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1.
Haematologica ; 2024 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-38841800

RESUMO

Diffuse large B-cell lymphoma (DLBCL) is the most common malignancy that develops in patients with ataxia-telangiectasia, a cancer-predisposing inherited syndrome characterized by inactivating germline ATM mutations. ATM is also frequently mutated in sporadic DLBCL. To investigate lymphomagenic mechanisms and lymphoma-specific dependencies underlying defective ATM, we applied ribonucleic acid (RNA)-seq and genome-scale loss-offunction clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 screens to systematically interrogate B-cell lymphomas arising in a novel murine model (Atm-/-nu-/-) with constitutional Atm loss, thymic aplasia but residual T-cell populations. Atm-/-nu-/-lymphomas, which phenotypically resemble either activated B-cell-like or germinal center Bcell-like DLBCL, harbor a complex karyotype, and are characterized by MYC pathway activation. In Atm-/-nu-/-lymphomas, we discovered nucleotide biosynthesis as a MYCdependent cellular vulnerability that can be targeted through the synergistic nucleotidedepleting actions of mycophenolate mofetil (MMF) and the WEE1 inhibitor, adavosertib (AZD1775). The latter is mediated through a synthetically lethal interaction between RRM2 suppression and MYC dysregulation that results in replication stress overload in Atm-/-nu-/-lymphoma cells. Validation in cell line models of human DLBCL confirmed the broad applicability of nucleotide depletion as a therapeutic strategy for MYC-driven DLBCL independent of ATM mutation status. Our findings extend current understanding of lymphomagenic mechanisms underpinning ATM loss and highlight nucleotide metabolism as a targetable therapeutic vulnerability in MYC-driven DLBCL.

2.
Cell ; 136(3): 420-34, 2009 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-19203578

RESUMO

The biological response to DNA double-strand breaks acts to preserve genome integrity. Individuals bearing inactivating mutations in components of this response exhibit clinical symptoms that include cellular radiosensitivity, immunodeficiency, and cancer predisposition. The archetype for such disorders is Ataxia-Telangiectasia caused by biallelic mutation in ATM, a central component of the DNA damage response. Here, we report that the ubiquitin ligase RNF168 is mutated in the RIDDLE syndrome, a recently discovered immunodeficiency and radiosensitivity disorder. We show that RNF168 is recruited to sites of DNA damage by binding to ubiquitylated histone H2A. RNF168 acts with UBC13 to amplify the RNF8-dependent histone ubiquitylation by targeting H2A-type histones and by promoting the formation of lysine 63-linked ubiquitin conjugates. These RNF168-dependent chromatin modifications orchestrate the accumulation of 53BP1 and BRCA1 to DNA lesions, and their loss is the likely cause of the cellular and developmental phenotypes associated with RIDDLE syndrome.


Assuntos
Dano ao DNA , Síndromes de Imunodeficiência/metabolismo , Transdução de Sinais , Ubiquitina/metabolismo , Linhagem Celular , Histonas/metabolismo , Humanos , Síndromes de Imunodeficiência/genética , Tolerância a Radiação , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
3.
Blood ; 137(22): 3064-3078, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-33512408

RESUMO

Chronic lymphocytic leukemia (CLL) remains incurable despite B-cell receptor-targeted inhibitors revolutionizing treatment. This suggests that other signaling molecules are involved in disease escape mechanisms and resistance. Toll-like receptor 9 (TLR9) is a promising candidate that is activated by unmethylated cytosine guanine dinucleotide-DNA. Here, we show that plasma from patients with CLL contains significantly more unmethylated DNA than plasma from healthy control subjects (P < .0001) and that cell-free DNA levels correlate with the prognostic markers CD38, ß2-microglobulin, and lymphocyte doubling time. Furthermore, elevated cell-free DNA was associated with shorter time to first treatment (hazard ratio, 4.0; P = .003). We also show that TLR9 expression was associated with in vitro CLL cell migration (P < .001), and intracellular endosomal TLR9 strongly correlated with aberrant surface expression (sTLR9; r = 0.9). In addition, lymph node-derived CLL cells exhibited increased sTLR9 (P = .016), and RNA-sequencing of paired sTLR9hi and sTLR9lo CLL cells revealed differential transcription of genes involved in TLR signaling, adhesion, motility, and inflammation in sTLR9hi cells. Mechanistically, a TLR9 agonist, ODN2006, promoted CLL cell migration (P < .001) that was mediated by p65 NF-κB and STAT3 transcription factor activation. Importantly, autologous plasma induced the same effects, which were reversed by a TLR9 antagonist. Furthermore, high TLR9 expression promoted engraftment and rapid disease progression in a NOD/Shi-scid/IL-2Rγnull mouse xenograft model. Finally, we showed that dual targeting of TLR9 and Bruton's tyrosine kinase (BTK) was strongly synergistic (median combination index, 0.2 at half maximal effective dose), which highlights the distinct role for TLR9 signaling in CLL and the potential for combined targeting of TLR9 and BTK as a more effective treatment strategy in this incurable disease.


