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1.
J Cell Sci ; 136(21)2023 11 01.
Article in English | MEDLINE | ID: mdl-37772773

ABSTRACT

Centrosome amplification (CA) is a prominent feature of human cancers linked to tumorigenesis in vivo. Here, we report mechanistic contributions of CA induction alone to tumour architecture and extracellular matrix (ECM) remodelling. CA induction in non-tumorigenic breast cells MCF10A causes cell migration and invasion, with underlying disruption of epithelial cell-cell junction integrity and dysregulation of expression and subcellular localisation of cell junction proteins. CA also elevates expression of integrin ß-3, its binding partner fibronectin-1 and matrix metalloproteinase enzymes, promoting cell-ECM attachment, ECM degradation, and a migratory and invasive cell phenotype. Using a chicken embryo xenograft model for in vivo validation, we show that CA-induced (+CA) MCF10A cells invade into the chick mesodermal layer, with inflammatory cell infiltration and marked focal reactions between chorioallantoic membrane and cell graft. We also demonstrate a key role of small GTPase Rap-1 signalling through inhibition using GGTI-298, which blocked various CA-induced effects. These insights reveal that in normal cells, CA induction alone (without additional oncogenic alterations) is sufficient to confer early pro-tumorigenic changes within days, acting through Rap-1-dependent signalling to alter cell-cell contacts and ECM disruption.


Subject(s)
Breast Neoplasms , Neoplasms , Chick Embryo , Humans , Animals , Female , Chickens , Neoplasms/metabolism , Signal Transduction , Cell Movement , Centrosome/metabolism , Cell Line, Tumor , Breast Neoplasms/genetics
2.
Curr Oncol ; 29(10): 7512-7523, 2022 10 07.
Article in English | MEDLINE | ID: mdl-36290868

ABSTRACT

Breast cancer represents a heterogeneous condition in which the interaction between host immune response and primary oncogenic events can impact disease progression. Ratios of systemic blood-based immunocytes have emerged as clinically-relevant prognostic biomarkers in cancer patients. The NLR (neutrophil-to-lymphocyte ratio) has been shown to be prognostic in a variety of cancers, including breast cancer. However, evaluation of the prognostic value for overall survival (OS) and disease-free survival (DFS) of other key immunocyte ratios-neutrophil-to-lymphocyte ratio (NLR), lymphocyte-to-monocyte ratio (LMR), neutrophil-to-white cell count ratio (NWR), lymphocyte-to-white cell count ratio (LWR), monocyte-to-white cell count ratio (MWR), platelet-to-lymphocyte (PLR)-by breast cancer subtypes in a neoadjuvant chemotherapy (NAC) cohort remains to be fully explored. An NAC-treated breast cancer cohort, comprised of Luminal A, Luminal B, HER2-positive, and triple negative/basal breast cancers, treated at a tertiary referral center (minimum 3-year follow-up), was used to calculate immunocyte ratios and immunocyte cut-off values, calculated with >80% specificity (using decision tree modeling). The association with subtype-specific OS, DFS, and tumor grade was analyzed using cut offs calculated using both receiver operating characteristic curves and decision tree modelling. Decision tree calculated ratios showed that LMR (5.29) and MWR (0.06) were significantly associated with Luminal A OS (p = 0.004 and p = 0.022) and DFS (p = 0.004 and p = 0.022), and Luminal B OS (p = 0.027 and p = 0.008) and DFS (p = 0.005 and p = 0.007). NLR (1.79) and LWR (0.30) were significantly associated with HER2-positive OS (p = 0.013 and p = 0.043). NLR (1.79) and NWR (0.62) were significantly associated with DFS (p = 0.035 and p = 0.021). No significant association we observed between any immunocyte ratio in the triple negative cohort. Our results demonstrate the subtype-specific prognostic value of immunocyte ratios in NAC-treated breast cancer patients. Further validation of immunocyte ratios will provide clinicians with a new prognostic aid for disease management and monitoring.


