RESUMEN

Alterations of TWIST-1 expression are often seen in solid tumors and contribute to tumorigenesis and cancer progression. However, studies concerning its pathogenic role in leukemia are scarce. Our study shows that TWIST-1 is overexpressed in bone marrow mononuclear cells of patients with acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). Gain-of-function and loss-of-function analyses demonstrate that TWIST-1 promotes cell growth, colony formation and drug resistance of AML and CML cell lines. Furthermore, TWIST-1 is aberrantly highly expressed in CD34+CD38- leukemia stem cell candidates and its expression declines with differentiation. Down-modulation of TWIST-1 in myeloid leukemia CD34+ cells impairs their colony-forming capacity. Mechanistically, c-MPL, which is highly expressed in myeloid leukemia cells and associated with poor prognosis, is identified as a TWIST-1 coexpressed gene in myeloid leukemia patients and partially contributes to TWIST-1-mediated leukemogenic effects. Moreover, patients with higher TWIST-1 expression have shorter overall and event-free survival (OS and EFS) in AML. Multivariate analysis further demonstrates that TWIST-1 overexpression is a novel independent unfavourable predictor for both OS and EFS in AML. These data highlight TWIST-1 as a new candidate gene contributing to leukemogenesis of myeloid leukemia, and propose possible new avenues for improving risk and treatment stratification in AML.

Asunto(s)

Resistencia a Antineoplásicos , Regulación Leucémica de la Expresión Génica , Leucemia Mieloide Aguda/metabolismo , Proteínas Nucleares/metabolismo , Proteína 1 Relacionada con Twist/metabolismo , ADP-Ribosil Ciclasa 1/metabolismo , Adolescente , Adulto , Anciano , Antígenos CD34/metabolismo , Proliferación Celular , Separación Celular , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Supervivencia sin Enfermedad , Femenino , Citometría de Flujo , Humanos , Células K562 , Masculino , Persona de Mediana Edad , Análisis Multivariante , Células Madre Neoplásicas/metabolismo , Pronóstico , Receptores de Trombopoyetina/metabolismo , Resultado del Tratamiento , Células U937 , Adulto Joven

RESUMEN

Tumor-associated macrophages are widely studied in solid tumors. The distribution of macrophages in lymph node samples was found to be associated with the prognosis of lymphoma patients. However, the role of macrophages in leukemia and their functional and phenotypic characteristics in hematopoietic malignancies have not been defined. In this study, we examined the distribution and functional and phenotypic characteristics of macrophages in a Notch1-induced mouse model of T cell acute lymphoblastic leukemia (T-ALL). The distribution of macrophages in bone marrow (BM) and spleen, which are proposed as BM and spleen leukemia-associated macrophages (LAMs), were different during the development of leukemia. LAMs stimulated the proliferation of T-ALL cells and had higher migration activity. RNA-sequencing analysis revealed that gene expression profiles of BM and spleen LAMs showed considerable differences. RT-PCR analysis showed that LAMs expressed both M1- and M2-associated phenotypic genes, but they expressed much lower levels of TGF-ß1, VEGF-A, and CSF-1 than did tumor-associated macrophages from B16 melanoma. Furthermore, spleen LAMs more potently stimulated the proliferation of T-ALL cells compared with BM LAMs. Moreover, LAMs could be subdivided into M1-like (CD206(-)) and M2-like (CD206(+)) groups. Both CD206(+) and CD206(-) LAMs stimulated the proliferation of T-ALL cells, although CD206(+) LAMs expressed higher levels of most M1- and M2-associated genes. These results suggested the functional and phenotypic characteristics of LAMs, which were modified by organ specific microenvironments. Our results broaden our knowledge about macrophages in malignant microenvironments from solid tumors to leukemia.

