68 results on '"Alexander L. Kovalchuk"'
Search Results
2. A Pentavalent Shigella flexneri LPS-Based Vaccine Candidate Is Safe and Immunogenic in Animal Models
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Vladimir A. Ledov, Marina E. Golovina, Biana I. Alkhazova, Vyacheslav L. Lvov, Alexander L. Kovalchuk, and Petr G. Aparin
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Shigella flexneri ,vaccine ,lipopolysaccharide ,preclinical studies ,Medicine - Abstract
A multivalent vaccine is much needed to achieve protection against predominant Shigella serotypes. Recently, we demonstrated the clinical applicability and immunogenic potential of tri-acylated S. flexneri 2a lipopolysaccharide (Ac3-S-LPS). Using a similar approach, we designed a pentavalent LPS candidate vaccine against S. flexneri 1b, 2a, 3a, 6, and Y (PLVF). In this study, we performed molecular and antigenic characterization of the vaccine candidate and its preclinical evaluation. There were no signs of acute toxicity after subcutaneous administration of PLVF in rabbits at a proposed human dose of 125 μg. No pyrogenic reactions and adverse effects associated with chronic toxicity after repeated administration of PLVF were revealed either. The immunization of mice with PLVF led to ≥16-fold increase in S. flexneri 1b-, 2a-, 3a-, 6-, and Y-specific antibodies. In a serum bactericidal antibody (SBA) assay, we registered 54%, 66%, 35%, 60%, and 60% killing of S. flexneri 1b, 2a, 3a, 6, and Y, respectively. In the guinea pig keratoconjunctivitis model, the efficacy was 50% to 75% against challenge with all five S. flexneri serotypes. These studies demonstrate that PLVF is safe, immunogenic over a wide range of doses, and provides protection against challenge with homologous S. flexneri strains, thus confirming the validity of pentavalent design of the combined vaccine.
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- 2023
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3. Molecular Lipopolysaccharide Di-Vaccine Protects from Shiga-Toxin Producing Epidemic Strains of Escherichia coli O157:H7 and O104:H4
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Ivan A. Dyatlov, Edward A. Svetoch, Anna A. Mironenko, Boris V. Eruslanov, Victoria V. Firstova, Nadezhda K. Fursova, Alexander L. Kovalchuk, Vyacheslav L. Lvov, and Petr G. Aparin
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STEC ,clinically applicable LPS ,antibody response ,intraperitoneal infection ,intragastric infection ,Medicine - Abstract
Background: Shiga toxin-producing Escherichia coli (STEC) O157:H7 and O104:H4 strains are important causative agents of food-borne diseases such as hemorrhagic colitis and hemolytic–uremic syndrome, which is the leading cause of kidney failure and death in children under 5 years as well as in the elderly. Methods: the native E. coli O157:H7 and O104:H4 lipopolysaccharides (LPS) were partially deacylated under alkaline conditions to obtain apyrogenic S-LPS with domination of tri-acylated lipid A species—Ac3-S-LPS. Results: intraperitoneal immunization of BALB/c mice with Ac3-S-LPS antigens from E. coli O157:H7 and O104:H4 or combination thereof (di-vaccine) at single doses ranging from 25 to 250 µg induced high titers of serum O-specific IgG (mainly IgG1), protected animals against intraperitoneal challenge with lethal doses of homologous STEC strains (60–100% survival rate) and reduced the E. coli O157:H7 and O104:H4 intestinal colonization under an in vivo murine model (6–8-fold for monovalent Ac3-S-LPS and 10-fold for di-vaccine). Conclusions: Di-vaccine induced both systemic and intestinal anti-colonization immunity in mice simultaneously against two highly virulent human STEC strains. The possibility of creating a multivalent STEC vaccine based on safe Ac3-S-LPS seems to be especially promising due to a vast serotype diversity of pathogenic E. coli.
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- 2022
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4. Characterization of ARF-BP1/HUWE1 Interactions with CTCF, MYC, ARF and p53 in MYC-Driven B Cell Neoplasms
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Herbert C. Morse III, Jiafang Sun, Delin Chen, William C. Cho, Wei Gu, Shao Xiang, Ziedulla Abdullaev, Ted A. Torrey, Siegfried Janz, Alexander L. Kovalchuk, Yong-Soo Kim, and Chen-Feng Qi
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ARF-BP1 ,B-cell lymphoma ,p53 ,MYC ,CTCF ,ARF ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Transcriptional activation of MYC is a hallmark of many B cell lineage neoplasms. MYC provides a constitutive proliferative signal but can also initiate ARF-dependent activation of p53 and apoptosis. The E3 ubiquitin ligase, ARF-BP1, encoded by HUWE1, modulates the activity of both the MYC and the ARF-p53 signaling pathways, prompting us to determine if it is involved in the pathogenesis of MYC-driven B cell lymphomas. ARF-BP1 was expressed at high levels in cell lines from lymphomas with either wild type or mutated p53 but not in ARF-deficient cells. Downregulation of ARF-BP1 resulted in elevated steady state levels of p53, growth arrest and apoptosis. Co-immunoprecipitation studies identified a multiprotein complex comprised of ARF-BP1, ARF, p53, MYC and the multifunctional DNA-binding factor, CTCF, which is involved in the transcriptional regulation of MYC, p53 and ARF. ARF-BP1 bound and ubiquitylated CTCF leading to its proteasomal degradation. ARF-BP1 and CTCF thus appear to be key cofactors linking the MYC proliferative and p53-ARF apoptotic pathways. In addition, ARF-BP1 could be a therapeutic target for MYC-driven B lineage neoplasms, even if p53 is inactive, with inhibition reducing the transcriptional activity of MYC for its target genes and stabilizing the apoptosis-promoting activities of p53.
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- 2012
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5. SupplementalFigures 1-4, Tables from Hypomorphic mTOR Downregulates CDK6 and Delays Thymic Pre-T LBL Tumorigenesis
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Beverly A. Mock, Joseph R. Testa, Wendy Dubois, R. Mark Simpson, Maudeline Etienne, Snehal Gaikwad, Matti Kiupel, Tuddow Thaiwong, Jin-Qiu Chen, Aleksandra M. Michalowski, Alexander L. Kovalchuk, Ke Zhang, Benjamin J. Gamache, Nicholas Watson, Tyler J. Peat, Shuling Zhang, Jinfei Xu, John K. Simmons, and Joy M. Gary
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Supplemental Figures S1-S4 + two supp. tables
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- 2023
6. Supplementary Data 1 from Anaplastic, Plasmablastic, and Plasmacytic Plasmacytomas of Mice: Relationships to Human Plasma Cell Neoplasms and Late-Stage Differentiation of Normal B Cells
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Herbert C. Morse, Siegfried Janz, Wendy F. Davidson, Derry C. Roopenian, Janet W. Hartley, Torgny N. Fredrickson, Alexander L. Kovalchuk, Shao Xiang, Zohreh Naghashfar, Chang Hoon Lee, Jeff X. Zhou, and Chen-Feng Qi
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Supplementary Data 1 from Anaplastic, Plasmablastic, and Plasmacytic Plasmacytomas of Mice: Relationships to Human Plasma Cell Neoplasms and Late-Stage Differentiation of Normal B Cells
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- 2023
7. Data from Anaplastic, Plasmablastic, and Plasmacytic Plasmacytomas of Mice: Relationships to Human Plasma Cell Neoplasms and Late-Stage Differentiation of Normal B Cells
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Herbert C. Morse, Siegfried Janz, Wendy F. Davidson, Derry C. Roopenian, Janet W. Hartley, Torgny N. Fredrickson, Alexander L. Kovalchuk, Shao Xiang, Zohreh Naghashfar, Chang Hoon Lee, Jeff X. Zhou, and Chen-Feng Qi
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We have compared histologic features and gene expression profiles of newly identified plasmacytomas from NFS.V+ congenic mice with plasmacytomas of IL6 transgenic, Fasl mutant, and SJL-β2M−/− mice. NFS.V+ tumors comprised an overlapping morphologic spectrum of high-grade/anaplastic, intermediate-grade/plasmablastic, and low-grade/plasmacytic cases with similarities to subsets of human multiple myeloma and plasmacytoma. Microarray and immunohistochemical analyses of genes expressed by the most prevalent tumors, plasmablastic plasmacytomas, showed them to be most closely related to immunoblastic lymphomas, less so to plasmacytomas of Fasl mutant and SJL mice, and least to plasmacytic plasmacytomas of IL6 transgenic mice. Plasmablastic tumors seemed to develop in an inflammatory environment associated with gene signatures of T cells, natural killer cells, and macrophages not seen with plasmacytic plasmacytomas. Plasmablastic plasmacytomas from NFS.V+ and SJL-β2M−/− mice did not have structural alterations in Myc or T(12;15) translocations and did not express Myc at high levels, regular features of transgenic and pristane-induced plasmacytomas. These findings imply that, as for human multiple myeloma, Myc-independent routes of transformation contribute to the pathogenesis of these tumors. These findings suggest that plasma cell neoplasms of mice and humans exhibit similar degrees of complexity. Mouse plasmacytomas, previously considered to be homogeneous, may thus be as diverse as their human counterparts with respect to oncogenic mechanisms of plasma cell transformation. Selecting specific types of mouse plasmacytomas that relate most closely to subtypes of human multiple myeloma may provide new opportunities for preclinical testing of drugs for treatment of the human disease. [Cancer Res 2007;67(6):2439–47]
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- 2023
8. Supplementary Figure 1 from Anaplastic, Plasmablastic, and Plasmacytic Plasmacytomas of Mice: Relationships to Human Plasma Cell Neoplasms and Late-Stage Differentiation of Normal B Cells
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Herbert C. Morse, Siegfried Janz, Wendy F. Davidson, Derry C. Roopenian, Janet W. Hartley, Torgny N. Fredrickson, Alexander L. Kovalchuk, Shao Xiang, Zohreh Naghashfar, Chang Hoon Lee, Jeff X. Zhou, and Chen-Feng Qi
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Supplementary Figure 1 from Anaplastic, Plasmablastic, and Plasmacytic Plasmacytomas of Mice: Relationships to Human Plasma Cell Neoplasms and Late-Stage Differentiation of Normal B Cells
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- 2023
9. Supplementary Figure 3 from Anaplastic, Plasmablastic, and Plasmacytic Plasmacytomas of Mice: Relationships to Human Plasma Cell Neoplasms and Late-Stage Differentiation of Normal B Cells
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Herbert C. Morse, Siegfried Janz, Wendy F. Davidson, Derry C. Roopenian, Janet W. Hartley, Torgny N. Fredrickson, Alexander L. Kovalchuk, Shao Xiang, Zohreh Naghashfar, Chang Hoon Lee, Jeff X. Zhou, and Chen-Feng Qi
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Supplementary Figure 3 from Anaplastic, Plasmablastic, and Plasmacytic Plasmacytomas of Mice: Relationships to Human Plasma Cell Neoplasms and Late-Stage Differentiation of Normal B Cells
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- 2023
10. Supplementary Figure 2 from Anaplastic, Plasmablastic, and Plasmacytic Plasmacytomas of Mice: Relationships to Human Plasma Cell Neoplasms and Late-Stage Differentiation of Normal B Cells
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Herbert C. Morse, Siegfried Janz, Wendy F. Davidson, Derry C. Roopenian, Janet W. Hartley, Torgny N. Fredrickson, Alexander L. Kovalchuk, Shao Xiang, Zohreh Naghashfar, Chang Hoon Lee, Jeff X. Zhou, and Chen-Feng Qi
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Supplementary Figure 2 from Anaplastic, Plasmablastic, and Plasmacytic Plasmacytomas of Mice: Relationships to Human Plasma Cell Neoplasms and Late-Stage Differentiation of Normal B Cells
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- 2023
11. CTCF mediates chromatin looping via N-terminal domain-dependent cohesin retention
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Alexander L. Kovalchuk, Alexander V. Strunnikov, Gabriel E. Zentner, Sungyun Kang, Tajmul, Victor V. Lobanenkov, Naoki Kubo, Dmitri Loukinov, Elena M. Pugacheva, and Bing Ren
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CCCTC-Binding Factor ,Cohesin complex ,Chromosomal Proteins, Non-Histone ,cohesin ,Breast Neoplasms ,Cell Cycle Proteins ,03 medical and health sciences ,0302 clinical medicine ,Protein Domains ,BORIS ,3D genome organization ,Tumor Cells, Cultured ,Humans ,Binding site ,030304 developmental biology ,Zinc finger ,0303 health sciences ,Binding Sites ,Multidisciplinary ,Cohesin ,Genome, Human ,Chemistry ,DNA, Neoplasm ,Cell Biology ,Biological Sciences ,CTCF ,Chromatin ,Cell biology ,DNA-Binding Proteins ,DNA binding site ,030220 oncology & carcinogenesis ,Female ,Chromatin Loop ,biological phenomena, cell phenomena, and immunity ,Protein Binding - Abstract
Significance The DNA-binding protein CCCTC-binding factor (CTCF) and the cohesin complex function together to establish chromatin loops and regulate gene expression in mammalian cells. It has been proposed that the cohesin complex moving bidirectionally along DNA extrudes the chromatin fiber and generates chromatin loops when it pauses at CTCF binding sites. To date, the mechanisms by which cohesin localizes at CTCF binding sites remain unclear. In the present study we define two short segments within the CTCF protein that are essential for localization of cohesin complexes at CTCF binding sites. Based on our data, we propose that the N-terminus of CTCF and 3D geometry of the CTCF–DNA complex act as a roadblock constraining cohesin movement and establishing long-range chromatin loops., The DNA-binding protein CCCTC-binding factor (CTCF) and the cohesin complex function together to shape chromatin architecture in mammalian cells, but the molecular details of this process remain unclear. Here, we demonstrate that a 79-aa region within the CTCF N terminus is essential for cohesin positioning at CTCF binding sites and chromatin loop formation. However, the N terminus of CTCF fused to artificial zinc fingers was not sufficient to redirect cohesin to non-CTCF binding sites, indicating a lack of an autonomously functioning domain in CTCF responsible for cohesin positioning. BORIS (CTCFL), a germline-specific paralog of CTCF, was unable to anchor cohesin to CTCF DNA binding sites. Furthermore, CTCF–BORIS chimeric constructs provided evidence that, besides the N terminus of CTCF, the first two CTCF zinc fingers, and likely the 3D geometry of CTCF–DNA complexes, are also involved in cohesin retention. Based on this knowledge, we were able to convert BORIS into CTCF with respect to cohesin positioning, thus providing additional molecular details of the ability of CTCF to retain cohesin. Taken together, our data provide insight into the process by which DNA-bound CTCF constrains cohesin movement to shape spatiotemporal genome organization.