Subject(s)
Neoadjuvant Therapy , Triple Negative Breast Neoplasms , Humans , Prognosis , Retrospective Studies , Lymphocytes/pathology , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/pathology , Biomarkers
3.
Front Cell Dev Biol ; 9: 747314, 2021.
Article in English | MEDLINE | ID: mdl-34805157

ABSTRACT

Discoidin domain receptor tyrosine kinases (DDRs) are a class of receptor tyrosine kinases (RTKs), and their dysregulation is associated with multiple diseases (including cancer, chronic inflammatory conditions, and fibrosis). The DDR family members (DDR1a-e and DDR2) are widely expressed, with predominant expression of DDR1 in epithelial cells and DDR2 in mesenchymal cells. Structurally, DDRs consist of three regions (an extracellular ligand binding domain, a transmembrane domain, and an intracellular region containing a kinase domain), with their kinase activity induced by receptor-specific ligand binding. Collagen binding to DDRs stimulates DDR phosphorylation activating kinase activity, signaling to MAPK, integrin, TGF-ß, insulin receptor, and Notch signaling pathways. Abnormal DDR expression is detected in a range of solid tumors (including breast, ovarian, cervical liver, gastric, colorectal, lung, and brain). During tumorigenesis, abnormal activation of DDRs leads to invasion and metastasis, via dysregulation of cell adhesion, migration, proliferation, secretion of cytokines, and extracellular matrix remodeling. Differential expression or mutation of DDRs correlates with pathological classification, clinical characteristics, treatment response, and prognosis. Here, we discuss the discovery, structural characteristics, organizational distribution, and DDR-dependent signaling. Importantly, we highlight the key role of DDRs in the development and progression of breast and ovarian cancer.

4.
Molecules ; 23(5)2018 May 13.
Article in English | MEDLINE | ID: mdl-29757235

ABSTRACT

Advances in technology have facilitated the molecular profiling (genomic and transcriptomic) of tumours, and has led to improved stratification of patients and the individualisation of treatment regimes. To fully realize the potential of truly personalised treatment options, we need targeted therapies that precisely disrupt the compensatory pathways identified by profiling which allow tumours to survive or gain resistance to treatments. Here, we discuss recent advances in novel therapies that impact the genome (chromosomes and chromatin), pathways targeted and the stage of the pathways targeted. The current state of research will be discussed, with a focus on compounds that have advanced into trials (clinical and pre-clinical). We will discuss inhibitors of specific DNA damage responses and other genome stability pathways, including those in development, which are likely to synergistically combine with current therapeutic options. Tumour profiling data, combined with the knowledge of new treatments that affect the regulation of essential tumour signalling pathways, is revealing fundamental insights into cancer progression and resistance mechanisms. This is the forefront of the next evolution of advanced oncology medicine that will ultimately lead to improved survival and may, one day, result in many cancers becoming chronic conditions, rather than fatal diseases.


Subject(s)
Genomic Instability/drug effects , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Biomarkers , Centrosome , Chromosomal Instability/drug effects , DNA Damage , DNA Repair , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylase Inhibitors/therapeutic use , Humans , Molecular Targeted Therapy/adverse effects , Molecular Targeted Therapy/methods , Phthalazines/pharmacology , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Signal Transduction
5.
Methods Mol Biol ; 1509: 195-208, 2017.
Article in English | MEDLINE | ID: mdl-27826929

ABSTRACT

The purpose of this chapter is to provide a starting point for the analysis of miRNA array data, using freely available online suites of tools. This chapter does not describe how to perform analysis of primary array data, rather how to use the top differentially regulated miRNA (returned from comparing one miRNA group to another) as the starting point for further practical analysis.Here we describe the methods and tools required to identify targets worthy of additional investigation, using the identified miRNA as a starting point. Importantly, this additional information (pathways targeted, gene expression, mRNA targets, miRNA families) can be used to positively inform any project.


