RESUMEN
Aggressive systemic mastocytosis (ASM) and mast cell leukemia (MCL) are advanced hematopoietic neoplasms with poor prognosis. In these patients, neoplastic mast cells (MCs) are resistant against various drugs. We examined the effects of 2 demethylating agents, 5-azacytidine and decitabine on growth and survival of neoplastic MCs and the MC line HMC-1. Two HMC-1 subclones were used, HMC-1.1 lacking KIT D816V and HMC-1.2 exhibiting KIT D816V. Both agents induced apoptosis in HMC-1.1 and HMC-1.2 cells. Decitabine, but not 5-azacytidine, also produced a G(2)/M cell-cycle arrest in HMC-1 cells. Drug-induced apoptosis was accompanied by cleavage of caspase-8 and caspase-3 as well as FAS-demethylation and FAS-re-expression in neoplastic MCs. Furthermore, both demethylating agents were found to synergize with the FAS-ligand in inducing apoptosis in neoplastic MCs. Correspondingly, siRNA against FAS was found to block drug-induced expression of FAS and drug-induced apoptosis in HMC-1 cells. Neither 5-azacytidine nor decitabine induced substantial apoptosis or growth arrest in normal MCs or normal bone marrow cells. Together, 5-azacytidine and decitabine exert growth-inhibitory and proapoptotic effects in neoplastic MCs. These effects are mediated through "FAS-re-expression" and are augmented by the FAS-ligand. Whether epigenetic drugs produce antineoplastic effects in vivo in patients with ASM and MCL remains to be determined.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Azacitidina/análogos & derivados , Azacitidina/farmacología , Leucemia de Mastocitos/patología , Mastocitos/efectos de los fármacos , Mastocitosis Sistémica/patología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Receptor fas/metabolismo , Adulto , Anciano , Secuencia de Bases , Línea Celular Tumoral/efectos de los fármacos , Islas de CpG , Metilación de ADN/efectos de los fármacos , ADN de Neoplasias/metabolismo , Decitabina , Sinergismo Farmacológico , Proteína Ligando Fas/fisiología , Femenino , Humanos , Masculino , Mastocitos/patología , Metilación/efectos de los fármacos , Persona de Mediana Edad , Datos de Secuencia Molecular , Proteínas de Neoplasias/metabolismo , Mutación Puntual , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas c-kit/genética , ARN Interferente Pequeño/farmacología , Receptor fas/antagonistas & inhibidores , Receptor fas/genéticaRESUMEN
Pollinosis to birch pollen is a common type I allergy in the Northern Hemisphere. Moreover, birch pollen-allergic individuals sensitized to the major birch pollen allergen Bet v 1 frequently develop allergic reactions to stone fruits, hazelnuts, and certain vegetables due to immunological cross-reactivity. The major T cell epitope Bet v 1(142-153) plays an important role in cross-reactivity between the respiratory allergen Bet v 1 and its homologous food allergens. In this study, we cloned and functionally analyzed a human αß TCR specific for the immunodominant epitope Bet v 1(142-153). cDNAs encoding TCR α- and ß-chains were amplified from a Bet v 1(142-153)-specific T cell clone, introduced into Jurkat T cells and peripheral blood T lymphocytes of allergic and nonallergic individuals, and evaluated functionally. The resulting TCR transgenic (TCRtg) T cells responded in an allergen-specific and costimulation-dependent manner to APCs either pulsed with Bet v 1(142-153) peptide or coexpressing invariant chain::Bet v 1(142-153) fusion proteins. TCRtg T cells responded to Bet v 1-related food and tree pollen allergens that were processed and presented by monocyte-derived dendritic cells. Bet v 1(142-153)-presenting but not Bet v 1(4-15)-presenting artificial APCs coexpressing membrane-bound IL-12 polarized allergen-specific TCRtg T cells toward a Th1 phenotype, producing high levels of IFN-γ. Coculture of such Th1-polarized T cells with allergen-specific Th2-differentiated T cells significantly suppressed Th2 effector cytokine production. These data suggest that human allergen-specific TCR can transfer the fine specificity of the original T cell clone to heterologous T cells, which in turn can be instructed to modulate the effector function of the disease initiating/perpetuating allergen-specific Th2-differentiated T cells.
