RESUMO
Ferroptosis is a form of cell death that has received considerable attention not only as a means to eradicate defined tumour entities but also because it provides unforeseen insights into the metabolic adaptation that tumours exploit to counteract phospholipid oxidation1,2. Here, we identify proferroptotic activity of 7-dehydrocholesterol reductase (DHCR7) and an unexpected prosurvival function of its substrate, 7-dehydrocholesterol (7-DHC). Although previous studies suggested that high concentrations of 7-DHC are cytotoxic to developing neurons by favouring lipid peroxidation3, we now show that 7-DHC accumulation confers a robust prosurvival function in cancer cells. Because of its far superior reactivity towards peroxyl radicals, 7-DHC effectively shields (phospho)lipids from autoxidation and subsequent fragmentation. We provide validation in neuroblastoma and Burkitt's lymphoma xenografts where we demonstrate that the accumulation of 7-DHC is capable of inducing a shift towards a ferroptosis-resistant state in these tumours ultimately resulting in a more aggressive phenotype. Conclusively, our findings provide compelling evidence of a yet-unrecognized antiferroptotic activity of 7-DHC as a cell-intrinsic mechanism that could be exploited by cancer cells to escape ferroptosis.
Assuntos
Linfoma de Burkitt , Desidrocolesteróis , Ferroptose , Neuroblastoma , Animais , Humanos , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patologia , Sobrevivência Celular , Desidrocolesteróis/metabolismo , Peroxidação de Lipídeos , Transplante de Neoplasias , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Oxirredução , Fenótipo , Reprodutibilidade dos TestesRESUMO
Experimental evidence suggests that ubiquitin-protein ligases regulate a number of cellular processes involved in tumorigenesis. We analysed the role of the E3 ubiquitin-protein ligase HUWE1 for pathobiology of multiple myeloma (MM), a still incurable blood cancer. mRNA expression analysis indicates an increase in HUWE1 expression levels correlated with advanced stages of myeloma. Pharmacologic as well as RNAi-mediated HUWE1 inhibition caused anti-proliferative effects in MM cell lines in vitro and in an MM1.S xenotransplantation mouse model. Cell cycle analysis upon HUWE1 inhibition revealed decreased S phase cell fractions. Analyses of potential HUWE1-dependent molecular functions did not show involvement in MYC-dependent gene regulation. However, HUWE1 depleted MM cells displayed increased DNA tail length by comet assay, as well as changes in the levels of DNA damage response mediators such as pBRCA1, DNA-polymerase ß, γH2AX and Mcl-1. Our finding that HUWE1 might thus be involved in endogenous DNA repair is further supported by strongly enhanced apoptotic effects of the DNA-damaging agent melphalan in HUWE1 depleted cells in vitro and in vivo. These data suggest that HUWE1 might contribute to tumour growth by endogenous repair of DNA, and could therefore potentially be exploitable in future treatment developments.
Assuntos
Regulação Neoplásica da Expressão Gênica , Mieloma Múltiplo/enzimologia , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Antineoplásicos Alquilantes , Linhagem Celular Tumoral , Reparo do DNA , Progressão da Doença , Feminino , Humanos , Melfalan , Camundongos SCID , Neoplasias Experimentais , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-myc/metabolismoRESUMO
Multiple Myeloma (MM) is an incurable hematological malignancy affecting millions of people worldwide. As in all tumor cells both glucose and more recently glutamine have been identified as important for MM cellular metabolism, however there is some dispute as to the role of glutamine in MM cell survival. Here we show that the small molecule inhibitor compound 968 effectively inhibits glutaminase and that this inhibition induces apoptosis in both human multiple myeloma cell lines (HMCLs) and primary patient material. The HMCL U266 which does not express MYC was insensitive to both glutamine removal and compound 968, but ectopic expression of MYC imparted sensitivity. Finally, we show that glutamine depletion is reflected by rapid loss of MYC protein which is independent of MYC transcription and post translational modifications. However, MYC loss is dependent on proteasomal activity, and this loss was paralleled by an equally rapid induction of apoptosis. These findings are in contrast to those of glucose depletion which largely affected rates of proliferation in HMCLs, but had no effects on either MYC expression or viability. Therefore, inhibition of glutaminolysis is effective at inducing apoptosis and thus serves as a possible therapeutic target in MM.