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- 2020
12. Highly homogenous tri-acylated S-LPS acts as a novel clinically applicable vaccine against Shigella flexneri 2a infection
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Vyacheslav L. L'vov, Marina Eduardovna Golovina, Anna Aleksandrovna Markina, Vladimir A. Ledov, Alexander L. Kovalchuk, Yuriy A. Knirel, and Petr G. Aparin
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Lipopolysaccharides ,Shigellosis ,Lipopolysaccharide ,Acylation ,Guinea Pigs ,030231 tropical medicine ,medicine.disease_cause ,Article ,Shigella flexneri ,Microbiology ,Lipid A ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Immune system ,Bacterial Proteins ,Shigella Vaccines ,Antigen ,Immunity ,Cell Line, Tumor ,medicine ,Animals ,Humans ,Shigella ,030212 general & internal medicine ,Dysentery, Bacillary ,General Veterinary ,General Immunology and Microbiology ,biology ,business.industry ,Public Health, Environmental and Occupational Health ,O Antigens ,U937 Cells ,medicine.disease ,biology.organism_classification ,Antibodies, Bacterial ,Infectious Diseases ,chemistry ,Molecular Medicine ,lipids (amino acids, peptides, and proteins) ,business - Abstract
Shigellosis, a major cause of diarrhea worldwide, exhibits high morbidity and mortality in children. Specificity of Shigella immunity is determined by the structure of the main protective O-antigen polysaccharide component incorporated into the lipopolysaccharide (LPS) molecule. Endotoxicity, however, precludes LPS clinical use. Thus, there is still no vaccine against the most prevalent shigellosis species (serotype S. flexneri 2a), despite ongoing efforts focused on inducing serotype-specific immunity. As LPS is highly heterogenous, we hypothesized that more homogenous pools of LPS might be less toxic. We developed a method to generate a homogenous S. flexneri 2a LPS subfraction, Ac3-S-LPS, containing long chain O-specific polysaccharide (S-LPS) and mainly tri-acylated lipid A, with no penta- and hexa-acylated, and rare tetra-acylated lipid A. Ac3-S-LPS had dramatically reduced pyrogenicity and protected guinea pigs from shigellosis. In volunteers, 50 µg of injected Ac3-S-LPS vaccine was safe, with low pyrogenicity, no severe and few minor adverse events, and did not induce pro-inflammatory cytokines. In spite of the profound lipid A modification, the vaccine induced a prevalence of IgG and IgA antibodies. Thus, we have developed the first safe immunogenic LPS-based vaccine candidate for human administration. Homogenous underacetylated LPSs may also be useful for treating other LPS-driven human diseases. Clinical trial registry: http://grls.rosminzdrav.ru/
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- 2019
13. 3’Igh enhancers hs3b/hs4 are dispensable for Myc deregulation in mouse plasmacytomas with T(12;15) translocations
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Michel Cogné, Tomomi Sakai, Herbert C. Morse, Wendy Du Bois, Alexander L. Kovalchuk, Wesley A. Dunnick, and Chen-Feng Qi
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0301 basic medicine ,Programmed cell death ,biology ,Breakpoint ,translocation ,Chromosomal translocation ,Myc ,medicine.disease_cause ,Molecular biology ,Phenotype ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Oncology ,030220 oncology & carcinogenesis ,biology.protein ,medicine ,Immunoglobulin heavy chain ,Antibody ,PCT ,Enhancer ,Carcinogenesis ,immunoglobulin ,Research Paper - Abstract
Myc-deregulating T(12;15) chromosomal translocations are the hallmark cytogenetic abnormalities of murine plasmacytomas (PCTs). In most PCTs, the immunoglobulin heavy chain (Igh) locus is broken between the Eμ enhancer and the 3’ regulatory region (3’RR), making the latter the major candidate for orchestrating Myc deregulation. To elucidate the role of the Igh3’RR in tumorigenesis, we induced PCTs in Bcl-xL-transgenic mice deficient for the major Igh3’RR enhancer elements, hs3b and hs4 (hs3b-4-/-). Contrary to previous observations using a mouse lymphoma model, which showed no tumors with peripheral B-cell phenotype in hs3b-4-/- mice, these animals developed T(12;15)-positive PCTs, although with a lower incidence than hs3b-4+/+ (wild-type, WT) controls. In heterozygous hs3b-4+/- mice there was no allelic bias in targeting Igh for T(12;15). Molecular analyses of Igh/Myc junctions revealed dominance of Sμ region breakpoints versus the prevalence of Sγ or Sα in WT controls. Myc expression and Ig secretion in hs3b-4-/- PCTs did not differ from WT controls. We also evaluated the effect of a complete Igh3’RR deletion on Myc expression in the context of an established Igh/Myc translocation in ARS/Igh11-transgenic PCT cell lines. Cre-mediated deletion of the Igh3’RR resulted in gradual reduction of Myc expression, loss of proliferative activity and increased cell death, confirming the necessity of the Igh3’RR for Myc deregulation by T(12;15).
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- 2018
14. Hypomorphic mTOR downregulates CDK6 and delays thymic Pre-T LBL tumorigenesis
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Wendy Dubois, Benjamin J. Gamache, Beverly A. Mock, Shuling Zhang, Matti Kiupel, Snehal M. Gaikwad, John K. Simmons, Ke Zhang, Aleksandra M. Michalowski, Tuddow Thaiwong, Joy Gary, Nicholas Watson, Tyler J Peat, Jinfei Xu, Joseph R. Testa, Jinqiu-Qiu Chen, Maudeline Etienne, Alexander L. Kovalchuk, and R. Mark Simpson
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0301 basic medicine ,Cancer Research ,Carcinogenesis ,Down-Regulation ,Mice, Transgenic ,Palbociclib ,medicine.disease_cause ,Article ,03 medical and health sciences ,Mice ,0302 clinical medicine ,medicine ,Animals ,Protein kinase B ,PI3K/AKT/mTOR pathway ,Gene knockdown ,Everolimus ,biology ,Chemistry ,Gene Expression Profiling ,TOR Serine-Threonine Kinases ,Cyclin-Dependent Kinase 6 ,medicine.disease ,Leukemia ,030104 developmental biology ,Oncology ,030220 oncology & carcinogenesis ,Cancer research ,biology.protein ,Cyclin-dependent kinase 6 ,medicine.drug - Abstract
PI3K/AKT/mTOR pathway hyperactivation is frequent in T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/LBL). To model inhibition of mTOR, pre–T-cell lymphoblastic leukemia/lymphoma (pre-T LBL) tumor development was monitored in mice with T lymphocyte–specific, constitutively active AKT (Lck-MyrAkt2) that were either crossed to mTOR knockdown (KD) mice or treated with the mTOR inhibitor everolimus. Lck-MyrAkt2;mTOR KD mice lived significantly longer than Lck-MyrAkt2;mTOR wild-type (WT) mice, although both groups ultimately developed thymic pre-T LBL. An increase in survival was also observed when Lck-MyrAkt2;mTOR WT mice were treated for 8 weeks with everolimus. The transcriptional profiles of WT and KD thymic lymphomas were compared, and Ingenuity Pathway Upstream Regulator Analysis of differentially expressed genes in tumors from mTOR WT versus KD mice identified let-7 and miR-21 as potential regulatory genes. mTOR KD mice had higher levels of let-7a and miR-21 than mTOR WT mice, and rapamycin induced their expression in mTOR WT cells. CDK6 was one of the most downregulated targets of both let-7 and miR21 in mTOR KD tumors. CDK6 overexpression and decreased expression of let-7 in mTOR KD cells rescued a G1 arrest phenotype. Combined mTOR (rapamycin) and CDK4/6 (palbociclib) inhibition decreased tumor size and proliferation in tumor flank transplants, increased survival in an intravenous transplant model of disseminated leukemia compared with single agent treatment, and cooperatively decreased cell viability in human T-ALL/LBL cell lines. Thus, mTOR KD mice provide a model to explore drug combinations synergizing with mTOR inhibitors and can be used to identify downstream targets of inhibition.
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- 2020
15. Insights into the Mechanism of Action of Highly Diluted Biologics
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Sergey A. Tarasov, Reno J. Groenestein, Jean-Pierre Tafani, Oleg I. Epstein, Peter H. van der Meide, Naveena Yanamala, Elena Don, Evgeniy A. Gorbunov, A. Sylvia S. Schleker, Alexander L. Kovalchuk, Judith Klein-Seetharaman, and Alexandra G. Emelyanova
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Drug ,media_common.quotation_subject ,Immunology ,Pharmacology ,medicine.disease_cause ,Autoimmunity ,Cell Line ,Madin Darby Canine Kidney Cells ,03 medical and health sciences ,Interferon-gamma ,Mice ,0302 clinical medicine ,Dogs ,Orthomyxoviridae Infections ,Cell Line, Tumor ,medicine ,Immunology and Allergy ,Animals ,Humans ,Amino Acids ,Receptor ,media_common ,chemistry.chemical_classification ,Biological Products ,Mice, Inbred BALB C ,U937 Cells ,Amino acid ,Transplantation ,Biopharmaceutical ,chemistry ,Mechanism of action ,Influenza A virus ,Leukocytes, Mononuclear ,Female ,medicine.symptom ,Function (biology) ,030215 immunology - Abstract
The therapeutic use of Abs in cancer, autoimmunity, transplantation, and other fields is among the major biopharmaceutical advances of the 20th century. Broader use of Ab-based drugs is constrained because of their high production costs and frequent side effects. One promising approach to overcome these limitations is the use of highly diluted Abs, which are produced by gradual reduction of an Ab concentration to an extremely low level. This technology was used to create a group of drugs for the treatment of various diseases, depending on the specificity of the used Abs. Highly diluted Abs to IFN-γ (hd-anti–IFN-γ) have been demonstrated to be efficacious against influenza and other respiratory infections in a variety of preclinical and clinical studies. In the current study, we provide evidence for a possible mechanism of action of hd-anti–IFN-γ. Using high-resolution solution nuclear magnetic resonance spectroscopy, we show that the drug induced conformational changes in the IFN-γ molecule. Chemical shift changes occurred in the amino acids located primarily at the dimer interface and at the C-terminal region of IFN-γ. These molecular changes could be crucial for the function of the protein, as evidenced by an observed hd-anti–IFN-γ–induced increase in the specific binding of IFN-γ to its receptor in U937 cells, enhanced induced production of IFN-γ in human PBMC culture, and increased survival of influenza A–infected mice.
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- 2020
16. Transcriptional activation of p21Waf1 contributes to suppression of HR by p53 in response to replication arrest induced by camptothecin
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Larisa Y. Romanova, Alexander L. Kovalchuk, and Frederick Mushinski
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p53 ,biology ,Chemistry ,Kinase ,DNA repair ,Protein subunit ,homologous recombination ,complex mixtures ,Cell biology ,enzymes and coenzymes (carbohydrates) ,Oncology ,Cyclin-dependent kinase ,medicine ,biology.protein ,RPA phosphorylation ,transcriptional activation ,Phosphorylation ,Homologous recombination ,Replication protein A ,Camptothecin ,Research Paper ,medicine.drug - Abstract
// Larisa Y. Romanova 1, 2 , Frederick Mushinski 1, * and Alexander L. Kovalchuk 2 1 The Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland, USA 2 The Virology and Cellular Immunology Section, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, NIH, Rockville, Maryland, USA * Deceased Correspondence to: Alexander L. Kovalchuk, email: kovalcha@niaid.nih.gov Keywords: RPA phosphorylation; p53; transcriptional activation; homologous recombination Received: January 24, 2018 Accepted: March 21, 2018 Published: May 22, 2018 ABSTRACT The inhibitory effect of p53 on homologous recombination (HR) is exerted through sequestration of replication protein A (RPA). Release of the p53/RPA complex in response to replication stress is crucially dependent on the phosphorylation status of both proteins and is required for efficient DNA repair by HR. Phosphorylation of RPA within its RPA2 subunit by cyclin-dependent kinases (CDK) is an early event in the replication stress response. Here we investigated the role of transcriptional activation of the p53 downstream target, p21 Waf1 , on RPA2 phosphorylation, the stability of the p53/RPA complex and HR in cells undergoing replication arrest induced by camptothecin (CPT). We show that in CPT-treated cells, activation of p53 and p21 Waf1 impedes RPA2 phosphorylation, while their depletion by siRNA stimulates it. The p53/RPA complex is more stable in wild-type cells than in cells depleted of p21 Waf1 . We used nocodazole-synchronized cells treated with CPT at the entrance to S phase to assess rates of HR. Regardless of their p53 or p21 Waf1 status, the cells proceed through S phase at a similar rate and enter G2. While HR is low in wild-type cells and high in p53-depleted cells, only partial inhibition of HR is observed in the p21 Waf1 -depleted cells. This correlates with the extent of RPA sequestration by p53. Thus, in CPT-treated cells, p53-induced transcriptional activation of p21 Waf1 regulates RPA2 phosphorylation, the stability of the p53/RPA complex and HR.