Asunto(s)
Antígenos de Plantas/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Rinitis Alérgica Estacional/inmunología , Células TH1/inmunología , Células Th2/inmunología , Secuencia de Aminoácidos , Secuencia de Bases , Separación Celular , Reacciones Cruzadas/inmunología , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Hipersensibilidad a los Alimentos/inmunología , Células HEK293 , Humanos , Epítopos Inmunodominantes/inmunología , Células Jurkat , Activación de Linfocitos/inmunología , Datos de Secuencia Molecular , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción Genética , TransgenesRESUMEN
BACKGROUND: Regulatory T (Treg) cells establish and maintain tolerance to self-antigens and many foreign antigens, such as allergens, by suppressing effector T-cell proliferation and function. We have previously shown that human T-cell receptor (TCR) αß-chains specific for allergen-derived epitopes confer allergen specificity on peripheral blood T cells of individuals with and without allergy. OBJECTIVE: To study the feasibility of generating allergen-specific human Treg cells by retroviral transduction of a transcription unit encoding forkhead box protein 3 (FOXP3) and allergen-specific TCR αß-chains. METHODS: cDNAs encoding the α and ß-chains of a Bet v 1(142-153)-specific TCR (TCR alpha variable region 6/TCR beta variable region 20) and human FOXP3 were linked via picornaviral 2A sequences and expressed as single translational unit from an internal ribosomal entry site-green fluorescence protein-containing retroviral vector. Retrovirally transduced peripheral blood T cells were tested for expression of transgenes, Treg phenotype, and regulatory capacity toward allergen-specific effector T cells. RESULTS: Transduced T cells displayed a Treg phenotype with clear-cut upregulation of CD25, CD39, and cytotoxic T-lymphocyte antigen 4. The transduced cells were hyporesponsive in cytokine production and secretion and, like naturally occurring Treg cells, did not proliferate after antigen-specific or antigen-mimetic stimulation. However, proliferation was inducible upon exposure to exogenous IL-2. In coculture experiments, TRAV6(+)TRBV20(+)FOXP3(+) transgenic T cells, unlike FOXP3(+) single transgenic T cells or naturally occurring Treg cells, highly significantly suppressed T cell cytokine production and proliferation of corresponding allergen-specific effector T cells in an allergen-specific, dose-dependent manner. CONCLUSION: We demonstrate a transgenic approach to engineer human allergen-specific Treg cells that exert their regulatory function in an activation-dependent manner. Customized Treg cells might become useful for tolerance induction therapies in individuals with allergic and other immune-mediated diseases.
