RESUMEN
We have observed a time-correlated frequency fluctuation in non-Markovian dephasing of excitons in InAs quantum dots using a six-wave mixing technique. In this measurement, the arrival times of the excitation pulses were controlled to eliminate the influence of Markovian dephasing and to measure the pure non-Markovian behavior. The experimental result shows that the time correlation of the frequency fluctuation due to exciton-phonon interactions was maintained in the quantum dots for over 10 ps. This long-time correlation is caused by the modification of the phonon coupling distribution.
RESUMEN
Surface dose monitoring in patients and physicians during 29 uterine artery embolisation (UAE) procedures was performed using photoluminescence dosemeters and thermo-luminescence dosemeters. Organ or tissue doses were measured with an anthropomorphic phantom using UAE exposure conditions averaged from the 29 cases, and effective doses were estimated for the patient. Entrance surface dose of the patients at the maximum dose position ranged from 121.5 to 1650 mGy. Estimated doses ranged from 3.16 to 43 mGy for the ovary and from 3.8 to 51.8 mGy for the uterus. The effective dose was 1.09-14.8 mSv. Monitored doses on the body surface of physicians were relatively high in the upper arm (5.41+/-1.52 to 163+/-17.25 microGy) and the hand and fingers (0.85+/-1.18 to 222+/-16.4 microGy).
Asunto(s)
Embolización Terapéutica , Fluoroscopía , Exposición Profesional , Ovario/efectos de la radiación , Médicos , Radiografía Intervencional , Dosimetría Termoluminiscente , Útero/irrigación sanguínea , Arterias , Femenino , Humanos , Dosis de Radiación , Útero/efectos de la radiaciónRESUMEN
The aim of this study is to estimate tube current modulation (TCM) profiles in paediatric computed tomography (CT) examinations with a TCM scheme (Volume-EC) and evaluate the estimation accuracy of TCM profiles. Another aim is to validate organ doses calculated using Monte Carlo-based CT dosimetry software and estimated TCM profiles by comparing them with those measured using 5-year-old and 10-year-old anthropomorphic phantoms and radio-photoluminescence glass dosemeters. Dose calculations were performed by inputting detailed descriptions of a CT scanner, scan parameters and CT images of the phantoms into the software. Organ doses were evaluated from the calculated dose distribution images. Average relative differences (RDs) between the estimated and actual TCM profiles ranged from -3.6 to 5.6%. RDs between the calculated and measured organ doses ranged from -4.2 to 13.0% and -18.1 to 4.9% for 5-year-old and 10-year-old phantoms, respectively. These results validate dose calculations for paediatric CT scans using TCM.
Asunto(s)
Dosis de Radiación , Radiometría/métodos , Tomografía Computarizada por Rayos X , Niño , Preescolar , Humanos , Método de Montecarlo , Fantasmas de Imagen , Programas Informáticos , Dosimetría TermoluminiscenteRESUMEN
Death receptor Fas transduces cell death signaling upon stimulation by Fas ligand, and this death signaling is mediated by caspase. Recently, we reported that the cell cycle regulator p21 interacts with procaspase 3 to resist Fas-mediated cell death. In the present study, the molecular characterization and functional region of the procaspase 3-p21 complex was further investigated. We observed the p21 expression in the mitochondrial fraction of HepG2 cells and detected Fas-mediated cell death only in the presence of actinomycin D. However, mitochondrial-DNA-lacking HepG2 (MDLH) cells showed this effect even in the absence of actinomycin D. Both p21 and procaspase 3 were expressed in MDLH cells, but the procaspase 3-p21 complex formation was not observed. Interestingly, the resistance to Fas-mediated cell death in the MDLH cells without actinomycin D was recovered after microinjection of HepG2-derived mitochondria into the MDLH cells. We conclude that mitochondria are necessary for procaspase 3-p21 complex formation and propose that the mitochondrial role during cell death is not only death induction but also death suppression.
