RESUMEN
Aplastic anemia (AA) is a hematologic disease characterized by pancytopenia and hypocellular bone marrow, potentially leading to chronic anemia, hemorrhage, and infection. The China Aplastic Anemia Committee and British Committee for Standards in Haematology guidelines recommend hematopoietic stem-cell transplantation (HSCT) or immunosuppressive therapy (IST) comprising antithymocyte globulin (ATG) with cyclosporine (CsA) as initial treatment for AA patients. With limited epidemiological data on the clinical management of AA in Asia, a prospective cohort registry study involving 22 AA treatment centers in China was conducted to describe the disease characteristics of newly diagnosed AA patients and investigate real-world treatment patterns and patient outcomes. Of 340 AA patients, 72.9, 12.6, and 3.5% were receiving IST, traditional Chinese medicine, and HSCT, respectively, at baseline; only 22.2% of IST-treated patients received guideline-recommended ATG with CsA initially. Almost all patients received supportive care (95.6%) as blood transfusion (97.8%), antibiotics (63.7%), and/or hematopoietic growth factors (58.2%). Overall, 64.8% achieved a partial or complete response, and 0.9% experienced relapse. No new safety concerns were identified; serious adverse events were largely unrelated to the treatment regimen. These results demonstrate the need to identify and minimize treatment barriers to standardize and align AA management in China with treatment guideline recommendations and further improve patient outcomes.
Asunto(s)
Anemia Aplásica , Suero Antilinfocítico/administración & dosificación , Ciclosporina/administración & dosificación , Trasplante de Células Madre Hematopoyéticas , Terapia de Inmunosupresión , Medicina Tradicional China , Sistema de Registros , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Aloinjertos , Anemia Aplásica/mortalidad , Anemia Aplásica/terapia , Niño , Preescolar , China/epidemiología , Supervivencia sin Enfermedad , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Tasa de SupervivenciaRESUMEN
ß-hemoglobinopathies are one of six groups of common illnesses affecting human health. Although the genetic mechanisms have been elucidated for several decades, curable treatment options, other than allogeneic bone marrow transplantation, are still lacking. In recent years, rapid development in genome editing technologies and their clinical applications have opened up new directions for treatment of ß-hemoglobinopathies. Genome editing technologies, as applied in autologous CD34 + hematopoietic stem and progenitor cells, represents a promising remedial means for the ß-globin disorders. Hemoglobin gene mutations could be corrected with homologous recombination-mediated DNA repair pathway to repair the genetic defects, while the nonhomologous end-joining pathway may be used to silence the key repressor of fetal globin expression and reactivate fetal hemoglobin expression, thereby alleviating the clinical symptoms of ß-hemoglobinopathies in patients. This review summarizes the recent advances on genome editing of ß-hemoglobinopathies from the bench design to the establishment of clinical translational platforms, thereby providing critical insights and references on the application of genome editing technologies for the development of therapeutic strategies for ß-hemoglobinopathies.
Asunto(s)
Edición Génica/métodos , Terapia Genética/métodos , Hemoglobinopatías/genética , Hemoglobinopatías/terapia , Sistemas CRISPR-Cas , Hemoglobina Fetal/genética , Humanos , Mutación , Investigación Biomédica Traslacional/métodos , Globinas beta/genéticaRESUMEN
This study compared 6-year follow-up data from patients undergoing reduced-intensity conditioning (RIC) transplantation with an HLA-matched related donor (MRD), an HLA-matched unrelated donor (MUD), or an HLA-haploidentical donor (HID) for leukemia. Four hundred and twenty-seven patients from the China RIC Cooperative Group were enrolled, including 301 in the MRD, 79 in the HID, and 47 in the MUD groups. The conditioning regimen involved fludarabine combined with anti-lymphocyte globulin and cyclophosphamide. Graft-versus-host disease (GVHD) prophylaxis was administered using cyclosporin A (CsA) and mycophenolate mofetil (MMF). Four hundred and nineteen patients achieved stable donor chimerism. The incidence of stage II-IV acute GVHD in the HID group was 44.3 %, significantly higher than that in the MRD (23.6 %) and MUD (19.1 %) groups. The 1-year transplantation-related mortality (TRM) rates were 44.3, 17.6, and 21.3, respectively. Event-free survival (EFS) at 6 years in the HID group was 36.7 %, significantly lower than that of the MRD and MUD groups (59.1 and 66.0 %, P < 0.001 and P = 0.001, respectively). For advanced leukemia, the relapse rate of the HID group was 18.5 %, lower than that of the MRD group (37.5 %, P = 0.05), but the EFS at 6 years was 31.7 and 30.4 % (P > 0.05), respectively. RIC transplantation with MRD and MUD had similar outcome in leukemia which is better than that with HID. RIC transplantation with HID had lower relapsed with higher TRM and GVHD rate, particularly in advanced leukemias. RIC transplantation with MRD and MUD had similar outcomes in leukemia and they were better than those with HID. RIC transplantation with HID had a lower relapse rate but higher TRM and GVHD rates, particularly in cases of advanced leukemia.