Subject(s)
Computational Biology , MicroRNAs/genetics , Databases, Genetic , Gene Ontology , Humans , Molecular Sequence Annotation , Online Systems , Software
6.
Biochem Soc Trans ; 44(4): 979-86, 2016 08 15.
Article in English | MEDLINE | ID: mdl-27528742

ABSTRACT

Two opposing enzyme classes regulate fundamental elements of genome maintenance, gene regulation and metabolism, either through addition of an acetyl moiety by histone acetyltransferases (HATs) or its removal by histone de-acetyltransferases (HDAC), and are exciting targets for drug development. Importantly, dysfunctional acetylation has been implicated in numerous diseases, including cancer. Within the HAT superfamily the MYST family holds particular interest, as its members are directly involved in the DNA damage response and repair pathways and crucially, several members have been shown to be down-regulated in common cancers (such as breast and prostate). In the present study we focus on the development of lysine (K) acetyltransferase inhibitors (KATi) targeting the MYST family member Tip60 (Kat5), an essential protein, designed or discovered through screening libraries. Importantly, Tip60 has been demonstrated to be significantly down-regulated in many cancers which urgently require new treatment options. We highlight current and future efforts employing these KATi as cancer treatments and their ability to synergize and enhance current cancer treatments. We investigate the different methods of KATi production or discovery, their mechanisms and their validation models. Importantly, the utility of KATi is based on a key concept: using KATi to abrogate the activity of an already down-regulated essential protein (effectively creating a lethal knockout) provides another innovative mechanism for targeting cancer cells, while significantly minimizing any off-target effects to normal cells. This approach, combined with the rapidly developing interest in KATi, suggests that KATi have a bright future for providing truly personalized therapies.


Subject(s)
Enzyme Inhibitors/pharmacology , Histone Acetyltransferases/antagonists & inhibitors , Neoplasms/drug therapy , Cell Survival/drug effects , Cell Survival/genetics , Enzyme Inhibitors/chemistry , Histone Acetyltransferases/genetics , Histone Acetyltransferases/metabolism , Humans , Lysine Acetyltransferase 5 , Molecular Structure , Molecular Targeted Therapy/methods , Mutation , Neoplasms/enzymology , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Treatment Outcome
7.
Stem Cell Res Ther ; 6: 136, 2015 Jul 24.
Article in English | MEDLINE | ID: mdl-26204937

ABSTRACT

INTRODUCTION: Bone marrow-derived stromal cells (BMSCs), also known as mesenchymal stem cells, are the focus of intensive efforts worldwide to elucidate their function and biology. Despite the importance of BMSC migration for their potential therapeutic uses, the mechanisms and signalling governing stem cell migration are still not fully elucidated. METHODS: We investigated and detailed the effects of MCP-1 activation on BMSCs by using inhibitors of G protein-coupled receptor alpha beta (GPCR αß), ROCK (Rho-associated, coiled-coil containing protein kinase), and PI3 kinase (PI3K). The effects of MCP-1 stimulation on intracellular signalling cascades were characterised by using immunoblotting and immunofluorescence. The effectors of MCP-1-mediated migration were investigated by using migration assays (both two-dimensional and three-dimensional) in combination with inhibitors. RESULTS: We established the kinetics of the MCP-1-activated signalling cascade and show that this cascade correlates with cell surface re-localisation of chemokine (C motif) receptor 2 (CCR2) (the MCP-1 receptor) to the cell periphery following MCP-1 stimulation. We show that MCP-1-initiated signalling is dependent on the activation of ßγ subunits from the GPCR αßγ complex. In addition, we characterise a novel role for PI3Kγ signalling for the activation of both PAK and ERK following MCP-1 stimulation. We present evidence that the Gßγ complex is responsible for PI3K/Akt, PAK, and ERK signalling induced by MCP-1 in BMSCs. Importantly, we found that, in BMSCs, inhibition of ROCK significantly inhibits MCP-1-induced chemotactic migration, in contrast to previous reports in other systems. CONCLUSIONS: Our results indicate differential chemotactic signalling in mouse BMSCs, which has important implications for the translation of in vivo mouse model findings into human trials. We identified novel components and interactions activated by MCP-1-mediated signalling, which are important for stem cell migration. This work has identified additional potential therapeutic targets that could be manipulated to improve BMSC delivery and homing.