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- 2018
17. ATP-degrading ENPP1 is required for survival (or persistence) of long-lived plasma cells
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Solomon Conteh, Javier Traba, Chen-Feng Qi, Dong-Mi Shin, Shweta Jain, Patrick E. Duffy, Jeongheon Yoon, Wendy Dubois, Ines Gonzalez-Garcia, Herbert C. Morse, Zohreh Naghashfar, Alexander L. Kovalchuk, Sadia Abbasi, Sungyun Kang, Hongsheng Wang, Michael N. Sack, Jiafang Sun, and Yuanyuan Gao
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0301 basic medicine ,Cell Survival ,Plasma Cells ,lcsh:Medicine ,Bone Marrow Cells ,Carbohydrate metabolism ,Article ,Mice ,03 medical and health sciences ,Adenosine Triphosphate ,Antigen ,Downregulation and upregulation ,Bone Marrow ,medicine ,Animals ,Humans ,Glycolysis ,Pyrophosphatases ,lcsh:Science ,Cells, Cultured ,B cell ,B-Lymphocytes ,Multidisciplinary ,Phosphoric Diester Hydrolases ,Chemistry ,lcsh:R ,Phosphodiesterase ,Germinal center ,Germinal Center ,Molecular biology ,Up-Regulation ,Mice, Inbred C57BL ,Glucose ,030104 developmental biology ,medicine.anatomical_structure ,Antibody Formation ,lcsh:Q ,Bone marrow ,Spleen - Abstract
Survival of antibody-secreting plasma cells (PCs) is vital for sustained antibody production. However, it remains poorly understood how long-lived PCs (LLPCs) are generated and maintained. Here we report that ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is preferentially upregulated in bone marrow LLPCs compared with their splenic short-lived counterparts (SLPCs). We studied ENPP1-deficient mice (Enpp1 −/− ) to determine how the enzyme affects PC biology. Although Enpp1 −/− mice generated normal levels of germinal center B cells and plasmablasts in periphery, they produced significantly reduced numbers of LLPCs following immunization with T-dependent antigens or infection with plasmodium C. chabaudi. Bone marrow chimeric mice showed B cell intrinsic effect of ENPP1 selectively on generation of bone marrow as well as splenic LLPCs. Moreover, Enpp1 −/− PCs took up less glucose and had lower levels of glycolysis than those of wild-type controls. Thus, ENPP1 deficiency confers an energetic disadvantage to PCs for long-term survival and antibody production.
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- 2017
18. Eef1a2 promotes cell growth, inhibits apoptosis and activates JAK/STAT and AKT signaling in mouse plasmacytomas.
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Zhaoyang Li, Chen-Feng Qi, Dong-Mi Shin, Adriana Zingone, Helen J Newbery, Alexander L Kovalchuk, Catherine M Abbott, and Herbert C Morse
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Medicine ,Science - Abstract
The canonical function of EEF1A2, normally expressed only in muscle, brain, and heart, is in translational elongation, but recent studies suggest a non-canonical function as a proto-oncogene that is overexpressed in a variety of solid tumors including breast and ovary. Transcriptional profiling of a spectrum of primary mouse B cell lineage neoplasms showed that transcripts encoding EEF1A2 were uniquely overexpressed in plasmacytomas (PCT), tumors of mature plasma cells. Cases of human multiple myeloma expressed significantly higher levels of EEF1A2 transcripts than normal bone marrow plasma cells. High-level expression was also a feature of a subset of cell lines developed from mouse PCT and from the human MM.Heightened expression of EEF1A2 was not associated with increased copy number or coding sequence mutations. shRNA-mediated knockdown of Eef1a2 transcripts and protein was associated with growth inhibition due to delayed G1-S progression, and effects on apoptosis that were seen only under serum-starved conditions. Transcriptional profiles and western blot analyses of knockdown cells revealed impaired JAK/STAT and PI3K/AKT signaling suggesting their contributions to EEF1A2-mediated effects on PCT induction or progression.EEF1A2 may play contribute to the induction or progression of some PCT and a small percentage of MM. Eef1a2 could also prove to be a useful new marker for a subset of MM and, ultimately, a possible target for therapy.
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- 2010
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19. Associations of Autoimmunity, Immunodeficiency, Lymphomagenesis, and Gut Microbiota in Mice with Knockins for a Pathogenic Autoantibody
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Alexander L. Kovalchuk, Zohreh Naghashfar, Hongsheng Wang, Dong-Mi Shin, Shweta Jain, Jerrold M. Ward, and Herbert C. Morse
- Subjects
Male ,0301 basic medicine ,Lymphoma, B-Cell ,Microarray ,Transgene ,Autoimmunity ,Mice, Transgenic ,Biology ,medicine.disease_cause ,Article ,Pathology and Forensic Medicine ,Mice ,03 medical and health sciences ,medicine ,Animals ,Receptor ,Immunodeficiency ,Autoantibodies ,B-Lymphocytes ,Immunologic Deficiency Syndromes ,Autoantibody ,Heterozygote advantage ,medicine.disease ,Gastrointestinal Microbiome ,Lymphoma ,030104 developmental biology ,Toll-Like Receptor 7 ,Immunology ,Female - Abstract
A number of mouse strains transgenic for B-cell receptors specific for nucleic acids or other autoantigens have been generated to understand how autoreactive B cells are regulated in normal and autoimmune mice. Previous studies of nonautoimmune C57BL/6 mice heterozygous for both the IgH and IgL knockins of the polyreactive autoantibody, 564, produced high levels of autoantibodies in a largely Toll-like receptor 7–dependent manner. Herein, we describe studies of mice homozygous for the knockins that also expressed high levels of autoantibodies but, unlike the heterozygotes, exhibited a high incidence of mature B-cell lymphomas and enhanced susceptibility to bacterial infections. Microarray analyses and serological studies suggested that lymphomagenesis might be related to chronic B-cell activation promoted by IL-21. Strikingly, mice treated continuously with antibiotic-supplemented water did not develop lymphomas or abscesses and exhibited less autoimmunity. This mouse model may help us understand the reasons for enhanced susceptibility to lymphoma development exhibited by humans with a variety of autoimmune diseases, such as Sjögren syndrome, systemic lupus erythematosus, and highly active rheumatoid arthritis.
- Published
- 2017
20. EBI2 overexpression in mice leads to B1 B-cell expansion and chronic lymphocytic leukemia–like B-cell malignancies
- Author
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Hongsheng Wang, Line Barington, Kristine Niss Arfelt, Allan Randrup Thomsen, Maria Rosaria Bassi, Mette M. Rosenkilde, Viktorija Daugvilaite, Thue W. Schwartz, Peter Johannes Holst, Alexander L. Kovalchuk, Tau Benned-Jensen, Kristoffer L. Egerod, Jan Pravsgaard Christensen, Katja Spiess, Herbert C. Morse, and Valentina Kubale
- Subjects
0301 basic medicine ,Lymphoma ,Chronic lymphocytic leukemia ,Immunology ,Population ,Biology ,CD5 Antigens ,Biochemistry ,Receptors, G-Protein-Coupled ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Downregulation and upregulation ,immune system diseases ,hemic and lymphatic diseases ,medicine ,Animals ,education ,B cell ,B-Lymphocytes ,education.field_of_study ,Lymphoid Neoplasia ,GPR183 ,Germinal center ,Cell Biology ,Hematology ,Germinal Center ,medicine.disease ,Leukemia, Lymphocytic, Chronic, B-Cell ,Up-Regulation ,Gene Expression Regulation, Neoplastic ,Leukemia ,030104 developmental biology ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,Cancer research ,CD5 - Abstract
Human and mouse chronic lymphocytic leukemia (CLL) develops from CD5+ B cells that in mice and macaques are known to define the distinct B1a B-cell lineage. B1a cells are characterized by lack of germinal center (GC) development, and the B1a cell population is increased in mice with reduced GC formation. As a major mediator of follicular B-cell migration, the G protein-coupled receptor Epstein-Barr virus-induced gene 2 (EBI2 or GPR183) directs B-cell migration in the lymphoid follicles in response to its endogenous ligands, oxysterols. Thus, upregulation of EBI2 drives the B cells toward the extrafollicular area, whereas downregulation is essential for GC formation. We therefore speculated whether increased expression of EBI2 would lead to an expanded B1 cell subset and, ultimately, progression to CLL. Here, we demonstrate that B-cell-targeted expression of human EBI2 (hEBI2) in mice reduces GC-dependent immune responses, reduces total immunoglobulin M (IgM) and IgG levels, and leads to increased proliferation and upregulation of cellular oncogenes. Furthermore, hEBI2 overexpression leads to an abnormally expanded CD5+ B1a B-cell subset (present as early as 4 days after birth), late-onset lymphoid cancer development, and premature death. These findings are highly similar to those observed in CLL patients and identify EBI2 as a promoter of B-cell malignancies.
- Published
- 2017
21. Transcription factors IRF8 and PU.1 are required for follicular B cell development and BCL6-driven germinal center responses
- Author
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Chen-Feng Qi, Yuanyuan Gao, Alexander L. Kovalchuk, Warren J. Leonard, Jian-Xin Lin, Peng Li, Hongsheng Wang, Shweta Jain, Jiafang Sun, Sadia Abbasi, Silvia Bolland, Tomomi Sakai, Herbert C. Morse, Jangsuk Oh, Zohreh Naghashfar, and Stephen L. Nutt
- Subjects
Mice, Knockout ,B-Lymphocytes ,Multidisciplinary ,Lymphocyte ,Germinal center ,Biology ,BCL6 ,Germinal Center ,Lymphocyte Activation ,Immunoglobulin Class Switching ,Cell biology ,Mice ,medicine.anatomical_structure ,Immunoglobulin class switching ,PNAS Plus ,Proto-Oncogene Proteins ,Interferon Regulatory Factors ,medicine ,Proto-Oncogene Proteins c-bcl-6 ,Trans-Activators ,Animals ,Follicular B cell ,IRF8 ,Transcription factor ,B cell - Abstract
The IRF and Ets families of transcription factors regulate the expression of a range of genes involved in immune cell development and function. However, the understanding of the molecular mechanisms of each family member has been limited due to their redundancy and broad effects on multiple lineages of cells. Here, we report that double deletion of floxed Irf8 and Spi1 (encoding PU.1) by Mb1-Cre (designated DKO mice) in the B cell lineage resulted in severe defects in the development of follicular and germinal center (GC) B cells. Class-switch recombination and antibody affinity maturation were also compromised in DKO mice. RNA-seq (sequencing) and ChIP-seq analyses revealed distinct IRF8 and PU.1 target genes in follicular and activated B cells. DKO B cells had diminished expression of target genes vital for maintaining follicular B cell identity and GC development. Moreover, our findings reveal that expression of B-cell lymphoma protein 6 (BCL6), which is critical for development of germinal center B cells, is dependent on IRF8 and PU.1 in vivo, providing a mechanism for the critical role for IRF8 and PU.1 in the development of GC B cells.
- Published
- 2019
22. Cutting Edge: Expression of IRF8 in Gastric Epithelial Cells Confers Protective Innate Immunity against Helicobacter pylori Infection
- Author
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Chengfu Xu, Warren J. Leonard, Alexander L. Kovalchuk, Yin Zhu, Jiafang Sun, Peng Li, Nonghua Lv, Sadia Abbasi, Hongsheng Wang, Wei Liao, Yan Sun, Herbert C. Morse, Ming Yan, and Jungsoo Joo
- Subjects
0301 basic medicine ,Chromatin Immunoprecipitation ,Cellular differentiation ,Blotting, Western ,Immunology ,Biology ,Real-Time Polymerase Chain Reaction ,Article ,Helicobacter Infections ,Interferon-gamma ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Immune system ,Immunity ,Gastric mucosa ,medicine ,Animals ,Immunology and Allergy ,Oligonucleotide Array Sequence Analysis ,Mice, Knockout ,Regulation of gene expression ,Innate immune system ,Helicobacter pylori ,Flow Cytometry ,biology.organism_classification ,Immunohistochemistry ,Immunity, Innate ,030104 developmental biology ,medicine.anatomical_structure ,Gene Expression Regulation ,Gastric Mucosa ,Interferon Regulatory Factors ,Cancer research ,Chromatin immunoprecipitation ,030215 immunology - Abstract
IFN regulatory factor 8 (IRF8) is expressed in many types of blood cells and plays critical roles in cellular differentiation and function. However, the role of IRF8 in nonhematopoietic systems remains poorly understood. In this study, we provide evidence that IRF8 is a transcriptional modulator of the gastric mucosa necessary for limiting Helicobacter pylori colonization. H. pylori infection significantly upregulated expression of IRF8, which, in turn, promoted IFN-γ expression by gastric epithelial cells. Mice deficient in IRF8 exhibited increased H. pylori colonization and aborted induction of mucosal IFN-γ. Genome-wide analyses of IFN-γ–treated gastric epithelial cells by chromatin immunoprecipitation sequencing and RNA sequencing led to the identification of IRF8 target genes, with many belonging to the IFN-regulated gene family that was observed previously in immune cells. Our results identify the IRF8–IFN-γ circuit as a novel gastric innate immune mechanism in the host defense against infection with H. pylori.