Asunto(s)

Médula Ósea/inmunología , Macrófagos/inmunología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/inmunología , Bazo/inmunología , Microambiente Tumoral/inmunología , Animales , Médula Ósea/metabolismo , Médula Ósea/patología , Movimiento Celular/genética , Movimiento Celular/inmunología , Femenino , Citometría de Flujo , Perfilación de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones Endogámicos C57BL , Mutación/inmunología , Análisis de Secuencia por Matrices de Oligonucleótidos , Especificidad de Órganos/inmunología , Fenotipo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Bazo/metabolismo , Bazo/patología , Análisis de Supervivencia , Microambiente Tumoral/genética

RESUMEN

This study was aimed at exploring the expression pattern of P2X family receptors (P2XR) in peritoneal macrophages and their relationship with the activation states of macrophages in Notch1-induced mouse T-ALL model. After establishment of the leukemia model, F4/80(+) peritoneal macrophages, F4/80(+)CD206(+) M2-like and F4/80(+)CD206(-) M1-like peritoneal macrophages were sorted by flow cytometry based on F4/80 and CD206 surface markers. The expression of P2XR in each cell population was detected by real time RT-PCR. The results showed that macrophages,M1-like and M2-like macrophages moderately expressed P2XR except for P2X5R. The expression of P2XR varied with the development of leukemia. The expression of P2X1R and P2X7R in peritoneal macrophages increased steadily; the expression of P2X2R and P2X3R decreased at late stage of leukemia;the expression of P2X4R slightly decreased at intermediate stage;the expression of P2X6R kept unchanged. At intermediate stage of leukemia, the expression of P2XR in M1-like and M2-like peritoneal macrophages varied. M1-like macrophages expressed higher level of P2X1R than M2-like macrophages, whereas M2-like macrophages expressed higher level of P2X7R than M1-like macrophages, which suggested that the expression of P2XR were related to the activation states. It is concluded that the expression of P2XR in peritoneal macrophages from leukemia mice is related to the progression of leukemia and the activation states of macrophages, which lay a foundation for further studying the role of macrophages in the development of leukemia.

Asunto(s)

Macrófagos Peritoneales/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Receptores Purinérgicos P2X/metabolismo , Animales , Citocinas/metabolismo , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , Receptor Notch1/metabolismo , Transducción de Señal

RESUMEN

This study aimed to construct the dual expression vectors of wide type or N187D mutant P2X7 receptor and intracellular domain of Notch1 (ICN1) linked by 2A peptide to coexpress them in leukemia cells so as to lay a foundation for further investigating the role of P2X7 in development of leukemia. Overlap PCR was used to construct the dual expression vectors encoding wide type or N187D mutant type P2X7 receptor and ICN1 linked by the self-cleaving 2A sequence. The results showed that stable expressing cell lines were obtained by retroviral infection followed by cell sorting after DNA sequence analysis. RT-PCR, Western blot, intracellular free calcium concentration analysis were used to verify the functionally successful construction of K562 cell line expressing P2X7 receptor alone or with ICN1. DNA sequence analysis revealed that all construction were right. The infection efficiency of packaged constructed virus ranged from 40% to 70% for K562 cells. Stable infected cell line was obtained by cell sorting. RT-PCR analysis revealed that P2X7 receptor and/or ICN1 could be detected at high level in their stable infected cell lines, respectively. Western blot analysis also showed that P2X7 receptor was highly expressed in cell line infected by virus with P2X7 receptor. Sustained increase in intracellular free calcium concentration ([Ca(2+)]i) could be observed in K562 cells overexpressing either type of P2X7 receptor upon stimulation with BzATP. It is concluded that the wide type or N187D mutant P2X7 receptor and ICN1 are simultaneously and functionally over-express in leukemia cells, which lay a foundation for further studying the role of P2X7 receptor in the development of leukemia.

Asunto(s)

Receptor Notch1/genética , Receptores Purinérgicos P2X7/genética , Expresión Génica , Vectores Genéticos , Humanos , Células K562 , ARN Mensajero/genética , Retroviridae/genética

RESUMEN

HSPB8 has been shown to be involved in regulation of cell proliferation and apoptosis, and it has also been found to have divergent properties in solid tumors. The purpose of this study was to investigate the expression and function of HSPB8 in hematopoietic malignancies. Expression and induced expression of HSPB8 was evaluated in hematopoietic tumor cell lines and bone marrow samples from patients with leukemia. Methylation status was investigated by methylation-specific polymerase chain reaction. The role of HSPB8 in hematopoietic malignancies was addressed by reintroducing HSPB8 expression into the K562 (leukemia) and Namalwa (lymphoma) cell lines. Expression of HSPB8 was absent in hematopoietic tumor cell lines and primary patient and normal volunteer samples. Promoter DNA methylation of HSPB8 was observed in these cells. HSPB8 expression could be restored after demethylation treatment with 5-aza-2'-deoxycytidine. Overexpression of HSPB8 reduced colony formation of both K562 and Namalwa cell lines, inhibited the cell growth of Namalwa in vitro, and suppressed tumor formation of K562 cells in vivo. The present study demonstrates that HSPB8 is silenced by DNA methylation in hematopoietic malignant and normal cells and its expression can be induced by treatment with 5-aza-2'-deoxycytidine. Overexpression of HSPB8 may have an antitumor activity in chronic myelogenous leukemia and lymphoma.