Assuntos
Movimento Celular/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Leucemia Linfocítica Crônica de Células B , Proteínas de Neoplasias , Oligodesoxirribonucleotídeos/farmacologia , Receptor Toll-Like 9 , Animais , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas de Neoplasias/agonistas , Proteínas de Neoplasias/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor Toll-Like 9/agonistas , Receptor Toll-Like 9/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Blood ; 135(6): 411-428, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-31794600

RESUMO

Spontaneous regression is a recognized phenomenon in chronic lymphocytic leukemia (CLL) but its biological basis remains unknown. We undertook a detailed investigation of the biological and clinical features of 20 spontaneous CLL regression cases incorporating phenotypic, functional, transcriptomic, and genomic studies at sequential time points. All spontaneously regressed tumors were IGHV-mutated with no restricted IGHV usage or B-cell receptor (BCR) stereotypy. They exhibited shortened telomeres similar to nonregressing CLL, indicating prior proliferation. They also displayed low Ki-67, CD49d, cell-surface immunoglobulin M (IgM) expression and IgM-signaling response but high CXCR4 expression, indicating low proliferative activity associated with poor migration to proliferation centers, with these features becoming increasingly marked during regression. Spontaneously regressed CLL displayed a transcriptome profile characterized by downregulation of metabolic processes as well as MYC and its downstream targets compared with nonregressing CLL. Moreover, spontaneous regression was associated with reversal of T-cell exhaustion features including reduced programmed cell death 1 expression and increased T-cell proliferation. Interestingly, archetypal CLL genomic aberrations including HIST1H1B and TP53 mutations and del(13q14) were found in some spontaneously regressing tumors, but genetic composition remained stable during regression. Conversely, a single case of CLL relapse following spontaneous regression was associated with increased BCR signaling, CLL proliferation, and clonal evolution. These observations indicate that spontaneously regressing CLL appear to undergo a period of proliferation before entering a more quiescent state, and that a complex interaction between genomic alterations and the microenvironment determines disease course. Together, the findings provide novel insight into the biological processes underpinning spontaneous CLL regression, with implications for CLL treatment.


Assuntos
Leucemia Linfocítica Crônica de Células B/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Proliferação de Células , Feminino , Regulação Leucêmica da Expressão Gênica , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Imunoglobulina M/genética , Antígeno Ki-67/genética , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Polimorfismo de Nucleotídeo Único , Receptores CXCR4/genética , Microambiente Tumoral
5.
Blood ; 130(2): 156-166, 2017 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-28495793