Asunto(s)
Apoptosis/genética , Caspasas/metabolismo , Ciclinas/metabolismo , Precursores Enzimáticos/metabolismo , Glicoproteínas de Membrana/metabolismo , Mitocondrias/metabolismo , Receptor fas/metabolismo , Caspasa 3 , Supervivencia Celular/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , ADN Mitocondrial/genética , Dactinomicina/farmacología , Activación Enzimática , Proteína Ligando Fas , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica/genética , Humanos , Microinyecciones , Mitocondrias/genética , Modelos Biológicos , Unión Proteica , Transducción de Señal/genética , Células Tumorales CultivadasRESUMEN
Malignant cells generally acquire some immune escape mechanisms for clonal expansion. Immune escape mechanisms also contribute to the failure of graft-versus-leukemia (GVL) effect after allogeneic hematopoietic stem cell transplantation (allo-SCT). Infant leukemias with mixed-lineage leukemia (MLL) rearrangement have a remarkably short latency, and GVL effect after allo-SCT has not been clearly evidenced in these leukemias. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)- and FasL-mediated cytotoxic pathways play important roles in cytotoxic T-lymphocyte- and natural killer cell-mediated antitumor immunity and optimal GVL activity. We investigated the in vitro sensitivity of MLL-rearranged acute lymphoblastic leukemia (ALL) and acute myeloblastic leukemia (AML) cells to TRAIL- and FasL-mediated cytotoxicity. Most of cell lines and primary leukemia cells were highly resistant to TRAIL primarily owing to low cell-surface expression of death receptors in ALL and simultaneous expression of decoy receptors in AML. Nearly half of cell lines and majority of primary leukemia cells showed low sensitivity to FasL. These results suggest that resistance to death-inducing ligands, particularly to TRAIL, could be one of the mechanisms for a rapid clonal expansion and a poor sensitivity to the GVL effect in infant leukemias with MLL rearrangement.
Asunto(s)
Reordenamiento Génico , Leucemia/inmunología , Proteína de la Leucemia Mieloide-Linfoide/genética , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Escape del Tumor , Resistencia a Antineoplásicos , Efecto Injerto vs Leucemia , N-Metiltransferasa de Histona-Lisina , Humanos , Lactante , Recién Nacido , Leucemia/tratamiento farmacológico , Leucemia/genética , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/análisisRESUMEN
The aims of this study were to estimate tube current values for each X-ray projection angle used in adult chest computed tomography (CT) and abdomen-pelvis CT examinations with tube current modulation (TCM) and to validate organ doses determined using Monte Carlo (MC) simulations through comparisons with the doses measured using in-phantom dosimetry. For dose simulations, dose distribution images were obtained by inputting the geometry of a CT scanner, scan parameters including estimated TCM curves and CT images of an adult anthropomorphic phantom into MC simulation software. Organ doses were then determined from the dose distribution images. For dose measurements, organ doses were evaluated using radio-photoluminescence glass dosemeters located at various organ positions within the phantom. Relative differences between the simulated and measured organ doses were -2.5 to 11.0% and -1.5 to 10.5% for organs in chest and abdomen-pelvis CT scan ranges, respectively. Thus, the simulated and measured doses agreed well.
Asunto(s)
Dosis de Radiación , Tomografía Computarizada por Rayos X , Humanos , Método de Montecarlo , Fantasmas de Imagen , RadiometríaRESUMEN
We previously found that tyrosine kinase 2 (TYK2) signaling through its downstream effector phospho-STAT1 acts to upregulate BCL2, which in turn mediates aberrant survival of T-cell acute lymphoblastic leukemia (T-ALL) cells. Here we show that pharmacologic inhibition of heat shock protein 90 (HSP90) with a small-molecule inhibitor, NVP-AUY922 (AUY922), leads to rapid degradation of TYK2 and apoptosis in T-ALL cells. STAT1 protein levels were not affected by AUY922 treatment, but phospho-STAT1 (Tyr-701) levels rapidly became undetectable, consistent with a block in signaling downstream of TYK2. BCL2 expression was downregulated after AUY922 treatment, and although this effect was necessary for AUY922-induced apoptosis, it was not sufficient because many T-ALL cell lines were resistant to ABT-199, a specific inhibitor of BCL2. Unlike ABT-199, AUY922 also upregulated the proapoptotic proteins BIM and BAD, whose increased expression was required for AUY922-induced apoptosis. Thus, the potent cytotoxicity of AUY922 involves the synergistic combination of BCL2 downregulation coupled with upregulation of the proapoptotic proteins BIM and BAD. This two-pronged assault on the mitochondrial apoptotic machinery identifies HSP90 inhibitors as promising drugs for targeting the TYK2-mediated prosurvival signaling axis in T-ALL cells.