- Published
- 2016
23. DNase-active TREX1 frame-shift mutants induce serologic autoimmunity in mice
- Author
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John P. Atkinson, Herbert C. Morse, Hongsheng Wang, Fred W. Perrino, Chen Feng Qi, Takuya Miyazaki, Tomomi Sakai, Yongsoo Kim, Dong-Mi Shin, Nan Yan, Alexander L. Kovalchuk, Jeeva Munasinghe, Robert N. Fariss, Charles S. Fermaintt, and Parul H. Kothari
- Subjects
0301 basic medicine ,Transgene ,Immunology ,Mutant ,Gene Expression ,Apoptosis ,Autoimmunity ,Mice, Transgenic ,Biology ,medicine.disease_cause ,Article ,Retina ,Frameshift mutation ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Antigen ,medicine ,Immunology and Allergy ,Animals ,Humans ,Genetic Predisposition to Disease ,Aclarubicin ,Frameshift Mutation ,Genetic Association Studies ,Autoantibodies ,Mutation ,B-Lymphocytes ,Thymocytes ,Autoantibody ,Phosphoproteins ,Molecular biology ,Phenotype ,Enzyme Activation ,030104 developmental biology ,Exodeoxyribonucleases ,Amino Acid Substitution ,Transcriptome ,030217 neurology & neurosurgery - Abstract
TREX1/DNASE III, the most abundant 3'-5' DNA exonuclease in mammalian cells, is tail-anchored on the endoplasmic reticulum (ER). Mutations at the N-terminus affecting TREX1 DNase activity are associated with autoimmune and inflammatory conditions such as Aicardi-Goutieres syndrome (AGS). Mutations in the C-terminus of TREX1 cause loss of localization to the ER and dysregulation of oligosaccharyltransferase (OST) activity, and are associated with retinal vasculopathy with cerebral leukodystrophy (RVCL) and in some cases with systemic lupus erythematosus (SLE). Here we investigate mice with conditional expression of the most common RVCL mutation, V235fs, and another mouse expressing a conditional C-terminal mutation, D272fs, associated with a case of human SLE. Mice homozygous for either mutant allele express the encoded human TREX1 truncations without endogenous mouse TREX1, and both remain DNase active in tissues. The two mouse strains are similar phenotypically without major signs of retinal, cerebral or renal disease but exhibit striking elevations of autoantibodies in the serum. The broad range of autoantibodies is primarily against non-nuclear antigens, in sharp contrast to the predominantly DNA-related autoantibodies produced by a TREX1-D18N mouse that specifically lacks DNase activity. We also found that treatment with an OST inhibitor, aclacinomycin, rapidly suppressed autoantibody production in the TREX1 frame-shift mutant mice. Together, our study presents two new mouse models based on TREX1 frame-shift mutations with a unique set of serologic autoimmune-like phenotypes.
- Published
- 2017
24. Rap2b, a novel p53 target, regulates p53-mediated pro-survival function
- Author
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Xinyue Zhang, Wendy Dubois, Xiaolin Wu, Kyoung-Hwa Lee, Jing Huang, Weimin Zhang, Ziqing Li, Alexander L. Kovalchuk, and Yunlong He
- Subjects
Chromatin Immunoprecipitation ,Cell cycle checkpoint ,DNA repair ,DNA damage ,Immunoblotting ,Apoptosis ,Biology ,Real-Time Polymerase Chain Reaction ,Mice ,Transcription (biology) ,Report ,Animals ,Humans ,Epigenetics ,Molecular Biology ,Regulation of gene expression ,Analysis of Variance ,Reverse Transcriptase Polymerase Chain Reaction ,Cell Cycle Checkpoints ,Cell Biology ,Fibroblasts ,HCT116 Cells ,Microarray Analysis ,Chromatin ,rap GTP-Binding Proteins ,Gene Expression Regulation ,Gene Knockdown Techniques ,Cancer research ,Tumor Suppressor Protein p53 ,DNA Damage ,Developmental Biology - Abstract
The tumor suppressor p53 is a critical regulator of apoptosis and cell cycle arrest/pro-survival. Upon DNA damage, p53 evokes both cell cycle arrest/pro-survival and apoptosis transcriptional programs. The ultimate cellular outcome depends on the balance of these two programs. However, the p53 downstream targets that mediate this cell fate decision remain to be identified. Using an integrative genomic approach, we identify Rap2b as a conserved p53-activated gene that counters p53-mediated apoptosis after DNA damage. Upon DNA damage, p53 directly binds to the promoter of Rap2b and activates its transcription. The reduction of Rap2b levels by small interference RNA sensitizes cells to DNA damage-induced apoptosis in a p53-dependent manner. Consistent with its pro-survival function, analysis of cancer genomic data reveals that Rap2b is overexpressed in many types of tumors. Anchorage-independent growth assays show that Rap2b has only weak transformation activity, suggesting that it is not an oncogene by itself. Together, our results identify Rap2b as a new player in the pro-survival program conducted by p53 and raise the possibility that targeting Rap2b could sensitize tumor cells to apoptosis in response to DNA damage.
- Published
- 2013
25. Expression of plasma cell alloantigen 1 defines layered development of B-1a B-cell subsets with distinct innate-like functions
- Author
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Sadia Abbasi, Alexander L. Kovalchuk, Herbert C. Morse, Ines Gonzalez-Garcia, Sophia Chen, Shweta Jain, Dong-Mi Shin, Natalie Beaty, and Hongsheng Wang
- Subjects
endocrine system ,T-Lymphocytes ,Cellular differentiation ,Plasma Cells ,Enzyme-Linked Immunosorbent Assay ,Biology ,Plasma cell ,Statistics, Nonparametric ,Mice ,Immune system ,Antigen ,medicine ,Animals ,Pyrophosphatases ,B cell ,Cell Proliferation ,B-Lymphocytes, Regulatory ,Multidisciplinary ,CD40 ,Phosphoric Diester Hydrolases ,Cell Differentiation ,Biological Sciences ,Flow Cytometry ,Adoptive Transfer ,Immunohistochemistry ,Molecular biology ,Immunity, Innate ,Mice, Inbred C57BL ,B-1 cell ,medicine.anatomical_structure ,Microscopy, Fluorescence ,biology.protein ,Interleukin 12 ,Cytokines - Abstract
Innate-like B-1a cells contribute significantly to circulating natural antibodies and mucosal immunity as well as to immunoregulation. Here we show that these classic functions of B-1a cells segregate between two unique subsets defined by expression of plasma cell alloantigen 1 (PC1), also known as ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1). These subsets, designated B-1a.PC1 lo and B-1a.PC1 hi , differ significantly in IgH chain utilization. Adoptively transferred PC1 lo cells secreted significantly more circulating natural IgM and intestinal IgA than PC1 hi cells. In contrast, PC1 hi cells produced more IL-10 than PC1 lo cells when stimulated with LPS and phorbol 12-myristate 13-acetate (PMA). PC1 hi cells were also more efficient than PC1 lo cells in regulating Th1 cell differentiation, even though both B-1a subsets were comparably active in stimulating T-cell proliferation. Furthermore, PC1 lo cells generated antigen-specific IgM responses to pneumococcal polysaccharide antigens, whereas PC1 hi cells do not. We found that PC1 lo cells develop from an early wave of B-1a progenitors in fetal life, whereas PC1 hi cells are generated from a later wave after birth. We conclude that identification of B-1a.PC1 lo and B-1a.PC1 hi cells extends the concept of a layered immune system with important implications for developing effective vaccines and promoting the generation of immunoregulatory B cells.
- Published
- 2012
26. TMEM4 is Highly Expressed in Plasma Cell Neoplasms and Associated with B Lymphocyte Differentiation
- Author
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Peng Liang, Zhengping Zhuang, Huabao Xiong, Min Sun Shin, Xingpei Hao, Herbert C. Morse, Shao Xiang, Guifang Ouyang, Alexander L. Kovalchuk, Chen-Feng Qi, Jeff X. Zhou, and Susan K Pierce
- Subjects
Pathology ,medicine.medical_specialty ,XBP1 ,medicine.medical_treatment ,CD38 ,Plasma cell ,Plasma cell neoplasm ,Biology ,medicine.disease ,Cytokine ,medicine.anatomical_structure ,Plasma cell differentiation ,medicine ,Cancer research ,Plasmacytoma ,B cell - Abstract
Aiming to identify biomarkers for plasma cell neoplasms, we analyzed gene expression profile and proteomic characteristics of mouse models of plasmacytomas along with other types of B-cell neoplasms. We found that transmembrane protein 4 (Tmem4) was highly expressed in plasmacytomas in comparison with early-stage B-cell neoplasms. The serum levels of Tmem4 were also significantly greater in mice with plasmacytoma comparing to those with early-stage B-cell neoplasms (P
- Published
- 2016
27. An essential role of transcription factors PU.1 and IRF8 in follicular B cell development and the germinal center response
- Author
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Hongsheng Wang, Shweta Jain, Jiafang Sun, Alexander L. Kovalchuk, and Herbert C. Morse
- Subjects
Immunology ,Immunology and Allergy - Abstract
The transcription factors PU.1 and IRF8 regulate an unknown number of gene programs for differentiation of many hematopoietic cells including B cells, dendritic cells and myeloid cells. Their roles in B cell development were previously studied using B cell-specific conditional deletion mouse models, such as PU.1flox/flox-CD19Cre, IRF8flox/flox-CD19Cre, IRF8−/−PU.1flox/flox-CD19Cre, or IRF8flox/flox-PU.1flox/flox-CD19Cre mice. While PU.1-deficient B cells are phenotypically normal, deletion of IRF8 in B cells caused a moderate expansion of marginal zone B cells. Double deletion of PU.1 and IRF8 caused moderate expansion of plasma cells (PCs) in vitro. In this report, we investigated IRF8 and PU.1 double deletion mice using Mb1-Cre - termed DKO mice. FACS analysis revealed normal levels of early stage B cells in the bone marrow and transitional B cells in the spleens of DKO mice. However, follicular B cells were markedly reduced and the MZ B cell compartment was modestly expanded in DKO mice. The peritoneal CD5+ B-1a cells, which are a major source of circulating IgM, were completely absent in DKO mice. Surprisingly, the serum levels of IgM were higher and the levels of IgG3 and IgA were slightly lower in DKO than control mice. While the levels of serum IgG1 were comparable between DKO and control mice, DKO mice had no serum IgG2b or IgG2c antibodies. More intriguingly, following immunization with NP-KLH/alum, although the DKO mice produced more IgM-secreting PCs early on, they failed to generate germinal centers and produced markedly reduced levels of NP-specific switched IgG antibodies. Taken together, these data revealed a critical role of IRF8 and PU.1 in differentiation of follicular and germinal center B cells.
- Published
- 2018
28. Anaplastic plasmacytomas: relationships to normal memory B cells and plasma cell neoplasms of immunodeficient and autoimmune mice
- Author
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Dong-Mi Shin, Chen-Feng Qi, Torgny N. Fredrickson, Alexander L. Kovalchuk, Hongsheng Wang, Janet W. Hartley, Herbert C. Morse, Jianxum Feng, and Zhaoyang Li
- Subjects
medicine.diagnostic_test ,Plasma cell neoplasm ,Plasma cell ,Biology ,medicine.disease ,Molecular biology ,Pathology and Forensic Medicine ,Flow cytometry ,Gene expression profiling ,medicine.anatomical_structure ,Cell culture ,medicine ,Plasmacytoma ,Memory B cell ,Fluorescence in situ hybridization - Abstract
Anaplastic plasmacytomas (APCTs) from NFS.V(+) congenic mice and pristane-induced plasmacytic PCTs from BALB/c mice were previously shown to be histologically and molecularly distinct subsets of plasma cell neoplasms (PCNs). Here we extended these comparisons, contrasting primary APCTs and PCTs by gene expression profiling in relation to the expression profiles of normal naive, germinal centre, and memory B cells and plasma cells. We also sequenced immunoglobulin genes from APCT and APCT-derived cell lines and defined surface phenotypes and chromosomal features of the cell lines by flow cytometry and by spectral karyotyping and fluorescence in situ hybridization. The results indicate that APCTs share many features with normal memory cells and the plasma cell-related neoplasms (PLs) of FASL-deficient mice, suggesting that APCTs and PLs are related and that both derive from memory B cells. Published in 2010 by John Wiley & Sons, Ltd.