Asunto(s)

Metilación de ADN , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Neoplasias Hematológicas/metabolismo , Neoplasias Hematológicas/patología , Leucemia Mielógena Crónica BCR-ABL Positiva/terapia , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Azacitidina/análogos & derivados , Azacitidina/farmacología , Metilación de ADN/genética , Decitabina , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HL-60 , Proteínas de Choque Térmico/biosíntesis , Proteínas de Choque Térmico/deficiencia , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/terapia , Humanos , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Linfoma/genética , Linfoma/metabolismo , Linfoma/patología , Linfoma/terapia , Chaperonas Moleculares , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Serina-Treonina Quinasas/deficiencia , Células Tumorales Cultivadas

RESUMEN

This study was purposed to investigate the expression of ADAR1 isoforms of P110 and P150 during the development of murine leukemia. A Notch1 over-expressing murine T cell acute lymphoblastic leukemia model was used to study the expression of ADAR1. BMMNC were isolated at different stages of disease and CD45.2(+)GFP(+) leukemia cells were sorted by flow cytometry at late stage. The expression of ADAR1 was detected by real time quantitative PCR. The results showed that mouse bone marrow cells from both leukemia and control groups expressed P110 and P150. Difference of P110 and P150 mRNA expression were observed during the development of leukemia. The expression of P110 dramatically increased and was significantly higher than that in control group. However, the expression level of P150 in leukemia group decreased stably and reached one-fourth of that in control group at 14 day. Furthermore, similar expression patterns could be detected in sorted CD45.2(+)GFP(+) leukemia cells. It is concluded that the mRNA expressions of P110 and P150 show diverse patterns in the development of leukemia, suggesting that RNA editing mediated by ADAR1 isoforms may play different roles in leukemia.

Asunto(s)

Adenosina Desaminasa/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Animales , Expresión Génica , Ratones , Isoformas de Proteínas/genética , Edición de ARN , ARN Mensajero/genética , Proteínas de Unión al ARN

RESUMEN

The posttranscriptional RNA editing by the type 1 adenosine deaminase acting on RNAs (ADAR1), expressed as p110 and p150 isoforms, is important for both physiological and pathological processes. Their expression and significance in leukemias remain unknown. Here, we investigated the expression of ADAR1 in Chinese pediatric acute leukemias by real-time PCR and Western blot. The results showed that significant high expression of p110 was detected in leukemias, especially in B-ALL, whereas a slight increase of p150 could be observed. Furthermore, the decrease of p110 expression was observed in B-ALL patients achieving complete remission. Moreover, among prognostic risk groups in ALL, the highest expressions of p110 and p150 were detected in standard-risk group, whereas their lowest expressions were in high-risk group. This observation was further confirmed in comparisons between good and poor prognostic groups based on prognostic related clinical features. These results demonstrated that ADAR1 isoforms showed different expression patterns, suggesting that they might play different roles in pediatric leukemias. Our results will help us for the better understanding of RNA editing, exploring the potential target for the treatment, and making prognostic evaluation in childhood leukemias.

Asunto(s)

Adenosina Desaminasa/biosíntesis , Leucemia Mieloide Aguda/enzimología , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimología , Edición de ARN , Adenosina Desaminasa/genética , Adolescente , Niño , Preescolar , China , Humanos , Isoenzimas/biosíntesis , Isoenzimas/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Proteínas de Unión al ARN , Inducción de Remisión

