RESUMEN
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
This study was purposed to construct prokaryotic expression vector and to investigate the expression of Notch ligand Jagged1 in E.coli. An expression vector pET-hJagged1 was constructed, which can be inserted in Jagged1 with different lengths, but the DSL domain of human Jagged1 should be contained. Then the recombinant plasmids were transformed into the competent cell of E.coli BL21, and the expression of the fusion protein was induced by IPTG. Fusion protein was purified from the supernatant of cell lysates via the Nickel affinity chromatography. The results showed that prokaryotic expression vectors pET-hJagged1 (Bgl II), pET-hJagged1 (Hind I) and pET-hJagged1 (Stu I) were successfully constructed, but only pET-hJagged1 (Stu I) could express the soluble TRX-hJagged1. The purified TRX-Jagged1 protein could be obtained via the Nickel affinity chromatography, and then confirmed by Western Blot. It is concluded that prokaryotic expression vector pET-hJagged1 is successfully constructed, but only pET-hJagged1 (Stu I) can express the soluble TRX-hJagged1 and the TRX-Jagged1 fusion protein is obtained through the prokaryotic expression system, which laid a solid foundation for further to explore the effects of Jagged1 in hematopoietic and lymphoid system.
Asunto(s)
Proteínas de Unión al Calcio/genética , Escherichia coli/metabolismo , Vectores Genéticos , Péptidos y Proteínas de Señalización Intercelular/genética , Proteínas de la Membrana/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas de Unión al Calcio/metabolismo , Clonación Molecular , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteína Jagged-1 , Proteínas de la Membrana/metabolismo , Plásmidos , Proteínas Recombinantes de Fusión/genética , Proteínas Serrate-JaggedRESUMEN
The purpose of this study was to compare the efficacy of CEP plus G-CSF and CVP plus G-CSF regimens in the mobilization and collection of peripheral blood hematopoietic stem cells (PBHSC), and in the hematopoietic recovery. 57 patients with non-Hodgkin's lymphoma (NHL) underwent autologous PBHSC transplantation were analyzed retrospectively. The PBHSC were mobilized and collected by using CEP plus G-CSF and CVP plus G-CSF respectively, and were retransfused into these NHL patients after preconditioning, then the mobilization efficacy, adverse reactions and hematopoietic recovery were analyzed. The results showed that the WBC count decreased to ≤ 1.0 × 10(9)/L, platelet amount dropped to ≤ 40 × 10(9)/L during peripheral blood stem cell mobilization of all patients, which indicated successful collection of PBHSC. The mean value of (4.38 ± 3.40) × 10(8)/kg mononuclear cells (MNC) containing (2.79 ± 2.53) × 10(6)/kg CD34(+) cells were collected in CEP plus G-CSF group, while the mean value of (3.31 ± 1.23) × 10(8)/kg MNC containing (2.02 ± 0.87) × 10(6)/kg CD34(+) cells were collected in CVP plus G-CSF group. The efficacy of mobilization in CEP plus G-CSF group was significantly higher than that in CVP plus G-CSF group (p < 0.05). After preconditioning, bone marrow was suppressed in all patients. The average time of WBC count recovery to ≥ 1.0 × 10(9)/L was 11.4 days in CEP plus G-CSF group and 12.3 days in CVP plus G-CSF group; the average time of platelet amount recovery to ≥ 50 × 10(9)/L was 18.6 days in CEP plus G-CSF group and 19.3 days in CVP plus G-CSF group. The statistical analysis showed no significant difference in the average time of hematopoietic recovery between 2 groups. It is concluded that autologous PBHSC transplantation shows significant effect for treatment of patients with NHL. Either modified CEP or CVP plus G-CSF regimen is safe and effective in PBHSC mobilization. The CEP plus G-CSF regimen is better than CVP plus G-CSF regimen.
Asunto(s)
Movilización de Célula Madre Hematopoyética/métodos , Linfoma no Hodgkin/terapia , Trasplante de Células Madre de Sangre Periférica , Adolescente , Adulto , Niño , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Trasplante Autólogo , Adulto JovenRESUMEN
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.