Asunto(s)
Haplotipos/genética , Trasplante de Células Madre Hematopoyéticas/tendencias , Leucemia/mortalidad , Leucemia/terapia , Estadística como Asunto , Donante no Emparentado , Adolescente , Adulto , Anciano , Niño , China/epidemiología , Femenino , Trasplante de Células Madre Hematopoyéticas/mortalidad , Humanos , Leucemia/genética , Masculino , Persona de Mediana Edad , Mortalidad/tendencias , Estudios Retrospectivos , Estadística como Asunto/tendencias , Factores de Tiempo , Donantes de Tejidos , Trasplante Homólogo/mortalidad , Trasplante Homólogo/tendencias , Resultado del Tratamiento , Adulto JovenRESUMEN
Vascular endothelial dysfunction, a central hallmark of diabetes, predisposes diabetic patients to numerous cardiovascular complications. The POZ/BTB and AT-hook-containing zinc finger protein 1 (PATZ1), is an important transcriptional regulatory factor and regulates divergent pathways depending on the cellular context, but its role in endothelial cells remains poorly understood. Herein, we report for the first time that endothelial PATZ1 expression was abnormally upregulated in diabetic endothelial cells (ECs) regardless of diabetes classification. This stimulatory effect was further confirmed in the high glucose-treated human umbilical vein endothelial cells (HUVECs). From a functional standpoint, transgenic overexpression of PATZ1 in endothelial colony forming cells (ECFCs) blunted angiogenesis in vivo and rendered endothelial cells unresponsive to established angiogenic factors. Mechanistically, PATZ1 acted as a potent transcriptional corepressor of fatty acid-binding protein 4 (FABP4), an essential convergence point for angiogenic and metabolic signaling pathways in ECs. Taken together, endothelial PATZ1 thus potently inhibits endothelial function and angiogenesis via inhibition of FABP4 expression, and abnormal induction of endothelial PATZ1 may contribute to multiple aspects of vascular dysfunction in diabetes.
Asunto(s)
Proteínas de Unión a Ácidos Grasos/metabolismo , Hiperglucemia/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Represoras/metabolismo , Transducción de Señal , Animales , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Proteínas de Unión a Ácidos Grasos/genética , Glucosa/administración & dosificación , Células Endoteliales de la Vena Umbilical Humana , Humanos , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Umbilical cord blood (UCB) is becoming an alternative cell source for hematopoietic stem cell transplantation (HSCT). However, umbilical cord blood transplantation (UCBT) has been severely limited by low and finite numbers of hematopoietic stem cells and their delayed engraftment. New strategies are needed to improve ex vivo expansion efficiency and in vivo haematopoietic recovery. METHODS: We produced an endothelium-targeted soluble Notch ligand, the Delta-Serrate-Lag-2 (DSL) domain of human Delta-like 1 fused with a RGD motif (hD1R), and tested the effects of this protein on human umbilical cord blood hematopoietic stem and progenitor cell (UCB-HSPC) ex vivo and in vivo. RESULTS: hD1R-mediated ex vivo expansion system was able to significantly increase the absolute number of UCB-HSPCs. The hD1R-expanded cells had the enhanced homing and maintained long-term hematopoietic stem cell repopulation capacity in the bone marrow of immunodeficient nonobese diabetic-severe combined immunodeficient (NOD/SCID) mice. Moreover, systemic administration of hD1R promoted the in vivo regeneration of donor cells in recipient mice and accelerated hematopoietic recovery, particularly in settings wherein the HSPCs dose was limiting. CONCLUSIONS: Our results indicated that hD1R might be applied in improving hematopoietic recovery and HSC engraftment in human UCBT.
