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1.
Nat Commun ; 13(1): 3743, 2022 06 29.
Article in English | MEDLINE | ID: mdl-35768435

ABSTRACT

Perturbation in the replication-stress response (RSR) and DNA-damage response (DDR) causes genomic instability. Genomic instability occurs in Wiskott-Aldrich syndrome (WAS), a primary immunodeficiency disorder, yet the mechanism remains largely uncharacterized. Replication protein A (RPA), a single-strand DNA (ssDNA) binding protein, has key roles in the RSR and DDR. Here we show that human WAS-protein (WASp) modulates RPA functions at perturbed replication forks (RFs). Following genotoxic insult, WASp accumulates at RFs, associates with RPA, and promotes RPA:ssDNA complexation. WASp deficiency in human lymphocytes destabilizes RPA:ssDNA-complexes, impairs accumulation of RPA, ATR, ETAA1, and TOPBP1 at genotoxin-perturbed RFs, decreases CHK1 activation, and provokes global RF dysfunction. las17 (yeast WAS-homolog)-deficient S. cerevisiae also show decreased ScRPA accumulation at perturbed RFs, impaired DNA recombination, and increased frequency of DNA double-strand break (DSB)-induced single-strand annealing (SSA). Consequently, WASp (or Las17)-deficient cells show increased frequency of DSBs upon genotoxic insult. Our study reveals an evolutionarily conserved, essential role of WASp in the DNA stress-resolution pathway, such that WASp deficiency provokes RPA dysfunction-coupled genomic instability.


Subject(s)
DNA Breaks, Double-Stranded , DNA Replication , DNA, Single-Stranded , Replication Protein A , Saccharomyces cerevisiae Proteins , Wiskott-Aldrich Syndrome Protein , Animals , Antigens, Surface/metabolism , DNA Repair , DNA, Single-Stranded/genetics , DNA, Single-Stranded/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Genomic Instability , Humans , Protein Binding , Replication Protein A/genetics , Replication Protein A/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Wiskott-Aldrich Syndrome Protein/genetics , Wiskott-Aldrich Syndrome Protein/metabolism
2.
Oncogene ; 40(2): 345-354, 2021 01.
Article in English | MEDLINE | ID: mdl-33139832

ABSTRACT

Immunodeficiency is associated with cancer risk. Accordingly, hematolymphoid cancers develop in Wiskott-Aldrich syndrome (WAS), an X-linked primary immunodeficiency disorder (PID) resulting from the deficiency of WAS-protein (WASp) expressed predominantly in the hematolymphoid cell lineages. Despite the correlation between WASp deficiency and hematolymphoid cancers, the molecular mechanism underlying the oncogenic role of WASp is incompletely understood. Employing the WASp-sufficient and WASp-deficient cell-pair model of human T and B lymphocytes, we show that WASp deficiency differentially influences hyperactivation versus inhibition of both CDC42:ERK1/2 and NF-κB:AP-1 pro-oncogenic signaling pathways in nonmalignant versus malignant T and B lymphocytes. Furthermore, WASp deficiency induces a cell-type specific up/down-modulation of the DNA-binding activities of NF-κB, AP-1, and multiple other transcription factors with known roles in oncogenesis. We propose that WASp functions as a putative "tumor-suppressor" protein in normal T and B cells, and "oncoprotein" in a subset of established T and B cell malignancies that are not associated with the NPM-ALK fusion.


Subject(s)
B-Lymphocytes/pathology , Oncogene Proteins/metabolism , T-Lymphocytes/pathology , Tumor Suppressor Proteins/metabolism , Wiskott-Aldrich Syndrome Protein/deficiency , Wiskott-Aldrich Syndrome/pathology , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Cell Proliferation , Cells, Cultured , Humans , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , NF-kappa B/metabolism , Signal Transduction , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Transcription Factor AP-1/metabolism , Wiskott-Aldrich Syndrome/immunology , Wiskott-Aldrich Syndrome/metabolism , cdc42 GTP-Binding Protein/metabolism
4.
J Allergy Clin Immunol ; 145(1): 324-334, 2020 01.
Article in English | MEDLINE | ID: mdl-31604087

ABSTRACT

BACKGROUND: Wiskott-Aldrich syndrome (WAS) is an X-linked primary immune deficiency disorder resulting from Wiskott-Aldrich syndrome protein (WASp) deficiency. Lymphocytes from patients with WAS manifest increased DNA damage and lymphopenia from cell death, yet how WASp influences DNA damage-linked cell survival is unknown. A recently described mechanism promoting cell survival after ionizing radiation (IR)-induced DNA damage involves fragmentation and dispersal of the Golgi apparatus, known as the Golgi-dispersal response (GDR), which uses the Golgi phosphoprotein 3 (GOLPH3)-DNA-dependent protein kinase (DNA-PK)-myosin XVIIIA-F-actin signaling pathway. OBJECTIVE: We sought to define WASp's role in the DNA damage-induced GDR and its disruption as a contributor to the development of radiosensitivity-linked immunodeficiency in patients with WAS. METHODS: In human TH and B-cell culture systems, DNA damage-induced GDR elicited by IR or radiomimetic chemotherapy was monitored in the presence or absence of WASp or GOLPH3 alone or both together. RESULTS: WASp deficiency completely prevents the development of IR-induced GDR in human TH and B cells, despite the high DNA damage load. Loss of WASp impedes nuclear translocation of GOLPH3 and its colocalization with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Surprisingly, however, depletion of GOLPH3 alone or depolymerization of F-actin in WASp-sufficient TH cells still allows development of robust GDR, suggesting that WASp, but not GOLPH3, is essential for GDR and cell survival after IR-induced DNA-damage in human lymphocytes. CONCLUSION: The study identifies WASp as a novel effector of the nucleus-to-Golgi cell-survival pathway triggered by IR-induced DNA damage in cells of the hematolymphoid lineage and proposes an impaired GDR as a new cause for development of a "radiosensitive" form of immune dysregulation in patients with WAS.