RESUMEN

Characterizing genes associated with leukemic cell differentiation may provide help for understanding mechanisms on the leukemia differentiation. The aim of this study is to investigate whether the expression of melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) could be induced during leukemia differentiation and whether mda-7/IL-24 plays a role in leukemia differentiation. We showed that the expression of mda-7/IL-24 and IL-24 delE5, an mda-7/IL-24 splice variant, was induced in U937 and HL60 cells during 12-O-tetradecanoylphorbol-13-acetate (TPA)-mediated monocytic differentiation. Activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway was required for their induction. Knockdown of mda-7/IL-24 and IL-24 delE5 resulted in significant inhibition of the monocytic differentiation induced by TPA. More importantly, ectopic overexpression of mda-7/IL-24 and IL-24 delE5 significantly induced U937 cells, HL60 cells, and blast cells from patients with acute myeloid leukemia-M5 to differentiate, whereas normal hematopoietic progenitors were not affected. Furthermore, the molecular effector associated with selective differentiation induction by mda-7/IL-24 and IL-24 delE5 may be reactive oxygen species (ROS), and the source of ROS generation was nicotinamide adenine dinucleotide phosphate oxidase. Taken together, our results reveal the mechanism by which TPA induces monocytic differentiation and show for the first time the specific differentiation-inducing effects of mda-7/IL-24 and IL-24 delE5 on human myeloid leukemic cells.

Asunto(s)

Diferenciación Celular/genética , Interleucinas/genética , Leucemia Mieloide/genética , Enfermedad Aguda , Empalme Alternativo , Animales , Western Blotting , Activación Enzimática/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HL-60 , Humanos , Interleucinas/metabolismo , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patología , Ratones , Ratones Desnudos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Neoplasias Experimentales/genética , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferencia de ARN , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Acetato de Tetradecanoilforbol/farmacología , Trasplante Heterólogo , Carga Tumoral , Células U937

RESUMEN

Nucleotides are new players in the intercellular communication network. P2X7 is a member of the P2X family of receptors, which are ATP-gated plasma membrane ion channels with diverse biological functions. Abnormal expression and dysfunction of P2X7 have been reported in leukemias. Here, we report a new P2X7 mutant (an A(559)-to-G substitution causing N187D P2X7) cloned from J6-1 leukemia cells. The characteristics of N187D P2X7 were studied by establishing stably transfected K562 cell lines. Our results show that N187D P2X7 required a higher concentration of agonist for its activation, leading to Ca(2+) influx (EC(50) = 293.3 ± 6.6 µm for the mutant and 93.6 ± 2.2 µm for wild-type P2X7) and ERK phosphorylation, which were not caused by differential cell-surface expression or related to high ATPase activity on the cell surface and in the extracellular space. K562 cells expressing this N187D mutant showed a proliferative advantage and reduced pro-apoptosis effects in vitro and in vivo. Furthermore, elevated angiogenesis and CD206-positive macrophage infiltration were found in tumor tissues formed by K562-M cells. In addition, higher expression of VEGF and MCP1 could be detected in tumor tissues formed by K562-M cells. Our results suggest that N187D P2X7, representing mutants hyposensitive to agonist, might be a positive regulator in the progression of hematopoietic malignancies.

Asunto(s)

Regulación Leucémica de la Expresión Génica , Leucemia Experimental/genética , Mutación Missense , Receptores Purinérgicos P2X7/genética , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Animales , Western Blotting , Calcio/metabolismo , Línea Celular Tumoral , Progresión de la Enfermedad , Activación Enzimática/efectos de los fármacos , Femenino , Humanos , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Células K562 , Leucemia Experimental/metabolismo , Leucemia Experimental/patología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Receptores Purinérgicos P2X7/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trasplante Heterólogo , Carga Tumoral/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo

RESUMEN

Nucleotides are new players in intercellular communication network. P2X family receptors are ATP-gated plasma membrane ion channels with diverse biological functions. Their distribution patterns and significance in pediatric leukemias have not been established. Here we investigated the expression of P2X receptors in BMMC samples from Chinese pediatric acute leukemias. Real-time PCR and Western blot results showed that P2X1, P2X4, P2X5 and P2X7 receptors were simultaneously over expressed in leukemias compared with controls, whereas P2X2, P2X3 and P2X6 were absent or marginally expressed in both groups. It was worth noting that the co-expression feature of them, especially between P2X4 and P2X7, could be observed and the highest expression of P2X7 was detected in relapsed patients. Moreover, concomitant decrease of P2X4, P2X5 and P2X7 expressions was observed at CR stage in a follow-up study. Functional P2X7 was also verified. These results suggested that P2X1, P2X4, P2X5 and P2X7 were hematopoiesis-related P2X receptors, and their signaling, especially for P2X7, might play important roles in pediatric leukemias. P2X receptors might co-operatively contribute to the malignant phenotype in human pediatric leukemias.