Asunto(s)
Endotelio/metabolismo , Sangre Fetal/citología , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de la Membrana/metabolismo , Regeneración , Secuencias de Aminoácidos , Animales , Antígenos CD34/metabolismo , Médula Ósea/metabolismo , Proteínas de Unión al Calcio , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Ratones Endogámicos NOD , Ratones SCID , Donantes de TejidosRESUMEN
BACKGROUND: Aberrantly activated Notch signaling has been found in more than 50% of patients with T-cell acute lymphoblastic leukemia (T-ALL). Current strategies that employ γ-secretase inhibitors (GSIs) to target Notch activation have not been successful. Many limitations, such as non-Notch specificity, dose-limiting gastrointestinal toxicity and GSI resistance, have prompted an urgent need for more effective Notch signaling inhibitors for T-ALL treatment. Human four-and-a-half LIM domain protein 1C (FHL1C) (KyoT2 in mice) has been demonstrated to suppress Notch activation in vitro, suggesting that FHL1C may be new candidate target in T-ALL therapy. However, the role of FHL1C in T-ALL cells remained unclear. METHODS: Using RT-PCR, we amplified full-length human FHL1C, and constructed full-length and various truncated forms of FHL1C. Using cell transfection, flow cytometry, transmission electron microscope, real-time RT-PCR, and Western blotting, we found that overexpression of FHL1C induced apoptosis of Jurkat cells. By using a reporter assay and Annexin-V staining, the minimal functional sequence of FHL1C inhibiting RBP-J-mediated Notch transactivation and inducing cell apoptosis was identified. Using real-time PCR and Western blotting, we explored the possible molecular mechanism of FHL1C-induced apoptosis. All data were statistically analyzed with the SPSS version 12.0 software. RESULTS: In Jurkat cells derived from a Notch1-associated T-ALL cell line insensitive to GSI treatment, we observed that overexpression of FHL1C, which is down-regulated in T-ALL patients, strongly induced apoptosis. Furthermore, we verified that FHL1C-induced apoptosis depended on the RBP-J-binding motif at the C-terminus of FHL1C. Using various truncated forms of FHL1C, we found that the RBP-J-binding motif of FHL1C had almost the same effect as full-length FHL1C on the induction of apoptosis, suggesting that the minimal functional sequence in the RBP-J-binding motif of FHL1C might be a new drug candidate for T-ALL treatment. We also explored the molecular mechanism of FHL1C overexpression-induced apoptosis, which suppressed downstream target genes such as Hes1 and c-Myc and key signaling pathways such as PI3K/AKT and NF-κB of Notch signaling involved in T-ALL progression. CONCLUSIONS: Our study has revealed that FHL1C overexpression induces Jurkat cell apoptosis. This finding may provide new insights in designing new Notch inhibitors based on FHL1C to treat T-ALL.
Asunto(s)
Apoptosis/genética , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas con Dominio LIM/genética , Proteínas Musculares/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Receptor Notch1/metabolismo , Secuencias de Aminoácidos , Estudios de Casos y Controles , Línea Celular Tumoral , Supervivencia Celular/genética , Progresión de la Enfermedad , Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas con Dominio LIM/química , Proteínas con Dominio LIM/metabolismo , Leucocitos Mononucleares/metabolismo , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transducción de SeñalRESUMEN
Dendritic cells (DCs) are professional antigen presenting cells that activate and modulate immune responses, but the mechanisms underlying DC activation have not been fully understood. In this study, we investigated the role of Notch signaling in DC activation by using murine bone marrow-derived DCs. Triggering of Toll-like receptors (TLRs) of DCs led to upregulated expression of Notch ligands. Disruption of Notch signaling by the deletion of RBP-J, the critical transcription factor mediating the canonical signaling from all Notch receptors, resulted in a reduced capacity of DCs in activating T cells. Moreover, RBP-J deficiency altered the polarization of T cell activation, as manifested by downregulated interferon-γ and upregulated interleukin-4 and -10 expressions after LPS or Poly(I:C) stimulation. Furthermore, we found that RBP-J(-/-) DCs had reduced intracellular calcium after TLR-triggering. Immunofluorescent staining showed that RBP-J deficient DCs exhibited attenuated cytoskeleton reorganization when contacting T cells. In summary, our results suggested that the canonical Notch signaling promotes the cytoskeleton reorganization and the TLR-mediated DC activation.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Células Dendríticas/metabolismo , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/genética , Receptores Toll-Like/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Comunicación Celular , Células Cultivadas , Células Dendríticas/inmunología , Células Dendríticas/ultraestructura , Eliminación de Gen , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/deficiencia , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores Notch/metabolismo , Proteínas Serrate-Jagged , Transducción de Señal , Linfocitos T/fisiología , Regulación hacia ArribaRESUMEN
Signal regulatory protein (SIRP) α, a transmembrane protein belonging to the immunoglobulin superfamily, is a receptor for CD47. The interaction between SIRPα and CD47 plays an important role in regulating the phagocytosis of leukemia cells and leukemia stem cells (LSCs) by macrophages. Blocking antibodies against CD47 have been shown to promote phagocytosis of LSCs by macrophages. Here, we consider an alternative way to interrupt the interaction between CD47 and SIRPα. We expressed the extracellular domains of the human SIRPα (hSIRP(ext)) and the human CD47 (hCD47(ext)) in Escherichia coli as Trx fusion proteins, and purified them by using affinity chromatography. We show that the purified fusion protein Trx-SIRP(ext) could interact in vitro with Trx-hCD47(ext). Moreover, Trx-SIRP(ext) could effectively bind to Jurkat T-ALL cells, which expressed CD47 at a high level. CD47(ext), on the other hand, bound to human macrophages. In vitro phagocytosis assay showed that these fusion proteins could enhance the phagocytosis of Jurkat cells by macrophage, with Trx-hSIRP(ext) showed a higher efficiency than Trx-CD47(ext). These results indicated that the soluble Trx-hSIRP(ext) and Trx-CD47(ext) polypeptides could be alternative molecules to interrupt CD47-SIRPα interaction between leukemia cells and macrophages, and might be potentially useful for the targeted therapy of leukemia.