Asunto(s)
Apoptosis/efectos de los fármacos , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Isoxazoles/uso terapéutico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Resorcinoles/uso terapéutico , TYK2 Quinasa/metabolismo , Proteínas Reguladoras de la Apoptosis/análisis , Proteína 11 Similar a Bcl2 , Línea Celular Tumoral , Humanos , Proteínas de la Membrana/análisis , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Proteína Letal Asociada a bcl/análisisRESUMEN
Secondary to the previous development of age-specific Japanese head phantoms, the authors designed Japanese torso phantoms for dose assessment in infant computed tomography (CT) examinations and completed a Japanese 3-y-old head-torso phantom. For design of age-specific torso phantoms (0, 0.5, 1 and 3 y old), anatomical structures were measured from CT images of Japanese infant patients. From the CT morphometry, it was found that rib cages of Japanese infants were smaller than those in Europeans and Americans. Radiophotoluminescence glass dosemeters were used for dose measurement of a 3-y-old head-torso phantom. To examine the validity of the developed phantom, organ and effective doses by the in-phantom dosimetry system were compared with simulation values in a web-based CT dose calculation system (WAZA-ARI). The differences in doses between the two systems were <20 % at the doses of organs within scan regions and effective doses in head, chest and abdominopelvic CT examinations.
Asunto(s)
Fantasmas de Imagen , Radiometría/métodos , Tomografía Computarizada por Rayos X/métodos , Antropometría/métodos , Preescolar , Simulación por Computador , Femenino , Cabeza , Humanos , Lactante , Recién Nacido , Japón , Masculino , Método de Montecarlo , Dosis de Radiación , Distribución TisularRESUMEN
Since diagnostic reference levels (DRLs) for children are not currently established in Japan, the authors determined local DRLs for the full range of paediatric CT examinations in a single tertiary care children's hospital. A retrospective review of 4801 CT performance records for paediatric patients (<15 y old) who had undergone CT examinations from 2008 to 2011 was conducted. The most frequent examinations were of the head (52 %), followed by cardiac (15 %), temporal bone (9 %), abdomen (7 %), chest (6 %) and others (11 %). Approximately one-third of children received two or more CT scans. The authors' investigation showed that mean CTDIvol and DLP for head, chest and abdomen increased as a function of age. Benchmarking of the results showed that CTDIvol, DLP and effective dose for chest and abdomen examinations in this hospital were below average, whereas those for the head tended to be at or slightly above average of established DRL values from five countries. The results suggest that CT examinations as performed in a tertiary children's hospital in Japan are well optimised.
Asunto(s)
Pediatría , Tomografía Computarizada por Rayos X/normas , Adolescente , Niño , Preescolar , Femenino , Hospitales Pediátricos , Humanos , Lactante , Japón , Masculino , Dosis de Radiación , Radiometría , Valores de Referencia , Estudios Retrospectivos , Centros de Atención TerciariaRESUMEN
The death mediator caspase acts as the dominant regulator during cell death induction. The CPP32 subfamily, including caspase 3 (CPP32/Yama/Apopain), is essential for the cell death signaling. We recently reported that activation of caspase 3 is regulated by complex formation with p21 or ILP. In the present study, we investigated the binding domain with p21 and ILP to further characterize the caspase 3 inactivation machinery. Our results show that caspase 3 contains p21 binding domain in the N-terminus and ILP binding domain in the active site. Further, the caspase 3 binding domain in p21 was independent of the Cdk- or PCNA-binding domain. We also found caspase 3 protection by p21 from the p3-site cleavage serineproteinase contributes to the suppression machinery. Here, we propose the caspase 3 inactivation system by p21 and ILP as new essential system in the regulation of cell death.