Asunto(s)
Antígenos de Plantas/genética , Factores de Transcripción Forkhead/genética , Ingeniería Genética/métodos , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Linfocitos T Reguladores/inmunología , Alérgenos/genética , Alérgenos/inmunología , Antígenos de Plantas/inmunología , Betula , Separación Celular , Citometría de Flujo , Factores de Transcripción Forkhead/inmunología , Vectores Genéticos , Células HEK293 , Humanos , Activación de Linfocitos/inmunología , Polen , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Retroviridae , Linfocitos T Reguladores/metabolismo , Transducción Genética , Transfección , TransgenesRESUMEN
BACKGROUND: In advanced systemic mastocytosis the response of neoplastic mast cells to conventional drugs is poor and the prognosis is bad. Current research is, therefore, attempting to identify novel drug targets in neoplastic mast cells. Polo-like kinase-1 is a serine/threonine kinase that plays an essential role in mitosis and has recently been introduced as a new target in myeloid leukemias and solid tumors. DESIGN AND METHODS: In the present study, we analyzed the expression and function of Polo-like kinase-1 in neoplastic mast cells in systemic mastocytosis. RESULTS: As determined by immunostaining, primary neoplastic mast cells as well as the human mast cell leukemia cell line HMC-1 displayed phosphorylated Polo-like kinase-1. In addition, neoplastic mast cells expressed Polo-like kinase-1 mRNA. Polo-like kinase-1-specific small interfering RNA induced apoptosis in neoplastic mast cells, whereas no effect was seen with a control small interfering RNA. BI 2536, a drug targeting Polo-like kinase-1, was found to inhibit proliferation in HMC-1 cells in a dose-dependent manner. BI 2536 also inhibited the growth of primary neoplastic mast cells and cells of the canine mastocytoma cell line C2. The growth-inhibitory effects of BI 2536 on neoplastic mast cells were found to be associated with mitotic arrest and subsequent apoptosis. Finally, BI 2536 was found to synergize with the KIT-targeting kinase inhibitor midostaurin (PKC412) in inhibiting the growth of neoplastic mast cells. In control experiments, BI 2536 did not induce apoptosis in normal cultured mast cells. CONCLUSIONS: Collectively, our data show that Polo-like kinase-1 is a potential therapeutic target in neoplastic mast cells. Targeting Polo-like kinase-1 may be an attractive pharmacological concept in the management of advanced systemic mastocytosis.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Mastocitos/metabolismo , Mastocitosis Sistémica/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Células HL-60 , Humanos , Inmunohistoquímica , Células K562 , Mastocitos/efectos de los fármacos , Mastocitos/patología , Mastocitosis Sistémica/genética , Mastocitosis Sistémica/patología , Fosforilación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Pteridinas/farmacología , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Timidina/metabolismo , Timidina/farmacocinética , Células Tumorales Cultivadas , Células U937 , Quinasa Tipo Polo 1RESUMEN
OBJECTIVE: Macrophages are the main drivers of obesity-induced adipose tissue (AT) inflammation that causes insulin resistance. Macrophages polarize toward different inflammatory (M1) or protective (M2) phenotypes. Osteopontin (OPN) is an inflammatory cytokine highly expressed in AT in obesity and known to be involved in chronic inflammatory processes. It was hypothesized that OPN polarizes macrophages into a proinflammatory phenotype. METHODS: AT macrophages (ATMs) of OPN-deficient (Spp1(-/-) ) and wild-type C57BL/6 (WT) mice with obesity and bone marrow-derived macrophages (BMDMs) of Spp1(-/-) and WT mice as well as human monocyte-derived macrophages (MDMs) polarized in the presence of OPN were investigated. RESULTS: While ATM infiltration was lower in Spp1(-/-) upon high-fat diet, Spp1(-/-) ATMs expressed more M1 and less M2 markers but less tumor necrosis factor-α compared with WT. There was no effect of OPN deficiency on BMDM polarization. In human MDMs, the presence of OPN during polarization ambiguously altered M1/M2-related marker expression and diminished LPS-induced inflammatory cytokine production. Strikingly, phagocytic activity was elevated by the presence of OPN during polarization in both human MDMs and murine BMDMs. CONCLUSIONS: In contradiction to our hypothesis, data indicated that OPN does not induce inflammatory macrophages but was a signal to induce phagocytosis, which may be required due to increased adipocyte death in obesity.