Asunto(s)
Antígenos de Diferenciación/genética , Antígenos de Diferenciación/farmacología , Antígeno CD47/genética , Antígeno CD47/farmacología , Escherichia coli/genética , Macrófagos/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Receptores Inmunológicos/genética , Antígenos de Diferenciación/química , Antígenos de Diferenciación/inmunología , Antígeno CD47/química , Antígeno CD47/inmunología , Humanos , Células Jurkat , Leucemia de Células T/tratamiento farmacológico , Leucemia de Células T/inmunología , Macrófagos/inmunología , Replegamiento Proteico , Estructura Terciaria de Proteína , Receptores Inmunológicos/química , Receptores Inmunológicos/inmunología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
Macrophage activation is modulated by both environmental cues and endogenous programs. In the present study, we investigated the role of a PAQR family protein, monocyte to macrophage differentiation-associated (MMD), in macrophage activation and unveiled its underlying molecular mechanism. Our results showed that while MMD expression could be detected in all tissues examined, its expression level is significantly up-regulated upon monocyte differentiation. Within cells, EGFP-MMD fusion protein could be co-localized to endoplasmic reticulum, mitochondria, Golgi apparatus, but not lysosomes and cytoplasm. MMD expression is up-regulated in macrophages after LPS stimulation, and this might be modulated by RBP-J, the critical transcription factor of Notch signaling. Overexpression of MMD in macrophages increased the production of TNF-α and NO upon LPS stimulation. We found that MMD overexpression enhanced ERK1/2 and Akt phosphorylation in macrophages after LPS stimulation. Blocking Erk or Akt by pharmacological agent reduced TNF-α or NO production in MMD-overexpressing macrophages, respectively. These results suggested that MMD modulates TNF-α and NO production in macrophages, and this process might involves Erk or Akt.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Linfocinas/metabolismo , Macrófagos/enzimología , Proteínas de la Membrana/metabolismo , Óxido Nítrico/biosíntesis , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesis , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular , Activación Enzimática/efectos de los fármacos , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Lipopolisacáridos/farmacología , Linfocinas/genética , Macrófagos/citología , Macrófagos/efectos de los fármacos , Ratones , Fosforilación/efectos de los fármacos , Receptores Notch/metabolismo , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
Cancer metastasis is the leading cause of cancer-related deaths all over the world at present. Accumulated researches have demonstrated that cancer metastasis is composed of a series of successive incidents, mainly including epithelial-mesenchymal transition (EMT), malignant cell migration, resistance to anoikis, and angiogenesis and lymphangiogenesis processes. However, the complicated cellular and molecular mechanisms underlying and modulating these processes have not been well elucidated. Thus, studies on cancer metastasis mechanism may propose possibilities to therapeutically interfere with signaling pathways required for each step of cancer metastasis, therefore inhibiting the outgrowth of distant metastasis of tumors. Recent insights have linked the Notch signaling pathway, a critical pathways governing embryonic development and maintaining tumor stemness, to cancer metastasis. This chapter highlights the current evidence for aberration of the Notch signaling in metastasis of tumors such as osteosarcoma, breast cancer, prostate cancer, and melanoma. In these studies, Notch activity seems to participate in cancer metastasis by modulating the EMT, tumor angiogenesis processes, and the anoikis-resistance of tumor cells. Therefore, manipulating Notch signaling may represent a promising alternative/ complement therapeutic strategy targeting cancer metastasis besides cancer stemness.