Asunto(s)
Apoptosis , Caspasas/metabolismo , Ciclinas/metabolismo , Receptor fas/metabolismo , Sitios de Unión , Caspasa 3 , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Precursores Enzimáticos/metabolismo , Humanos , Fragmentos de Péptidos/metabolismo , Unión Proteica , Células Tumorales CultivadasRESUMEN
The death receptor Fas transduces apoptotic death signaling mediated by caspases. In the present study, human hepatoma HepG2 cells showed the Fas-mediated apoptosis mediated by caspase, especially caspase 3, only in the presence of actinomycin D. Interestingly, cytosolic proteins extracted from intact HepG2 cells induced caspase 3 inactivation. Our results reveal that this inactivation was triggered by the direct inhibition of activated caspase 3 by IAP gene family ILP. In addition, a 53 kDa protein was co-immunoprecipitated with anti-human caspase 3 antibody from intact HepG2 cells. This protein was a complex-protein of procaspase 3 and the cell cycle regulator p21WAF1 (p21). P21 bound to only procaspase 3, but not to activated caspase 3. We also demonstrate that p21 protein-loaded HepG2 cells resist to Fas-mediated apoptosis even in the presence of actinomycin D. Here we report that caspase 3 inactivation for the resistance to Fas-mediated apoptosis is induced by a procaspase 3/p21 complex formation and direct inhibition of activated caspase 3 by ILP.
Asunto(s)
Apoptosis/fisiología , Caspasas , Ciclinas/fisiología , Cisteína Endopeptidasas/metabolismo , Proteínas/fisiología , Receptor fas/fisiología , Anticuerpos Monoclonales/fisiología , Apoptosis/inmunología , Carcinoma Hepatocelular/enzimología , Carcinoma Hepatocelular/patología , Caspasa 3 , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/inmunología , Ciclinas/metabolismo , Cisteína Endopeptidasas/inmunología , Cisteína Endopeptidasas/fisiología , Inhibidores de Cisteína Proteinasa/fisiología , Activación Enzimática/efectos de los fármacos , Precursores Enzimáticos/inmunología , Precursores Enzimáticos/metabolismo , Precursores Enzimáticos/fisiología , Humanos , Proteínas Inhibidoras de la Apoptosis , Familia de Multigenes , Pruebas de Precipitina , Proteínas/genética , Células Tumorales Cultivadas , Proteína Inhibidora de la Apoptosis Ligada a X , Receptor fas/inmunologíaRESUMEN
Survivin is observed uniquely in tumor cells and developmental cells, which undergo either inappropriate or programmed cell growth. In the current study, we investigated the influence of Survivin on cell cycle. Overexpression of Survivin resulted in accelerated S phase shift, resistance to G1 arrest, and activated Cdk2/Cyclin E complex leading Rb phosphorylation. In addition, nuclear translocation of Survivin followed by an interaction with Cdk4 was detected. Interestingly, Survivin nuclear translocation coincided with S phase shift, and prevention of nuclear transport suppressed Survivin nuclear translocation and S phase shift. Further, we also observed that Survivin competitively interacted with the Cdk4/p16(INK4a) complex in a cell free system and in vivo. These results suggest that Survivin initiates the cell cycle entry as a result of nuclear translocation followed by an interaction with Cdk4.