Asunto(s)

Biomarcadores de Tumor/metabolismo , Leucemia/metabolismo , Receptores Purinérgicos P2/biosíntesis , Adolescente , Pueblo Asiatico , Niño , Preescolar , Humanos , Receptores Purinérgicos P2X

RESUMEN

The membrane form of macrophage colony-stimulating factor (mM-CSF) is an alternative splicing variant of this cytokine. Although its high expression was detected in hematopoietic malignancies, its physiologic and pathologic roles in hematopoietic system have not been established. In this report, stable transfectant clones expressing mM-CSF (Namalwa-M and Ramos-M) were obtained, which showed reduced proliferation potential in vitro. Moreover, the in vivo study showed that Namalwa-M and Ramos-M exhibited enhanced oncogenicity in tumor size in nude mice model, which could be inhibited by M-CSF monoclonal antibody. A remarkable increase in infiltrating macrophage and the vessel densities was found in tumor tissues formed by lymphoma cell lines that stably expressed mM-CSF, which suggested the involvement of macrophages in this process. The in vitro results using coculture system showed that macrophages could promote Namalwa-M and Ramos-M proliferation and activate extracellular signal-regulated kinase/mitogen-activated protein kinase signal pathway. In addition, the expression of murine origin vascular endothelial growth factor, basic fibroblast growth factor, and hepatocyte growth factor was elevated in Namalwa-M formed tumor tissues. These results suggested that mM-CSF should be a positive regulator in the development of hematopoietic malignancies by abnormally activating infiltrating macrophages, which in turn promote the malignant development. Thus, mM-CSF may be a critical linker between macrophages and malignant cells in the development of hematopoietic malignancies.

Asunto(s)

Membrana Celular/metabolismo , Sistema Hematopoyético/citología , Sistema Hematopoyético/metabolismo , Factor Estimulante de Colonias de Macrófagos/metabolismo , Macrófagos/metabolismo , Animales , Línea Celular Tumoral , Femenino , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/metabolismo

RESUMEN

OBJECTIVE: Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) has been consistently shown to exert growth inhibitory effects on various tumor types. However, the majority of these reports were limited to solid tumors. The purpose of this study was to investigate the antitumor activity of mda-7/IL-24 and the underlying mechanism in hematopoietic malignancies. MATERIALS AND METHODS: We determined the expression of mda-7/IL24 and its heterodimeric receptors in hematopoietic tumor cell lines and then stably transfected mda-7/IL-24 into K562 (leukemia) and Namalwa (lymphoma) cell lines to assess the effects of mda-7/IL-24 on cell proliferation, cell cycle, apoptosis, colony-forming ability, and tumor growth in vivo. Microarray analysis was performed to determine the genes that were differentially regulated by mda-7/IL-24 in K562 cells. RESULTS: Expression of mda-7/IL-24 or its intact receptor pairs was not detected in the 11 cell lines tested. Ectopic expression of mda-7/IL-24 induced significant (p < 0.05) inhibition of cell growth and colony formation in both K562 and Namalwa cells, and the growth inhibition in K562 cells was associated with G(0)/G(1) cell-cycle arrest. Results of in vivo studies showed good correlation with in vitro inhibition of tumor cell proliferation in both the cell lines. We also showed that the increase in p21(WAF-1) and BCCIP and decrease in cdk6, smurf2, and phosphorylated pRb, which are regulators of cell-cycle progression, might account for G(0)/G(1) cell-cycle arrest in K562 cells. CONCLUSIONS: The present study demonstrated for the first time the potential antitumor activity of mda-7/IL-24 in chronic myelogenous leukemia and lymphoma.