RESUMO
BACKGROUND: Plasmacytosis (ie, an expansion of plasma cell populations to much greater than the homeostatic level) occurs in the context of various immune disorders and plasma cell neoplasia. This condition is often associated with immunodeficiency that causes increased susceptibility to severe infections. Yet a causative link between plasmacytosis and immunodeficiency has not been established. OBJECTIVE: Because recent studies have identified plasma cells as a relevant source of the immunosuppressive cytokine IL-10, we sought to investigate the role of IL-10 during conditions of polyclonal and neoplastic plasmacytosis for the regulation of immunity and its effect on inflammation and immunodeficiency. METHODS: We used flow cytometry, IL-10 reporter (Vert-X) and B cell-specific IL-10 knockout mice, migration assays, and antibody-mediated IL-10 receptor blockade to study plasmacytosis-associated IL-10 expression and its effect on inflammation and Streptococcus pneumoniae infection in mice. ELISA was used to quantify IL-10 levels in patients with myeloma. RESULTS: IL-10 production was a common feature of normal and neoplastic plasma cells in mice, and IL-10 levels increased with myeloma progression in patients. IL-10 directly inhibited neutrophil migration toward the anaphylatoxin C5a and suppressed neutrophil-dependent inflammation in a murine model of autoimmune disease. MOPC.315.BM murine myeloma leads to an increased incidence of bacterial infection in the airways, which was reversed after IL-10 receptor blockade. CONCLUSION: We provide evidence that plasmacytosis-associated overexpression of IL-10 inhibits neutrophil migration and neutrophil-mediated inflammation but also promotes immunodeficiency.
Assuntos
Interleucina-10/imunologia , Plasmócitos/imunologia , Animais , Linhagem Celular Tumoral , Complemento C5a/imunologia , Humanos , Doenças do Sistema Imunitário/imunologia , Inflamação/imunologia , Interleucina-10/genética , Transtornos Leucocíticos/imunologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mieloma Múltiplo/imunologia , Neutrófilos/imunologia , Infecções Pneumocócicas/imunologiaRESUMO
Multiple myeloma is a bone marrow plasma cell tumor which is supported by the external growth factors APRIL and IL-6, among others. Recently, we identified eosinophils and megakaryocytes to be functional components of the micro-environmental niches of benign bone marrow plasma cells and to be important local sources of these cytokines. Here, we investigated whether eosinophils and megakaryocytes also support the growth of tumor plasma cells in the MOPC315.BM model for multiple myeloma. As it was shown for benign plasma cells and multiple myeloma cells, IL-6 and APRIL also supported MOPC315.BM cell growth in vitro, IL-5 had no effect. Depletion of eosinophils in vivo by IL-5 blockade led to a reduction of the early myeloma load. Consistent with this, myeloma growth in early stages was retarded in eosinophil-deficient ΔdblGATA-1 mice. Late myeloma stages were unaffected, possibly due to megakaryocytes compensating for the loss of eosinophils, since megakaryocytes were found to be in contact with myeloma cells in vivo and supported myeloma growth in vitro. We conclude that eosinophils and megakaryocytes in the niches for benign bone marrow plasma cells support the growth of malignant plasma cells. Further investigations are required to test whether perturbation of these niches represents a potential strategy for the treatment of multiple myeloma.
Assuntos
Medula Óssea/patologia , Proliferação de Células , Eosinófilos/citologia , Megacariócitos/citologia , Mieloma Múltiplo/patologia , Animais , Técnicas de Cocultura , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Multiple myeloma (MM) is a lethal human cancer characterized by a clonal expansion of malignant plasma cells in bone marrow. Mouse models of human MM are technically challenging and do not always recapitulate human disease. Therefore, new mouse models for MM are needed. Mineral-oil induced plasmacytomas (MOPC) develop in the peritoneal cavity of oil-injected BALB/c mice. However, MOPC typically grow extramedullary and are considered poor models of human MM. Here we describe an in vivo-selected MOPC315 variant, called MOPC315.BM, which can be maintained in vitro. When injected i.v. into BALB/c mice, MOPC315.BM cells exhibit tropism for bone marrow. As few as 10(4) MOPC315.BM cells injected i.v. induced paraplegia, a sign of spinal cord compression, in all mice within 3-4 weeks. MOPC315.BM cells were stably transfected with either firefly luciferase (MOPC315.BM.Luc) or DsRed (MOPC315.BM.DsRed) for studies using noninvasive imaging. MOPC315.BM.Luc cells were detected in the tibiofemoral region already 1 hour after i.v. injection. Bone foci developed progressively, and as of day 5, MM cells were detected in multiple sites in the axial skeleton. Additionally, the spleen (a hematopoietic organ in the mouse) was invariably affected. Luminescent signals correlated with serum myeloma protein concentration, allowing for easy tracking of tumor load with noninvasive imaging. Affected mice developed osteolytic lesions. The MOPC315.BM model employs a common strain of immunocompetent mice (BALB/c) and replicates many characteristics of human MM. The model should be suitable for studies of bone marrow tropism, development of osteolytic lesions, drug testing, and immunotherapy in MM.