RESUMO

The role of deubiquitylase ubiquitin-specific protease 7 (USP7) in the regulation of the p53-dependent DNA damage response (DDR) pathway is well established. Whereas previous studies have mostly focused on the mechanisms underlying how USP7 directly controls p53 stability, we recently showed that USP7 modulates the stability of the DNA damage responsive E3 ubiquitin ligase RAD18. This suggests that targeting USP7 may have therapeutic potential even in tumors with defective p53 or ibrutinib resistance. To test this hypothesis, we studied the effect of USP7 inhibition in chronic lymphocytic leukemia (CLL) where the ataxia telangiectasia mutated (ATM)-p53 pathway is inactivated with relatively high frequency, leading to treatment resistance and poor clinical outcome. We demonstrate that USP7 is upregulated in CLL cells, and its loss or inhibition disrupts homologous recombination repair (HRR). Consequently, USP7 inhibition induces significant tumor-cell killing independently of ATM and p53 through the accumulation of genotoxic levels of DNA damage. Moreover, USP7 inhibition sensitized p53-defective, chemotherapy-resistant CLL cells to clinically achievable doses of HRR-inducing chemotherapeutic agents in vitro and in vivo in a murine xenograft model. Together, these results identify USP7 as a promising therapeutic target for the treatment of hematological malignancies with DDR defects, where ATM/p53-dependent apoptosis is compromised.


Assuntos
Regulação Neoplásica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/genética , Reparo de DNA por Recombinação/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética , Proteases Específicas de Ubiquitina/genética , Adenina/análogos & derivados , Animais , Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Dano ao DNA , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Camundongos , Camundongos Endogâmicos NOD , Piperidinas , Cultura Primária de Células , Pirazóis/farmacologia , Pirimidinas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , Peptidase 7 Específica de Ubiquitina , Proteases Específicas de Ubiquitina/antagonistas & inibidores , Proteases Específicas de Ubiquitina/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Blood ; 127(5): 582-95, 2016 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-26563132

RESUMO

TP53 and ataxia telangiectasia mutated (ATM) defects are associated with genomic instability, clonal evolution, and chemoresistance in chronic lymphocytic leukemia (CLL). Currently, therapies capable of providing durable remissions in relapsed/refractory TP53- or ATM-defective CLL are lacking. Ataxia telangiectasia and Rad3-related (ATR) mediates response to replication stress, the absence of which leads to collapse of stalled replication forks into chromatid fragments that require resolution through the ATM/p53 pathway. Here, using AZD6738, a novel ATR kinase inhibitor, we investigated ATR inhibition as a synthetically lethal strategy to target CLL cells with TP53 or ATM defects. Irrespective of TP53 or ATM status, induction of CLL cell proliferation upregulated ATR protein, which then became activated in response to replication stress. In TP53- or ATM-defective CLL cells, inhibition of ATR signaling by AZD6738 led to an accumulation of unrepaired DNA damage, which was carried through into mitosis because of defective cell cycle checkpoints, resulting in cell death by mitotic catastrophe. Consequently, AZD6738 was selectively cytotoxic to both TP53- and ATM-defective CLL cell lines and primary cells. This was confirmed in vivo using primary xenograft models of TP53- or ATM-defective CLL, where treatment with AZD6738 resulted in decreased tumor load and reduction in the proportion of CLL cells with such defects. Moreover, AZD6738 sensitized TP53- or ATM-defective primary CLL cells to chemotherapy and ibrutinib. Our findings suggest that ATR is a promising therapeutic target for TP53- or ATM-defective CLL that warrants clinical investigation.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/genética , Inibidores de Proteínas Quinases/uso terapêutico , Proteína Supressora de Tumor p53/genética , Adenina/análogos & derivados , Animais , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Dano ao DNA/efeitos dos fármacos , Humanos , Leucemia Linfocítica Crônica de Células B/metabolismo , Camundongos Endogâmicos NOD , Piperidinas , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Células Tumorais Cultivadas
7.
Blood ; 127(17): 2122-30, 2016 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-26837699