Subject(s)
B-Lymphocytes/immunology , DNA Damage/immunology , Golgi Apparatus/immunology , Signal Transduction/immunology , T-Lymphocytes/immunology , Wiskott-Aldrich Syndrome Protein Family/immunology , DNA Damage/genetics , DNA-Activated Protein Kinase/genetics , DNA-Activated Protein Kinase/immunology , Golgi Apparatus/genetics , Humans , Membrane Proteins/genetics , Membrane Proteins/immunology , Wiskott-Aldrich Syndrome/genetics , Wiskott-Aldrich Syndrome/immunology , Wiskott-Aldrich Syndrome Protein Family/genetics
5.
J Allergy Clin Immunol ; 142(1): 219-234, 2018 07.
Article in English | MEDLINE | ID: mdl-29248492

ABSTRACT

BACKGROUND: Wiskott-Aldrich syndrome (WAS), X-linked thrombocytopenia (XLT), and X-linked neutropenia, which are caused by WAS mutations affecting Wiskott-Aldrich syndrome protein (WASp) expression or activity, manifest in immunodeficiency, autoimmunity, genomic instability, and lymphoid and other cancers. WASp supports filamentous actin formation in the cytoplasm and gene transcription in the nucleus. Although the genetic basis for XLT/WAS has been clarified, the relationships between mutant forms of WASp and the diverse features of these disorders remain ill-defined. OBJECTIVE: We sought to define how dysfunctional gene transcription is causally linked to the degree of TH cell deficiency and genomic instability in the XLT/WAS clinical spectrum. METHODS: In human TH1- or TH2-skewing cell culture systems, cotranscriptional R-loops (RNA/DNA duplex and displaced single-stranded DNA) and DNA double-strand breaks (DSBs) were monitored in multiple samples from patients with XLT and WAS and in normal T cells depleted of WASp. RESULTS: WASp deficiency provokes increased R-loops and R-loop-mediated DSBs in TH1 cells relative to TH2 cells. Mechanistically, chromatin occupancy of serine 2-unphosphorylated RNA polymerase II is increased, and that of topoisomerase 1, an R-loop preventing factor, is decreased at R-loop-enriched regions of IFNG and TBX21 (TH1 genes) in TH1 cells. These aberrations accompany increased unspliced (intron-retained) and decreased spliced mRNA of IFNG and TBX21 but not IL13 (TH2 gene). Significantly, increased cellular load of R-loops and DSBs, which are normalized on RNaseH1-mediated suppression of ectopic R-loops, inversely correlates with disease severity scores. CONCLUSION: Transcriptional R-loop imbalance is a novel molecular defect causative in TH1 immunodeficiency and genomic instability in patients with WAS. The study proposes that cellular R-loop load could be used as a potential biomarker for monitoring symptom severity and prognostic outcome in the XLT-WAS clinical spectrum and could be targeted therapeutically.


Subject(s)
Genomic Instability/genetics , Th1 Cells/pathology , Wiskott-Aldrich Syndrome/genetics , Cells, Cultured , DNA Damage/genetics , Humans , Transcription, Genetic , Wiskott-Aldrich Syndrome/pathology
6.
Blood ; 126(14): 1670-82, 2015 Oct 01.
Article in English | MEDLINE | ID: mdl-26261240

ABSTRACT

In Wiskott-Aldrich syndrome (WAS), immunodeficiency and autoimmunity often comanifest, yet how WAS mutations misregulate chromatin-signaling in Thelper (TH) cells favoring development of auto-inflammation over protective immunity is unclear. Previously, we identified an essential promoter-specific, coactivator role of nuclear-WASp in TH1 gene transcription. Here we identify small ubiquitin-related modifier (SUMO)ylation as a novel posttranslational modification of WASp, impairment of which converts nuclear-WASp from a transcriptional coactivator to a corepressor of nuclear factor (NF)-κB response genes in human (TH)1-differentiating cells. V75M, one of many disease-causing mutations occurring in SUMO*motif (72-ψψψψKDxxxxSY-83) of WASp, compromises WASp-SUMOylation, associates with COMMD1 to attenuate NF-κB signaling, and recruits histone deacetylases-6 (HDAC6) to p300-marked promoters of NF-κB response genes that pattern immunity but not inflammation. Consequently, proteins mediating adaptive immunity (IFNG, STAT1, TLR1) are deficient, whereas those mediating auto-inflammation (GM-CSF, TNFAIP2, IL-1ß) are paradoxically increased in TH1 cells expressing SUMOylation-deficient WASp. Moreover, SUMOylation-deficient WASp favors ectopic development of the TH17-like phenotype (↑IL17A, IL21, IL22, IL23R, RORC, and CSF2) under TH1-skewing conditions, suggesting a role for WASp in modulating TH1/TH17 plasticity. Notably, pan-histone deacetylase inhibitors lift promoter-specific repression imposed by SUMOylation-deficient WASp and restore misregulated gene expression. Our findings uncovering a SUMOylation-based mechanism controlling WASp's dichotomous roles in transcription may have implications for personalized therapy for patients carrying mutations that perturb WASp-SUMOylation.