Asunto(s)
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
Epigallocatechin-3-gallate (EGCG), the major component of green tea polyphenol, has potent efficiency to prevent the growth of a variety of cancer cells. As a novel anticancer agent for treatment of cancers, EGCG is promising and the mechanism has not been fully understood. Laryngeal squamous cell carcinoma (LSCC) is one common tumor in head and neck cancers. In the present study, we assess the effects of EGCG on LSCC cell line Hep-2, and their possible involvement in EGCG-induced apoptosis. The result showed that treatment of Hep-2 cells with EGCG decreased the cell viability, inhibited the growth and proliferation, induced apoptosis and increased the activity of caspase-3 in a dose-dependent manner. Furthermore, we found that EGCG-treatment repressed telomerase activity effectively in a concentration-dependent manner. The combined results show that EGCG induced apoptosis in Hep-2 cells via inhibiting the telomerase activity.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Catequina/análogos & derivados , Neoplasias Laríngeas/tratamiento farmacológico , Telomerasa/antagonistas & inhibidores , Caspasa 3/metabolismo , Catequina/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Fase G1/efectos de los fármacos , Humanos , Neoplasias Laríngeas/patología , Telomerasa/genéticaRESUMEN
The green tea polyphenol (GTP) has been shown to possess cancer therapeutic effect through induction of apoptosis, while the underlying molecular mechanism of its anticancer effect is not well understood. PUMA (p53-upregulated modulator of apoptosis) plays an important role in the process of apoptosis induction in a variety of human tumor cells in both p53-dependent and -independent manners. However, whether or not PUMA is involved in the process of GTP-induced apoptosis in cancer cells has not been well reported. In the present study, we treated HT-29 (mutant p53) and LoVo (wild type p53) human colorectal cancer cells with different concentrations of GTP, which led to repression of cell proliferation and induction of apoptosis in both cell lines. Meanwhile, we also observed increased PUMA expression and decreased ERK (extracellular signal-regulated kinase) activity in both of GTP-treated tumor cell lines carrying different genotypes of p53. To determine the role of PUMA in GTP-induced apoptosis, we used stable RNA interference (RNAi) to suppress PUMA expression. As a result, apoptosis was abrogated in response to GTP-treatment. We also found that suppression of ERK activity by either RNAi or its specific inhibitor significantly enhanced GTP-induced PUMA expression. All these results indicate that PUMA plays a critical role in GTP-induced apoptosis pathway in human colorectal cancer cells and can be regulated partly by ERK inactivation. Demonstration of the molecular mechanism involved in the anti-cancer effect of GTP may be useful in the therapeutic target selection for p53 deficient colorectal cancer.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/biosíntesis , Apoptosis , Neoplasias Colorrectales/metabolismo , Flavonoides/farmacología , Regulación Neoplásica de la Expresión Génica , Fenoles/farmacología , Proteínas Proto-Oncogénicas/biosíntesis , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Guanosina Trifosfato/química , Humanos , Fenotipo , Polifenoles , ARN Interferente Pequeño/metabolismo , Té , Factores de Tiempo , TransfecciónRESUMEN
OBJECTIVE: To study the effect of nonmyeloablative allogeneic peripheral blood stem cell (NST) transplantation combined with imatinib in the treatment of chronic myeloid leukemia (CML). METHODS: Ten CML patients, 5 males and 5 females, aged 21-41, 3 in chronic phase (CP), 4 in accelerated phase (AP) and 3 in blast crisis phase (BP), were treated with imatinib (400-1500 mg/d) before (n = 10) and/or after (n = 6) NST transplantation. The donors were HLA-identical (n = 4), 5/6 antigen-matched (n = 2), 4/6 antigen-matched (n = 2), 3/6 antigen-matched (n = 1) siblings or haplo-identical mothers (n = 2). The preparative regimen included cytoxin (CTX), Ara-C, and fludarabine combined with antithymocyte globulin (ATG) or anti-CD3 monoclonal antibody. Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporine (CSA) and mycophenolate mofetil (MMF), or with low-dose methotrexate (MTX) or zenapax. RESULTS: All the 10 patients showed donor cell chimerism at different degree: three had full chimerism (> 95%) and seven mixed chimerism (44%-95%). Mixed chimerism in 6 cases had been transformed into full chimerism during 1.5-10 months after NST transplantation through immunosuppressive agent withdrawal, donor peripheral blood stem cell/donor lymphocyte infusion or treatment of imatinib. The time needed for increase of the number of neutrophils to more than 0.5 x 10(9)/L was 16 d days (10-21 days). The time needed for increase of the number of platelets more than 20 x 10(9)/L was 10 days (4-15 days). 6 cases had I-II degrees acute and chronic GVHD of skin. 2 case had III-IV degrees chronic GVHD. 2 cases died of transplantation-related complication 27 and 45 days after transplantation respectively. One patient died of III-IV degrees cGVHD. Seven patients remained alive after a median follow-up of 14.5 months (7-23 months). The time needed for bcr/abl becoming negative was 33-130 days. None case relapsed during the following-up. CONCLUSION: An effective and safer method for CML, especially advanced CML treatment of NST transplantation combined with imatinib before and after transplantation reduces the leukemic cell load before transplantation, inhibits the proliferation of residual leukemic cells, promotes full chimerism change and enhanced the effect of graft versus leukemia.