RESUMO

Genomic studies have revealed the complex clonal heterogeneity of chronic lymphocytic leukemia (CLL). The acquisition and selection of genomic aberrations may be critical to understanding the progression of this disease. In this study, we have extensively characterized the mutational status of TP53, SF3B1, BIRC3, NOTCH1, and ATM in 406 untreated CLL cases by ultra-deep next-generation sequencing, which detected subclonal mutations down to 0.3% allele frequency. Clonal dynamics were examined in longitudinal samples of 48 CLL patients. We identified a high proportion of subclonal mutations, isolated or associated with clonal aberrations. TP53 mutations were present in 10.6% of patients (6.4% clonal, 4.2% subclonal), ATM mutations in 11.1% (7.8% clonal, 1.3% subclonal, 2% germ line mutations considered pathogenic), SF3B1 mutations in 12.6% (7.4% clonal, 5.2% subclonal), NOTCH1 mutations in 21.8% (14.2% clonal, 7.6% subclonal), and BIRC3 mutations in 4.2% (2% clonal, 2.2% subclonal). ATM mutations, clonal SF3B1, and both clonal and subclonal NOTCH1 mutations predicted for shorter time to first treatment irrespective of the immunoglobulin heavy-chain variable-region gene (IGHV) mutational status. Clonal and subclonal TP53 and clonal NOTCH1 mutations predicted for shorter overall survival together with the IGHV mutational status. Clonal evolution in longitudinal samples mainly occurred in cases with mutations in the initial samples and was observed not only after chemotherapy but also in untreated patients. These findings suggest that the characterization of the subclonal architecture and its dynamics in the evolution of the disease may be relevant for the management of CLL patients.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Genes p53 , Proteínas Inibidoras de Apoptose/genética , Leucemia Linfocítica Crônica de Células B/genética , Proteínas de Neoplasias/genética , Fosfoproteínas/genética , Fatores de Processamento de RNA/genética , Receptor Notch1/genética , Ubiquitina-Proteína Ligases/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas Mutadas de Ataxia Telangiectasia/fisiologia , Proteína 3 com Repetições IAP de Baculovírus , Células Clonais , Análise Mutacional de DNA , Progressão da Doença , Evolução Molecular , Feminino , Humanos , Proteínas Inibidoras de Apoptose/fisiologia , Estimativa de Kaplan-Meier , Leucemia Linfocítica Crônica de Células B/terapia , Masculino , Pessoa de Meia-Idade , Mutação , Proteínas de Neoplasias/fisiologia , Células-Tronco Neoplásicas , Fosfoproteínas/fisiologia , Prognóstico , Fatores de Processamento de RNA/fisiologia , Receptor Notch1/fisiologia , Tempo para o Tratamento , Resultado do Tratamento , Proteína Supressora de Tumor p53/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Adulto Jovem
8.
Haematologica ; 100(8): 1076-85, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25840602

RESUMO

Inactivation of the Ataxia Telangiectasia Mutated gene in chronic lymphocytic leukemia results in resistance to p53-dependent apoptosis and inferior responses to treatment with DNA damaging agents. Hence, p53-independent strategies are required to target Ataxia Telangiectasia Mutated-deficient chronic lymphocytic leukemia. As Ataxia Telangiectasia Mutated has been implicated in redox homeostasis, we investigated the effect of the Ataxia Telangiectasia Mutated-null chronic lymphocytic leukemia genotype on cellular responses to oxidative stress with a view to therapeutic targeting. We found that in comparison to Ataxia Telangiectasia Mutated-wild type chronic lymphocytic leukemia, pro-oxidant treatment of Ataxia Telangiectasia Mutated-null cells led to reduced binding of NF-E2 p45-related factor-2 to antioxidant response elements and thus decreased expression of target genes. Furthermore, Ataxia Telangiectasia Mutated-null chronic lymphocytic leukemia cells contained lower levels of antioxidants and elevated mitochondrial reactive oxygen species. Consequently, Ataxia Telangiectasia Mutated-null chronic lymphocytic leukemia, but not tumors with 11q deletion or TP53 mutations, exhibited differentially increased sensitivity to pro-oxidants both in vitro and in vivo. We found that cell death was mediated by a p53- and caspase-independent mechanism associated with apoptosis inducing factor activity. Together, these data suggest that defective redox-homeostasis represents an attractive therapeutic target for Ataxia Telangiectasia Mutated-null chronic lymphocytic leukemia.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Homozigoto , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Mutação , Oxidantes/metabolismo , Fenótipo , Animais , Antioxidantes/metabolismo , Apoptose , Caspases/metabolismo , Modelos Animais de Doenças , Regulação Leucêmica da Expressão Gênica , Humanos , Mitocôndrias/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Ligação Proteica , Espécies Reativas de Oxigênio/metabolismo , Elementos de Resposta , Superóxidos/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Br J Haematol ; 182(3): 429-433, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-28643365
10.
Nat Genet ; 55(8): 1311-1323, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37524790