Asunto(s)
Quinasas CDC2-CDC28 , Proteínas Portadoras/metabolismo , Ciclina E/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Proteínas Asociadas a Microtúbulos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogénicas , Secuencia de Aminoácidos , Animales , Apoptosis , Unión Competitiva , Transporte Biológico , Ciclo Celular , Núcleo Celular/metabolismo , Quinasa 2 Dependiente de la Ciclina , Quinasa 4 Dependiente de la Ciclina , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Activación Enzimática , Humanos , Proteínas Inhibidoras de la Apoptosis , Datos de Secuencia Molecular , Proteínas de Neoplasias , Pruebas de Neutralización , Fosforilación , Proteínas/genética , Proteínas/inmunología , Conejos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Proteína de Retinoblastoma/metabolismo , Survivin , Células Tumorales CultivadasRESUMEN
Caspase 3 is an essential death factor for the Fas-mediated cell death, and its inactivation in cells is initiated by an interaction with p21 on mitochondria or with IAP family member ILP. Survivin is also a member of IAP family and is specifically expressed during embryogenesis and in tumor cells and suppresses cell death signaling. In our current study, we demonstrated that Survivin translocation into the nucleus is dependent on Fas stimulation and cell proliferation. Survivin also interacts with the cell cycle regulator Cdk4, leading to Cdk2/Cyclin E activation and Rb phosphorylation. As a result of Survivin/Cdk4 complex formation, p21 is released from its complex with Cdk4 and interacts with mitochondrial procaspase 3 to suppress Fas-mediated cell death. Here, we propose that Survivin supports procaspase 3/p21 complex formation as a result of interaction with Cdk4 resulting in suppression of cell death signaling.
Asunto(s)
Caspasas/metabolismo , Muerte Celular , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Proteínas Asociadas a Microtúbulos , Proteínas/metabolismo , Proteínas Proto-Oncogénicas , Receptor fas/metabolismo , Caspasa 3 , Supervivencia Celular , Quinasa 4 Dependiente de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Precursores Enzimáticos/metabolismo , Proteínas Inhibidoras de la Apoptosis , Modelos Biológicos , Proteínas de Neoplasias , Unión Proteica , SurvivinRESUMEN
The amino-acid sequence of 96 residues in the N-terminal region of rat pepsinogen I was determined and the first 46 residues were found to constitute the activation peptide segment. There was high degree of homology between the activation segments of rat pepsinogen and some pepsinogens A (pig, cow, Japanese monkey and human). However, the number of residues substituted between rat and the other pepsinogens were considerably larger than those among pepsinogens A. In the N-terminal 24 residues of active pepsin, homology (88%) between rat pepsin and human gastricsin was higher than that (50%) between rat pepsin and pepsin A from human or pig. This strongly suggests that rat pepsin should be classified as pepsin C.
Asunto(s)
Pepsinógenos , Secuencia de Aminoácidos , Animales , Mucosa Gástrica/enzimología , Conformación Proteica , RatasRESUMEN
Caspase 3 is an essential factor for Fas-mediated cell death and exists endogenously in cells where its activation is suppressed by p21 and ILP. Inside the cell, procaspase 3 interacts with p21 on mitochondria. In the present study, we investigated the molecular basis for procaspase 3/p21 complex formation. During Fas-mediated cell death, mitochondria are damaged, accompanied by decreased mitochondrial membrane-potential and decreased intracellular ATP levels. This mitochondrial damage occurs before an estrangement of the procaspase 3/p21 complex, and we demonstrate that intracellular ATP-deprivation also initiates an estrangement of procaspase 3/p21 complex formation and accelerates Fas-mediated cell death. In addition, our current results revealed that the phosphorylated p21 by PKA interacts with procaspase 3. Here, we report that the mitochondrial role, especially for ATP synthesis, and PKA are necessary for the procaspase 3/p21 complex formation to resist Fas-mediated cell death.