Asunto(s)

Neoplasias Hematológicas/terapia , Interleucinas/genética , Animales , Western Blotting , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Terapia Genética/métodos , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/metabolismo , Humanos , Interleucinas/biosíntesis , Ratones , Ratones Desnudos , Plásmidos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ensayo de Tumor de Célula Madre , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Murine beta-defensin 2 (MBD2) is a small antimicrobial peptide of the innate immune system. Recent study showed that MBD2 could not only recruit immature dendritic cells but also activate them by Toll-like receptor 4 and thus may provide a critical link between the innate immune system and the adaptive immune response. In this report, we examined the antileukemia activity of MBD2 in a murine model of acute lymphoid leukemia (ALL) L1210. L1210 cells were engineered to secrete biologically functional MBD2. MBD2-modified L1210 (L1210-MBD2) showed significantly reduced leukemogenecity, resulting in a 80% rate of complete leukemia rejection. Inoculation of mice with L1210-MBD2 induced enhanced CTL and natural killer (NK) activity and augmented interleukin-12 and IFN-gamma production. All the recovered mice from the inoculation showed a protective immunity to the following challenge with parental L1210 cells and generate leukemia-specific memory CTL. Vaccines with irradiated L1210-MBD2 cells could cure 50% leukemia-bearing mice. Depletion of CD8+ T cells but not CD4+ T cells completely abrogated the antileukemia activity of MBD2. Interestingly, NK cells were also required for the MBD2-mediated antileukemia response, although ALL generally display a high degree of resistance to NK-mediated lysis. Our results suggest that MBD2 can activate both innate and adaptive immunity to generate potent antileukemia response, and MBD2 immunotherapy warrants further evaluation as a potential treatment for ALL.

Asunto(s)

Vacunas contra el Cáncer/inmunología , Leucemia Linfoide/inmunología , Leucemia Linfoide/terapia , beta-Defensinas/inmunología , Enfermedad Aguda , Animales , Formación de Anticuerpos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Células Dendríticas , Femenino , Inmunidad Innata , Memoria Inmunológica , Células Asesinas Naturales , Ratones , Transducción Genética

RESUMEN

The cDNA encoding N-terminal three immunoglobin-like domains of human M-CSFR was linked to His-tag and endoplasmic reticulum retention sequence (KDEL) before being inserted into the genome of tobacco plant, Nicotiana tabacum cv. NC-89, by Agrobacterium tumefaciens-mediated transformation. The insertion and expression of target gene were confirmed by PCR, ELISA, and Western blot. The recombinant M-CSFsR reached a maximum expression level of 1.92% of total soluble protein in transgenic tobacco plant leaf tissues. The recombinant M-CSFsR could be purified through a one-step IMAC process and its bioactivity was confirmed by the inhibition of colony formation of J6-1 cells. The results suggested that we successfully expressed a high level of bioactive human M-CSFsR in tobacco plants.

Asunto(s)

Expresión Génica , Nicotiana/genética , Plantas Modificadas Genéticamente/genética , Receptor de Factor Estimulante de Colonias de Macrófagos/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Línea Celular , Humanos , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Estructura Terciaria de Proteína/genética , Receptor de Factor Estimulante de Colonias de Macrófagos/genética , Receptor de Factor Estimulante de Colonias de Macrófagos/aislamiento & purificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/farmacología , Nicotiana/metabolismo

RESUMEN

OBJECTIVE: To clone the entire coding sequence and analyze the function of P2X7 receptor of J6-1 human leukemia cells. METHODS: The entire coding sequence of P2X7 receptor was amplified by RT-PCR and then inserted into pTARGET plasmid to construct an eukaryotic expressing plasmid followed by DNA sequencing. HEK293 cells stably expressing P2X7 receptor were obtained after transfection and screening, and confirmed by RT-PCR and Western blotting. The bleb formation upon agonist stimulation was observed under phase contrast microscope. RESULTS: The entire coding sequence of P2X7 receptor of J6-1 cells was successfully cloned. DNA sequencing analysis revealed a substitution of G559, for A559, causing a substitution of Glu187 for Gln187. The P2X7 receptor derived from J6-1 cells could be functionally expressed in HEK293 cells, in which bleb formation could be detected upon stimulation. CONCLUSIONS: The entire coding sequence of P2X7 receptors was successfully cloned from J6-1 leukemia cells. Other unknown mechanism may contribute to the dysfunction of P2X7 receptor in these cells.