RESUMO

SF3B1 hotspot mutations are associated with a poor prognosis in several tumor types and lead to global disruption of canonical splicing. Through synthetic lethal drug screens, we identify that SF3B1 mutant (SF3B1MUT) cells are selectively sensitive to poly (ADP-ribose) polymerase inhibitors (PARPi), independent of hotspot mutation and tumor site. SF3B1MUT cells display a defective response to PARPi-induced replication stress that occurs via downregulation of the cyclin-dependent kinase 2 interacting protein (CINP), leading to increased replication fork origin firing and loss of phosphorylated CHK1 (pCHK1; S317) induction. This results in subsequent failure to resolve DNA replication intermediates and G2/M cell cycle arrest. These defects are rescued through CINP overexpression, or further targeted by a combination of ataxia-telangiectasia mutated and PARP inhibition. In vivo, PARPi produce profound antitumor effects in multiple SF3B1MUT cancer models and eliminate distant metastases. These data provide the rationale for testing the clinical efficacy of PARPi in a biomarker-driven, homologous recombination proficient, patient population.


Assuntos
Neoplasias , Inibidores de Poli(ADP-Ribose) Polimerases , Humanos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Mutação , Fatores de Transcrição/genética , Neoplasias/tratamento farmacológico , Neoplasias/genética , Proteína BRCA1/genética , Linhagem Celular Tumoral , Fatores de Processamento de RNA/genética , Fosfoproteínas/genética
11.
Blood ; 116(22): 4578-87, 2010 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-20739657

RESUMO

The Ataxia Telangiectasia Mutated (ATM) gene is frequently inactivated in lymphoid malignancies such as chronic lymphocytic leukemia (CLL), T-prolymphocytic leukemia (T-PLL), and mantle cell lymphoma (MCL) and is associated with defective apoptosis in response to alkylating agents and purine analogues. ATM mutant cells exhibit impaired DNA double strand break repair. Poly (ADP-ribose) polymerase (PARP) inhibition that imposes the requirement for DNA double strand break repair should selectively sensitize ATM-deficient tumor cells to killing. We investigated in vitro sensitivity to the poly (ADP-ribose) polymerase inhibitor olaparib (AZD2281) of 5 ATM mutant lymphoblastoid cell lines (LCL), an ATM mutant MCL cell line, an ATM knockdown PGA CLL cell line, and 9 ATM-deficient primary CLLs induced to cycle and observed differential killing compared with ATM wildtype counterparts. Pharmacologic inhibition of ATM and ATM knockdown confirmed the effect was ATM-dependent and mediated through mitotic catastrophe independently of apoptosis. A nonobese diabetic/severe combined immunodeficient (NOD/SCID) murine xenograft model of an ATM mutant MCL cell line demonstrated significantly reduced tumor load and an increased survival of animals after olaparib treatment in vivo. Addition of olaparib sensitized ATM null tumor cells to DNA-damaging agents. We suggest that olaparib would be an appropriate agent for treating refractory ATM mutant lymphoid tumors.


Assuntos
Antineoplásicos/uso terapêutico , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Linfoma de Célula do Manto/tratamento farmacológico , Ftalazinas/uso terapêutico , Piperazinas/uso terapêutico , Inibidores de Poli(ADP-Ribose) Polimerases , Proteínas Serina-Treonina Quinases/genética , Proteínas Supressoras de Tumor/genética , Animais , Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Dano ao DNA/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Linfoma de Célula do Manto/genética , Camundongos , Camundongos SCID , Mutação , Ftalazinas/farmacologia , Piperazinas/farmacologia
12.
J Cell Biol ; 167(6): 1161-70, 2004 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-15611337