Assuntos
Medula Óssea/patologia , Modelos Animais de Doenças , Camundongos , Mieloma Múltiplo/patologia , Osteólise/patologia , Animais , Linhagem Celular Tumoral , Expressão Gênica , Genes Reporter , Medições Luminescentes , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Imagem Molecular , Mieloma Múltiplo/induzido quimicamente , Mieloma Múltiplo/mortalidade , Osteólise/diagnóstico por imagem , Radiografia , TransfecçãoRESUMO
The P-type calcium current is mediated by a voltage-sensing CaV2.1 alpha subunit in combination with modulatory auxiliary subunits. In Purkinje neurones, this current has distinctively slow inactivation kinetics that may depend on alternative splicing of the alpha subunit and/or association with different CaVbeta subunits. To better understand the molecular components of P-type calcium current, we cloned a CaV2.1 cDNA from total mouse brain. The full-length CaV2.1 isoform that we isolated (GenBank AY714490) contains sequences recently shown to be present in Purkinje neurones. In agreement with previously published work, the alternatively spliced amino acid V421, implicated in slow inactivation, was not encoded in AY714490 and was absent from reverse transcription-polymerase chain reaction products generated from single Purkinje cells. Next, we studied the expression of the four known mouse auxiliary CaVbeta2 isoforms in Purkinje neurones. Confirmation of the presence of CaVbeta2a in Purkinje cells, previously shown by others to slow CaV2.1 kinetics, led us to characterize its influence on current dynamics. We studied currents generated by the clone AY714490 coexpressed in tsA201 cells with four different CaVbeta subunits. In addition to the well-documented slowing of open-state inactivation kinetics, coexpression with the CaVbeta2a subunit also protected CaV2.1 channels from closed-state inactivation and prevented the channel from inactivating during physiological trains of action potential-like stimuli. This strong resistance to inactivation parallels the property of Purkinje neurone P-type currents and is suggestive of a role for CaVbeta2a in modulating the inactivation properties of P-type calcium currents in Purkinje neurones.
Assuntos
Canais de Cálcio Tipo N/fisiologia , Potenciais da Membrana/fisiologia , Células de Purkinje/fisiologia , Animais , Encéfalo/citologia , Bloqueadores dos Canais de Cálcio/farmacologia , Linhagem Celular Transformada , Clonagem Molecular/métodos , Relação Dose-Resposta à Radiação , Estimulação Elétrica/métodos , Proteínas de Fluorescência Verde/metabolismo , Humanos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/efeitos da radiação , Camundongos , Dados de Sequência Molecular , Técnicas de Patch-Clamp , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Células de Purkinje/efeitos dos fármacos , Alinhamento de Sequência/métodos , Transfecção/métodos , ômega-Agatoxina IVA/farmacologiaRESUMO
The sodium pump (Na+/K+-ATPase), maintains intracellular and extracellular concentrations of sodium and potassium by catalysing ATP. Three sodium pump alpha subunits, ATP1A1, ATP1A2 and ATP1A3, are expressed in brain. We compared their role in pyramidal cells and a subset of interneurones in the subiculum. Interneurones were identified by their expression of GFP under the GAD-65 promoter. We used the sensitivity to the cardiac glycoside, ouabain, to discriminate between different alpha subunit isoforms. GFP-positive interneurones were depolarized by nanomolar doses of ouabain, but higher concentrations were needed to depolarize pyramidal cells. Comparison of pump currents in these cells revealed a current sensitive to low doses of ouabain in interneurones, while micromolar doses of ouabain were needed to suppress the pump current in subicular pyramidal cells. As predicted, nanomolar doses of ouabain increased the frequency but not the amplitudes of IPSPs in pyramidal cells. Immunostaining confirmed a differential distribution of alpha-subunits of the Na+/K+-ATPase in subicular interneurones and pyramidal cells. In conclusion, these data suggest that while ATP1A3-isoforms regulate sodium and potassium homeostasis in subicular interneurones, ATP1A1-isoforms assume this function in pyramidal cells. This differential expression of sodium pump isoforms may contribute to differences in resting membrane potential of subicular interneurones and pyramidal cells.