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Neoplasias/metabolismo , Neoplasias/patología , Receptores Notch/metabolismo , Transducción de Señal , Animales , Humanos , Metástasis de la NeoplasiaRESUMEN
UNLABELLED: Liver sinusoid (LS) endothelial cells (LSECs) support hepatocytes in resting livers and proliferate during liver regeneration to revascularize regenerated liver parenchyma. We report that recombination signal-binding protein-Jkappa (RBP-J), the critical transcription factor mediating Notch signaling, regulates both resting and regenerating LSECs. Conditional deletion of RBP-J resulted in LSEC proliferation and a veno-occlusive disease-like phenotype in the liver, as manifested by liver congestion, deposition of fibrin-like materials in LSs, edema in the space of Disse, and increased apoptosis of hepatocytes. Regeneration of liver was remarkably impaired, with reduced LSEC proliferation and destroyed sinusoidal structure. LSEC degeneration was obvious in the regenerating liver of RBP-J-deficient mice, with some LSECs losing cytoplasm, and organelles protruding into the remnant plasma-membrane of LSs to hamper the microcirculation and intensify veno-occlusive disease during liver regeneration. Hepatocytes were also degenerative, as shown by dilated endoplasmic reticulum, decreased proliferation, and increased apoptosis during liver regeneration. Molecular analyses revealed that the dynamic expression of several related molecules-such as vascular endothelial growth factor, vascular endothelial growth factor receptors 1 and 2, interleukin-6, and hepatocyte growth factor-was disturbed. CONCLUSION: Notch/RBP-J signaling may play dual roles in LSECs: in resting liver it represses proliferation, and in regenerating liver it supports proliferation and functional differentiation.
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Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/fisiología , Enfermedad Veno-Oclusiva Hepática/etiología , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/fisiología , Regeneración Hepática/fisiología , Animales , Apoptosis , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/efectos de los fármacos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/biosíntesis , Proliferación Celular , Células Endoteliales/patología , Células Endoteliales/fisiología , Femenino , Eliminación de Gen , Hepatectomía , Proteínas de Homeodominio/biosíntesis , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/efectos de los fármacos , Ratones , Poli I-C/farmacología , Receptores Notch/fisiología , Factor de Transcripción HES-1 , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/biosíntesis , Receptor 2 de Factores de Crecimiento Endotelial Vascular/biosíntesisRESUMEN
The Notch signaling pathway has been implicated in the development of several leukemia and lymphoma. In order to investigate the relationship between Notch signaling and acute myeloid leukemia (AML), in this study, we expressed a recombinant Notch ligand protein, the DSL domain of the human Jagged1 fused with GST (GST-Jag1). GST-Jag1 could activate Notch signaling in the human promyelocytic leukemia cell line HL60, as shown by a reporter assay and the induced expression of Notch effector gene Hes1 and Hes5. However, GST-Jag1 had no effect on the proliferation and survival of HL60 cells. HL60 cells expressed both Notch ligands and receptors, and had a potential of reciprocal stimulation of Notch signaling between cells. We, therefore, blocked Notch signaling in cultured HL60 cells using a gamma-secretase inhibitor (GSI). We found that GSI inhibited the proliferation of HL60 cells significantly by blocking the cell-cycle progression in the G1 phase. Furthermore, GSI induced remarkably apoptosis of HL60 cells. These changes in GSI-treated HL60 cells correlated with the down-regulation of c-Myc and Bcl2, and the low phosphorylation of the Rb protein. These results suggested that reciprocal Notch signaling might be necessary for the proliferation and survival of AML cells, possibly through the maintenance of the expression of c-Myc and Bcl2, as well as the phosphorylation of the Rb protein.