Subject(s)
Gene Expression Regulation/immunology , Mutation , NF-kappa B/immunology , Th1 Cells/immunology , Transcriptional Activation/physiology , Wiskott-Aldrich Syndrome Protein/genetics , Adaptive Immunity/genetics , Adaptive Immunity/immunology , Blotting, Western , Cell Line , Electrophoretic Mobility Shift Assay , Flow Cytometry , Humans , Immunoprecipitation , Mass Spectrometry , Mutagenesis, Site-Directed , NF-kappa B/genetics , Polymerase Chain Reaction , Signal Transduction/genetics , Signal Transduction/immunology , Sumoylation , Transfection
7.
Exp Hematol Oncol ; 4: 9, 2015.
Article in English | MEDLINE | ID: mdl-25838973

ABSTRACT

BACKGROUND: Comparative genetic and biological studies on malignant tumor counterparts in human beings and laboratory mice may be powerful gene discovery tools for blood cancers, including neoplasms of mature B-lymphocytes and plasma cells such as Burkitt lymphoma (BL) and multiple myeloma (MM). METHODS: We used EMSA to detect constitutive NF-κB/STAT3 activity in BL- and MM-like neoplasms that spontaneously developed in single-transgenic IL6 (interleukin-6) or MYC (c-Myc) mice, or in double-transgenic IL6MYC mice. qPCR measurements and analysis of clinical BL and MM datasets were employed to validate candidate NF-κB/STAT3 target genes. RESULTS: qPCR demonstrated that IL6- and/or MYC-dependent neoplasms in mice invariably contain elevated mRNA levels of the NF-κB target genes, Cdkn1a and Fancd2. Clinical studies on human CDKN1A, which encodes the cell cycle inhibitor and tumor suppressor p21, revealed that high p21 message predicts poor therapy response and survival in BL patients. Similarly, up-regulation of FANCD2, which encodes a key member of the Fanconi anemia and breast cancer pathway of DNA repair, was associated with poor outcome of patients with MM, particularly those with high-risk disease. CONCLUSIONS: Our findings suggest that CDKN1A and FANCD2 are potential oncotargets in BL and MM, respectively. Additionally, the IL-6- and/or MYC-driven mouse models of human BL and MM used in this study may lend themselves to the biological validation of CDKN1A and FANCD2 as molecular targets for new approaches to cancer therapy and prevention.

8.
Blood ; 124(23): 3409-19, 2014 Nov 27.
Article in English | MEDLINE | ID: mdl-25253772

ABSTRACT

Wiskott-Aldrich syndrome (WAS), an immunodeficiency disorder, and X-linked thrombocytopenia (XLT), a bleeding disorder, both arise from nonsynonymous mutations in WAS, which encodes a hematopoietic-specific WASp. Intriguingly, XLT evolves into WAS in some patients but not in others; yet the biological basis for this cross-phenotype (CP) effect remains unclear. Using human T-helper (TH) cells expressing different disease-causing WAS mutations, we demonstrated that hSWI/SNF-like complexes require nuclear-WASp to execute their chromatin-remodeling activity at promoters of WASp-target, immune function genes during TH1 differentiation. Hot-spot WAS mutations Thr45Met and Arg86Cys, which result in XLT-to-WAS disease progression, impair recruitment of hBRM- but not BRG1-enriched BAF complexes to IFNG and TBX21 promoters. Moreover, promoter enrichment of histone H2A.Z and its catalyzing enzyme EP400 are both impaired. Consequently, activation of Notch signaling, a hBRM-regulated event, and its downstream effector NF-κB are both compromised, along with decreased accessibility of nucleosomal DNA and inefficient transcription-elongation of WASp-target TH1 genes. In contrast, patient mutations Ala236Gly and Arg477Lys that manifest in XLT without progressing to WAS do not disrupt chromatin remodeling or transcriptional reprogramming of TH1 genes. Our study defines an indispensable relationship between nuclear-WASp- and hSWI/SNF-complexes in gene activation and reveals molecular distinctions in TH cells that might contribute to disease severity in the XLT/WAS clinical spectrum.


Subject(s)
Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Genetic Diseases, X-Linked/diagnosis , T-Lymphocytes/metabolism , Thrombocytopenia/diagnosis , Transcription Factors/genetics , Transcription Factors/metabolism , Wiskott-Aldrich Syndrome Protein/genetics , Wiskott-Aldrich Syndrome/diagnosis , Cell Nucleus/genetics , Cells, Cultured , Diagnosis, Differential , Genetic Diseases, X-Linked/genetics , Humans , Mutation , Promoter Regions, Genetic , Th1 Cells/metabolism , Thrombocytopenia/genetics , Wiskott-Aldrich Syndrome/genetics , Wiskott-Aldrich Syndrome Protein/metabolism
9.
Biochem Biophys Res Commun ; 452(3): 669-75, 2014 Sep 26.
Article in English | MEDLINE | ID: mdl-25193702

ABSTRACT

Piperlongumine (PL), a pepper plant alkaloid from Piper longum, has anti-inflammatory and anti-cancer properties. PL selectively kills both solid and hematologic cancer cells, but not normal counterparts. Here we evaluated the effect of PL on the proliferation and survival of B-cell acute lymphoblastic leukemia (B-ALL), including glucocorticoid (GC)-resistant B-ALL. Regardless of GC-resistance, PL inhibited the proliferation of all B-ALL cell lines, but not normal B cells, in a dose- and time-dependent manner and induced apoptosis via elevation of ROS. Interestingly, PL did not sensitize most of B-ALL cell lines to dexamethasone (DEX). Only UoC-B1 exhibited a weak synergistic effect between PL and DEX. All B-ALL cell lines tested exhibited constitutive activation of multiple transcription factors (TFs), including AP-1, MYC, NF-κB, SP1, STAT1, STAT3, STAT6 and YY1. Treatment of the B-ALL cells with PL significantly downregulated these TFs and modulated their target genes. While activation of AURKB, BIRC5, E2F1, and MYB mRNA levels were significantly downregulated by PL, but SOX4 and XBP levels were increased by PL. Intriguingly, PL also increased the expression of p21 in B-ALL cells through a p53-independent mechanism. Given that these TFs and their target genes play critical roles in a variety of hematological malignancies, our findings provide a strong preclinical rationale for considering PL as a new therapeutic agent for the treatment of B-cell malignancies, including B-ALL and GC-resistant B-ALL.


Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Dioxolanes/pharmacology , Gene Expression Regulation, Leukemic , Neoplasm Proteins/genetics , Piper/chemistry , Antineoplastic Agents, Phytogenic/isolation & purification , Apoptosis/drug effects , Aurora Kinase B/genetics , Aurora Kinase B/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Dexamethasone/pharmacology , Dioxolanes/isolation & purification , Dose-Response Relationship, Drug , Drug Resistance, Neoplasm , E2F1 Transcription Factor/genetics , E2F1 Transcription Factor/metabolism , Glucocorticoids/pharmacology , Humans , Inhibitor of Apoptosis Proteins/genetics , Inhibitor of Apoptosis Proteins/metabolism , Neoplasm Proteins/metabolism , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Proto-Oncogene Proteins c-myb/genetics , Proto-Oncogene Proteins c-myb/metabolism , Regulatory Factor X Transcription Factors , SOXC Transcription Factors/genetics , SOXC Transcription Factors/metabolism , Signal Transduction , Survivin , Transcription Factors/genetics , Transcription Factors/metabolism , rho GTP-Binding Proteins/genetics , rho GTP-Binding Proteins/metabolism
10.
J Exp Med ; 211(5): 869-86, 2014 May 05.
Article in English | MEDLINE | ID: mdl-24799533

ABSTRACT

IκB kinase ß (IKKß), a central coordinator of inflammatory responses through activation of NF-κB, has been implicated in vascular pathologies, but its role in atherogenesis remains elusive. Here, we demonstrate that IKKß functions in smooth muscle cells (SMCs) to regulate vascular inflammatory responses and atherosclerosis development. IKKß deficiency in SMCs driven by a SM22Cre-IKKß-flox system rendered low density lipoprotein receptor-null mice resistant to vascular inflammation and atherosclerosis induced by high-fat feeding. Unexpectedly, IKKß-deficient mice were also resistant to diet-induced obesity and metabolic disorders. Cell lineage analysis revealed that SM22Cre is active in primary adipose stromal vascular cells and deficiency of IKKß diminished the ability of these cells to differentiate, leading to accumulation of adipocyte precursor cells in adipose tissue. Mechanistically, reduction of IKKß expression or pharmacological inhibition of IKKß inhibited proteasome-mediated ß-catenin ubiquitination and degradation in murine preadipocytes, resulting in elevated ß-catenin levels and impaired adipocyte differentiation. Further, chronic treatment of mice with a potent IKKß inhibitor decreased adipogenesis and ameliorated diet-induced obesity. Our findings demonstrate a pivotal role of IKKß in linking vascular inflammation to atherosclerosis and adipose tissue development, and provide evidence for using appropriate IKKß inhibitors in the treatment of obesity and metabolic disorders.


Subject(s)
Atherosclerosis/metabolism , I-kappa B Kinase/metabolism , Myocytes, Smooth Muscle/metabolism , Obesity/metabolism , Vasculitis/metabolism , Absorptiometry, Photon , Adipogenesis/physiology , Animals , Blotting, Western , Body Composition , Body Weight , Cell Lineage , Cholesterol/blood , Chromatography, Liquid , Electrophoretic Mobility Shift Assay , Gene Expression Regulation/physiology , I-kappa B Kinase/antagonists & inhibitors , Magnetic Resonance Spectroscopy , Mice , Microfilament Proteins/deficiency , Microfilament Proteins/genetics , Microfilament Proteins/metabolism , Muscle Proteins/deficiency , Muscle Proteins/genetics , Muscle Proteins/metabolism , Triglycerides/blood , Ubiquitination , beta Catenin/metabolism
11.
PLoS One ; 8(10): e76889, 2013.
Article in English | MEDLINE | ID: mdl-24130802

ABSTRACT

Comparative genome-wide expression profiling of malignant tumor counterparts across the human-mouse species barrier has a successful track record as a gene discovery tool in liver, breast, lung, prostate and other cancers, but has been largely neglected in studies on neoplasms of mature B-lymphocytes such as diffuse large B cell lymphoma (DLBCL) and Burkitt lymphoma (BL). We used global gene expression profiles of DLBCL-like tumors that arose spontaneously in Myc-transgenic C57BL/6 mice as a phylogenetically conserved filter for analyzing the human DLBCL transcriptome. The human and mouse lymphomas were found to have 60 concordantly deregulated genes in common, including 8 genes that Cox hazard regression analysis associated with overall survival in a published landmark dataset of DLBCL. Genetic network analysis of the 60 genes followed by biological validation studies indicate FOXM1 as a candidate DLBCL and BL gene, supporting a number of studies contending that FOXM1 is a therapeutic target in mature B cell tumors. Our findings demonstrate the value of the "mouse filter" for genomic studies of human B-lineage neoplasms for which a vast knowledge base already exists.