Subject(s)
Chemotaxis , GTP-Binding Protein beta Subunits/metabolism , Mesenchymal Stem Cells/physiology , rho-Associated Kinases/metabolism , Animals , Antigens/metabolism , Cells, Cultured , Chemokine CCL2/physiology , Extracellular Signal-Regulated MAP Kinases/metabolism , Humans , Mice, Inbred BALB C , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Protein Processing, Post-Translational , Receptors, G-Protein-Coupled/metabolism , Signal Transduction
8.
Sci Rep ; 4: 5372, 2014 Jun 20.
Article in English | MEDLINE | ID: mdl-24947938

ABSTRACT

Histone acetylation is required for many aspects of gene regulation, genome maintenance and metabolism and dysfunctional acetylation is implicated in numerous diseases, including cancer. Acetylation is regulated by histone acetyltransferases (HATs) and histone deacetylases and currently, few general HAT inhibitors have been described. We identified the HAT Tip60 as an excellent candidate for targeted drug development, as Tip60 is a key mediator of the DNA damage response and transcriptional co-activator. Our modeling of Tip60 indicated that the active binding pocket possesses opposite charges at each end, with the positive charges attributed to two specific side chains. We used structure based drug design to develop a novel Tip60 inhibitor, TH1834, to fit this specific pocket. We demonstrate that TH1834 significantly inhibits Tip60 activity in vitro and treating cells with TH1834 results in apoptosis and increased unrepaired DNA damage (following ionizing radiation treatment) in breast cancer but not control cell lines. Furthermore, TH1834 did not affect the activity of related HAT MOF, as indicated by H4K16Ac, demonstrating specificity. The modeling and validation of the small molecule inhibitor TH1834 represents a first step towards developing additional specific, targeted inhibitors of Tip60 that may lead to further improvements in the treatment of breast cancer.


Subject(s)
Antineoplastic Agents/administration & dosage , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Histone Acetyltransferases/antagonists & inhibitors , Models, Chemical , Molecular Docking Simulation/methods , Antineoplastic Agents/chemical synthesis , Apoptosis/drug effects , Binding Sites , Breast Neoplasms/pathology , Cell Line, Tumor , Computer Simulation , Drug Design , Enzyme Activation , Enzyme Inhibitors/administration & dosage , Enzyme Inhibitors/chemical synthesis , Humans , Lysine Acetyltransferase 5 , Protein Binding , Treatment Outcome
9.
Cell Stem Cell ; 7(2): 174-85, 2010 Aug 06.
Article in English | MEDLINE | ID: mdl-20619762

ABSTRACT

Most adult stem cells, including hematopoietic stem cells (HSCs), are maintained in a quiescent or resting state in vivo. Quiescence is widely considered to be an essential protective mechanism for stem cells that minimizes endogenous stress caused by cellular respiration and DNA replication. We demonstrate that HSC quiescence can also have detrimental effects. We found that HSCs have unique cell-intrinsic mechanisms ensuring their survival in response to ionizing irradiation (IR), which include enhanced prosurvival gene expression and strong activation of p53-mediated DNA damage response. We show that quiescent and proliferating HSCs are equally radioprotected but use different types of DNA repair mechanisms. We describe how nonhomologous end joining (NHEJ)-mediated DNA repair in quiescent HSCs is associated with acquisition of genomic rearrangements, which can persist in vivo and contribute to hematopoietic abnormalities. Our results demonstrate that quiescence is a double-edged sword that renders HSCs intrinsically vulnerable to mutagenesis following DNA damage.