Asunto(s)
Apoptosis , Caspasas/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Ciclinas/metabolismo , Precursores Enzimáticos/metabolismo , Receptor fas/metabolismo , Adenosina Trifosfato/metabolismo , Caspasa 3 , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Humanos , Líquido Intracelular , Mitocondrias/fisiología , Fosforilación , Células Tumorales CultivadasRESUMEN
Fluoride has been used to prevent caries in the dentition, but the possible underlying mechanisms of cytotoxicity induction by this compound are still unclear. Since fluoride is known as an inhibitor of glycolytic enzymes, we investigated the possible connection between NaF-induced apoptosis and glycolysis in human promyelocytic leukemia HL-60 cells. NaF-induced apoptotic cell death is characterized by caspase activation, internucleosomal DNA fragmentation, loss of mitochondrial membrane potential, and production of apoptotic bodies. Higher activation of caspases-3 and -9, as compared with that of caspase-8, suggested the involvement of an extrinsic pathway. Utilization of glucose was nearly halted by NaF, whereas that of glutamine was rather enhanced. NaF enhanced the expression of Bad protein, but not that of Bcl-2 and Bax proteins, and reduced HIF-1alpha mRNA expression. Analysis of these data suggests a possible link between glycolysis and apoptosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Cariostáticos/farmacología , Glucosa/metabolismo , Glucólisis/efectos de los fármacos , Fluoruro de Sodio/farmacología , Proteína Letal Asociada a bcl/efectos de los fármacos , Caspasas/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Glutamina/metabolismo , Células HL-60 , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Estadísticas no Paramétricas , Proteína Letal Asociada a bcl/metabolismoRESUMEN
The purpose of this study was to compare hard copy images from a flat-panel detector digital radiography system with conventional radiography, photofluorographic radiography and storage phosphor radiography for the detection of simulated lung adenocarcinoma lesions and also for radiation dose. To test the diagnostic performance of these four systems, the authors used 15 types of lung adenocarcinoma phantom according to Noguchi's classification and an anthropomorphic chest phantom. The visual evaluation of tumour detectability by four radiologists and two general thoracic surgeons was examined with a five-level confidence scale. Lung doses were measured with glass dosemeters for the chest radiology systems under the conditions used by each hospital and centre. Our results indicated that flat-panel detector digital radiography and storage phosphor radiography are not necessarily superior to conventional radiography and photofluorographic radiography for detecting lung adenocarcinomas when only hard copy images are used, and this suggests a need to carefully optimize chest radiography.
Asunto(s)
Adenocarcinoma/diagnóstico por imagen , Neoplasias Pulmonares/diagnóstico por imagen , Fotofluorografía/instrumentación , Humanos , Variaciones Dependientes del Observador , Fantasmas de Imagen , Fotofluorografía/métodos , Fotofluorografía/normas , Sensibilidad y Especificidad , Película para Rayos X/normas , Pantallas Intensificadoras de Rayos X/normasRESUMEN
Vascular endothelial cells can affect hematopoiesis by releasing granulocyte-macrophage colony-stimulating factor (GM-CSF). In the present study, we show that GM-CSF production in murine aorta-derived vascular endothelial cells is regulated by two cytokines:interleukin-1 (IL-1), which induces the production of GM-CSF, and interferon gamma (IFN-gamma), which downregulates this production. The GM-CSF gene is constitutively transcribed in these cells and the transcription rate of the GM-CSF gene does not change with either IL-1 alone or IL-1 plus IFN-gamma. Whereas IL-1 treatment increases the GM-CSF mRNA half-life, simultaneous treatment with IL-1 plus IFN-gamma results in a decrease in the mRNA half-life. IL-1 also enhances IL-6 mRNA accumulation in these cells, although by increasing its transcription rate. In this case, IFN-gamma does not affect IL-6 mRNA expression. These data suggest that IL-1-induced GM-CSF expression in endothelial cells is regulated at the posttranscriptional level and that IFN-gamma specifically inhibits GM-CSF expression via destabilization of the mRNA. These suppressive effects of IFN-gamma provide evidence for an additional role of IFN-gamma in regulating GM-CSF production.