Asunto(s)

Clonación Molecular , Leucemia/genética , Receptores Purinérgicos P2/fisiología , Línea Celular Tumoral , ADN Complementario/genética , Expresión Génica , Humanos , Leucemia/metabolismo , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X7 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección

RESUMEN

The P 2 X 7 nucleotide receptor is an adenosine 5'-triphosphate (ATP)-gated ion channel, which induces cation channel opening imparting significant permeability to Ca(2+), and is widely expressed in cells of hematopoietic origin. Our previous report showed that P 2 X 7-mediated calcium response was absent in three Epstein-Barr virus (EBV)-positive and P 2 X 7 positive cell lines. In this report, we detected the cell surface ATPase activity, which contributes to the hydrolysis of extracellular ATP, and the expression of CD 39, which is the main source of ATPase on hematopoietic cells, in these cell lines. Then, we tried to restore the P 2 X 7-mediated calcium response in LCL-H and J 6-1 cells by either increasing the concentration of agonist or suppressing the ATPase activity by betagammaMeATP, a synthetic poorly metabolizable ATP analogue. The results showed that LCL-H and J 6-1 cells had higher levels of ATPase activity and CD 39 expression. The treatment of 300 microM betagammaMeATP efficiently inhibited the ATPase activity on LCL-H and J 6-1 cells. Both elevation of agonist concentration (10mM ATP or 1mM BzATP) and pretreatment with 300 microM betagammaMeATP followed by stimulation with normal concentration of agonists (1mM ATP or 0.1mM BzATP) could cause P 2 X 7-mediated calcium response in LCL-H but neither in J 6-1 cells. These results suggested that multiple mechanisms contributed to the loss of the P 2 X 7-mediated calcium response. CD 39-associated high ATPase activity contributed to the loss of the P 2 X 7-mediated calcium response in LCL-H cells, while additional mechanism(s) existed in J 6-1 cells.

Asunto(s)

Adenosina Trifosfatasas/metabolismo , Antígenos CD/metabolismo , Apirasa/metabolismo , Calcio/metabolismo , Células Madre Hematopoyéticas/metabolismo , Receptores Purinérgicos P2/metabolismo , Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Antígenos CD/biosíntesis , Apirasa/biosíntesis , Línea Celular Tumoral , Células HL-60 , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Receptores Purinérgicos P2X7 , Células U937

RESUMEN

Regulation of P2X7 receptor expression is of interest because activation of this receptor by extracellular ATP triggers a wide variety of cell functions in leukocytes. However, its expression and modulation in human peripheral blood mononuclear cells (PBMC) and monocytes remain unclear. RT-PCR was used to detect the constitutive level of P2X7 receptor and the levels upon stimulation with bacteria, bacterial product, mitogen and various cytokines in human PBMC and monocytes. P2X7 receptor mRNA was detected in PBMC and monocytes. P2X7 receptor expression in PBMC was up-regulated by interleukin-2, -4, -6 (IL-2, IL-4, IL-6) tumour necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS) and heat-inactivated Staphylococcus aureus Cowan strain I (SAC). However, interferon-gamma (IFN-gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF) and phytohemagglutinin-M (PHA-M) had little effect on the expression of P2X7 receptor. Furthermore, LPS and M-CSF could up-regulate P2X7 receptor expression in monocytes, while IFN-gamma, TNF-alpha and GM-CSF had weak effects, but pretreatment with these inducers could not further enhance LPS-stimulated P2X7 receptor expression in monocytes. The results obtained demonstrate that inflammatory stimuli drive P2X7 expression, thus supporting the hypothesis that P2X7 receptor may play a role in the inflammatory responses against bacteria infection, which need further verification.

Asunto(s)

Interleucina-2/fisiología , Leucocitos Mononucleares/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Factor de Necrosis Tumoral alfa/fisiología , Humanos , Interleucina-4/fisiología , Leucocitos Mononucleares/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Purinérgicos P2X7/genética

RESUMEN

DNA vaccine against M-CSFR(J6-1) (macrophage colony-stimulating factor receptor cloned from the J6-1 leukemic cell line) has shown both protective and therapeutic effects. In this study, to explore the adjuvant effects of LL-37 to M-CSFR(J6-1) DNA vaccines, we constructed genetically fused vaccines encoding M-CSFR(J6-1) and LL-37(pF). After immunizing BALB/c mice, specific humoral and cellular immune responses were detected. Compared with pR (encoding the extracellular region of M-CSFR(J6-1)), pF was more effective in inducing humoral and cytotoxic immune response, prolonging survival of mice challenged with SP2/0-CSFR(J6-1) tumor cells, and inducing IFN-gamma and IL-4 release by splenocytes. In this study, we also constructed pLL37 (encoding the mature LL-37) and coadministrated pLL37 and pR to see whether the genetic fusion was necessary. We found that compared with pR alone, pLL37+pR could not prolong survival of mice challenged with SP2/0-CSFR(J6-1) tumor cells. Our results suggest that when genetically fused with M-CSFR(J6-1), LL-37 could enhance adaptive immune response against M-CSFR(J6-1) in a murine model challenged with tumor cells bearing M-CSFR(J6-1).