- Published
- 2010
29. Restricting activation-induced cytidine deaminase tumorigenic activity in B lymphocytes
- Author
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Michael Potter, Rafael Casellas, Alexander L. Kovalchuk, and Arito Yamane
- Subjects
Immunoglobulin gene ,Genes, Immunoglobulin Heavy Chain ,Immunology ,Genes, myc ,Somatic hypermutation ,Review Article ,Biology ,Lymphocyte Activation ,medicine.disease_cause ,Translocation, Genetic ,Mice ,chemistry.chemical_compound ,Cytidine Deaminase ,medicine ,Activation-induced (cytidine) deaminase ,Animals ,Immunology and Allergy ,Gene ,B-Lymphocytes ,Cytidine deaminase ,Molecular biology ,Cell Transformation, Neoplastic ,chemistry ,Immunoglobulin class switching ,biology.protein ,Somatic Hypermutation, Immunoglobulin ,Carcinogenesis ,DNA ,Plasmacytoma - Abstract
DNA breaks play an essential role in germinal centre B cells as intermediates to immunoglobulin class switching, a recombination process initiated by activation-induced cytidine deaminase (AID). Immunoglobulin gene hypermutation is likewise catalysed by AID but is believed to occur via single-strand DNA breaks. When improperly repaired, AID-mediated lesions can promote chromosomal translocations (CTs) that juxtapose the immunoglobulin loci to heterologous genomic sites, including oncogenes. Two of the most studied translocations are the t(8;14) and T(12;15), which deregulate cMyc in human Burkitt’s lymphomas and mouse plasmacytomas, respectively. While a complete understanding of the aetiology of such translocations is lacking, recent studies using diverse mouse models have shed light on two important issues: (1) the extent to which non-specific or AID-mediated DNA lesions promote CTs, and (2) the safeguard mechanisms that B cells employ to prevent AID tumorigenic activity. Here we review these advances and discuss the usage of pristane-induced mouse plasmacytomas as a tool to investigate the origin of Igh–cMyc translocations and B-cell tumorigenesis.
- Published
- 2009
30. Anaplastic, Plasmablastic, and Plasmacytic Plasmacytomas of Mice: Relationships to Human Plasma Cell Neoplasms and Late-Stage Differentiation of Normal B Cells
- Author
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Zohreh Naghashfar, Torgny N. Fredrickson, Jeff X. Zhou, Chang Hoon Lee, Janet W. Hartley, Alexander L. Kovalchuk, Chen-Feng Qi, Siegfried Janz, Herbert C. Morse, Wendy F. Davidson, Derry C. Roopenian, and Shao Xiang
- Subjects
Genetically modified mouse ,Cancer Research ,Pathology ,medicine.medical_specialty ,Cellular differentiation ,Genes, myc ,Mice, Transgenic ,Plasma cell ,Biology ,Mice ,immune system diseases ,hemic and lymphatic diseases ,medicine ,Animals ,Humans ,Cell Lineage ,neoplasms ,Multiple myeloma ,Neoplasm Staging ,Mice, Knockout ,B-Lymphocytes ,Mice, Inbred BALB C ,Interleukin-6 ,Gene Expression Profiling ,Cancer ,Cell Differentiation ,Plasma cell neoplasm ,medicine.disease ,Immunohistochemistry ,Gene expression profiling ,medicine.anatomical_structure ,Oncology ,Plasmacytoma - Abstract
We have compared histologic features and gene expression profiles of newly identified plasmacytomas from NFS.V+ congenic mice with plasmacytomas of IL6 transgenic, Fasl mutant, and SJL-β2M−/− mice. NFS.V+ tumors comprised an overlapping morphologic spectrum of high-grade/anaplastic, intermediate-grade/plasmablastic, and low-grade/plasmacytic cases with similarities to subsets of human multiple myeloma and plasmacytoma. Microarray and immunohistochemical analyses of genes expressed by the most prevalent tumors, plasmablastic plasmacytomas, showed them to be most closely related to immunoblastic lymphomas, less so to plasmacytomas of Fasl mutant and SJL mice, and least to plasmacytic plasmacytomas of IL6 transgenic mice. Plasmablastic tumors seemed to develop in an inflammatory environment associated with gene signatures of T cells, natural killer cells, and macrophages not seen with plasmacytic plasmacytomas. Plasmablastic plasmacytomas from NFS.V+ and SJL-β2M−/− mice did not have structural alterations in Myc or T(12;15) translocations and did not express Myc at high levels, regular features of transgenic and pristane-induced plasmacytomas. These findings imply that, as for human multiple myeloma, Myc-independent routes of transformation contribute to the pathogenesis of these tumors. These findings suggest that plasma cell neoplasms of mice and humans exhibit similar degrees of complexity. Mouse plasmacytomas, previously considered to be homogeneous, may thus be as diverse as their human counterparts with respect to oncogenic mechanisms of plasma cell transformation. Selecting specific types of mouse plasmacytomas that relate most closely to subtypes of human multiple myeloma may provide new opportunities for preclinical testing of drugs for treatment of the human disease. [Cancer Res 2007;67(6):2439–47]
- Published
- 2007
31. AID-deficient Bcl-xL transgenic mice develop delayed atypical plasma cell tumors with unusual Ig/Myc chromosomal rearrangements
- Author
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Carsten Hirt, Nicole McNeil, Timothy W. Behrens, Wendy Dubois, Zhaoyang Li, Michael Potter, Tasuku Honjo, Thomas Ried, E. B. Mushinski, Chen-Feng Qi, Siegfried Janz, Alexander L. Kovalchuk, and Masamichi Muramatsu
- Subjects
Immunology ,Genes, myc ,bcl-X Protein ,Somatic hypermutation ,Chromosomal translocation ,Mice, Transgenic ,Biology ,Article ,Mice ,Cytidine Deaminase ,medicine ,Immunology and Allergy ,Animals ,Neoplasms, Plasma Cell ,Gene Rearrangement ,Mice, Knockout ,Mice, Inbred BALB C ,medicine.diagnostic_test ,Genes, Immunoglobulin ,Gene rearrangement ,Cytidine deaminase ,Articles ,Molecular biology ,Mice, Inbred C57BL ,Immunoglobulin class switching ,Mice, Inbred DBA ,biology.protein ,Immunoglobulin heavy chain ,Antibody ,Fluorescence in situ hybridization - Abstract
Activation-induced cytidine deaminase (AID) is required for immunoglobulin (Ig) class switch recombination and somatic hypermutation, and has also been implicated in translocations between Ig switch regions and c-Myc in plasma cell tumors in mice. We asked if AID is required for accelerated tumor development in pristane-treated Bcl-xL transgenic BALB/c mice deficient in AID (pBxAicda−/−). pBxAicda−/− mice developed tumors with a lower frequency (24 vs. 62%) and a longer mean latency (108 vs. 36 d) than AID-sufficient mice. The tumors appeared in oil granuloma tissue and did not form ascites. By interphase fluorescence in situ hybridization, six out of nine pBxAicda−/− primary tumors had T(12;15) and one had T(6;15) chromosomal translocations. Two tumors were transplantable and established as stable cell lines. Molecular and cytogenetic analyses showed that one had an unusual unbalanced T(12;15) translocation, with IgH Cμ and Pvt-1 oriented head to tail at the breakpoint, resulting in an elevated expression of c-Myc. In contrast, the second was T(12;15) negative, but had an elevated N-Myc expression caused by a paracentric inversion of chromosome 12. Thus, novel mechanisms juxtapose Ig and Myc-family genes in AID-deficient plasma cell tumors.
- Published
- 2007
32. IL-21-driven neoplasms in SJL mice mimic some key features of human angioimmunoblastic T-cell lymphoma
- Author
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Tomomi Sakai, Herbert C. Morse, Alina Nicolae, Derry C. Roopenian, Caroline M. Leeth, Jing Chen, Dong-Mi Shin, Jerold E. Rehg, Shweta Jain, Elaine S. Jaffe, Mark Raffeld, Alexander L. Kovalchuk, Elisabeth B Adkins, Thomas A. Waldmann, Jerrold M. Ward, Hongsheng Wang, and Thomas J. Sproule
- Subjects
CD4-Positive T-Lymphocytes ,Angioimmunoblastic T-cell lymphoma ,Lymphoma, B-Cell ,Microarray ,Spleen ,Biology ,Lymphoma, T-Cell ,Immunoglobulin G ,Pathology and Forensic Medicine ,Mice ,medicine ,Animals ,Humans ,Oligonucleotide Array Sequence Analysis ,B-Lymphocytes ,Gene Expression Profiling ,Interleukins ,Germinal center ,Interleukin-21 Receptor alpha Subunit ,Regular Article ,Sequence Analysis, DNA ,medicine.disease ,Germinal Center ,Lymphoma ,Gene expression profiling ,Mice, Inbred C57BL ,Disease Models, Animal ,medicine.anatomical_structure ,Interleukin-21 receptor ,Immunoblastic Lymphadenopathy ,Immunology ,biology.protein ,Cytokines ,Female ,Lymph Nodes ,Signal Transduction - Abstract
SJL/J mice exhibit a high incidence of mature B-cell lymphomas that require CD4(+) T cells for their development. We found that their spleens and lymph nodes contained increased numbers of germinal centers and T follicular helper (TFH) cells. Microarray analyses revealed high levels of transcripts encoding IL-21 associated with high levels of serum IL-21. We developed IL-21 receptor (IL21R)-deficient Swiss Jim Lambart (SJL) mice to determine the role of IL-21 in disease. These mice had reduced numbers of TFH cells, lower serum levels of IL-21, and few germinal center B cells, and they did not develop B-cell tumors, suggesting IL-21-dependent B-cell lymphomagenesis. We also noted a series of features common to SJL disease and human angioimmunoblastic T-cell lymphoma (AITL), a malignancy of TFH cells. Gene expression analyses of AITL showed that essentially all cases expressed elevated levels of transcripts for IL21, IL21R, and a series of genes associated with TFH cell development and function. These results identify a mouse model with features of AITL and suggest that patients with the disease might benefit from therapeutic interventions that interrupt IL-21 signaling.
- Published
- 2015
33. Isotype switch-mediatedCH deletions are a recurrent feature ofMyc/CH translocations in peritoneal plasmacytomas in mice
- Author
-
Siegfried Janz and Alexander L. Kovalchuk
- Subjects
Genetically modified mouse ,Cancer Research ,Transgene ,Genes, myc ,Mice, Transgenic ,Chromosomal translocation ,Plasma cell ,Biology ,Translocation, Genetic ,Mice ,medicine ,Animals ,Humans ,Peritoneal Neoplasms ,Sequence Deletion ,Genetics ,Mice, Inbred BALB C ,Base Sequence ,Oncogene ,Interleukin-6 ,Chromosome Mapping ,medicine.disease ,Immunoglobulin Class Switching ,Isotype ,Immunoglobulin Isotypes ,medicine.anatomical_structure ,Oncology ,Immunoglobulin class switching ,Cancer research ,Plasmacytoma ,Immunoglobulin Heavy Chains - Abstract
Oncogene activating chromosomal translocations that interrupt IGH switch (S) regions at 14q32 are thought to be caused by misguided IGH isotype switching in postgerminal center B-cell lymphomas and plasma cell myelomas in humans. Aberrant switching also seems to be involved in altering the fine structure of the translocation in some of these tumors, but the significance of these changes is not known. Here we report on 3 cases of IL-6 transgenic mouse plasmacytomas (PCT) that harbor T(12;15) translocations that had been modified by frustrated switch attempts that result in C(H) deletions. When considered together with 6 similar cases of PCT described previously, our observations suggest that secondary deletions in C(H) are a regular feature in the molecular evolution of T(12;15) translocations and, thereby, in the progression of PCT. We propose that the T(12;15)(+) mouse PCT offers a uniquely valuable model system for elucidating the dual role of abnormal isotype switching in causation and 'remodeling' of chromosomal translocations.
- Published
- 2002
34. Disruption of Transforming Growth Factor β Signaling by a Novel Ligand-dependent Mechanism
- Author
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Alexander L. Kovalchuk, Michael Potter, Tania Fernandez, Valery Bliskovski, Stephanie R. Amoroso, Shellyann Sharpe, Gary M. Jones, Lalage M. Wakefield, John J. Letterio, and Seong-Jin Kim
- Subjects
signal-transduction ,receptor ,Blotting, Western ,Immunology ,Smad2 Protein ,Protein Serine-Threonine Kinases ,Ligands ,Article ,Mice ,Paracrine signalling ,plasmacytoma ,trafficking ,Transforming Growth Factor beta ,Animals ,Immunology and Allergy ,RNA, Antisense ,Autocrine signalling ,R-SMAD ,biology ,Terpenes ,Cell growth ,Cell Membrane ,Receptor, Transforming Growth Factor-beta Type II ,Transforming growth factor beta ,intracellular ,Cell biology ,DNA-Binding Proteins ,Autocrine Communication ,Protein Transport ,Trans-Activators ,biology.protein ,Signal transduction ,Receptors, Transforming Growth Factor beta ,Intracellular ,Protein Binding ,Signal Transduction ,Transforming growth factor - Abstract
Transforming growth factor (TGF)-beta is the prototype in a family of secreted proteins that act in autocrine and paracrine pathways to regulate cell development and function. Normal cells typically coexpress TGF-beta receptors and one or more isoforms of TGF-beta, thus the synthesis and secretion of TGF-beta as an inactive latent complex is considered an essential step in regula-ting the activity of this pathway. To determine whether intracellular activation of TGF-beta results in TGF-beta ligand-receptor interactions within the cell, we studied pristane-induced plasma cell tumors (PCTs). We now demonstrate that active TGF-beta1 in the PCT binds to intracellular TGF-beta type II receptor (TbetaRII). Disruption of the expression of TGF-beta1 by antisense TGF-beta1 mRNA restores localization of TbetaRII at the PCT cell surface, indicating a ligand-induced impediment in receptor trafficking. We also show that retroviral expression of a truncated, dominant-negative TbetaRII (dnTbetaRII) effectively competes for intracellular binding of active ligand in the PCT and restores cell surface expression of the endogenous TbetaRII. Analysis of TGF-beta receptor-activated Smad2 suggests the intracellular ligand-receptor complex is not capable of signaling. These data are the first to demonstrate the formation of an intracellular TGF-beta-receptor complex, and define a novel mechanism for modulating the TGF-beta signaling pathway.