Subject(s)
Burkitt Lymphoma/genetics , Gene Expression Profiling , Genes, Neoplasm/genetics , Lymphoma, Large B-Cell, Diffuse/genetics , Animals , Burkitt Lymphoma/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Forkhead Box Protein M1 , Forkhead Transcription Factors/antagonists & inhibitors , Forkhead Transcription Factors/genetics , Gene Regulatory Networks , Humans , Lymphoma, Large B-Cell, Diffuse/pathology , Mice , Species Specificity , Thiostrepton/pharmacology
12.
Biochem Biophys Res Commun ; 436(4): 660-5, 2013 Jul 12.
Article in English | MEDLINE | ID: mdl-23764397

ABSTRACT

Piperlongumine (PL), isolated from the fruit of Long pepper, Piper longum, is a cancer-inhibiting compound that selectively kills tumor cells while sparing their normal counterparts. Here we evaluated the efficacy with which PL suppresses malignant B cells derived from a newly developed, double-transgenic mouse model of human endemic Burkitt lymphoma (BL), designated mCD40-LMP1/iMyc(Eµ). PL inhibited tumor cell proliferation in a concentration-dependent manner and induced apoptosis of neoplastic but not normal B cells. Treatment with PL resulted in downregulation of EBV-encoded LMP1, cellular Myc, constitutive NF-κB activity, and a host of LMP1-Myc-NF-κB-regulated target genes including Aurka, Bcat1, Bub1b, Ccnb1, Chek1, Fancd2, Tfrc and Xrcc6. Of note, p21(Cip1)-encoding Cdkn1a was suppressed independent of changes in Trp53 mRNA levels and p53 DNA-binding activity. Considering the central role of the LMP1-NF-κB-Myc axis in B-lineage neoplasia, these findings further our understanding of the mechanisms by which PL inhibits B-lymphoma and provide a preclinical rationale for the inclusion of PL in new interventions in blood cancers.


Subject(s)
Dioxolanes/pharmacology , Genes, myc , Lymphoma, B-Cell/pathology , Viral Matrix Proteins/physiology , Animals , Cell Line, Tumor , Lymphoma, B-Cell/genetics , Mice , Mice, Inbred C57BL , NF-kappa B/metabolism
13.
Leuk Res ; 37(2): 146-54, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23237561

ABSTRACT

Piperlongumine (PL), a pepper plant alkaloid from Piper longum, kills solid tumor cells in a highly selective, potent fashion. To evaluate whether PL may have similar effects on malignant blood cells, we determined the efficacy with which PL inhibits the B-lymphocyte derived neoplasm, Burkitt lymphoma (BL). Low micromolar concentrations of PL (IC(50) = 2.8 µM × 8.5 µM) curbed growth and survival of two EBV(+) BL cell lines (Daudi, Raji) and two EBV BL cell lines (Ramos, DG-75), but left normal peripheral blood B-lymphocytes unharmed. PL-dependent cytotoxicity was effected in part by reduced NF-κB and MYC activity, with the former being caused by inhibition of IκBα degradation, nuclear translocation of p65, and binding of NF-κB dimers to cognate DNA sequences in gene promoters. In 4 of 4 BL cell lines, the NF-κB/MYC-regulated cellular target genes, E2F1 and MYB, were down regulated, while the stress sensor gene, GADD45B, was up regulated. The EBV-encoded oncogene, LMP-1, was suppressed in Daudi and Raji cells. Considering that NF-κB, MYC and LMP-1 play a crucial role in the biology of many blood cancers including BL, our results provide a strong preclinical rationale for considering PL in new intervention approaches for patients with hematologic malignancies.


Subject(s)
Antineoplastic Agents/pharmacology , Burkitt Lymphoma/metabolism , Dioxolanes/pharmacology , Antigens, Differentiation/genetics , Antineoplastic Agents/chemistry , Apoptosis/drug effects , Burkitt Lymphoma/genetics , Burkitt Lymphoma/virology , Caspase 3/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Dioxolanes/chemistry , E2F1 Transcription Factor/genetics , Enzyme Activation/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Genes, myb , Humans , Inhibitory Concentration 50 , NF-kappa B/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Viral Matrix Proteins/genetics
14.
Arterioscler Thromb Vasc Biol ; 32(12): 2869-76, 2012 Dec.
Article in English | MEDLINE | ID: mdl-23023371

ABSTRACT

OBJECTIVE: Inflammatory responses are the driving force of atherosclerosis development. IκB kinase ß (IKKß), a central coordinator in inflammation through regulation of nuclear factor-κB, has been implicated in the pathogenesis of atherosclerosis. Macrophages play an essential role in the initiation and progression of atherosclerosis, yet the role of macrophage IKKß in atherosclerosis remains elusive and controversial. This study aims to investigate the impact of IKKß expression on macrophage functions and to assess the effect of myeloid-specific IKKß deletion on atherosclerosis development. METHODS AND RESULTS: To explore the issue of macrophage IKKß involvement of atherogenesis, we generated myeloid-specific IKKß-deficient low-density lipoprotein receptor-deficient mice (IKKß(ΔMye)LDLR(-/-)). Deficiency of IKKß in myeloid cells did not affect plasma lipid levels but significantly decreased diet-induced atherosclerotic lesion areas in the aortic root, brachiocephalic artery, and aortic arch of low-density lipoprotein receptor-deficient mice. Ablation of myeloid IKKß attenuated macrophage inflammatory responses and decreased atherosclerotic lesional inflammation. Furthermore, deficiency of IKKß decreased adhesion, migration, and lipid uptake in macrophages. CONCLUSIONS: The present study demonstrates a pivotal role for myeloid IKKß expression in atherosclerosis by modulating macrophage functions involved in atherogenesis. These results suggest that inhibiting nuclear factor-κB activation in macrophages may represent a feasible approach to combat atherosclerosis.