Asunto(s)
Leucemia Mielógena Crónica BCR-ABL Positiva/terapia , Trasplante de Células Madre de Sangre Periférica , Piperazinas/uso terapéutico , Pirimidinas/uso terapéutico , Adulto , Benzamidas , Femenino , Estudios de Seguimiento , Humanos , Mesilato de Imatinib , MasculinoRESUMEN
To explore the clinical features, risk factors an d treatment of retinoic acid syndrome (RAS) in patients with acute promyelocytic leukemia (APL) treated with retinoic acid, the clinical and laboratory data of 11 APL patients with RAS were retrospectively analysed. The results showed that earlier and more common symptoms of RAS were successively dyspnea (11/11), fever (10/11) and hydrothorax (6/11). Higher WBC count (> or = 15.0 x 10(9)/L) in the course of treatment of all-trans retinoic acid susceptible to develop RAS (9/11). The RAS patients were treated with dexamethasone without discontinuing the treatment of retinoic acid, complete remission was achieved in 10 cases and one patient died from disseminated intravascular coagulation. It is concluded that the identification and dexamethasone treatment of RAS in earlier period are extremely important for obtaining better clinical curative effect, and it does not influence therapeutic effect of continuing application of retinoic acid.
Asunto(s)
Leucemia Promielocítica Aguda/tratamiento farmacológico , Tretinoina/efectos adversos , Adolescente , Adulto , Niño , Disnea/etiología , Femenino , Fiebre/etiología , Humanos , Hidrotórax/etiología , Masculino , Persona de Mediana Edad , SíndromeRESUMEN
OBJECTIVE: To study the activating effect of protein transduction domain (PTD) mediated BCR/ABL protein on T cells from CML patients. METHODS: The plasmid containing PTD and b3a2 bcr/abl of CML was constructed by genetic engineering and expressed in E. coli. The peripheral blood mononuclear cells from CML patients were stimulated in vitro with purified PTD-BCR/ABL protein and the expression of the early activation antigen CD(69) on CD(8)(+) and CD(4)(+) T cells was detected by flow cytometry (FCM). RESULTS: The optimal concentration of PTD-BCR/ABL protein for activating CD(8)(+) T cells in vitro was 100 micro g/ml, CD(69) expression peaked in three days stimulation. CD(8)(+) T cells were activated in 10 of 15 CML patients, the expression rate of CD(69) was (15.01 +/- 3.75)%. CD(4)(+) T cells were activated in 4 of 15 patients, the expression rate of CD(69) was (10.32 +/- 3.08)%. Both CD(8)(+) and CD(4)(+) T cells were activated simultaneously in 3 of them. However, neither CD(4)(+) nor CD(8)(+) T cells was activated by stimulation with BCR/ABL protein in all 15 specimens, the expression rate of CD(69) on CD(8)(+) and CD(4)(+) T cells was (1.36 +/- 0.31)% and (1.41 +/- 0.43)%, respectively. There was no difference compared with that of PBS control group (P > 0.05). CONCLUSION: By using a PTD-mediated antigen delivering system, exogenous BCR/ABL protein can be delivered into APC, processed and presented onto surface of APC to activate Ag-specific CD(8)(+) and CD(4)(+) T cells in vitro.