Assuntos
Hipocampo/fisiologia , Interneurônios/fisiologia , Células Piramidais/fisiologia , ATPase Trocadora de Sódio-Potássio/genética , Animais , Inibidores Enzimáticos/farmacologia , Feminino , Expressão Gênica/fisiologia , Glutamato Descarboxilase/genética , Proteínas de Fluorescência Verde/genética , Hipocampo/citologia , Imuno-Histoquímica , Hibridização In Situ , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ouabaína/farmacologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Sinapses/fisiologiaRESUMO
The combined blockade of the IL-6R/STAT3 and the MAPK signaling pathways has been shown to inhibit bone marrow microenvironment (BMM)-mediated survival of multiple myeloma (MM) cells. Here, we identify the molecular chaperones heat shock proteins (Hsp) 90alpha and beta as target genes of both pathways. The siRNA-mediated knockdown of Hsp90 or treatment with the novel Hsp90 inhibitor 17-DMAG attenuated the levels of STAT3 and phospho-ERK and decreased the viability of MM cells. Although knockdown of Hsp90beta-unlike knockdown of Hsp90alpha-was sufficient to induce apoptosis, this effect was strongly increased when both Hsp90s were targeted, indicating a cooperation of both. Given the importance of the BMM for drug resistance and MM-cell survival, apoptosis induced by Hsp90 inhibition was not mitigated in the presence of bone marrow stromal cells, osteoclasts, or endothelial cells. These observations suggest that a positive feedback loop consisting of Hsp90alpha/beta and major signaling pathways supports the survival of MM cells. Finally, in situ overexpression of both Hsp90 proteins was observed in most MMs but not in monoclonal gammopathy of undetermined significance (MGUS) or in normal plasma cells. Our results underpin a role for Hsp90alpha and beta in MM pathogenesis.
Assuntos
Proteínas de Choque Térmico HSP90/biossíntese , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mieloma Múltiplo/metabolismo , Fator de Transcrição STAT3/metabolismo , Apoptose/efeitos dos fármacos , Benzoquinonas/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/imunologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Humanos , Lactamas Macrocíclicas/farmacologia , Sistema de Sinalização das MAP Quinases , Mieloma Múltiplo/patologia , Fosforilação , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
The main factors that govern the pathophysiology and malignant growth of multiple myeloma (MM) are genetic defects within the tumour and the interaction between myeloma cells and the bone marrow microenvironment (BMM). This interaction leads to the activation of signalling pathways that promote the expansion of the malignant clone and stimulate neoangiogenesis and osteoclastogenesis. For many years, the cytokine interleukin-6 (IL-6) was considered a central growth factor and was thus believed to play a pivitol role in the pathogenesis of MM. However, increasing numbers of cytokines, chemokines and cell-to-cell contacts provided by the BMM have since been found to support MM cells. It has consistently been demonstrated that oncogenic mutations as well as the BMM stimulate IL-6-independent signalling pathways that protect MM cells from apoptosis. Consequently, multiple targeting of a complex signalling network rather than inhibition of a single pathway or growth factor is required to effectively induce myeloma cell death. Because the tumour suppressor p53 is rarely mutated in MM, non-genotoxic activation of the p53-dependent death pathway could be another attractive therapeutic strategy for this disease. Even though a number of promising new drugs are currently being tested in MM, a comprehensive knowledge of the signalling and survival pathways should pinpoint additional molecular targets and lead to the development of novel and hopefully more effective treatment strategies.