Subject(s)
DNA Repair , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Mutagenesis/genetics , Animals , Cell Death/radiation effects , Cell Proliferation/radiation effects , Cell Survival/radiation effects , DNA Damage , DNA Repair/radiation effects , Hematopoietic Stem Cells/radiation effects , Mice , Mice, Inbred C57BL , Mutagenesis/radiation effects , Myeloid Progenitor Cells/cytology , Myeloid Progenitor Cells/metabolism , Myeloid Progenitor Cells/radiation effects , Radiation Tolerance/radiation effects , Radiation, Ionizing , Recombination, Genetic/radiation effects , Signal Transduction/radiation effects , Tumor Suppressor Protein p53/metabolism
10.
Environ Mol Mutagen ; 50(8): 725-32, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19274769

ABSTRACT

Altered centrosome numbers are seen in tumor cells in response to DNA damaging treatments and are hypothesised to contribute to cancer development. The mechanism by which the centrosome and chromosome cycles become disconnected after DNA damage is not yet clear. Here, we show that centrosome amplification occurs after ionising radiation (IR) in chicken DT40 cells that lack DNA-PK, Ku70, H2AX, Xpa, and Scc1, demonstrating that these activities are not required for centrosome amplification. We show that inhibition of topoisomerase II induces Chk1-dependent centrosome amplification, a similar response to that seen after IR. In the immortalised, nontransformed hTERT-RPE1 line, we observed centriole splitting, followed by dose-dependent centrosome amplification, after IR. We found that IR results in the formation of single, not multiple, daughter centrioles during centrosome amplification in U2OS osteosarcoma cells. Analysis of BRCA1 and BRCA2 mutant tumor cells showed high levels of centriole splitting in the absence of any treatment. IR caused pronounced levels of centrosome amplification in BRCA1 mutant breast cancer cells. These data show that centrosome amplification occurs after different forms of DNA damage in chicken cells, in nontransformed human cells and in human tumor cell lines, indicating that this is a general response to DNA damaging treatments. Together, our data suggest that centriole splitting is a key step in potentiation of the centrosome amplification that is a general response to DNA damage.


Subject(s)
Centrioles , Centrosome , DNA Damage , Animals , Cell Line, Transformed , Genes, BRCA1 , Genes, BRCA2 , Humans
11.
DNA Repair (Amst) ; 7(5): 713-24, 2008 May 03.
Article in English | MEDLINE | ID: mdl-18308646

ABSTRACT

Telomere repeat sequences are added to linear chromosome ends by telomerase, an enzyme comprising a reverse transcriptase (TERT) and an RNA template component (TR). We aimed to investigate TR in the DT40 B-cell tumour line using gene targeting, but were unable to generate TR nulls, suggesting a requirement for TR in DT40 proliferation. Disruption of one TR allele reduced telomerase activity and caused a progressive decline in telomere and G-strand overhang length. We then examined the interactions between TR and cellular DNA double-strand break (DSB) repair. Deletion in TR+/- cells of the gene encoding the non-homologous end-joining protein, Ku70, caused rapid loss of G-strand overhangs. Ku70-/-TR+/- cells proliferated more slowly than either single mutant and showed frequent mitotic aberrations. Activation of the DNA damage response was observed in TR-deficient cells and was exacerbated by Ku deficiency, although frequent telomeric DNA damage signals were not observed until late passages. This activation of the DNA damage response was suppressed by deletion of Rad54, a key homologous recombination gene. These findings suggest that Ku and telomerase cooperate to block homologous recombination from acting on telomeres.


Subject(s)
Antigens, Nuclear/metabolism , Chickens/genetics , Chickens/metabolism , DNA-Binding Proteins/metabolism , Genomic Instability/genetics , RNA/genetics , Telomerase/genetics , Animals , Cell Line, Tumor , Cell Proliferation , Chromosome Aberrations , DNA Damage , Heterozygote , Ku Autoantigen , RNA/metabolism , Telomerase/metabolism , Telomere/genetics , Telomere-Binding Proteins/metabolism
12.
EMBO Rep ; 8(6): 603-9, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17468739

ABSTRACT

Centrosomal abnormalities are frequently observed in cancers and in cells with defective DNA repair. Here, we used light and electron microscopy to show that DNA damage induces centrosome amplification, not fragmentation, in human cells. Caffeine abrogated this amplification in both ATM (ataxia telangiectasia, mutated)- and ATR (ATM and Rad3-related)-defective cells, indicating a complementary role for these DNA-damage-responsive kinases in promoting centrosome amplification. Inhibition of checkpoint kinase 1 (Chk1) by RNA-mediated interference or drug treatment suppressed DNA-damage-induced centrosome amplification. Radiation-induced centrosome amplification was abrogated in Chk1(-/-) DT40 cells, but occurred at normal levels in Chk1(-/-) cells transgenically expressing Chk1. Expression of kinase-dead Chk1, or Chk1S345A, through which the phosphatidylinositol-3-kinase cannot signal, failed to restore centrosome amplification, showing that signalling to Chk1 and Chk1 catalytic activity are necessary to promote centrosome overduplication after DNA damage.