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Proliferación Celular , Leucemia Promielocítica Aguda/metabolismo , Receptores Notch/metabolismo , Proteína de Retinoblastoma/metabolismo , Transducción de Señal , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Apoptosis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Unión al Calcio/metabolismo , Ciclo Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Células HL-60 , Células HeLa , Proteínas de Homeodominio/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteína Jagged-1 , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/patología , Proteínas de la Membrana/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/metabolismo , Proteínas Serrate-Jagged , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Factor de Transcripción HES-1 , TransfecciónRESUMEN
PURPOSE: The role of antithymocyte globulin (ATG) in preventing acute graft-versus-host disease (aGVHD) after HLA-matched sibling donor transplantation (MSDT) is still controversial. PATIENTS AND METHODS: We performed a prospective, multicenter, open-label, randomized controlled trial (RCT) across 23 transplantation centers in China. Patients ages 40-60 years with standard-risk hematologic malignancies with an HLA-matched sibling donor were randomly assigned to an ATG group (4.5 mg/kg thymoglobulin plus cyclosporine [CsA], methotrexate [MTX], and mycophenolate mofetil [MMF]) and a control group (CsA, MTX, and MMF). The primary end point of this study was grade 2-4 aGVHD on day 100. RESULTS: From November 2013 to April 2018, 263 patients were enrolled. The cumulative incidence rate of grade 2-4 aGVHD was significantly reduced in the ATG group (13.7%; 95% CI, 13.5% to 13.9%) compared with the control group (27.0%; 95% CI, 26.7% to 27.3%; P = .007). The ATG group had significantly lower incidences of 2-year overall chronic GVHD (27.9% [95% CI, 27.6% to 28.2%] v 52.5% [95% CI, 52.1% to 52.9%]; P < .001) and 2-year extensive chronic GVHD (8.5% [95% CI, 8.4% to 8.6%] v 23.2% [95% CI, 22.9% to 23.5%]; P = .029) than the control group. There were no differences between the ATG and control groups with regard to cytomegalovirus reactivation, Epstein-Barr virus reactivation, 3-year nonrelapse mortality (NRM), 3-year cumulative incidence of relapse (CIR), 3-year overall survival, or 3-year leukemia-free survival. Three-year GVHD relapse-free survival was significantly improved in the ATG group (38.7%; 95% CI, 29.9% to 47.5%) compared with the control group (24.5%; 95% CI, 16.9% to 32.1%; P = .003). CONCLUSION: Our study is the first prospective RCT in our knowledge to demonstrate that ATG can effectively decrease the incidence of aGVHD after MSDT in the CsA era without affecting the CIR or NRM.
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Suero Antilinfocítico/administración & dosificación , Neoplasias Hematológicas/terapia , Trasplante de Células Madre Hematopoyéticas/métodos , Acondicionamiento Pretrasplante/métodos , Adulto , Ciclosporina/administración & dosificación , Femenino , Enfermedad Injerto contra Huésped/etiología , Enfermedad Injerto contra Huésped/prevención & control , Antígenos HLA/inmunología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Humanos , Inmunosupresores/administración & dosificación , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Hermanos , Donantes de TejidosRESUMEN
Previously, we have shown that KyoT2, an isoform of the four and a half LIM domain protein 1 (FHL1), modulates Notch signaling via repressing RBP-J-mediated transactivation. In this study, we investigated the effect of another isoform of FHL1, KyoT3, on transactivation of a RBP-J-dependent promoter. We found that KyoT3 was expressed widely in a variety of tissues. By constructing EGFP fusion proteins, we showed that KyoT3 locates preferentially in nucleus. KyoT3 interacted with RBP-J, as shown by co-immunoprecipitation assays. Moreover, we demonstrated by a reporter assay that KyoT3 repressed transactivation of a RBP-J-dependent promoter, which was activated by both the Notch intracellular domain and Epstein-Barr virus nuclear antigen 2, an EB virus-encoded oncoprotein. These results suggest a multi-elemental control of the Notch signaling pathway, which is critical for cell differentiation in development.
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Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/fisiología , Regulación de la Expresión Génica , Proteínas Musculares/fisiología , Activación Transcripcional , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Diferenciación Celular , Núcleo Celular/metabolismo , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Genes Reporteros , Células HeLa , Humanos , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas , Péptidos y Proteínas de Señalización Intracelular , Proteínas con Dominio LIM , Ratones , Modelos Biológicos , Proteínas Musculares/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Virales/metabolismoRESUMEN
Notch signaling functions in the development of some types of leukemia and lymphoma, but the relationship between Notch signaling and chronic myeloid leukemia (CML) remains to be elucidated. In this study, we examined the expression of Notch receptors and ligands in the human CML cell line K562. When the active form of Notch1, the Notch intra-cellular domain (NIC), was over-expressed in K562, the proliferation of K562 was mildly but significantly inhibited, accompanied by increased Hes1 mRNA level. On the other hand, when Notch signaling was attenuated by over-expression of a dominant-negative RBP-J, RBP-J(R218H), in K562 cells, the proliferation of K562 was increased. Moreover, we found that activation of Notch signaling inhibited while repression of Notch signaling promoted the colony-forming activity of K562 cells. We examined cell cycle-related molecules in K562 transfected with NIC or RBP-J(R218H), and found that the protein level of the retinoblastoma gene product (the Rb protein) was induced in K562 expressing NIC, and down-regulated in K562 expressing RBP-J(R218H). These data suggest that the Notch signaling may function as a tumor inhibitor in human CML cells.