- Published
- 2002
35. IL-6 transgenic mouse model for extraosseous plasmacytoma
- Author
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Herbert C. Morse, Siegfried Janz, Sung Sup Park, Jerrold M. Ward, Tadamitsu Kishimoto, Allen E. Coleman, Michael Potter, Alexander L. Kovalchuk, and Joong Su Kim
- Subjects
Pathology ,medicine.medical_specialty ,Lymphoma, B-Cell ,Time Factors ,Cell Survival ,Genes, myc ,Mice, Transgenic ,Biology ,Translocation, Genetic ,Mice ,Extraosseous Plasmacytoma ,parasitic diseases ,Plasma Cell Myeloma ,medicine ,Animals ,Humans ,In Situ Hybridization, Fluorescence ,B cell ,Multiple myeloma ,Granuloma ,Multidisciplinary ,Interleukin-6 ,Large cell ,Plasmacytosis ,Chromosome Mapping ,Cell Differentiation ,Biological Sciences ,Plasma cell neoplasm ,medicine.disease ,Mice, Inbred C57BL ,Disease Models, Animal ,medicine.anatomical_structure ,Immunology ,Plasmacytoma ,Cell Division ,Neoplasm Transplantation ,hormones, hormone substitutes, and hormone antagonists - Abstract
Plasma cell neoplasms in humans comprise plasma cell myeloma, otherwise known as multiple myeloma, Ig deposition and heavy chain diseases, and plasmacytoma (PCT). A subset of PCT, designated extramedullary PCT, is distinguished from multiple myeloma and solitary PCT of bone by its distribution among various tissue sites but not the bone marrow. Extramedullary (extraosseus) PCT are rare spontaneous neoplasms of mice but are readily induced in a susceptible strain, BALB/c, by treatment with pristane. The tumors develop in peritoneal granulomas and are characterized by Myc -activating T(12;15) chromosomal translocations and, most frequently, by secretion of IgA. A uniting feature of human and mouse plasma cell neoplasms is the critical role played by IL-6, a B cell growth, differentiation, and survival factor. To directly test the contribution of IL-6 to PCT development, we generated BALB/c mice carrying a widely expressed IL-6 transgene. All mice exhibited lymphoproliferation and plasmacytosis. By 18 months of age, over half developed readily transplantable PCT in lymph nodes, Peyer's patches, and sometimes spleen. These neoplasms also had T(12;15) translocations, but remarkably, none expressed IgA. Unexpectedly, ≈30% of the mice developed follicular and diffuse large cell B cell lymphomas that often coexisted with PCT. These findings provide a unique model of extramedullary PCT for studies on pathogenesis and treatment and suggest a previously unappreciated role for IL-6 in the genesis of germinal center-derived lymphomas.
- Published
- 2002
36. Abstract 499: IgH enhancers hs3b/hs4 are dispensable for c-Myc deregulation in mouse plasmacytomas with T(12;15) translocations
- Author
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Alexander L. Kovalchuk and Herbert C. Morse
- Subjects
Cancer Research ,Oncology ,Chromosomal translocation ,Biology ,Enhancer ,Molecular biology - Abstract
c-Myc deregulating t(12;15) chromosomal translocation is the hallmark cytogenetic abnormality of murine plasmacytomas (PCTs). In the great majority of PCTs, the immunoglobulin heavy chain (IgH) locus is broken between Eμ enhancer and the 3’ regulatory region (3’RR), making the latter the major candidate for orchestrating c-Myc deregulation. To elucidate the role of 3’RR in tumorigenesis, we induced PCTs in mice deficient for the major 3’RR enhancer elements, namely hs3b and hs4 (3’KO). Unexpectedly, and contrary to the previous observations made in a mouse lymphoma model, Hs3b/Hs4 3’KO mice did develop t(12;15)-positive PCTs, although with a lower incidence than wild-type controls. In heterozygous mice, there was no allelic bias in targeting IgH for the t(12;15). Molecular analysis of IgH/Myc junctions revealed dominance of Sμ region breakpoints compared to the prevalence of hits into Sγ or Sα in the controls. Analysis of c-Myc expression as well as Ig secretion in 3’KO PCT cell lines revealed no significant differences from the controls. Our current findings highlight the complexity of the 3’RR and the potential of its components to compensate for each other in the context of differentiation from B-lymphocyte to plasma cell. We next extended our observations to include a PCT model where Cre-mediated deletion can remove all four 3'IgH enhancers: hs3a, hs1,2, hs3b and hs4. In PCT cell lines where c-Myc is translocated to an IgH BAC transgene with a floxed 3’IgH enhancer region, induction of retrovirally-expressed Cre-recombinase causes gradual Myc downregulation and complete cell death within 96 hours. The later observation confirms the requirement of the full 3’RR for c-Myc deregulation by T(12;15). Citation Format: Alexander L. Kovalchuk, Herbert C. Morse. IgH enhancers hs3b/hs4 are dispensable for c-Myc deregulation in mouse plasmacytomas with T(12;15) translocations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 499. doi:10.1158/1538-7445.AM2017-499
- Published
- 2017
37. Transgenic mouse model of IgM+ lymphoproliferative disease mimicking Waldenström macroglobulinemia
- Author
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Herbert C. Morse, Jerold E. Rehg, Susan A. Walsh, Michael R. Acevedo, Sunderland Js, Van S. Tompkins, Ramakrishna Sompallae, Timothy R. Rosean, Siegfried Janz, Xuefang Jing, Seong-Su Han, Carol J. Holman, Alexander L. Kovalchuk, and Herms S
- Subjects
0301 basic medicine ,Genetically modified mouse ,biology ,business.industry ,Waldenstrom macroglobulinemia ,Hematology ,Cytidine deaminase ,medicine.disease ,3. Good health ,Lymphoplasmacytic Lymphoma ,Lymphoma ,03 medical and health sciences ,Leukemia ,030104 developmental biology ,Oncology ,immune system diseases ,Immunoglobulin M ,hemic and lymphatic diseases ,B-cell leukemia ,Immunology ,biology.protein ,Medicine ,business - Abstract
Waldenström macroglobulinemia (WM) is a low-grade incurable immunoglobulin M+ (IgM+) lymphoplasmacytic lymphoma for which a genetically engineered mouse model of de novo tumor development is lacking. On the basis of evidence that the pro-inflammatory cytokine, interleukin 6 (IL6), and the survival-enhancing oncoprotein, B cell leukemia 2 (BCL2), have critical roles in the natural history of WM, we hypothesized that the enforced expression of IL6 and BCL2 in mice unable to perform immunoglobulin class switch recombination may result in a lymphoproliferative disease that mimics WM. To evaluate this possibility, we generated compound transgenic BALB/c mice that harbored the human BCL2 and IL6 transgenes, EμSV-BCL2-22 and H2-Ld-hIL6, on the genetic background of activation-induced cytidine deaminase (AID) deficiency. We designated these mice BCL2+IL6+AID− and found that they developed—with full genetic penetrance (100% incidence) and suitably short latency (93 days median survival)—a severe IgM+ lymphoproliferative disorder that recapitulated important features of human WM. However, the BCL2+IL6+AID− model also exhibited shortcomings, such as low serum IgM levels and histopathological changes not seen in patients with WM, collectively indicating that further refinements of the model are required to achieve better correlations with disease characteristics of WM.
- Published
- 2016
38. Non-Hodgkin Lymphomas of Mice
- Author
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Sisir K. Chattopadhyay, Siegfried Janz, Jerrold M. Ward, Alexander L. Kovalchuk, Georg W. Bornkamm, Torgny N. Fredrickson, Herbert C. Morse, Janet W. Hartley, Chen Feng Qi, Neal G. Copeland, Mitsuo Hori, Nancy A. Jenkins, and Shao Xiang
- Subjects
Pathology ,medicine.medical_specialty ,Lymphoma, B-Cell ,Transgene ,Biology ,Lymphoma, T-Cell ,Immunophenotyping ,Mice ,immune system diseases ,hemic and lymphatic diseases ,Tumor Cells, Cultured ,medicine ,Animals ,Humans ,Lymphoid neoplasms ,Molecular Biology ,Genetically engineered ,Lymphoma, Non-Hodgkin ,Mouse Lymphoma ,Cell Biology ,Hematology ,BCL6 ,medicine.disease ,Burkitt Lymphoma ,Lymphoma ,Karyotyping ,Models, Animal ,Splenic Marginal Zone ,Molecular Medicine ,Immunoglobulin Heavy Chains - Abstract
Studies of lymphoid neoplasms occurring in normal or genetically engineered mice have revealed parallels and differences to non-Hodgkin lymphomas (NHL) of humans. Some mouse lymphomas have strong histologic similarities to the human NHL subsets including precursor B- and T-cell lymphoblastic, small lymphocytic, splenic marginal zone, and diffuse large-cell B-cell lymphomas (DLCL); whether molecular parallels also exist is under study. Others mouse types such as sIg+ lymphoblastic B-cell lymphoma have no histologic equivalent in human NHL even though they share molecular deregulation of BCL6 with human DLCL. Finally, Burkitt lymphoma does not appear to occur naturally in mice, but it can be induced with appropriately engineered transgenes.
- Published
- 2001
39. Translocation remodeling in the primary BALB/c plasmacytoma TEPC 3610
- Author
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Thomas Ried, Christoph Cremer, Sung S. Park, Alexander L. Kovalchuk, Allen E. Coleman, Arif Esa, and Siegfried Janz
- Subjects
Cancer Research ,biology ,Promoter ,Chromosomal translocation ,biology.organism_classification ,medicine.disease ,Molecular biology ,Isotype ,DNA sequencing ,BALB/c ,Tumor progression ,Genetics ,medicine ,Plasmacytoma ,Gene - Abstract
Myc-activating chromosomal 12;15 translocations, the hallmark mutations of inflammation-induced BALB/c plasmacytomas, have recently been shown to undergo remodeling by isotype switch-like genetic recombinations that remove approximately 180 kb of immunoglobulin heavy-chain sequence in the vicinity of the rearranged, expressed Myc gene. Here we combine cytogenetic data on the 12;15 translocation (SKY and FISH) with the molecular analysis of key junction sites (long-range PCR followed by DNA sequencing) to demonstrate that translocation remodeling occurred as an infrequent, stepwise, and disomic tumor progression event in the tetraploid, fully transformed, and transplantable plasmacytoma TEPC 3610. This result was used, in conjunction with previously obtained molecular data on five other primary plasmacytomas, to devise a hypothesis that predicts that the selective pressure to undergo translocation remodeling may be predetermined by the location of the break site in Myc. The pressure may be low if the break occurs 5' of the normal promoter region of Myc, but it may be considerably stronger if the break occurs 3' of the Myc promoter. Published 2001 Wiley-Liss, Inc.
- Published
- 2001
40. Interactome maps of mouse gene regulatory domains reveal basic principles of transcriptional regulation
- Author
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Jason Qian, Songjoon Baek, Guoliang Li, Vittorio Sartorelli, Chia-Lin Wei, Yijun Ruan, Ewy Mathé, Hossein Zare, Arito Yamane, Zhonghui Tang, J. Keith Joung, Hirotaka Nakahashi, Nathanael Pruett, Rafael Casellas, Ofir Hakim, Xiaoan Ruan, Kyong-Rim Kieffer-Kwon, Gordon L. Hager, Deepak Reyon, Jizhong Zou, Laura Vian, Alexander L. Kovalchuk, Myong-Hee Sung, Wolfgang Resch, Lars Grøntved, and Steevenson Nelson
- Subjects
Transcription, Genetic ,Regulome ,Enhancer RNAs ,Computational biology ,Biology ,Interactome ,Regulon ,General Biochemistry, Genetics and Molecular Biology ,Article ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Transcriptional regulation ,Animals ,Cell Lineage ,Enhancer ,Promoter Regions, Genetic ,Transcription factor ,Cells, Cultured ,Embryonic Stem Cells ,030304 developmental biology ,Genetics ,Regulation of gene expression ,0303 health sciences ,B-Lymphocytes ,Biochemistry, Genetics and Molecular Biology(all) ,Gene Expression Regulation, Developmental ,DNA Methylation ,Enhancer Elements, Genetic ,Genetic Techniques ,Organ Specificity ,DNA methylation ,CpG Islands ,RNA, Long Noncoding ,030217 neurology & neurosurgery ,Transcription Factors - Abstract
SummaryA key finding of the ENCODE project is that the enhancer landscape of mammalian cells undergoes marked alterations during ontogeny. However, the nature and extent of these changes are unclear. As part of the NIH Mouse Regulome Project, we here combined DNaseI hypersensitivity, ChIP-seq, and ChIA-PET technologies to map the promoter-enhancer interactomes of pluripotent ES cells and differentiated B lymphocytes. We confirm that enhancer usage varies widely across tissues. Unexpectedly, we find that this feature extends to broadly transcribed genes, including Myc and Pim1 cell-cycle regulators, which associate with an entirely different set of enhancers in ES and B cells. By means of high-resolution CpG methylomes, genome editing, and digital footprinting, we show that these enhancers recruit lineage-determining factors. Furthermore, we demonstrate that the turning on and off of enhancers during development correlates with promoter activity. We propose that organisms rely on a dynamic enhancer landscape to control basic cellular functions in a tissue-specific manner.
