RESUMEN
OBJECTIVES: To describe the appearance and size of the ganglionic eminence (GE) in normal fetuses on midtrimester three-dimensional (3D) neurosonography and to report on the association between GE alterations (cavitation or enlargement) and malformation of cortical development (MCD). METHODS: This was a prospective multicenter cohort study of normal fetuses and a retrospective analysis of pathological cases with MCD. From January 2022 to June 2022, patients attending our tertiary centers for an expert fetal brain scan were recruited for the purpose of the study. A 3D volume of the fetal head, starting from the sagittal plane, was acquired in apparently normal fetuses using a transabdominal or transvaginal approach. Stored volume datasets were then evaluated independently by two expert operators. Two measurements (longitudinal diameter and transverse diameter) of the GE in the coronal view were obtained twice by each operator. Intra- and interobserver measurement variation was calculated. Reference ranges for GE measurements were calculated in the normal population. A previously stored volume dataset of 60 cases with MCD was also analyzed independently by the two operators using the same method in order to assess if GE abnormalities (cavitation or enlargement) were present. Postnatal follow-up was obtained in all cases. RESULTS: In the study period, 160 normal fetuses between 19 and 22 weeks of gestation were included in the study. The GE was visible in the coronal plane on 3D neurosonography in 144 (90%) cases and was not clearly visible in the remaining 16 (10%) cases. The intra- and interobserver agreement was almost perfect for the longitudinal diameter, with an intraclass correlation coefficient (ICC) of 0.90 (95% CI, 0.83-0.93) and 0.90 (95% CI, 0.86-0.92), respectively, and substantial for the transverse diameter, with an ICC of 0.80 (95% CI, 0.70-0.87) and 0.64 (95% CI, 0.53-0.72), respectively. A retrospective analysis of 50 cases with MCD in the second trimester showed that GE enlargement was present in 12 cases and GE cavitation was present in four cases. CONCLUSIONS: Systematic assessment of the GE in fetuses at 19-22 weeks of gestation is feasible on 3D neurosonography, with good reproducibility in normal cases. Cavitation or enlargement of the GE can be demonstrated in fetuses with MCD. © 2023 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.
Asunto(s)
Feto , Ultrasonografía Prenatal , Femenino , Embarazo , Humanos , Segundo Trimestre del Embarazo , Estudios Retrospectivos , Estudios Prospectivos , Reproducibilidad de los Resultados , Estudios de Cohortes , Ultrasonografía Prenatal/métodos , Feto/anomalías , Edad GestacionalRESUMEN
Despite the heterogeneity of acute myeloid leukemia (AML), overexpression of the interleukin-3 receptor-α (CD123) on both the more differentiated leukemic blast and leukemic stem cells (LSCs) provides a therapeutic target for antibody treatment. Here we present data on the potential clinical activity of the monoclonal antibody CSL362, which binds to CD123 with high affinity. We first validated the expression of CD123 by 100% (52/52) of patient samples and the correlation of NPM1 and FLT3-ITD mutations with the high frequency of CD123 in AML. In vitro studies demonstrated that CSL362 potently induced antibody-dependent cell cytotoxicity (ADCC) of AML blasts including CD34+CD38-CD123+ LSCs by natural killer cells (NKs). Importantly, compared with healthy donor (HD) NKs, NKs drawn from AML patients in remission had a comparable ADCC activity against leukemic cells; of note, during remission, immature NKs were five times higher in AML patients than that in HDs. Significantly, we report a case where leukemic cells were resistant to autologous ADCC; however, the blasts were effectively lysed by CSL362 together with donor-derived NKs after allogeneic hematopoietic stem cell transplantation. These studies highlight CSL362 as a promising therapeutic option following chemotherapy and transplant so as to improve the outcome of AML patients.
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Citotoxicidad Celular Dependiente de Anticuerpos/genética , Subunidad alfa del Receptor de Interleucina-3/metabolismo , Células Asesinas Naturales/inmunología , Leucemia Mieloide Aguda/genética , Adulto , Anciano , Anciano de 80 o más Años , Humanos , Leucemia Mieloide Aguda/patología , Persona de Mediana Edad , Nucleofosmina , Inducción de Remisión , Adulto JovenRESUMEN
Early molecular response (EMR, BCR-ABL1 (IS)⩽10% at 3 months) is a strong predictor of outcome in imatinib-treated chronic phase chronic myeloid leukemia (CP-CML) patients, but for patients who transform early, 3 months may be too late for effective therapeutic intervention. Here, we employed multiplex cytokine profiling of plasma samples to test newly diagnosed CP-CML patients who subsequently received imatinib treatment. A wide range of pro-inflammatory and angiogenesis-promoting cytokines, chemokines and growth factors were elevated in the plasma of CML patients compared with that of healthy donors. Most of these normalized after tyrosine kinase inhibitor treatment while others remained high in remission samples. Importantly, we identified TGF-α and IL-6 as novel biomarkers with high diagnostic plasma levels strongly predictive of subsequent failure to achieve EMR and deep molecular response, as well as transformation to blast crisis and event-free survival. Interestingly, high TGF-α alone can also delineate a poor response group raising the possibility of a pathogenic role. This suggests that the incorporation of these simple measurements to the diagnostic work-up of CP-CML patients may enable therapy intensity to be individualized early according to the cytokine-risk profile of the patient.