Assuntos
Doenças Ósseas/etiologia , Medula Óssea/patologia , Mieloma Múltiplo/complicações , Apoptose/fisiologia , Doenças Ósseas/metabolismo , Doenças Ósseas/patologia , Comunicação Celular , Proteínas de Choque Térmico/fisiologia , Interleucina-6/fisiologia , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Receptores de Interleucina-6/fisiologia , Fator de Transcrição STAT3/fisiologia , Transdução de Sinais , Proteína Supressora de Tumor p53/fisiologiaRESUMO
Hodgkin/Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL) display unique characteristics that discriminate cHL from other B-cell lymphomas and normal B cells. Therefore, comparative gene expression profiling of Hodgkin and non-Hodgkin B cells could lead to the identification of candidate genes that are critical for the pathogenesis of cHL. We performed microarray analysis of Hodgkin and non-Hodgkin cell lines and identified activating transcription factor 3 (ATF3), a member of the cyclic AMP response element binding protein (CREB)/ATF family, as a differentially expressed candidate gene. Extensive analysis of a large panel of cell lines, primary tumor samples, and normal tissues revealed that high expression of ATF3 is found in nearly all cases of cHL and is almost exclusively restricted to it. Selective knock-down of ATF3 by RNA interference suppressed proliferation and strongly reduced viability of Hodgkin cells. Thus, overexpression of ATF3 is a molecular hallmark of cHL that contributes to the malignant growth of HRS cells.
Assuntos
Fator 3 Ativador da Transcrição/genética , Regulação Neoplásica da Expressão Gênica , Doença de Hodgkin/genética , Células de Reed-Sternberg/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Perfilação da Expressão Gênica , Doença de Hodgkin/patologia , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , RNA Interferente Pequeno/farmacologia , Células de Reed-Sternberg/metabolismoRESUMO
B cell differentiation is controlled by a complex network of lineage-restricted transcription factors. How perturbations to this network alter B cell fate remains poorly understood. Here we show that classical Hodgkin lymphoma tumor cells, which originate from mature B cells, have lost the B cell phenotype as a result of aberrant expression of transcriptional regulators. The B cell-specific transcription factor program was disrupted by overexpression of the helix-loop-helix proteins ABF-1 and Id2. Both factors antagonized the function of the B cell-determining transcription factor E2A. As a result, expression of genes specific to B cells was lost and expression of genes not normally associated with the B lineage was upregulated. These data demonstrate the plasticity of mature human lymphoid cells and offer an explanation for the unique classical Hodgkin lymphoma phenotype.
Assuntos
Linfócitos B/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Doença de Hodgkin/metabolismo , Proteína 2 Inibidora de Diferenciação/metabolismo , Linfócitos B/imunologia , Linfócitos B/patologia , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linhagem Celular Tumoral , Dimerização , Expressão Gênica , Doença de Hodgkin/genética , Doença de Hodgkin/imunologia , Doença de Hodgkin/patologia , Humanos , Proteína 2 Inibidora de Diferenciação/genética , Complexos Multiproteicos , Fator de Transcrição PAX5/genética , Fator de Transcrição PAX5/metabolismo , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismoRESUMO
Transcription factor nuclear factor kappa B (NF-kappaB) plays a central role in the pathogenesis of classical Hodgkin lymphoma (cHL). In anaplastic large-cell lymphomas (ALCLs), which share molecular lesions with cHL, the NF-kappaB system has not been equivalently investigated. Here we describe constitutive NF-kappaB p50 homodimer [(p50)2] activity in ALCL cells in the absence of constitutive activation of the IkappaB kinase (IKK) complex. Furthermore, (p50)2 contributes to the NF-kappaB activity in Hodgkin/Reed-Sternberg (HRS) cells. Bcl-3, which is an inducer of nuclear (p50)2 and is associated with (p50)2 in ALCL and HRS cell lines, is abundantly expressed in ALCL and HRS cells. Notably, a selective overexpression of Bcl-3 target genes is found in ALCL cells. By immunohistochemical screening of 288 lymphoma cases, a strong Bcl-3 expression in cHL and in peripheral T-cell non-Hodgkin lymphoma (T-NHL) including ALCL was found. In 3 of 6 HRS cell lines and 25% of primary ALCL, a copy number increase of the BCL3 gene locus was identified. Together, these data suggest that elevated Bcl-3 expression has an important function in cHL and peripheral T-NHL, in particular ALCL.