Asunto(s)
Macrófagos/fisiología , Obesidad/fisiopatología , Osteopontina/metabolismo , Adipocitos/fisiología , Tejido Adiposo/metabolismo , Animales , Dieta Alta en Grasa , Inflamación/fisiopatología , Resistencia a la Insulina , Masculino , Ratones , Ratones Endogámicos C57BL , Fagocitosis/fisiología , Fenotipo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
OBJECTIVE: Osteopontin (OPN) is upregulated in adipose tissue (AT) in obesity and contributes to subclinical inflammation, adipocyte dysfunction, and insulin resistance. OPN effects can be increased by cleavage by matrix metalloproteinases (MMP). This study aimed at investigating the presence of OPN cleavage products in human AT in obesity and their impact on adipocyte function and immunological blockade of these effects. METHODS: AT of severely obese and control donors was investigated for OPN and MMP expression and the presence of OPN cleavage fragments. Primary adipocytes were isolated from human donors for in vitro investigation of cleaved OPN effects. RESULTS: OPN and MMP-9 expression was highly correlated in AT from obese donors, and increased levels of cleaved OPN were detected in AT from obese individuals. The in vitro effect of OPN on adipocyte inflammation and insulin resistance was enhanced by protease cleavage, which could finally be blocked with a monoclonal antibody directed against the MMP cleavage site of OPN. CONCLUSIONS: These findings show that MMP cleavage of OPN in AT occurs in obesity, thereby enhancing OPN's inflammatory and pro-diabetic activity on adipocytes. Specifically targeting MMP-cleaved OPN opens avenues for prevention and treatment of obesity-induced type 2 diabetes.
Asunto(s)
Adipocitos/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Obesidad/inmunología , Osteopontina/metabolismo , Tejido Adiposo/metabolismo , Humanos , Resistencia a la Insulina , Obesidad/fisiopatología , Obesidad/prevención & controlRESUMEN
Advanced SM is an incurable neoplasm with short survival time. So far, no effective therapy is available for these patients. We and others have shown recently that neoplastic MC in ASM and MCL express antiapoptotic Mcl-1, Bcl-2, and Bcl-xL. In this study, we examined the effects of the pan-Bcl-2 family blocker obatoclax (GX015-070) on primary neoplastic MC, the human MC leukemia cell line HMC-1, and the canine mastocytoma cell line C2. Obatoclax was found to inhibit proliferation in primary human neoplastic MC (IC50: 0.057 µM), in HMC-1.2 cells expressing KIT D816V (IC50: 0.72 µM), and in HMC-1.1 cells lacking KIT D816V (IC50: 0.09 µM), as well as in C2 cells (IC50: 0.74 µM). The growth-inhibitory effects of obatoclax in HMC-1 cells were accompanied by an increase in expression of Puma, Noxa, and Bim mRNA, as well as by apoptosis, as evidenced by microscopy, TUNEL assay, and caspase cleavage. Viral-mediated overexpression of Mcl-1, Bcl-xL, or Bcl-2 in HMC-1 cells was found to introduce partial resistance against apoptosis-inducing effects of obatoclax. We were also able to show that obatoclax synergizes with several other antineoplastic drugs, including dasatinib, midostaurin, and bortezomib, in producing apoptosis and/or growth arrest in neoplastic MC. Together, obatoclax exerts major growth-inhibitory effects on neoplastic MC and potentiates the antineoplastic activity of other targeted drugs. Whether these drug effects can be translated to application in patients with advanced SM remains to be determined.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/genética , Apoptosis/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Sarcoma de Mastocitos/genética , Proteínas de la Membrana/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas/genética , Pirroles/farmacología , Adulto , Anciano , Anciano de 80 o más Años , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Proteína 11 Similar a Bcl2 , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Sinergismo Farmacológico , Femenino , Humanos , Indoles , Masculino , Sarcoma de Mastocitos/tratamiento farmacológico , Sarcoma de Mastocitos/mortalidad , Persona de Mediana Edad , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Pirroles/uso terapéutico , ARN Mensajero/genética , Proteína bcl-X/genéticaRESUMEN