Subject(s)
Centrosome/metabolism , DNA Damage , Protein Kinases/metabolism , Animals , Cell Cycle/radiation effects , Cell Line , Centrosome/radiation effects , Checkpoint Kinase 1 , Chickens , Humans , Radiation, Ionizing
13.
EMBO J ; 23(19): 3864-73, 2004 Oct 01.
Article in English | MEDLINE | ID: mdl-15359281

ABSTRACT

Centrosomes are the principal microtubule organising centres in somatic cells. Abnormal centrosome number is common in tumours and occurs after gamma-irradiation and in cells with mutations in DNA repair genes. To investigate how DNA damage causes centrosome amplification, we examined cells that conditionally lack the Rad51 recombinase and thereby incur high levels of spontaneous DNA damage. Rad51-deficient cells arrested in G2 phase and formed supernumerary functional centrosomes, as assessed by light and serial section electron microscopy. This centrosome amplification occurred without an additional DNA replication round and was not the result of cytokinesis failure. G2-to-M checkpoint over-ride by caffeine or wortmannin treatment strongly reduced DNA damage-induced centrosome amplification. Radiation-induced centrosome amplification was potentiated by Rad54 disruption. Gene targeting of ATM reduced, but did not abrogate, centrosome amplification induced by DNA damage in both the Rad51 and Rad54 knockout models, demonstrating ATM-dependent and -independent components of DNA damage-inducible G2-phase centrosome amplification. Our data suggest DNA damage-induced centrosome amplification as a mechanism for ensuring death of cells that evade the DNA damage or spindle assembly checkpoints.


Subject(s)
Cell Cycle Proteins/physiology , Centrosome/physiology , DNA Damage , DNA-Binding Proteins/physiology , G2 Phase , Gene Amplification , Nuclear Proteins/physiology , Protein Serine-Threonine Kinases/physiology , Tumor Suppressor Proteins/physiology , Androstadienes/pharmacology , Animals , Ataxia Telangiectasia Mutated Proteins , Avian Proteins , Caffeine/pharmacology , Cell Cycle Proteins/genetics , Cell Division , Central Nervous System Stimulants/pharmacology , Chickens , Chromosomal Instability , Cytokinesis , DNA Helicases , DNA Replication , DNA-Binding Proteins/genetics , Gene Targeting , Humans , Laser Scanning Cytometry , Mice , Mitosis , Mycotoxins/pharmacology , Nuclear Proteins/genetics , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/genetics , Rad51 Recombinase , Tumor Suppressor Proteins/genetics , Wortmannin
14.
J Immunol ; 170(12): 5999-6005, 2003 Jun 15.
Article in English | MEDLINE | ID: mdl-12794127