Assuntos
Regulação Neoplásica da Expressão Gênica , Doença de Hodgkin/genética , Doença de Hodgkin/metabolismo , Linfoma de Células T Periférico/genética , Linfoma de Células T Periférico/metabolismo , Subunidade p50 de NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteína 3 do Linfoma de Células B , Linhagem Celular , Dimerização , Humanos , Proteínas I-kappa B/metabolismo , Interleucina-9/metabolismo , Linfoma de Células T Periférico/patologia , Análise em Microsséries , Proteínas Oncogênicas v-rel/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Fatores de TranscriçãoRESUMO
Mutation of p53 is a rare event in multiple myeloma, but it is unknown if p53 signaling is functional in myeloma cells, and if targeted nongenotoxic activation of the p53 pathway is sufficient to kill tumor cells. Here, we demonstrate that treatment of primary tumor samples with a small-molecule inhibitor of the p53-murine double minute 2 (MDM2) interaction increases the level of p53 and induces p53 targets and apoptotic cell death. Significantly, given the importance of the bone marrow microenvironment for the support and drug resistance of myeloma cells, tumor cells undergo effective apoptosis also in the presence of stromal cells, which themselves appear to tolerate exposure to nutlin-3. The in vitro toxicity of nutlin-3 was similar to that of the genotoxic drug melphalan. Because nutlin-mediated p53 activation is not dependent on DNA damage, MDM2 antagonists may help to avoid or reduce the severe genotoxic side effects of chemotherapeutic agents currently used to treat multiple myeloma. Therefore, MDM2 antagonists may offer a new treatment option for this disease.
Assuntos
Apoptose/efeitos dos fármacos , Imidazóis/farmacologia , Mieloma Múltiplo/metabolismo , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Animais , Antineoplásicos Alquilantes/efeitos adversos , Antineoplásicos Alquilantes/farmacologia , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Humanos , Imidazóis/uso terapêutico , Melfalan/efeitos adversos , Melfalan/farmacologia , Camundongos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Mutagênicos/farmacologia , Piperazinas/uso terapêutico , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Células Estromais/metabolismo , Proteína Supressora de Tumor p53/genéticaRESUMO
The interleukin-6 receptor (IL-6R)/signal transducer and activator of transcription 3 (STAT3) pathway contributes to the pathogenesis of multiple myeloma (MM) and protects MM cells from apoptosis. However, MM cells survive the IL-6R blockade if they are cocultured with bone marrow stromal cells (BMSCs), suggesting that the BM microenvironment stimulates IL-6-independent pathways that exert a pro-survival effect. The goal of this study was to investigate the underlying mechanism. Detailed pathway analysis revealed that BMSCs stimulate STAT3 via the IL-6R, and mitogen-activated protein (MAP) kinases via IL-6R-independent mechanisms. Abolition of MEK1,2 activity with PD98059, or ERK1,2 small interfering RNA knockdown, was insufficient to induce apoptosis. However, the combined disruption of the IL-6R/STAT3 and MEK1,2/ERK1,2 pathways led to strong induction of apoptosis even in the presence of BMSCs. This effect was observed with MM cell lines and with primary MM cells, suggesting that the BMSC-induced activation of MEK1,2/ERK1,2 renders MM cells IL-6R/STAT3 independent. Therefore, in the presence of cells from the BM micro-environment, combined targeting of different (and independently activated) pathways is required to efficiently induce apoptosis of MM cells. This might have direct implications for the development of future therapeutic strategies for MM.
Assuntos
Proteínas de Ligação a DNA/metabolismo , MAP Quinase Quinase 1/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Mieloma Múltiplo/patologia , Receptores de Interleucina-6/metabolismo , Células Estromais/fisiologia , Transativadores/metabolismo , Idoso , Apoptose , Células da Medula Óssea , Técnicas de Cocultura , Proteínas de Ligação a DNA/antagonistas & inibidores , Feminino , Humanos , MAP Quinase Quinase 1/antagonistas & inibidores , Masculino , Pessoa de Meia-Idade , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , RNA Interferente Pequeno/farmacologia , Receptores de Interleucina-6/antagonistas & inibidores , Fator de Transcrição STAT3 , Transdução de Sinais , Células Estromais/citologia , Transativadores/antagonistas & inibidores , Células Tumorais CultivadasRESUMO
A recently developed bispecific antibody construct, directed against CD19 and CD3 (bscCD19xCD3), induces T-cell-mediated lysis of allogeneic and autologous B cells in a specific and highly efficient manner. Since knowledge of the molecular mechanisms underlying this lysis is limited, a study on bscCD19xCD3-activated T-cell-effector pathways was performed. BscCD19xCD3-induced lysis of target B-cell lines Nalm-6, Daudi, and Raji and of autologous primary B cells is caused by the perforin-dependent granule-exocytosis pathway but not by the death ligands FasL, TRAIL, or TNF-alpha. When activated by bscCD19xCD3 and Raji cells, T cells express FasL mRNA, but incubation of Raji cells with cell-free supernatants from cytotoxicity experiments caused an upregulation of c-Flipl, possibly accounting for the cells' insensitivity toward death-receptor-mediated lysis. In addition to granule exocytosis, Raji cells are lysed by at least one mechanism independent of perforin, which requires transport through the T cell's Golgi apparatus.