Asunto(s)

Péptidos Catiónicos Antimicrobianos/genética , Sistema Inmunológico/fisiología , Leucemia/terapia , Receptor de Factor Estimulante de Colonias de Macrófagos/genética , Proteínas Recombinantes de Fusión/genética , Vacunas de ADN/uso terapéutico , Adaptación Fisiológica , Adyuvantes Inmunológicos , Animales , Células COS , Quimiotaxis , Chlorocebus aethiops , Citocinas/metabolismo , Femenino , Inmunización , Leucemia/inmunología , Leucemia/metabolismo , Lipopolisacáridos , Ratones , Ratones Endogámicos BALB C , Plásmidos , Tasa de Supervivencia , Linfocitos T Citotóxicos/inmunología , Catelicidinas

RESUMEN

We detected LL-37/hCAP-18 expression in the peripheral blood smears of 50 healthy donors and 143 patients with various hematological diseases. Compared with that in the healthy donors, expression of the protein in the neutrophils was significantly lower in patients with acute myeloid leukemia (AML), especially those with infection, but no significant difference was detected in messenger RNA level. We did not detect increased LL-37/hCAP-18 protein expression in U937 cells treated with lipopolysaccharide or Staphylococcus aureus Cowan strain. Furthermore, LL-37/hCAP-18 protein production was not restored in differentiated myeloid cell lines NB4 or HL-60 induced by all-trans retinoic acid. LL-37/hCAP-18 has been shown to play a role in host defense, and its deficiency in AML may be one of the explanations for susceptibility to infection among these patients.

Asunto(s)

Péptidos Catiónicos Antimicrobianos/metabolismo , Leucemia Mieloide/metabolismo , Neutrófilos/metabolismo , Enfermedad Aguda , Adolescente , Adulto , Anciano , Niño , Preescolar , Femenino , Células HL-60 , Humanos , Masculino , Persona de Mediana Edad , Células U937 , Catelicidinas

RESUMEN

IL-23 is a novel cytokine composed of an IL-12 p40 and a p19 subunit. We analyzed human peripheral blood mononuclear cells (PBMC) and hematopoietic cell lines for constitutive expression of IL-23 and IL-12 subunits and expression following exposure to various stimuli, and investigated the mechanisms of their induction by LPS and SAC. IL-23 p19 and IL-12 p35 mRNAs were expressed in fresh PBMC, and expression of all IL-23 and IL-12 subunits was up-regulated by LPS and SAC. LPS-induced increase of IL-23 and IL-12 subunits expression was CD14-dependent, while CD14 was not involved in SAC-induced p19 transcription. Both LPS- and SAC-induced subunits expression required p38 MAPK pathway. PHA effected an increase of p19 mRNA in both CD4+ and CD8+ T cells, whereas p35 was minimally regulated by PHA. IFN-gamma primed monocytes for LPS stimulation of p19, p35 and p40 expression. p19 mRNA was detectable in most hematopoietic cell lines tested but p35 distribution was more restricted. A phorbol diester enhanced p19, p35 and p40 expression in EBV-positive cell lines. Our results suggest that both the subunits of IL-23 are tightly regulated; the expression pattern and regulation mode of IL-23 p19 is similar to as well as distinct from that of IL-12 p35.

Asunto(s)

Células Madre Hematopoyéticas/metabolismo , Interleucina-12/biosíntesis , Interleucinas/biosíntesis , Leucocitos Mononucleares/metabolismo , Subunidades de Proteína/biosíntesis , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Interleucina-12/genética , Subunidad p40 de la Interleucina-12 , Interleucina-23 , Subunidad p19 de la Interleucina-23 , Interleucinas/genética , Interleucinas/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Lipopolisacáridos/farmacología , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Staphylococcus aureus/fisiología