Asunto(s)
Proteínas de Fusión bcr-abl/genética , Productos del Gen tat/genética , Proteínas Recombinantes de Fusión/farmacología , Linfocitos T/efectos de los fármacos , Adolescente , Adulto , Anciano , Secuencia de Aminoácidos , Antígenos CD/análisis , Antígenos de Diferenciación de Linfocitos T/análisis , Antígenos CD4/análisis , Antígenos CD8/análisis , Relación Dosis-Respuesta a Droga , Femenino , Citometría de Flujo , Proteínas de Fusión bcr-abl/metabolismo , Productos del Gen tat/metabolismo , Humanos , Lectinas Tipo C , Leucemia Mielógena Crónica BCR-ABL Positiva/sangre , Masculino , Persona de Mediana Edad , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Linfocitos T/inmunologíaRESUMEN
AIM: The transcription factor EGR-1 (early growth response gene-1) plays an important role in cell growth, differentiation and development. It has identified that EGR-1 has significant transformation suppression activity in some neoplasms, such as fibrosarcoma, breast carcinoma. This experiment was designed to investigate the role of egr-1 in the cancerous process of hepatocellular carcinoma (HCC) and esophageal carcinoma (EC), and then to appraise the effects of EGR-1 on the growth of these tumor cells. METHODS: Firstly, the transcription and expression of egr-1 in HCC and EC, paracancerous tissues and their normal counterpart parts were detected by in situ hybridization and immunohistochemistry, with normal human breast and mouse brain tissues as positive controls. Egr-1 gene was then transfected into HCC (HHCC, SMMC7721) and EC (ECa109) cell lines in which no egr-1 transcription and expression were present. The cell growth speed, FCM cell cycle, plate clone formation and tumorigenicity in nude mice were observed and the controls were the cell lines transfected with vector only. RESULTS: Little or no egr-1 transcription and expression were detected in HCC, EC and normal liver tissues. The expression of egr-1 were found higher in hepatocellular paracancerous tissue (transcription level P=0.000; expression level P=0.143, probably because fewer in number of cases) and dysplastic tissue of esophageal cancer (transcription level P=0.000; expression level P=0.001). The growth rate of egr-1-transfected HHCC (HCC cell line) cells and ECa109 (EC cell line) cells was much slower than that of the controls. The proportion of S phase cell, clone formation and tumorigenicity were significantly lower than these of the controls' (decreased 45.5% in HHCC cells and 34.1% in ECa109 cells; 46.6% and 41.8%; 80.4% and 72.6% respectively). There were no obvious differences between SMMC7721 (HCC) egr-1-transfected cells and the controls with regard to the above items. CONCLUSION: The decreased expression of egr-1 might play a role in the dysregulation of normal growth in the cancerous process of HCC and EC. Egr-1 gene of transfected HHCC and ECa109 cells showed obvious suppression of the cell growth and malignant phenotypes, but no suppression in SMMC7721 (HCC cell line) cells.
Asunto(s)
Carcinoma Hepatocelular/patología , Proteínas de Unión al ADN/metabolismo , Neoplasias Esofágicas/patología , Neoplasias Hepáticas/patología , Factores de Transcripción/metabolismo , Animales , Carcinoma Hepatocelular/metabolismo , División Celular/fisiología , Trasplante de Células , Proteínas de Unión al ADN/genética , Proteína 1 de la Respuesta de Crecimiento Precoz , Humanos , Proteínas Inmediatas-Precoces/metabolismo , Hibridación in Situ , Neoplasias Hepáticas/metabolismo , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Factores de Transcripción/genética , Células Tumorales CultivadasRESUMEN
To study the alteration of the cell cycle during the differentiation of human myeloid leukemia cell line HL-60 induced with all trans-retinoic acid (RA), the flow cytometry was used to assay the various phases of cell cycle in HL-60 cells treated with RA. The results showed: (1) S + G(2)/M phase proportion kept relative invariability during the 48 hours incubation of HL-60 cells with RA, however, the proportion alteration of S-phase cells was associated with the RA concentrations. At 10(-6) mol/L RA, the proportion of S-phase cells appeared a temporarily increasing peak followed by persistent decrease of S-phase proportion. At 10(-5) mol/L RA, S-phase cell proportion only appeared the persistent decreasing tendency. (2) Re-culture of HL-60 cells without RA showed the decrease of S + G(2)/M and S-phase cells was associated with the increase of differentiated cells, but not all HL-60 cells were triggered into differentiation at the same time. Once the cells start to differentiate, even if there is no RA presence, the HL-60 cells were still differentiated until maturation. In conclusion, HL-60 cells are able to differentate and maturate after exposure to RA for a period of time. S-phase proportion is related to the concentrations of RA. Once the cells start to differentiate, even if there is no RA presence, the HL-60 cells still differentiatiated until maturation.