Assuntos
Anticorpos Biespecíficos/imunologia , Antígenos CD19/imunologia , Linfócitos B/imunologia , Complexo CD3/imunologia , Grânulos Citoplasmáticos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Glicoproteínas de Membrana/metabolismo , Linfócitos T/imunologia , Apoptose , Proteínas Reguladoras de Apoptose , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Proteínas de Transporte/metabolismo , Citotoxicidade Imunológica , Exocitose , Proteína Ligante Fas , Humanos , Imunoterapia , Ligantes , Ativação Linfocitária , Glicoproteínas de Membrana/genética , Perforina , Proteínas Citotóxicas Formadoras de Poros , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/patologia , Ligante Indutor de Apoptose Relacionado a TNF , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/metabolismo , Regulação para CimaRESUMO
Resistance to death receptor-mediated apoptosis is supposed to be important for the deregulated growth of B cell lymphoma. Hodgkin/Reed-Sternberg (HRS) cells, the malignant cells of classical Hodgkin's lymphoma (cHL), resist CD95-induced apoptosis. Therefore, we analyzed death receptor signaling, in particular the CD95 pathway, in these cells. High level CD95 expression allowed a rapid formation of the death-inducing signaling complex (DISC) containing Fas-associated death domain-containing protein (FADD), caspase-8, caspase-10, and most importantly, cellular FADD-like interleukin 1beta-converting enzyme-inhibitory protein (c-FLIP). The immunohistochemical analysis of the DISC members revealed a strong expression of CD95 and c-FLIP overexpression in 55 out of 59 cases of cHL. FADD overexpression was detectable in several cases. Triggering of the CD95 pathway in HRS cells is indicated by the presence of CD95L in cells surrounding them as well as confocal microscopy showing c-FLIP predominantly localized at the cell membrane. Elevated c-FLIP expression in HRS cells depends on nuclear factor (NF)-kappaB. Despite expression of other NF-kappaB-dependent antiapoptotic proteins, the selective down-regulation of c-FLIP by small interfering RNA oligoribonucleotides was sufficient to sensitize HRS cells to CD95 and tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis. Therefore, c-FLIP is a key regulator of death receptor resistance in HRS cells.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Apoptose/fisiologia , Proteínas de Transporte/fisiologia , Doença de Hodgkin/patologia , Doença de Hodgkin/fisiopatologia , Peptídeos e Proteínas de Sinalização Intracelular , Células de Reed-Sternberg/patologia , Células de Reed-Sternberg/fisiologia , Receptor fas/fisiologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose , Sequência de Bases , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Caspase 10 , Caspase 8 , Caspases/metabolismo , Linhagem Celular Tumoral , Cicloeximida/farmacologia , Proteína de Domínio de Morte Associada a Fas , Humanos , Glicoproteínas de Membrana/fisiologia , NF-kappa B/metabolismo , RNA Interferente Pequeno/genética , Células de Reed-Sternberg/efeitos dos fármacos , Ligante Indutor de Apoptose Relacionado a TNF , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
Apoptosis of lymphocytes recognizing self-antigens is an essential mechanism to protect the organism against autoimmune diseases. Programmed cell death of susceptible B cells occurs in response to surface IgM cross-linking mediated by self-antigens. This effect can be mimicked in the Burkitt's lymphoma line BL60-2 by addition of anti-IgM antibodies. In order to identify genes with differential expression in response to the apoptotic stimulus, total RNA prepared from BL60-2 cells before and at different points in time after IgM cross-linking was used for Atlas arrays, high-density oligonucleotide microarrays (GeneChip arrays, Affymetrix) and in RNase protection assays (RPA). One of our major observations was the downregulation of six genes involved in the ligation of DNA strand breaks, like DNA ligases and DNA-PK, indicating a shutdown of DNA repair mechanisms in apoptotic cells. In addition, we found changes on mRNA level for several transcription regulators, including early growth response genes 1 and 2, TAFII30 and topoisomerase I. Furthermore, we show accumulation of mRNA for the phosphatases CD45 and DUSP5 in anti-IgM stimulated BL60-2 cells. Our data provide a basis for further analysis of the differentially expressed genes and their roles in IgM-induced B cell death as well as in apoptosis in other cellular systems.