RESUMO

Interaction of macrophages with apoptotic cells involves multiple steps including recognition, tethering, phagocytosis, and anti-inflammatory macrophage responses. Defective apoptotic cell clearance is associated with pathogenesis of autoimmune disease. CD14 is a surface receptor that functions in vitro in the removal of apoptotic cells by human and murine macrophages, but its mechanism of action has not been defined. Here, we demonstrate that CD14 functions as a macrophage tethering receptor for apoptotic cells. Significantly, CD14(-/-) macrophages in vivo are defective in clearing apoptotic cells in multiple tissues, suggesting a broad role for CD14 in the clearance process. However, the resultant persistence of apoptotic cells does not lead to inflammation or increased autoantibody production, most likely because, as we show, CD14(-/-) macrophages retain the ability to generate anti-inflammatory signals in response to apoptotic cells. We conclude that CD14 plays a broad tethering role in apoptotic cell clearance in vivo and that apoptotic cells can persist in the absence of proinflammatory consequences.


Assuntos
Apoptose/fisiologia , Doenças Autoimunes/imunologia , Inflamação/patologia , Receptores de Lipopolissacarídeos/fisiologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Células COS , Linhagem Celular Tumoral , Dexametasona/farmacologia , Humanos , Ionomicina/farmacologia , Receptores de Lipopolissacarídeos/genética , Receptores de Lipopolissacarídeos/imunologia , Macrófagos/imunologia , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Timo/citologia , Timo/efeitos dos fármacos , Fatores de Tempo
13.
Neurochem Int ; 52(8): 1394-401, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18406496

RESUMO

Nitric oxide is a diffusible messenger that plays a multitude of roles within the nervous system including modulation of cell viability. However, its role in regulating neuronal survival during a defined period of neurodevelopment has never been investigated. We discovered that expression of the messenger RNA for both neuronal and endothelial nitric oxide synthase increased in the early postnatal period in the cerebellum in vivo, whilst the expression of inducible nitric oxide synthase remained constant throughout this time in development. Whilst scavenging of nitric oxide was deleterious to the survival of early postnatal cerebellar granule neurons in vitro, this effect was lost in cultures derived at increasing postnatal ages. Conversely, sensitivity to exogenous nitric oxide increased with advancing postnatal age. Thus, we have shown that as postnatal development proceeds, cerebellar granule cells alter their in vitro survival responses to both nitric oxide inhibition and donation, revealing that the nitric oxide's effects on developing neurons vary with the stage of development studied. These findings have important consequences for our understanding of the role of nitric oxide during neuronal development.


Assuntos
Córtex Cerebelar/enzimologia , Córtex Cerebelar/crescimento & desenvolvimento , Neurônios/enzimologia , Óxido Nítrico/biossíntese , Envelhecimento/metabolismo , Animais , Animais Recém-Nascidos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Córtex Cerebelar/citologia , Regulação Enzimológica da Expressão Gênica/genética , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Óxido Nítrico/farmacologia , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Ácido Peroxinitroso/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo
14.
Neurosci Lett ; 438(1): 17-21, 2008 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-18472337

RESUMO

As recent evidence has revealed a pro-survival role for the anti-obesity hormone leptin in the nervous system, we investigated the generality of this finding on cerebellar Purkinje and granule neurons in vitro. We found that whilst leptin promoted cerebellar Purkinje neuron survival, it had no affect on cerebellar granule cells. In addition, we discovered that leptin promoted both the outgrowth of neurites from cerebellar Purkinje neurons and increased the complexity of the neurite arbor. Thus, leptin has different effects on two neighbouring populations of neurons within the cerebellum implying specificity of its actions in the central nervous system.


Assuntos
Diferenciação Celular/fisiologia , Córtex Cerebelar/crescimento & desenvolvimento , Córtex Cerebelar/metabolismo , Leptina/metabolismo , Fatores de Crescimento Neural/metabolismo , Células de Purkinje/metabolismo , Animais , Animais Recém-Nascidos , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Córtex Cerebelar/citologia , Dendritos/efeitos dos fármacos , Dendritos/metabolismo , Relação Dose-Resposta a Droga , Leptina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Crescimento Neural/farmacologia , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Células de Purkinje/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Receptores para Leptina/agonistas , Receptores para Leptina/genética , Receptores para Leptina/metabolismo
15.
Neurosci Lett ; 413(1): 52-7, 2007 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-17157438