ABSTRACT

IL-1 elicits its cellular effects by binding a heterodimeric receptor consisting of IL-1RI and the accessory protein, IL-1RAcPr. In addition, it binds to IL-1RII, which lacking signaling function has been ascribed a decoy role. The fate of the ligand following interaction with the decoy receptor was examined in human polymorphonuclear cells (PMN), which express predominantly (>90%) IL-1RII. Incubation of PMN with IL-1beta results in a rapid decrease in cell surface-associated ligand accompanied by a concomitant increase in internalized IL-1 with 50-60% of IL-1beta located intracellularly within 1 h at 37 degrees C. The use of blocking Abs revealed that IL-1 internalization is mediated exclusively by the decoy receptor. The results of inhibitor analysis demonstrate that internalization requires ATP synthesis and involves clathrin-mediated endocytosis. Following removal of the ligand, the receptor was rapidly re-expressed on the cell surface. Cyclohexamide, a protein synthesis inhibitor, had no effect upon the process, suggesting that the re-expressed receptor was recycled. In addition, human keratinocytes stably transfected with IL-1RII (HaCAT 811) also internalized the IL-1RII with 43% cell surface receptor internalized after 90 min. Immunofluorescence microscopy revealed colocalization of the internalized receptor with wheat germ agglutinin-labeled internalized glycoproteins and early endosome Ag-1, a protein associated with the early endosome compartments, indicative of cellular uptake of IL-1RII by endocytosis. In contrast, little or no internalization was observed in other cells of immune origin. These results suggest that the decoy receptor IL-1RII can act as a scavenger of IL-1, representing a novel autoregulatory mechanism of the IL-1 system.


Subject(s)
Cadaverine/analogs & derivatives , Interleukin-1/metabolism , Neutrophils/immunology , Neutrophils/metabolism , Receptors, Interleukin-1/metabolism , Arsenicals/pharmacology , Autoantigens/metabolism , Cadaverine/pharmacology , Cell Line , Clathrin/pharmacology , Endocytosis/drug effects , Endocytosis/immunology , Endosomes/immunology , Endosomes/metabolism , Humans , Iodine Radioisotopes/metabolism , Ligands , Membrane Proteins/metabolism , Neutrophils/cytology , Neutrophils/drug effects , Receptors, Interleukin-1/antagonists & inhibitors , Receptors, Interleukin-1/physiology , Receptors, Interleukin-1 Type II , Sodium Azide/pharmacology , Time Factors , Vesicular Transport Proteins
15.
Blood ; 102(3): 956-63, 2003 Aug 01.
Article in English | MEDLINE | ID: mdl-12689944

ABSTRACT

The present study was designed to investigate the expression of members of the toll-like receptor (TLR) family in human B cells. High-density, resting, and low-density activated tonsillar B cells expressed TLR9 and TLR10 mRNA transcripts at the highest levels. Expression was higher in activated B cells than in resting cells. Analysis of a range of resting and activated human leukocyte populations revealed that mRNA expression of TLR10 was restricted to B cells. Stimulation of resting B cells with anti-mu and anti-CD40 antibodies or with Staphylococcus aureus Cowan I bacteria (SAC) increased expression of TLR9 and TLR10. TLR1 and TLR4 expression were not significantly induced by B-cell activation. Interestingly, a CpG oligonucleotide, a TLR9 agonist, also stimulated TLR9 expression in B cells. Exposure to anti-mu antibodies augmented TLR9 expression, concomitantly and dramatically increasing the responsiveness of B cells to CpG oligonucleotides in terms of proliferation and chemokine (CC chemokine ligand 3 [CCL3] and CCL22) production. Epstein-Barr virus (EBV)-transformed cell lines and other cell lines representative of mature B-cell neoplasias (Burkitt lymphoma, follicular lymphoma, multiple myeloma) expressed TLR9 and/or TLR10, whereas pre-B cell lines were negative. These results show that normal and neoplastic human B lymphocytes express a distinct TLR repertoire including TLR9 and TLR10 and that expression is increased upon engagement of the antigen receptor complex or TLR9 itself. Regulated expression of selected TLRs in B cells is likely to play an important role in linking innate and adaptive immune responses in normal and pathologic conditions.


Subject(s)
B-Lymphocytes/metabolism , Gene Expression Regulation , Membrane Glycoproteins/biosynthesis , Receptors, Cell Surface/biosynthesis , Cell Line, Transformed , CpG Islands , DNA-Binding Proteins/agonists , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/genetics , Humans , Lymphocyte Activation , Membrane Glycoproteins/genetics , Oligonucleotides/pharmacology , RNA, Messenger/analysis , Receptors, Cell Surface/agonists , Receptors, Cell Surface/genetics , Toll-Like Receptor 1 , Toll-Like Receptor 10 , Toll-Like Receptor 4 , Toll-Like Receptor 9 , Toll-Like Receptors
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