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Interleucina-6/sangre , Leucemia Mielógena Crónica BCR-ABL Positiva/sangre , Inducción de Remisión , Factor de Crecimiento Transformador alfa/sangre , Crisis Blástica , Citocinas/análisis , Citocinas/sangre , Supervivencia sin Enfermedad , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/diagnóstico , Activación de Linfocitos , Medicina de Precisión , Pronóstico , Factores de TiempoRESUMEN
Acute myeloid leukemia (AML) is a biologically heterogeneous group of related diseases in urgent need of better therapeutic options. Despite this heterogeneity, overexpression of the interleukin (IL)-3 receptor α-chain (IL-3 Rα/CD123) on both the blast and leukemic stem cell (LSC) populations is a common occurrence, a finding that has generated wide interest in devising new therapeutic approaches that target CD123 in AML patients. We report here the development of CSL362, a monoclonal antibody to CD123 that has been humanized, affinity-matured and Fc-engineered for increased affinity for human CD16 (FcγRIIIa). In vitro studies demonstrated that CSL362 potently induces antibody-dependent cell-mediated cytotoxicity of both AML blasts and CD34(+)CD38(-)CD123(+) LSC by NK cells. Importantly, CSL362 was highly effective in vivo reducing leukemic cell growth in AML xenograft mouse models and potently depleting plasmacytoid dendritic cells and basophils in cynomolgus monkeys. Significantly, we demonstrated CSL362-dependent autologous depletion of AML blasts ex vivo, indicating that CSL362 enables the efficient killing of AML cells by the patient's own NK cells. These studies offer a new therapeutic option for AML patients with adequate NK-cell function and warrant the clinical development of CSL362 for the treatment of AML.
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Anticuerpos Monoclonales Humanizados/farmacología , Subunidad alfa del Receptor de Interleucina-3/inmunología , Leucemia Eritroblástica Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/tratamiento farmacológico , Animales , Anticuerpos Monoclonales Humanizados/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Modelos Animales de Enfermedad , Femenino , Proteínas Ligadas a GPI/inmunología , Humanos , Células Asesinas Naturales/inmunología , Leucemia Eritroblástica Aguda/inmunología , Leucemia Mieloide Aguda/inmunología , Macaca fascicularis , Ratones Endogámicos NOD , Ratones SCID , Trasplante de Neoplasias , Ingeniería de Proteínas , Receptores de IgG/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
AIM: Ultrasonography is the most common noninvasive method for the evaluation of body organs and systems. However, the feasibility and potential advantages of ultrasound scanning by emergency physicians have not yet been fully explored. We therefore wanted to determine the impact of ultrasound scanning by emergency physicians on patient management in the Emergency Department, length of hospital stay, and related costs. METHODS: From a data-base search at our hospital we selected 111 patients and divided them into 3 groups according to symptoms: right hypochondriac region pain (Group A), flank pain (Group B), abdominal pain and hemodynamic instability (Group C). Patients were further stratified into 3 subgroups according to whether they were treated by an emergency physician or a radiologist or did not undergo ultrasonography. For each group the mean length of stay in the emergency department, the complications rate, the recurrence rate (defined as return visit to the emergency department for the same pathology) and the related costs were calculated. RESULTS: Of the 111 patients, 76 received ultrasound scanning, of which 43 were treated by an emergency physician. The length of hospital stay for this group was shorter than that of the other 2 subgroups. The recurrence rate was highest in the group that did not undergo ultrasonography. The costs were lower for the group that received ultrasound scanning by an emergency physician than for the group treated by a radiologist (Euro 20 vs Euro 38). CONCLUSION: Ultrasound scanning by emergency physicians can shorten length of hospital stay for emergency patients, reduce recurrence rates for the same pathology and lower patient management costs.
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Servicio de Urgencia en Hospital/economía , Ultrasonografía/economía , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Análisis Costo-Beneficio , Medicina de Emergencia/economía , Femenino , Humanos , Tiempo de Internación , Masculino , Persona de Mediana Edad , Radiología/economía , Ultrasonografía/estadística & datos numéricosRESUMEN
An 91 years old woman was hospitalized because of lethargy, shortness of breath and diffuse subcutaneous hemorrhage of legs. Clinical features were consistent with the diagnosis of vasculitis with systemic involvement. However dermatologic characteristics of the legs, in association with malnutrition, suggested vitamin C deficiency which was confirmed by laboratory test. Ascorbic acid supplement dramatically improved her clinical symptoms. This case remarks how scurvy may mimmick a systemic vasculitis.
Asunto(s)
Escorbuto/diagnóstico , Vasculitis/diagnóstico , Anciano , Anciano de 80 o más Años , Deficiencia de Ácido Ascórbico/complicaciones , Diagnóstico Diferencial , Femenino , Humanos , Escorbuto/etiologíaRESUMEN
Constrictive pericarditis is an infrequent complication of cardiac surgery. We report the case of a young woman who developed dyspnea and ascites 3 years after surgical closure of an atrial septal defect, and the findings at chest X-ray, computed tomographic scan and Doppler echocardiography are described. Epidemiology of the disease, new pathophysiologic concepts, diagnostic features, and therapeutic targets are reviewed.