RESUMO

Whilst a plethora of studies that describe the toxicity of homocysteine to CNS neurons have been published, the effects of homocysteine on the Purkinje neurons of the cerebellum that play a vital role in motor function remain wholly unexplored. We have therefore established cultures of embryonic cerebellar Purkinje neurons and exposed them to a range of concentrations of homocysteine and determined its effects on their survival. The experiments revealed that all concentrations of homocysteine studied, from 50 to 500microM, caused a significant decrease in cerebellar Purkinje neuron number. This loss could be counteracted by the pan-caspase inhibitor z-VAD-fmk in the first 24h following homocysteine exposure, revealing that the initial loss was apoptotic. However, z-VAD-fmk could not prevent homocysteine-mediated loss of cerebellar Purkinje neurons in the longer term, after 6 days in vitro. In addition to its effects on Purkinje neuron survival, homocysteine markedly reduced both the overall magnitude and the complexity of the neurite arbor extended by the cerebellar Purkinje neurons, following 6 days incubation with this agent in vitro. Taken together our data reveal that homocysteine is toxic to cerebellar Purkinje neurons in vitro, inhibiting both their survival and the outgrowth of neurites.


Assuntos
Cerebelo/citologia , Homocisteína/metabolismo , Neurotoxinas/farmacologia , Células de Purkinje/efeitos dos fármacos , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Calbindinas , Morte Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Interações Medicamentosas , Embrião de Mamíferos , Imuno-Histoquímica , Camundongos , Neuritos/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Células de Purkinje/citologia , Proteína G de Ligação ao Cálcio S100/metabolismo
16.
Oncotarget ; 8(27): 44749-44760, 2017 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-28496009

RESUMO

Subclonal heterogeneity and clonal selection influences disease progression in chronic lymphocytic leukemia (CLL). It is therefore important that therapeutic decisions are made based on an understanding of the CLL clonal architecture and its dynamics in individual patients. Identification of cytogenetic abnormalities by FISH remains the cornerstone of contemporary clinical practice and provides a simple means for prognostic stratification. Here, we demonstrate that multiplexed-FISH can enhance recognition of CLL subclonal repertoire and its dynamics during disease progression, both in patients and CLL patient-derived xenografts (PDX). We applied a combination of patient-specific FISH probes to 24 CLL cases before treatment and at relapse, and determined putative ancestral relationships between subpopulations with different cytogenetic features. We subsequently established 7 CLL PDX models in NOD/Shi-SCID/IL-2Rγctm1sug/Jic (NOG) mice. Application of multiplexed-FISH to these models demonstrated that all of the identified cytogenetic subpopulations had leukemia propagating activity and that changes in their representation during disease progression could be spontaneous, accelerated by treatment or treatment-induced. We conclude that multiplexed-FISH in combination with PDX models have the potential to distinguish between spontaneous and treatment-induced clonal selection, and therefore provide a valuable tool for the pre-clinical evaluation of novel therapies.


Assuntos
Aberrações Cromossômicas , Evolução Clonal/genética , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Animais , Terapia Combinada , Modelos Animais de Doenças , Progressão da Doença , Feminino , Xenoenxertos , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Leucemia Linfocítica Crônica de Células B/mortalidade , Leucemia Linfocítica Crônica de Células B/terapia , Masculino , Camundongos , Prognóstico , Análise de Célula Única , Resultado do Tratamento
17.
Oncotarget ; 7(42): 68513-68526, 2016 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-27655680

RESUMO

Chronic lymphocytic leukemia (B-CLL) and small lymphocytic lymphoma (SLL) are part of the same disease classification but are defined by differential distribution of tumor cells. B-CLL is characterized by significant immune suppression and dysregulation but this is not typical of patients with SLL. Natural killer cells (NK) are important mediators of immune function but have been poorly studied in patients with B-CLL/SLL. Here we report for the first time the NK cell phenotype and function in patients with B-CLL and SLL alongside their transcriptional profile. We show for the first time impaired B-CLL NK cell function in a xenograft model with reduced activating receptor expression including NKG2D, DNAM-1 and NCRs in-vitro. Importantly, we show these functional differences are associated with transcriptional downregulation of cytotoxic pathway genes, including activating receptors, adhesion molecules, cytotoxic molecules and intracellular signalling molecules, which remain intact in patients with SLL. In conclusion, NK cell function is markedly influenced by the anatomical site of the tumor in patients with B-CLL/SLL and lymphocytosis leads to marked impairment of NK cell activity. These observations have implications for treatment protocols which seek to preserve immune function by limiting the exposure of NK cells to tumor cells within the peripheral circulation.