Assuntos
Apoptose/imunologia , Reparo do DNA/genética , Regulação para Baixo/genética , Regulação da Expressão Gênica , Monoéster Fosfórico Hidrolases/genética , Apoptose/genética , Linhagem Celular Tumoral , Fragmentação do DNA , Primers do DNA , Humanos , Immunoblotting , Imunoglobulina M/imunologia , Medições Luminescentes , Análise de Sequência com Séries de Oligonucleotídeos , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , RibonucleasesRESUMO
The pathogenesis of Hodgkin lymphoma (HL) is still unclear. Previous investigations have demonstrated constitutive nuclear activity of the transcription factor NF kappa B (NF-kappaB) in Hodgkin/Reed-Sternberg (HRS) cells as an important prerequisite in protecting these cells from apoptosis. As a molecular mechanism leading to constitutive NF-kappaB activity in HRS cells, mutations of the NF-kappaB inhibitor I kappa B alpha (IkappaBalpha) have recently been identified in classical (c) HL-derived cell lines in a patient with cHL. In the present study, the NF-kappaB inhibitor I kappa B epsilon (IkappaBepsilon) has been analysed for somatic mutations in the same group of six patients already studied for IkappaBalpha mutations, as well as in cHL-derived cell lines. In one cHL-derived cell line (L428), a hemizygous frame-shift mutation generating a pre-terminal stop codon resulting in a severely truncated protein was found. Moreover, in the HRS cells of one patient, a hemizygous mutation affecting the 5'-splicing site of intron 1 of the IkappaBepsilon gene was found. These results, in combination with recently described IkappaBalpha mutations, indicate that defective NF-kappaB inhibitors appear more frequent than previously thought and might explain the constitutive nuclear activity of NF-kappaB in a significant proportion of cHL cases.
Assuntos
Doença de Hodgkin/genética , Proteínas I-kappa B/genética , NF-kappa B/antagonistas & inibidores , Western Blotting/métodos , Mapeamento Cromossômico/métodos , Clonagem Molecular/métodos , Éxons/genética , Regulação Neoplásica da Expressão Gênica/genética , Doença de Hodgkin/patologia , Humanos , Mutação/genética , Proteínas de Neoplasias/análise , Reação em Cadeia da Polimerase/métodos , RNA Neoplásico/análise , Células de Reed-Sternberg , Células Tumorais CultivadasRESUMO
Arsenic can induce apoptosis and is an efficient drug for the treatment of acute promyelocytic leukemia. Currently, clinical studies are investigating arsenic as a therapeutic agent for a variety of malignancies. In this study, Hodgkin/Reed-Sternberg (HRS) cell lines served as model systems to characterize the role of nuclear factor-kappaB (NF-kappaB) in arsenic-induced apoptosis. Arsenic rapidly down-regulated constitutive IkappaB kinase (IKK) as well as NF-kappaB activity and induced apoptosis in HRS cell lines containing functional IkappaB proteins. In these cell lines, apoptosis was blocked by inhibition of caspase-8 and caspase-3-like activity. Furthermore, arsenic treatment down-regulated NF-kappaB target genes, including tumor necrosis factor-alphareceptor-associated factor 1 (TRAF1), c-IAP2, interleukin-13 (IL-13), and CCR7. In contrast, cell lines with mutated, functionally inactive IkappaB proteins or with a weak constitutive IKK/NF-kappaB activity showed no alteration of the NF-kappaB activity and were resistant to arsenic-induced apoptosis. A direct role of the NF-kappaB pathway in arsenic-induced apoptosis is shown by transient overexpression of NF-kappaB-p65 in L540Cy HRS cells, which protected the cells from arsenic-induced apoptosis. In addition, treatment of NOD/SCID mice with arsenic trioxide induced a dramatic reduction of xenotransplanted L540Cy Hodgkin tumors concomitant with NF-kappaB inhibition. We conclude that inhibition of NF-kappaB contributes to arsenic-induced apoptosis. Furthermore, pharmacologic inhibition of the IKK/NF-kappaB activity might be a powerful treatment option for Hodgkin lymphoma.