- Published
- 2013
41. Lymph nodes and Peyer's patches of IL-6 transgenic BALB/c mice harbor T(12;15) translocated plasma cells that contain illegitimate exchanges between the immunoglobulin heavy-chain μ locus and c-myc
- Author
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Tadamitsu Kishimoto, Alexander L. Kovalchuk, and Siegfried Janz
- Subjects
Cancer Research ,Transgene ,Plasma Cells ,Genes, myc ,Chromosomal translocation ,Biology ,Polymerase Chain Reaction ,Sensitivity and Specificity ,Translocation, Genetic ,BALB/c ,Mice ,Peyer's Patches ,medicine ,Animals ,Humans ,DNA Primers ,Recombination, Genetic ,Chromosomes, Human, Pair 15 ,Mice, Inbred BALB C ,Chromosomes, Human, Pair 12 ,Base Sequence ,Immunoglobulin mu-Chains ,Interleukin-6 ,Hematology ,Gene rearrangement ,biology.organism_classification ,medicine.disease ,Molecular biology ,Oncology ,Immunology ,biology.protein ,Plasmacytoma ,Immunoglobulin heavy chain ,Lymph Nodes ,Lymph ,Antibody - Abstract
Hyperplastic plasmacytotic lymph nodes and Peyer's patches of 12 of 25 (48%) BALB/c mice that carried a human IL-6 transgene under the transcriptional control of the histocompatibility H-2L(D) promoter (BALB/c.IL-6 mice) were found to harbor 15 cell clones that contained in their T(12;15) translocation breakpoint regions illegitimate genetic recombinations between the upstream flank of the immunoglobulin heavy-chain C mu locus (5'-C mu) and c-myc (5'-C mu/c-myc+ clones). Similar 5'-C mu/c-myc+ clones were also detected in pristane-induced peritoneal granulomata (a significant source of IL-6 in situ) of three of 13 (13%) conventional BALB/c mice, but not in lymphoid tissues of pristane-treated BALB/c mice, nor in any tissue of untreated BALB/c mice. These findings provided strong evidence that IL-6 may be able to promote the growth and/or survival of clones that contained rearrangements between 5'-C mu and c-myc. Taken in conjunction with our previous observation that 5'-C mu/c-myc+ clones are the precursors for pristane-induced BALB/c plasmacytomas, the findings further suggested that IL-6 may play a pivotal role in the early stage of plasmacytoma development, by promoting tumor precursor cells. The BALB/c.IL-6 model of plasmacytomagenesis may be superior to the conventional BALA/c model because the putative plasmacytoma precursors appear to be more prevalent and in their development independent of treating the mice with inflammation-inducing plasmacytomagenic agents, such as pristane or silicone polymers.
- Published
- 2000
42. Cytogenetic analysis of the bipotential murine pre-B cell lymphoma, P388, and its derivative macrophage-like tumor, P388D1, using SKY and CGH
- Author
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Alexander L. Kovalchuk, Allen E. Coleman, Thomas Ried, Nicole McNeil, ST Forest, and Siegfried Janz
- Subjects
Cancer Research ,medicine.medical_specialty ,Lymphoma, B-Cell ,Myeloid ,Biology ,Polymerase Chain Reaction ,Mice ,medicine ,Animals ,Cell Lineage ,B-cell lymphoma ,Genetics ,Leukemia P388 ,Macrophages ,Lymphoblastic lymphoma ,Cytogenetics ,Nucleic Acid Hybridization ,Cell Differentiation ,Karyotype ,Hematology ,medicine.disease ,DNA Fingerprinting ,Molecular biology ,Lymphoma ,Mice, Inbred C57BL ,medicine.anatomical_structure ,Oncology ,Karyotyping ,Ploidy ,Comparative genomic hybridization - Abstract
Spectral karyotyping (SKY) and comparative genomic hybridization (CGH) were used to elucidate the divergent cytogenetic make-up of the prototypical bilineage lymphoblastic pre-B lymphoma, P388, and its progenitor macrophage-like tumor, P388D1. P388 was found to be diploid and genomically stable. P388D1 was triploid, highly unstable and characterized by numerous marker chromosomes (Chrs) and composite rearrangements. The karyotype of P388D1 was so complex that its clonal relatedness to P388 would have remained questionable without confirmation by molecular analysis of the clonotypic immunoglobulin heavy-chain and light-chain gene recombinations that coexisted in both tumors. The intrinsic instability of the P388D1 genome was indicated by the observation that only four out of 42 aberrations uncovered by SKY (in a total of 27 metaphases) occurred consistently (100% incidence), whereas 27 changes occurred non-randomly (27 to 96% incidence) and 11 alterations randomly (4 to 11% incidence). Persistent cytogenetic instability was also observed in P388 'macrophages' after phorbol ester- and ionomycin-induced conversion in vitro of P388 lymphoma cells. The 'cytogenetic noise' in these cells was manifested by a multiplicity of sporadic chromosomal aberrations; ie 25 distinct changes were identified by SKY in 40 metaphases. The results in P388D1 and P388 'macrophages' were interpreted to indicate that the myeloid differentiation program in the bipotential pre-B cell lymphoma P388 is invariably characterized by karyotypic instability. The study presented here demonstrates the power of the combined SKY and CGH approach to resolve complicated karyotypes of important and widely used mouse tumors.
- Published
- 1999
43. Deletional remodeling of c-myc-deregulating chromosomal translocations
- Author
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Jürgen R. Müller, Siegfried Janz, and Alexander L. Kovalchuk
- Subjects
Cancer Research ,Sequence analysis ,Molecular Sequence Data ,Genes, myc ,Chromosomal translocation ,Locus (genetics) ,Biology ,Translocation, Genetic ,Mice ,immune system diseases ,hemic and lymphatic diseases ,Precursor cell ,Genetics ,medicine ,Animals ,Molecular Biology ,Recombination, Genetic ,Mice, Inbred BALB C ,Base Sequence ,Immunoglobulin mu-Chains ,Breakpoint ,Gene rearrangement ,medicine.disease ,Immunoglobulin Class Switching ,Molecular biology ,Clone Cells ,biology.protein ,Plasmacytoma ,Antibody - Abstract
Evidence is presented for the existence of a novel remodeling-by-deletion mechanism that alters the fine structure of c-myc-deregulating chromosomal translocations in t(12;15)-positive BALB/c plasmacytomas. DNA sequence analysis of the t(12;15) in five primary tumors revealed the co-existence of precursor cells harboring genetic recombinations between the immunoglobulin heavy-chain mu locus (Igh mu) and c-myc with clonally related progenitors containing rearrangements between the immunoglobulin heavy-chain alpha locus (Igh alpha) and c-myc. Clonal relatedness was based upon unique junction fragments between the switch region of Igh mu and c-myc. S mu/c-myc junctions are thus useful clonotypic markers for monitoring the conversion of Igh mu/c-myc-positive tumor precursor clones into Igh alpha/c-myc-positive plasmacytomas. Aberrant isotype switch recombination appears to be the most likely mechanism effecting this conversion event (other possibilities are discussed) which may help to explain the preferred usage of the Igh alpha locus in recombinations with c-myc in t(12;15)-positive plasma cell tumors in BALB/c mice.
- Published
- 1997
44. Homeostatic defects in B cells deficient in the E3 ubiquitin ligase ARF-BP1 are restored by enhanced expression of MYC
- Author
-
Ruihua Zhang, Herbert C. Morse, Tomomi Sakai, Zhaoyang Li, Wei Gu, Jiafang Sun, Huabao Xiong, Dong-Mi Shin, Ning Kon, Chen-Feng Qi, Hongsheng Wang, and Alexander L. Kovalchuk
- Subjects
Cancer Research ,Cellular differentiation ,Ubiquitin-Protein Ligases ,Genes, myc ,Gene Expression ,Transfection ,environment and public health ,Article ,Mice ,Downregulation and upregulation ,Gene expression ,Animals ,Homeostasis ,Mice, Knockout ,Transcriptional activity ,B-Lymphocytes ,biology ,Tumor Suppressor Proteins ,Cell Differentiation ,Hematology ,Genes, p53 ,Molecular biology ,Ubiquitin ligase ,Up-Regulation ,Mice, Inbred C57BL ,enzymes and coenzymes (carbohydrates) ,Oncology ,biology.protein ,biological phenomena, cell phenomena, and immunity ,Function (biology) - Abstract
The E3 ligase ARF-BP1 governs the balance of life and death decisions by directing the degradation of p53 and enhancing the transcriptional activity of MYC. We find B cells selectively deficient in ARF-BP1 have many defects in developing and mature B cells associated with increased expression of p53 and reduced expression of Myc. Overexpression of Myc results in suppression of p53 and complete reversal of defects induced by ARF-BP1 deficiency. These findings indicate that the dynamic balance between MYC and p53 required for normal B cell maturation and function is finely tuned and critically dependent on the activities of ARF-BP1.
- Published
- 2013
45. Abstract 2833: Genetic and pharmacologic inhibition of mTOR delays mortality due to thymc lymphoma formation in mice and is associated with decreases in cell cycle proteins
- Author
-
Jin-Qiu Chen, Alexander L. Kovalchuk, Nicholas Watson, Jinfei Xu, Aleksandra M. Michalowski, Joseph R. Testa, Ke Zhang, Beverly A. Mock, Tuddow Thaiwong, Michelle A. Herrmann, Benjamin J. Gamache, Matti Kiupel, Joy Gary, John K. Simmons, Shuling Zhang, and Wendy Dubois
- Subjects
Cancer Research ,medicine.medical_specialty ,Everolimus ,biology ,business.industry ,RPTOR ,Palbociclib ,medicine.disease ,Lymphoma ,Leukemia ,Endocrinology ,Oncology ,Internal medicine ,Cancer research ,biology.protein ,Medicine ,Cyclin-dependent kinase 6 ,business ,Protein kinase B ,PI3K/AKT/mTOR pathway ,medicine.drug - Abstract
The AKT/mTOR pathway is frequently hyperactivated in T-cell acute lymphoblastic leukemia (T-ALL). To model inhibition of this pathway in lymphoma, mice with T-lymphocyte-specific, constitutively active AKT (Lck-MyrAkt2) were crossed to mice with genetically reduced mTOR expression (knock-down, KD). Mice with genetic reduction of mTOR had increased survival by 10 weeks relative to wild type mTOR mice, though both developed thymic pre-T-cell lymphoblastic leukemia/lymphoma (pre-T LBL). Similarly, when mTOR wild type Lck-MyrAkt2 mice were treated for 8 weeks with the rapamycin analog, everolimus, an inhibitor of the mTOR TORC1 complex, survival was also increased. Gene expression profiling of thymic lymphomas from the mice revealed that mTOR KD was associated with decreased expression of Cdk6, a critical proliferative control node in T-cell development and oncogenic transformation. Pharmacologic inhibition of mTOR in tumor cells also decreased CDK6. The combination of a mTOR inhibitor (rapamycin) and a CDK4/6 inhibitor (PD-0332991, Palbociclib) synergistically decreased the overall viability and signaling downstream of drug targets in mouse lymphoma cells and in human T-ALL/LBL cell lines. This combination was also evaluated in mice using a disseminated leukemia model. In vivo treatment with this combination not only reduced tumor size by inhibiting tumor cell proliferation and arresting tumor cell cycle, but also increased overall survival. We are currently validating upstream regulators of Cdk6 as well as downstream targets in the pre-T LBL tumors from the mTOR deficient mice. Citation Format: Shuling Zhang, Joy M. Gary, John K. Simmons, Jinfei Xu, Benjamin J. Gamache, Ke Zhang, Nicholas Watson, Alexander L. Kovalchuk, Aleksandra M. Michalowski, Jin-Qiu Chen, Michelle A. Herrmann, Tuddow Thaiwong, Matti Kiupel, Wendy Dubois, Joseph R. Testa, Beverly A. Mock. Genetic and pharmacologic inhibition of mTOR delays mortality due to thymc lymphoma formation in mice and is associated with decreases in cell cycle proteins. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2833.