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División Celular , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Receptores Notch/metabolismo , Transducción de Señal , Western Blotting , Humanos , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Proteína de Retinoblastoma/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
In adults, angiogenic abnormalities are involved in not only tumor growth but several human inherited diseases as well. It is unclear, however, concerning how the normal vascular structure is maintained and how angiogenesis is initiated in normal adults. Using the Cre-LoxP-mediated conditional gene deletion, we show in the present study that in adult mice disruption of the transcription factor recombination signal-binding protein Jkappa (RBP-J) in endothelial cells strikingly induced spontaneous angiogenesis in multiple tissues, including retina and cornea, as well as in internal organs, such as liver and lung. In a choroidal neovascularization model, which mimics the angiogenic process in tumor growth and age-related macular degeneration, RBP-J deficiency induced a more intensive angiogenic response to injury. This could be transmitted by bone marrow, indicating that RBP-J could modulate bone marrow-derived endothelial progenitor cells in adult angiogenesis. In addition, in the absence of RBP-J, proliferation of endothelial cells increased significantly, leading to accumulative vessel outgrowth. These findings suggest that in adults RBP-J-mediated Notch signaling may play an essential role in the maintenance of vascular homeostasis by repressing endothelial cell proliferation.
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Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/fisiología , Neovascularización Fisiológica/fisiología , Receptor Notch1/fisiología , Animales , Proliferación Celular , Coroides/irrigación sanguínea , Homeostasis/efectos de los fármacos , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas , Mesodermo/irrigación sanguínea , Ratones , Ratones Noqueados , Transducción de Señal , Regulación hacia Arriba , Receptor 2 de Factores de Crecimiento Endotelial Vascular/biosíntesisRESUMEN
The Notch signaling pathway plays a pivotal role in proliferation, apoptosis, and cell fate specification in both embryonic and postnatal development, and is a potential therapeutic target for human diseases such as cancer. To express in Escherichia coli and purify soluble fragment of human Delta-like1 (hDll1), we cloned two extracellular fragments of hDll1 [hDll1 (127-225) and hDll1 (26-225)]. The hDll1 (127-225) fragment was successfully expressed in E. coli as a GST fusion protein (GST-hDll1). The GST-hDll1 protein, which was expressed as inclusion bodies after induction by IPTG, was refolded and purified simultaneously using affinity chromatography and size exclusion chromatography. The purified GST-hDll1 was of more than 95% purity, and had a molecular weight of 39kDa. Reporter assay showed that GST-hDll1 could activate a reporter gene that is dependent on Notch activation. Therefore, using the E. coli expression system and different chromatography systems, we successfully expressed, refolded, and purified a biologically active GST-hDll1, which might be potentially useful for therapy and studying the Notch pathway.
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Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Péptidos y Proteínas de Señalización Intercelular/aislamiento & purificación , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/aislamiento & purificación , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas de Unión al Calcio , Cromatografía Líquida de Alta Presión , Escherichia coli/genética , Genes Reporteros , Glutatión Transferasa/biosíntesis , Glutatión Transferasa/química , Glutatión Transferasa/aislamiento & purificación , Humanos , Péptidos y Proteínas de Señalización Intercelular/química , Ligandos , Espectrometría de Masas , Proteínas de la Membrana/química , Pliegue de Proteína , Receptores Notch/metabolismo , Proteínas Recombinantes de Fusión/químicaRESUMEN
The Notch pathway plays critical roles in the development and functional modulation of myeloid cells. Previous studies have demonstrated that Notch activation promotes M1 polarization and phagocytosis of macrophages; however, the downstream molecular mechanisms mediating Notch signal remain elusive. In an attempt to identify Notch downstream targets in bone marrow-derived macrophages (BMDMs) using mass spectrometry, the signal regulatory protein α (SIRPα) appeared to respond to knockout of recombination signal-binding protein Jk (RBP-J), the critical transcription factor of Notch pathway, in macrophages. In this study, we validated that Notch activation could repress SIRPα expression likely via the Hes family co-repressors. SIRPα promoted macrophage M2 polarization, which was dependent on the interaction with CD47 and mediated by intracellular signaling through SHP-1. We provided evidence that Notch signal regulated macrophage polarization at least partially through SIRPα. Interestingly, Notch signal regulated macrophage phagocytosis of tumor cells through SIRPα but in a SHP-1-independent way. To access the translational value of our findings, we expressed the extracellular domains of the mouse SIRPα (mSIRPαext) to block the interaction between CD47 and SIRPα. We demonstrated that the soluble mSIRPαext polypeptides could promote M1 polarization and increase phagocytosis of tumor cells by macrophages. Taken together, our results provided new insights into the molecular mechanisms of notch-mediated macrophage polarization and further validated SIRPα as a target for tumor therapy through modulating macrophage polarization and phagocytosis.