OBJECTIVE: In mast cell (MC) neoplasms, clinical problems requiring therapy include local aggressive and sometimes devastating growth of MCs and mediator-related symptoms. A key mediator of MCs responsible for clinical symptoms is histamine. Therefore, use of histamine receptor (HR) antagonists is an established approach to block histamine effects in these patients. MATERIALS AND METHODS: We screened for additional beneficial effects of HR antagonists and asked whether any of these agents would also exert growth-inhibitory effects on primary neoplastic MCs, the human MC line HMC-1, and on two canine MC lines, C2 and NI-1. RESULTS: We found that the HR1 antagonists terfenadine and loratadine suppress spontaneous growth of HMC-1, C2, and NI-1 cells, as well as growth of primary neoplastic MCs in all donors tested (human patients, n = 5; canine patients, n = 8). The effects of both drugs were found to be dose-dependent (IC(50): terfenadine, 1-20 µM; loratadine, 10-50 µM). Both agents also produced apoptosis in neoplastic MCs and augmented apoptosis-inducing effects of two KIT-targeting drugs, PKC412 and dasatinib. The other HR1 antagonists (fexofenadine, diphenhydramine) and HR2 antagonists (famotidine, cimetidine, ranitidine) tested did not exert substantial growth-inhibitory effects on neoplastic MCs. None of the histamine receptor blockers were found to modulate cell-cycle progression in neoplastic MCs. CONCLUSIONS: The HR1 antagonists terfenadine and loratadine, in addition to their antimediator activity, exert in vitro growth-inhibitory effects on neoplastic MCs. Whether these drugs (terfenadine) alone, or in combination with KIT inhibitors, can also affect in vivo neoplastic MC growth remains to be determined.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Antagonistas de los Receptores Histamínicos H1 no Sedantes/farmacología , Loratadina/farmacología , Terfenadina/farmacología , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Caspasa 3/metabolismo , Gatos , Línea Celular Tumoral , Dasatinib , Perros , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Citometría de Flujo , Humanos , Concentración 50 Inhibidora , Mastocitoma/tratamiento farmacológico , Mastocitoma/metabolismo , Mastocitoma/patología , Mastocitosis Sistémica/tratamiento farmacológico , Mastocitosis Sistémica/metabolismo , Mastocitosis Sistémica/patología , Proteínas Proto-Oncogénicas c-kit/metabolismo , Pirimidinas/farmacología , Estaurosporina/análogos & derivados , Estaurosporina/farmacología , Tiazoles/farmacología , Células Tumorales CultivadasRESUMEN
OBJECTIVE: In most patients with systemic mastocytosis (SM), including aggressive SM (ASM) and mast cell (MC) leukemia (MCL), neoplastic cells express the oncogenic KIT mutation D816V, which confers resistance to imatinib. Cladribine (2CdA) is a nucleoside analog that has been introduced as a promising agent for treatment of advanced SM. MATERIALS AND METHODS: We examined the in vitro effects of 2CdA on growth of neoplastic MC, and the in vivo effects of 2CdA (0.13 mg/kg/day intravenously, days 1-5; three to eight cycles) in seven patients with advanced SM. RESULTS: Cladribine was found to inhibit growth of primary MC and the MC line HMC-1 in a dose-dependent manner, with lower IC(50) values recorded in HMC-1.2 cells harboring KIT D816V (IC(50): 10 ng/mL) compared to HMC-1.1 cells lacking KIT D816V (IC(50): 300 ng/mL). In two patients with progressive smoldering SM, 2CdA produced a long-lasting response with a sustained decrease in serum tryptase levels, whereas in patients with progressive ASM or MCL, 2CdA showed little if any effects. The drug was well-tolerated in most cases. However, one patient developed a massive generalized purulent long-lasting skin rash. The antiproliferative effects of 2CdA on MC were found to be associated with morphologic signs of apoptosis and caspase cleavage. Cladribine did not counteract the kinase activity of KIT D816V or KIT-downstream signaling molecules. CONCLUSIONS: Cladribine may be a promising agent for treatment of progressive smoldering KIT D816V(+) SM. In rapidly progressing ASM or MCL, additional or alternative drugs are required to induce long-lasting antineoplastic effects.