- Published
- 2016
46. Oncogenic Myc translocations are independent of chromosomal location and orientation of the immunoglobulin heavy chain locus
- Author
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Herbert C. Morse, David O. Ferguson, Rafael Casellas, Wesley A. Dunnick, Elizabeth Spehalski, Alexander L. Kovalchuk, Genqing Liang, Wendy Dubois, and John Collins
- Subjects
Lymphoma, B-Cell ,Transgene ,Genes, Immunoglobulin Heavy Chain ,Molecular Sequence Data ,Chromosomal translocation ,Locus (genetics) ,Biology ,Genome ,Proto-Oncogene Mas ,Translocation, Genetic ,Proto-Oncogene Proteins c-myc ,Mice ,Cell Line, Tumor ,medicine ,Animals ,Humans ,Transgenes ,Gene ,Genetics ,Mice, Inbred BALB C ,Multidisciplinary ,Models, Genetic ,Chromosome Mapping ,Biological Sciences ,medicine.disease ,Molecular biology ,Lymphoma ,Gene Expression Regulation, Neoplastic ,Plasmacytoma - Abstract
Many tumors are characterized by recurrent translocations between a tissue-specific gene and a proto-oncogene. The juxtaposition of the Ig heavy chain gene and Myc in Burkitt’s lymphoma and in murine plasmacytoma is a classic example. Regulatory elements within the heavy chain constant region locus are required for Myc translocation and/or deregulation. However, many genes are regulated by cis-acting elements at distances up to 1,000 kb outside the locus. Such putative distal elements have not been examined for the heavy chain locus, particularly in the context of Myc translocations. We demonstrate that a transgene containing the Ig heavy chain constant region locus, inserted into five different chromosomal locations, can undergo translocations involving Myc . Furthermore, these translocations are able to generate plasmacytomas in each transgenic line. We conclude that the heavy chain constant region locus itself includes all of the elements necessary for both the translocation and the deregulation of the proto-oncogene.
- Published
- 2012
47. Mouse model of endemic Burkitt translocations reveals the long-range boundaries of Ig-mediated oncogene deregulation
- Author
-
Wendy Dubois, Gordon L. Hager, Rafael Casellas, Alexander L. Kovalchuk, Wolfgang Resch, Helena Tolarová, Isaac A. Klein, Makiko Takizawa, Ofir Hakim, Herbert C. Morse, Camilo Ansarah-Sobrinho, Michel C. Nussenzweig, Michael Potter, and Arito Yamane
- Subjects
Oncogene Proteins ,Endemic Diseases ,Oncogene Proteins, Fusion ,Genes, Immunoglobulin Heavy Chain ,Plasma Cells ,Genes, myc ,Cytidine ,Biology ,Translocation, Genetic ,Epigenesis, Genetic ,Mice ,hemic and lymphatic diseases ,Oncogene MYCN ,Animals ,Humans ,Epigenetics ,Enhancer ,Gene Rearrangement, B-Lymphocyte ,Uracil-DNA Glycosidase ,Cells, Cultured ,Genetics ,Regulation of gene expression ,B-Lymphocytes ,Multidisciplinary ,Oncogene ,Gene rearrangement ,Biological Sciences ,Burkitt Lymphoma ,Immunoglobulin Class Switching ,Chromatin ,Gene Expression Regulation, Neoplastic ,Disease Models, Animal ,Enhancer Elements, Genetic ,Transcription Initiation Site - Abstract
Human Burkitt lymphomas are divided into two main clinical variants: the endemic form, affecting African children infected with malaria and the Epstein-Barr virus, and the sporadic form, distributed across the rest of the world. However, whereas sporadic translocations decapitate Myc from 5′ proximal regulatory elements, most endemic events occur hundreds of kilobases away from Myc . The origin of these rearrangements and how they deregulate oncogenes at such distances remain unclear. We here recapitulate endemic Burkitt lymphoma-like translocations in plasmacytomas from uracil N -glycosylase and activation-induced cytidine deaminase-deficient mice. Mapping of translocation breakpoints using an acetylated histone H3 lysine 9 chromatin immunoprecipitation sequencing approach reveals Igh fusions up to ∼350 kb upstream of Myc or the related oncogene Mycn . A comprehensive analysis of epigenetic marks, PolII recruitment, and transcription in tumor cells demonstrates that the 3′ Igh enhancer (Eα) vastly remodels ∼450 kb of chromatin into translocated sequences, leading to significant polymerase occupancy and constitutive oncogene expression. We show that this long-range epigenetic reprogramming is directly proportional to the physical interaction of Eα with translocated sites. Our studies thus uncover the extent of epigenetic remodeling by Ig 3′ enhancers and provide a rationale for the long-range deregulation of translocated oncogenes in endemic Burkitt lymphomas. The data also shed light on the origin of endemic-like chromosomal rearrangements.
- Published
- 2012
48. Characterization of ARF-BP1/HUWE1 Interactions with CTCF, MYC, ARF and p53 in MYC-Driven B Cell Neoplasms
- Author
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Yongsoo Kim, Delin Chen, Ziedulla Abdullaev, Wei Gu, Alexander L. Kovalchuk, Jiafang Sun, Herbert C. Morse, Shao Xiang, William C. Cho, Siegfried Janz, Ted A. Torrey, and Chen-Feng Qi
- Subjects
p53 ,CCCTC-Binding Factor ,MYC ,lcsh:Chemistry ,Mice ,0302 clinical medicine ,Transcriptional regulation ,Proto-Oncogene Proteins c-myc ,ARF-BP1 ,B-cell lymphoma ,CTCF ,ARF ,lcsh:QH301-705.5 ,Spectroscopy ,0303 health sciences ,biology ,General Medicine ,3. Good health ,Computer Science Applications ,Ubiquitin ligase ,Gene Expression Regulation, Neoplastic ,030220 oncology & carcinogenesis ,Signal transduction ,Signal Transduction ,Lymphoma, B-Cell ,Ubiquitin-Protein Ligases ,Catalysis ,Article ,Inorganic Chemistry ,03 medical and health sciences ,Downregulation and upregulation ,Cell Line, Tumor ,Animals ,Humans ,Physical and Theoretical Chemistry ,Molecular Biology ,030304 developmental biology ,Tumor Suppressor Proteins ,Organic Chemistry ,HEK 293 cells ,Wild type ,Neoplasms, Experimental ,Repressor Proteins ,HEK293 Cells ,lcsh:Biology (General) ,lcsh:QD1-999 ,biology.protein ,Cancer research ,Tumor Suppressor Protein p53 - Abstract
Transcriptional activation of MYC is a hallmark of many B cell lineage neoplasms. MYC provides a constitutive proliferative signal but can also initiate ARF-dependent activation of p53 and apoptosis. The E3 ubiquitin ligase, ARF-BP1, encoded by HUWE1, modulates the activity of both the MYC and the ARF-p53 signaling pathways, prompting us to determine if it is involved in the pathogenesis of MYC-driven B cell lymphomas. ARF-BP1 was expressed at high levels in cell lines from lymphomas with either wild type or mutated p53 but not in ARF-deficient cells. Downregulation of ARF-BP1 resulted in elevated steady state levels of p53, growth arrest and apoptosis. Co-immunoprecipitation studies identified a multiprotein complex comprised of ARF-BP1, ARF, p53, MYC and the multifunctional DNA-binding factor, CTCF, which is involved in the transcriptional regulation of MYC, p53 and ARF. ARF-BP1 bound and ubiquitylated CTCF leading to its proteasomal degradation. ARF-BP1 and CTCF thus appear to be key cofactors linking the MYC proliferative and p53-ARF apoptotic pathways. In addition, ARF-BP1 could be a therapeutic target for MYC-driven B lineage neoplasms, even if p53 is inactive, with inhibition reducing the transcriptional activity of MYC for its target genes and stabilizing the apoptosis-promoting activities of p53.
- Published
- 2012
- Full Text
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49. Phosphorylation of paxillin at threonine 538 by PKCdelta regulates LFA1-mediated adhesion of lymphoid cells
- Author
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Svenja K. Bahte, Yvona Ward, Alexander L. Kovalchuk, Gibran Holmes, Faikah Gueler, Larisa Y. Romanova, J. Frederic Mushinski, and Patrick J. Nelson
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inorganic chemicals ,Integrin ,Endogeny ,macromolecular substances ,environment and public health ,Cell Line ,Mice ,Two-Hybrid System Techniques ,Cell Adhesion ,Animals ,Humans ,Immunoprecipitation ,Lymphocytes ,Threonine ,Phosphorylation ,Cell Shape ,Paxillin ,Cytoskeleton ,Research Articles ,biology ,Cell Biology ,Actin cytoskeleton ,Molecular biology ,In vitro ,Actins ,Lymphocyte Function-Associated Antigen-1 ,Cell biology ,enzymes and coenzymes (carbohydrates) ,Protein Kinase C-delta ,Phosphothreonine ,Second messenger system ,biology.protein ,Tetradecanoylphorbol Acetate ,Interleukin-3 ,biological phenomena, cell phenomena, and immunity ,Protein Binding ,Signal Transduction - Abstract
We investigated the PKCδ-mediated phosphorylation of paxillin within its LIM4 domain and the involvement of this phosphorylation in activation of LFA-1 integrins of the Baf3 pro-B lymphocytic cell line. Using phosphorylated-threonine-specific antibodies, phosphorylated amino acid analysis and paxillin phosphorylation mutants, we demonstrated that TPA, the pharmacological analog of the endogenous second messenger diacyl glycerol, stimulates paxillin phosphorylation at threonine 538 (T538). The TPA-responsive PKC isoform PKCδ directly binds paxillin in a yeast two-hybrid assay and phosphorylates paxillin at T538 in vitro and also co-immunoprecipitates with paxillin and mediates phosphorylation of this residue in vivo. Recombinant wild-type paxillin, its phospho-inhibitory T538A or phospho-mimetic T538E mutants were expressed in the cells simultaneously with siRNA silencing of the endogenous paxillin. These experiments suggest that phosphorylation of paxillin T538 contributes to dissolution of the actin cytoskeleton, redistribution of LFA-1 integrins and an increase in their affinity. We also show that phosphorylation of T538 is involved in the activation of LFA-1 integrins by TPA.
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- 2010
50. Eef1a2 promotes cell growth, inhibits apoptosis and activates JAK/STAT and AKT signaling in mouse plasmacytomas
- Author
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Helen J Newbery, Chen-Feng Qi, Dong-Mi Shin, Herbert C. Morse, Catherine M. Abbott, Zhaoyang Li, Adriana Zingone, and Alexander L. Kovalchuk
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lcsh:Medicine ,Apoptosis ,Monoclonal Gammopathy of Undetermined Significance ,Proto-Oncogene Mas ,Culture Media, Serum-Free ,Mice ,0302 clinical medicine ,Peptide Elongation Factor 1 ,lcsh:Science ,Medicine(all) ,0303 health sciences ,Gene knockdown ,Multidisciplinary ,Agricultural and Biological Sciences(all) ,Hematology/Myeloma ,Cell Cycle ,JAK-STAT signaling pathway ,Cell cycle ,Gene Expression Regulation, Neoplastic ,STAT Transcription Factors ,030220 oncology & carcinogenesis ,Gene Knockdown Techniques ,Hematology/Lymphomas and Chronic Lymphoblastic Leukemia ,Signal transduction ,Multiple Myeloma ,Signal Transduction ,Research Article ,Plasma Cells ,Bone Marrow Cells ,Biology ,03 medical and health sciences ,Cell Line, Tumor ,Animals ,Humans ,Gene Silencing ,Protein kinase B ,PI3K/AKT/mTOR pathway ,Oncology/Myelomas and Lymphoproliferative Diseases ,030304 developmental biology ,Cell Proliferation ,Janus Kinases ,Cell growth ,Biochemistry, Genetics and Molecular Biology(all) ,lcsh:R ,Enzyme Activation ,Cancer research ,lcsh:Q ,Translational elongation ,Proto-Oncogene Proteins c-akt ,Plasmacytoma - Abstract
Background: The canonical function of EEF1A2, normally expressed only in muscle, brain, and heart, is in translational elongation, but recent studies suggest a non-canonical function as a proto-oncogene that is overexpressed in a variety of solid tumors including breast and ovary. Transcriptional profiling of a spectrum of primary mouse B cell lineage neoplasms showed that transcripts encoding EEF1A2 were uniquely overexpressed in plasmacytomas (PCT), tumors of mature plasma cells. Cases of human multiple myeloma expressed significantly higher levels of EEF1A2 transcripts than normal bone marrow plasma cells. High-level expression was also a feature of a subset of cell lines developed from mouse PCT and from the human MM.Methodology/Principal Findings: Heightened expression of EEF1A2 was not associated with increased copy number or coding sequence mutations. shRNA-mediated knockdown of Eef1a2 transcripts and protein was associated with growth inhibition due to delayed G1-S progression, and effects on apoptosis that were seen only under serum-starved conditions. Transcriptional profiles and western blot analyses of knockdown cells revealed impaired JAK/STAT and PI3K/AKT signaling suggesting their contributions to EEF1A2-mediated effects on PCT induction or progression.Conclusions/Significance: EEF1A2 may play contribute to the induction or progression of some PCT and a small percentage of MM. Eef1a2 could also prove to be a useful new marker for a subset of MM and, ultimately, a possible target for therapy.
- Published
- 2009
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