Subject(s)
Atherosclerosis/prevention & control , I-kappa B Kinase/deficiency , Myeloid Cells/metabolism , Receptors, LDL/deficiency , Animals , Atherosclerosis/metabolism , Atherosclerosis/pathology , Cell Adhesion , Cell Movement , Disease Models, Animal , I-kappa B Kinase/genetics , I-kappa B Kinase/metabolism , Lipid Metabolism , Macrophages/metabolism , Macrophages/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Receptors, LDL/genetics , Receptors, LDL/metabolism
15.
Biochem Biophys Res Commun ; 427(2): 349-54, 2012 Oct 19.
Article in English | MEDLINE | ID: mdl-22995306

ABSTRACT

Piperlongumine (piplartine, PL) is an alkaloid found in the long pepper (Piper longum L.) and has well-documented anti-platelet aggregation, anti-inflammatory, and anti-cancer properties; however, the role of PL in prevention of atherosclerosis is unknown. We evaluated the anti-atherosclerotic potential of PL in an in vivo murine model of accelerated atherosclerosis and defined its mechanism of action in aortic vascular smooth muscle cells (VSMCs) in vitro. Local treatment with PL significantly reduced atherosclerotic plaque formation as well as proliferation and nuclear factor-kappa B (NF-κB) activation in an in vivo setting. PL treatment in VSMCs in vitro showed inhibition of migration and platelet-derived growth factor BB (PDGF-BB)-induced proliferation to the in vivo findings. We further identified that PL inhibited PDGF-BB-induced PDGF receptor beta activation and suppressed downstream signaling molecules such as phospholipase Cγ1, extracellular signal-regulated kinases 1 and 2 and Akt. Lastly, PL significantly attenuated activation of NF-κB-a downstream transcriptional regulator in PDGF receptor signaling, in response to PDGF-BB stimulation. In conclusion, our findings demonstrate a novel, therapeutic mechanism by which PL suppresses atherosclerosis plaque formation in vivo.


Subject(s)
Dioxolanes/administration & dosage , Muscle, Smooth, Vascular/drug effects , Myocytes, Smooth Muscle/drug effects , Plaque, Atherosclerotic/prevention & control , Receptors, Platelet-Derived Growth Factor/agonists , Animals , Apolipoproteins E/genetics , Carotid Arteries , Cell Proliferation/drug effects , Disease Models, Animal , Ligation , Mice , Mice, Knockout , Muscle, Smooth, Vascular/pathology , Myocytes, Smooth Muscle/pathology , NF-kappa B/metabolism , Phosphorylation/drug effects , Receptors, Platelet-Derived Growth Factor/metabolism , Signal Transduction
16.
Toxicol Appl Pharmacol ; 261(2): 181-8, 2012 Jun 01.
Article in English | MEDLINE | ID: mdl-22521609

ABSTRACT

Tea flavonoids such as epigallocatechin gallate (EGCG) protect against vascular diseases such as atherosclerosis via their antioxidant and anti-inflammatory functions. Persistent and widespread environmental pollutants, including polychlorinated biphenyls (PCB), can induce oxidative stress and inflammation in vascular endothelial cells. Even though PCBs are no longer produced, they are still detected in human blood and tissues and thus considered a risk for vascular dysfunction. We hypothesized that EGCG can protect endothelial cells against PCB-induced cell damage via its antioxidant and anti-inflammatory properties. To test this hypothesis, primary vascular endothelial cells were pretreated with EGCG, followed by exposure to the coplanar PCB 126. Exposure to PCB 126 significantly increased cytochrome P450 1A1 (Cyp1A1) mRNA and protein expression and superoxide production, events which were significantly attenuated following pretreatment with EGCG. Similarly, EGCG also reduced DNA binding of NF-κB and downstream expression of inflammatory markers such as monocyte chemotactic protein-1 (MCP-1) and vascular cell adhesion protein-1 (VCAM-1) after PCB exposure. Furthermore, EGCG decreased endogenous or base-line levels of Cyp1A1, MCP-1 and VCAM-1 in endothelial cells. Most of all, treatment of EGCG upregulated expression of NF-E2-related factor 2 (Nrf2)-controlled antioxidant genes, including glutathione S transferase (GST) and NAD(P)H:quinone oxidoreductase 1 (NQO1), in a dose-dependent manner. In contrast, silencing of Nrf2 increased Cyp1A1, MCP-1 and VCAM-1 and decreased GST and NQO1 expression, respectively. These data suggest that EGCG can inhibit AhR regulated genes and induce Nrf2-regulated antioxidant enzymes, thus providing protection against PCB-induced inflammatory responses in endothelial cells.


Subject(s)
Catechin/analogs & derivatives , Endothelial Cells/drug effects , Gene Expression Regulation/drug effects , NF-E2-Related Factor 2/physiology , Polychlorinated Biphenyls/toxicity , Receptors, Aryl Hydrocarbon/antagonists & inhibitors , Animals , Catechin/pharmacology , Cells, Cultured , Chemokine CCL2/genetics , Cytochrome P-450 CYP1A1/biosynthesis , Cytoprotection , Glutathione Transferase/genetics , NAD(P)H Dehydrogenase (Quinone)/genetics , NF-kappa B/metabolism , Oxidative Stress/drug effects , Receptors, Aryl Hydrocarbon/metabolism , Swine , Vascular Cell Adhesion Molecule-1/genetics
17.
Mol Cancer ; 9: 97, 2010 Apr 30.
Article in English | MEDLINE | ID: mdl-20433747

