses on the relationship between LGL leukemia, rheumatoid disease, and Felty’s syndrome, and the complex interplay of defects in neutrophil production, distribution, destruction, and apoptosis that underly the development of neutropenia in those syndromes.
- Published
- 2004
50. The impact of adenovirus infection on the immunocompromised host
- Author
-
Marshall S. Horwitz, Phyllis Flomenberg, and Tsoline Kojaoghlanian
- Subjects
Cellular immunity ,Genetic enhancement ,HIV Infections ,Context (language use) ,Disease ,Biology ,medicine.disease_cause ,Virus ,Adenovirus Infections, Human ,Immunocompromised Host ,Virology ,medicine ,Humans ,Adenovirus infection ,Antigens, Viral ,Acquired Immunodeficiency Syndrome ,medicine.disease ,Adenoviridae ,Infectious Diseases ,Antibody Formation ,Immunology ,Heart Transplantation ,Viral disease ,Immunosuppressive Agents ,Lung Transplantation - Abstract
Adenovirus (Ad) infections in immunocompromised hosts have increased in frequency as the number of patients with transplants of bone marrow, liver, kidney, heart and other organs increase in number and survive longer. The numbers of such patients have also increased because of the emergence of the HIV epidemic. Ad infections with the 51 different serotypes recognised to date have few pathognomonic signs and symptoms, and thus require a variety of laboratory-based procedures to confirm infection. These viruses have the ability to target various organs with relative serotype specificity and can cause diverse manifestations including serious life-threatening diseases characteristic of the organs involved. Ads have cytolytic and immunoregulatory properties. The clinical dilemma remains the prompt recognition of Ad-related disease, the differentiation of Ad infection from Ad disease and the differentiation from other causative agents. Since the armamentarium of effective antiviral agents available to treat Ads is unproven by controlled trials and the virus is often not acquired de novo, it is difficult to prevent reactivation in immunodeficient hosts or new acquisition from donor organs. Timely discontinuation of immunosuppressive agents is necessary to prevent morbid outcomes. The clinical diseases, diagnostic tests, antiviral agents and biological aspects of the Ads as pathogens in immunocompromised patients are discussed in the context of this review. Some of the newer diagnostic tests are based on the well-studied molecular biology of Ads, which also have been attenuated by selective viral DNA deletions for use as vectors in numerous gene therapy trials in humans.
- Published
- 2003
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