Asunto(s)
Endotelio Vascular/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Interferón gamma/farmacología , Interleucina-1/farmacología , ARN Mensajero/metabolismo , Animales , Aorta , Células Cultivadas , Dactinomicina/farmacología , Estabilidad de Medicamentos , Expresión Génica , Semivida , Cinética , Ratones , Proteínas Recombinantes , Transcripción GenéticaRESUMEN
Recombinant human insulin-like growth factor I (IGF-I) increased human and murine erythropoietic colony formation in serum-free culture. In order to investigate the effects of purified factors such as IGF-I on hemopoietic progenitor cells, we have established a serum-free culture system which supports the clonal growth of CFU-E- and BFU-E-derived colonies. Exogenously supplied ingredients were bovine serum albumin (BSA), transferrin, lipid suspensions, 2-mercaptoethanol, and recombinant human erythropoietin (epo). Among these, BSA and cholesterol were found to be essential ingredients. The optimum concentration of BSA sufficient to grow BFU-E was 3%. Erythroid colony and burst formation of human and murine marrow cells was enhanced twofold (p less than 0.05) by a physiological concentration of recombinant human IGF-I. Potentiation was observed in a dose-dependent manner between 10(-9) and 10(-7) M. A few murine CFU-E colonies were formed in the absence of epo. These results suggest that IGF-I has a supportive effect on the proliferation and differentiation of erythroid precursor cells stimulated by epo and that its action is synergistic with that of epo.
Asunto(s)
Eritropoyesis/efectos de los fármacos , Factor I del Crecimiento Similar a la Insulina/farmacología , Proteínas Recombinantes/farmacología , Somatomedinas/farmacología , Animales , Sangre , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Relación Dosis-Respuesta a Droga , Eritropoyetina/farmacología , Humanos , Mercaptoetanol/farmacología , Ratones , Albúmina Sérica Bovina/farmacología , Transferrina/farmacologíaRESUMEN
We investigated the in vitro and in vivo effects of MDP-Lys(L18), a derivative of muramyl dipeptide (MDP), on megakaryocyte progenitor cells (megakaryocyte colony-forming units, CFU-Meg) in the mouse bone marrow and spleen. When CFU-Meg culture was performed with a suboptimum concentration (2%) of pokeweed mitogen-stimulated mouse spleen-conditioned medium (PWM-SCM), addition of 0.1-20 micrograms/ml of MDP-Lys(L18) increased the number of megakaryocyte colonies. The size of the megakaryocyte colonies (the number of megakaryocytes per colony) was also significantly increased by the addition of MDP-Lys(L18) under the same culture conditions in comparison with cultures without MDP-Lys(L18). MDP-Lys(L18) itself did not stimulate megakaryocyte colony formation without PWM-SCM, and it failed to enhance megakaryocyte colony formation in cultures with an optimum PWM-SCM concentration (10%). Furthermore, no effect of MDP-Lys(L18) was observed in cultures of phagocytic cell-depleted bone marrow cells. However, MDP-Lys(L18) enhanced megakaryocyte colony formation in cultures of T-lymphocyte-depleted bone marrow cells. The culture supernatant from a macrophage cell line, J774.1, plus MDP-Lys(L18) enhanced in vitro megakaryocyte colony formation in cultures with a suboptimum PWM-SCM concentration. Although interleukin 1 (IL-1)beta in the culture supernatant of J774.1 plus MDP-Lys(L18) was increased in a dose-dependent manner in response to MDP-Lys(L18), the effect of the culture supernatant was not blocked by an anti-IL-1 antibody, and IL-1 beta failed to enhance megakaryocyte colony formation in the presence of suboptimum PWM-SCM levels. The enhancement of megakaryocyte colony formation by MDP-Lys(L18) could be neutralized, however, by an anti-interleukin 6 (IL-6) antibody. Intraperitoneal administration of MDP-Lys(L18) (100 micrograms daily for 3 days) significantly increased the number of bone marrow and spleen megakaryocyte colonies at 24 to 72 h after the final injection. These in vitro and in vivo observations strongly suggest that MDP-Lys(L18) indirectly enhances the proliferation and differentiation of mouse CFU-Meg via colony-stimulating factor(s) other than IL-1, probably as a result of the stimulation of macrophages to produce IL-6.