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Regulación de la Expresión Génica , Activación de Macrófagos/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Fagocitosis , Receptores Inmunológicos/genética , Receptores Notch/metabolismo , Animales , Antígeno CD47/metabolismo , Proteínas Portadoras , Línea Celular Tumoral , Inmunomodulación , Ratones , Ratones Transgénicos , Fosforilación , Unión Proteica , Receptores Inmunológicos/metabolismoRESUMEN
OBJECTIVE: To express and purify the mouse endothelial cell-targeted recombinant Notch ligand protein mD1R, and to investigate its effect on hematopoiesis after carbon tetrachloride damage. METHODS: PCR was performed to clone and construct the expression vector pET22b(+)-mD1R. The mD1R successfully transformed into E. coli was induced by IPTG, and purified with Ni2+-beads affinity chromatography. The target protein was detected by SDS-PAGE. The fluorescence-activated cell sorting analysis (FACS), cell adhesion test, immunofluorescence staining and quantitative real-time PCR were employed to detect the endothelial cell-targeted and Notch signaling-activated biological characteristics of mD1R. The carbon tetrachloride mouse model was established to observe the effects of mD1R on the hematopoietic stem cell (HSC), myeloid cells and lymphoid cells by flow cytometry. The Lin-Scal-1+c-Kit+ cells were sorted by magnetic bead, FACS was performed to analyze the cell cycle, and RT-PCR was employed to observe the expression of interleukin (IL)-10. RESULTS: The prokaryotic expression vector was successfully cloned and constructed. The purity and the activity were confirmed in mD1R recombinant protein. The purified mD1R activated the Notch signaling pathway of hematopoietic stem cells in carbon tetrachloride damaged mouse, and internally elevated the number of HSC and long-term HSC to 2.96-fold and 6.18-fold. In addition, mD1R improved the amplification of the myeloid progenitor cells and the myeloid-derived suppressor cells, particularly the granulocyte/monocyte into blood. Mechanistically, the further analyses suggested that Notch pathway could increase the proliferation of HSC and enhance expression of IL-10 after stress injury. CONCLUSIONS: A new and activated recombinant Notch ligand protein has been obtained successfully to communicate hematopoietic stem cells and hematopoietic microenvironment. The Notch- mediated intrinsic hematopoiesis has been regulated by the anti-inflammatory factor after stress injury.
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Hematopoyesis , Animales , Tetracloruro de Carbono , Escherichia coli , Células Madre Hematopoyéticas , Ligandos , Ratones , Receptores Notch , Transducción de SeñalRESUMEN
This study was designed to investigate the pharmacological effects and mechanisms of polysaccharides from Dendrobium officinal (DOP) on premature ovarian failure (POF) in natural aging mice. Fifteen months old female mice (nâ¯â¯=â¯â¯28) and young adult female mice (nâ¯â¯=â¯â¯14, 6 weeks) were used. DOP (70â¯mg/kg) was administrated to mice by oral gavage for 10 weeks and the protection effects of DOP on ovaries were investigated in vivo. The results showed that DOP reduced body weight, ovary and uterus/body weight parameters to normal level and alleviated ovarian pathological damage. Moreover, DOP could reduce pro-inflammatory cytokines (TNF-α, IL-6) and MDA levels and improve estradiol, SOD, GSH-Px, T-AOC and IL-10 levels in serum. These results suggested that DOP may alleviate the damage caused by aging through the inhibition of the nuclear factor -κB (NF-κB) and p53/Bcl-2-mediate signaling pathways. Moreover, we found that DOP can increase the numbers of mitochondria and endoplasmic reticulum. Moreover, DOP increased the numbers of different stages of follicular cells and improved mitochondrial membrane potential in ovaries. These results indicated that DOP may relieve ovarian damage through the protection of mitochondria in the ovaries. These findings suggest that DOP may be a promising drug for treating POF caused by natural aging in females.