ABSTRACT

BACKGROUND: Myc is a well known driver of lymphomagenesis, and Myc-activating chromosomal translocation is the recognized hallmark of Burkitt lymphoma, an aggressive form of non-Hodgkin's lymphoma. We developed a model that mimics this translocation event by inserting a mouse Myc cDNA gene into the immunoglobulin heavy chain locus, just upstream of the intronic Emu enhancer. These mice, designated iMyc E mu, readily develop B-cell lymphoma. To study the mechanism of Myc-induced lymphoma, we analyzed signaling pathways in lymphoblastic B-cell lymphomas (LBLs) from iMyc E mu mice, and an LBL-derived cell line, iMyc E mu-1. RESULTS: Nuclear factor-kappaB (NF-kappaB) and signal transducer and activator of transcription 3 (STAT3) were constitutively activated in iMyc E mu mice, not only in LBLs but also in the splenic B-lymphocytes of young animals months before tumors developed. Moreover, inhibition of either transcription factor in iMyc E mu-1 cells suppressed growth and caused apoptosis, and the abrogation of NF-kappaB activity reduced DNA binding by both STAT3 and Myc, as well as Myc expression. Inhibition of STAT3 signaling eliminated the activity of both NF-kappaB and Myc, and resulted in a corresponding decrease in the level of Myc. Thus, in iMyc E mu-1 cells NF-kappaB and STAT3 are co-dependent and can both regulate Myc. Consistent with this, NF-kappaB and phosphorylated STAT3 were physically associated with one another. In addition, LBLs and iMyc E mu-1 cells also showed constitutive AKT phosphorylation. Blocking AKT activation by inhibiting PI3K reduced iMyc E mu-1 cell proliferation and caused apoptosis, via downregulation of NF-kappaB and STAT3 activity and a reduction of Myc levels. Co-treatment with NF-kappaB, STAT3 or/and PI3K inhibitors led to additive inhibition of iMyc E mu-1 cell proliferation, suggesting that these signaling pathways converge. CONCLUSIONS: Our findings support the notion that constitutive activation of NF-kappaB and STAT3 depends on upstream signaling through PI3K, and that this activation is important for cell survival and proliferation, as well as for maintaining the level of Myc. Together, these data implicate crosstalk among NF-kappaB, STAT3 and PI3K in the development of iMyc E mu B-cell lymphomas.


Subject(s)
Lymphoma, B-Cell/metabolism , NF-kappa B/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Receptor Cross-Talk/physiology , STAT3 Transcription Factor/metabolism , Signal Transduction/genetics , Animals , Apoptosis/physiology , Blotting, Western , Cell Line, Tumor , DNA Fragmentation , Disease Models, Animal , Electrophoretic Mobility Shift Assay , Enhancer Elements, Genetic , Enzyme Activation/physiology , Enzyme-Linked Immunosorbent Assay , Gene Expression/genetics , Gene Expression Regulation, Neoplastic/genetics , Genes, Immunoglobulin Heavy Chain , Genes, myc , Immunoprecipitation , Lymphoma, B-Cell/genetics , Mice , NF-kappa B/genetics , Phosphatidylinositol 3-Kinases/genetics , Reverse Transcriptase Polymerase Chain Reaction , STAT3 Transcription Factor/genetics
18.
Cancer Res ; 67(1): 246-53, 2007 Jan 01.
Article in English | MEDLINE | ID: mdl-17185378

ABSTRACT

Deletion or mutation of the androgen receptor (AR) renders prostate tumors refractory to apoptosis by androgen ablation, the mainstay of prostate cancer therapy. To identify novel therapeutics that can induce apoptosis regardless of the AR status of prostate cancer cells, we screened dietary herbal compounds using a reporter assay for the prostate apoptosis response-4 (Par-4) gene, which induces p53- and PTEN-independent and cancer-selective apoptosis. One of the compounds, withaferin A (WA), a major constituent of the dietary compound Withania somnifera, induced Par-4-dependent apoptosis in androgen-refractory prostate cancer cells and regression of PC-3 xenografts in nude mice. Interestingly, restoration of wild-type AR in PC-3 (AR negative) cells abrogated both Par-4 induction and apoptosis by WA. Individually, WA and anti-androgens induced neither Par-4 nor apoptosis in androgen-responsive prostate cancer cells, yet in combination, WA and anti-androgen synergistically induced Par-4 and apoptosis in androgen-responsive prostate cancer cells. Thus, when judiciously combined with anti-androgens, WA inhibits survival of both androgen-responsive and androgen-refractory prostate cancer cells by a Par-4-dependent mechanism. As Par-4 up-regulation induces apoptosis in most tumor cells, our findings can be extended to high-throughput screens to identify synergistic combinations for both therapy-sensitive and therapy-resistant cancers.


Subject(s)
Apoptosis Regulatory Proteins/physiology , Apoptosis/drug effects , Ergosterol/analogs & derivatives , Prostatic Neoplasms/drug therapy , Androgen Receptor Antagonists , Animals , Apoptosis/physiology , Apoptosis Regulatory Proteins/biosynthesis , Apoptosis Regulatory Proteins/genetics , Cell Line, Tumor , Ergosterol/pharmacology , Humans , Male , Mice , Mice, Nude , Neoplasms, Hormone-Dependent/drug therapy , Neoplasms, Hormone-Dependent/pathology , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Receptors, Androgen/physiology , Transfection , Withanolides , Xenograft Model Antitumor Assays
19.
Mol Cancer ; 5: 22, 2006 Jun 07.
Article in English | MEDLINE | ID: mdl-16759389