Assuntos
Citotoxicidade Imunológica/imunologia , Células Matadoras Naturais/imunologia , Leucemia Linfocítica Crônica de Células B/imunologia , Linfoma de Células B/imunologia , Idoso , Idoso de 80 Anos ou mais , Animais , Citotoxicidade Imunológica/genética , Citometria de Fluxo , Perfilação da Expressão Gênica/métodos , Regulação Leucêmica da Expressão Gênica/imunologia , Humanos , Imunofenotipagem , Células K562 , Células Matadoras Naturais/metabolismo , Leucemia Linfocítica Crônica de Células B/genética , Linfoma de Células B/genética , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Pessoa de Meia-Idade , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
18.
Brain Res Dev Brain Res ; 160(1): 85-9, 2005 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-16154637

RESUMO

Hyperhomocysteinemia is a risk factor for a range of neurodegenerative conditions, yet its effects in the developing nervous system have been poorly elucidated. We studied the in vitro response of cerebellar granule neurons (CGCs) to homocysteine. We have shown that embryonic CGCs are resistant to homocysteine-induced neurotoxicity, whilst postnatal CGCs are not. This is the first demonstration of a neuronal population undergoing a developmental switch in their response to homocysteine. Greater understanding of this change may have important implications for both neurodegenerative conditions and neurodevelopmental disorders.


Assuntos
Envelhecimento/metabolismo , Homocisteína/metabolismo , Hiper-Homocisteinemia/complicações , Necrose/induzido quimicamente , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Suscetibilidade a Doenças/metabolismo , Suscetibilidade a Doenças/fisiopatologia , Homocisteína/toxicidade , Camundongos , Microscopia Eletrônica de Transmissão , Necrose/metabolismo , Necrose/patologia , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/fisiopatologia , Neurônios/efeitos dos fármacos , Neurônios/patologia
19.
Dis Model Mech ; 8(11): 1401-12, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26398941

RESUMO

Chronic lymphocytic leukaemia (CLL) cells require microenvironmental support for their proliferation. This can be recapitulated in highly immunocompromised hosts in the presence of T cells and other supporting cells. Current primary CLL xenograft models suffer from limited duration of tumour cell engraftment coupled with gradual T-cell outgrowth. Thus, a greater understanding of the interaction between CLL and T cells could improve their utility. In this study, using two distinct mouse xenograft models, we investigated whether xenografts recapitulate CLL biology, including natural environmental interactions with B-cell receptors and T cells, and whether manipulation of autologous T cells can expand the duration of CLL engraftment. We observed that primary CLL xenografts recapitulated both the tumour phenotype and T-cell repertoire observed in patients and that engraftment was significantly shorter for progressive tumours. A reduction in the number of patient T cells that were injected into the mice to 2-5% of the initial number or specific depletion of CD8(+) cells extended the limited xenograft duration of progressive cases to that characteristic of indolent disease. We conclude that manipulation of T cells can enhance current CLL xenograft models and thus expand their utility for investigation of tumour biology and pre-clinical drug assessment.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Hospedeiro Imunocomprometido , Leucemia Linfocítica Crônica de Células B/imunologia , Linfócitos do Interstício Tumoral/imunologia , Subpopulações de Linfócitos T/imunologia , Microambiente Tumoral , Animais , Linfócitos T CD8-Positivos/patologia , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Técnicas de Cocultura , Citotoxicidade Imunológica , Sobrevivência de Enxerto , Xenoenxertos , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Ativação Linfocitária , Depleção Linfocítica , Linfócitos do Interstício Tumoral/patologia , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , Fenótipo , Baço/imunologia , Subpopulações de Linfócitos T/patologia , Fatores de Tempo
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