ABSTRACT

BACKGROUND: Gene-targeted iMycEmu mice that carry a His6-tagged mouse Myc(c-myc)cDNA, MycHis, just 5' of the immunoglobulin heavy-chain enhancer, Emu, are prone to B cell and plasma cell neoplasms, such as lymphoblastic B-cell lymphoma (LBL) and plasmacytoma (PCT). Cell lines derived from Myc-induced neoplasms of this sort may provide a good model system for the design and testing of new approaches to prevent and treat MYC-driven B cell and plasma cell neoplasms in human beings. To test this hypothesis, we used the LBL-derived cell line, iMycEmu-1, and the newly established PCT-derived cell line, iMycEmu-2, to evaluate the growth inhibitory and death inducing potency of the cancer drug candidate, CDDO-imidazolide (CDDO-Im). METHODS: Morphological features and surface marker expression of iMycEmu-2 cells were evaluated using cytological methods and FACS, respectively. mRNA expression levels of the inserted MycHis and normal Myc genes were determined by allele-specific RT-PCR and qPCR. Myc protein was detected by immunoblotting. Cell cycle progression and apoptosis were analyzed by FACS. The expression of 384 "pathway" genes was assessed with the help of Superarray cDNA macroarrays and verified, in part, by RT-PCR. RESULTS: Sub-micromolar concentrations of CDDO-Im caused growth arrest and apoptosis in iMycEmu-1 and iMycEmu-2 cells. CDDO-Im-dependent growth inhibition and apoptosis were associated in both cell lines with the up-regulation of 30 genes involved in apoptosis, cell cycling, NFkappaB signaling, and stress and toxicity responses. Strongly induced (> or = 10 fold) were genes encoding caspase 14, heme oxygenase 1 (Hmox1), flavin-containing monooxygenase 4 (Fmo4), and three members of the cytochrome P450 subfamily 2 of mixed-function oxygenases (Cyp2a4, Cyp2b9, Cyp2c29). CDDO-Im-dependent gene induction coincided with a decrease in Myc protein. CONCLUSION: Growth arrest and killing of neoplastic mouse B cells and plasma cells by CDDO-Im, a closely related derivative of the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid, appears to be caused, in part, by drug-induced stress responses and reduction of Myc.


Subject(s)
B-Lymphocytes/pathology , Imidazolidines/chemistry , Imidazolidines/pharmacology , Oleanolic Acid/analogs & derivatives , Plasmacytoma/metabolism , Plasmacytoma/pathology , Proto-Oncogene Proteins c-myc/metabolism , Animals , B-Lymphocytes/drug effects , B-Lymphocytes/metabolism , Cell Line , Cell Proliferation/drug effects , Cell Survival/drug effects , DNA, Complementary/genetics , Gene Expression Regulation, Neoplastic , Mice , Mice, Transgenic , NF-kappa B/genetics , Oleanolic Acid/chemistry , Oligonucleotide Array Sequence Analysis , Plasmacytoma/genetics , Proto-Oncogene Proteins c-myc/genetics , Transcriptional Activation , Up-Regulation
20.
Mol Cancer ; 4: 40, 2005 Nov 09.
Article in English | MEDLINE | ID: mdl-16277667

ABSTRACT

BACKGROUND: Myc-induced lymphoblastic B-cell lymphoma (LBL) in iMycEmu mice may provide a model system for the study of the mechanism by which human MYC facilitates the initiation and progression of B cell and plasma cell neoplasms in human beings. We have recently shown that gene-targeted iMycEmu mice that carry a His6-tagged mouse Myc cDNA, MycHis, just 5' of the immunoglobulin heavy-chain enhancer, Emu, are prone to B cell and plasma cell tumors. The predominant tumor (approximately 50%) that arose in the iMycEmu mice on the mixed genetic background of segregating C57BL/6 and 129/SvJ alleles was LBL. The purpose of this study was to establish and characterize a cell line, designated iMycEmu-1, for the in-depth evaluation of LBL in vitro. METHODS: The morphological features and the surface marker expression profile of the iMycEmu-1 cells were evaluated using cytological methods and FACS, respectively. The cytogenetic make-up of the iMycEmu-1 cells was assessed by spectral karyotyping (SKY). The expression of the inserted MycHis gene was determined using RT-PCR and qPCR. Clonotypic immunoglobulin gene arrangements were detected by Southern blotting. The global gene expression program of the iMycEmu-1 cells and the expression of 768 "pathway" genes were determined with the help of the Mouse Lymphochip(c) and Superarray(c) cDNA micro- and macroarrays, respectively. Array results were verified, in part, by RT-PCR and qPCR. RESULTS: Consistent with their derivation from LBL, the iMycEmu-1 cells were found to be neoplastic IgMhighIgDlow lymphoblasts that expressed typical B-cell surface markers including CD40, CD54 (ICAM-1), CD80 (B7-1) and CD86 (B7-2). The iMycEmu-1 cells harbored a reciprocal T(9;11) and three non-reciprocal chromosomal translocations, over-expressed MycHis at the expense of normal Myc, and exhibited gene expression changes on Mouse Lymphochip microarrays that were consistent with MycHis-driven B-cell neoplasia. Upon comparison to normal B cells using eight different Superarray cDNA macroarrays, the iMycEmu-1 cells showed the highest number of changes on the NFkappaB array. CONCLUSION: The iMycEmu-1 cells may provide a uniquely useful model system to study the growth and survival requirements of Myc-driven mouse LBL in vitro.


Subject(s)
Lymphoma, B-Cell/metabolism , Lymphoma, B-Cell/pathology , Proto-Oncogene Proteins c-myc/metabolism , Animals , Cell Line, Tumor , Cell Proliferation , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/genetics , Karyotyping , Lymphoma, B-Cell/genetics , Mice , Mice, Transgenic , Oligonucleotide Array Sequence Analysis , Proto-Oncogene Proteins c-myc/genetics
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