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Procedimientos Quirúrgicos Cardíacos/efectos adversos , Defectos del Tabique Interatrial/cirugía , Pericarditis Constrictiva/etiología , Adulto , Femenino , Defectos del Tabique Interatrial/diagnóstico , Defectos del Tabique Interatrial/fisiopatología , HumanosRESUMEN
Activation of macrophages by bacterial lipopolysaccharide (LPS) is accompanied by the secretion of type I interferons (IFNs) which can act in an autocrine manner. We examined the role of type I IFNs in macrophage responses to LPS using bone marrow-derived macrophages (BMM) from IFNAR1-/- mice, which lack a component of the type I IFN receptor and do not respond to type I IFNs. We found that, unlike wild-type (WT) BMM, LPS-treated IFNAR1-/- cells failed to produce nitric oxide (NO), or express inducible NO synthase (iNOS), indicating that type I IFNs are essential for all LPS-stimulated NO production in BMM. Exogenously added type II IFN (IFNgamma) rescued these responses in LPS-treated IFNAR1-/- BMM. In contrast to effects on NO, type I IFNs negatively regulated respiratory burst activity in LPS-primed BMM. We also found that while type I IFNs mediated the anti-proliferative effects of lower concentrations of LPS, at higher concentrations LPS acted in a type I IFNs-independent manner. Finally, we report that type I IFNs are a survival factor for BMM. Despite this, the ability of LPS to also prevent apoptosis in BMM was independent of type I IFNs. These findings highlight the diverse roles of type I IFNs in mediating LPS-stimulated macrophage responses.
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Interferón Tipo I/fisiología , Lipopolisacáridos/metabolismo , Activación de Macrófagos , Animales , Apoptosis , Western Blotting , Células de la Médula Ósea/metabolismo , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , ADN/biosíntesis , ADN/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Mutantes , Óxido Nítrico/metabolismo , Óxido Nítrico/farmacología , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Consumo de Oxígeno , Proteínas Recombinantes/metabolismo , Superóxidos/metabolismoRESUMEN
Independent of its antiapoptotic function, Bcl-2 can, through an undetermined mechanism, retard entry into the cell cycle. Cell cycle progression requires the phosphorylation by cyclin-dependent kinases (Cdks) of retinoblastoma protein (pRB) family members to free E2F transcription factors. We have explored whether retarded cycle entry is mediated by the Cdk inhibitor p27 or the pRB family. In quiescent fibroblasts, enforced Bcl-2 expression elevated levels of both p27 and the pRB relative p130. Bcl-2 still slowed G(1) progression in cells deficient in pRB but not in those lacking p27 or p130. Hence, pRB is not required, but both p27 and p130 are essential mediators. The ability of p130 to form repressive complexes with E2F4 is implicated, because the retardation by Bcl-2 was accentuated by coexpressed E2F4. A plausible relevant target of p130/E2F4 is the E2F1 gene, because Bcl-2 expression delayed E2F1 accumulation during G(1) progression and overexpression of E2F1 overrode the Bcl-2 inhibition. Hence, Bcl-2 appears to retard cell cycle entry by increasing p27 and p130 levels and maintaining repressive complexes of p130 with E2F4, perhaps to delay E2F1 expression.
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Proteínas Portadoras , Ciclo Celular/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Fosfoproteínas/metabolismo , Proteínas , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Factor de Transcripción E2F4 , Linfocitos/citología , Linfocitos/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismo , Proteína 1 de Unión a Retinoblastoma , Proteína p107 Similar a la del Retinoblastoma , Proteína p130 Similar a la del Retinoblastoma , Factor de Transcripción DP1 , Factores de Transcripción/genéticaRESUMEN
The mechanism leading to the expanding population of maturing myeloid cells which characterises chronic myeloid leukemia (CML) remains obscure. Because of its ability to mimic the proliferative and cell survival functions of hematopoietic growth factors, we hypothesized that the oncogene activated in CML, BCR-ABL, might also influence differentiation. To test this hypothesis, we examined the effects of expressing BCR-ABL on the myeloid differentiation of murine M1 leukemic cells, which cease dividing and differentiate into macrophages in the presence of the cytokines leukemia inhibitory factor (LIF) or interleukin (IL)-6. We found that BCR-ABL induced macrophage differentiation in M1 cells, accompanied by increased expression of macrophage cell surface markers and the acquisition of phagocytic ability. interestingly, clones of M1 cells which expressed BCR-ABL remained in cell cycle and were refractory to the growth inhibition and apoptosis induced by IL-6 or LIF in parental M1 cells. These cells also expressed inappropriately high levels of c-MYC mRNA for their degree of differentiation, which may have been important in maintaining cellular proliferation. These data suggest that BCR-ABL can stimulate both differentiation and proliferation and that these characteristics may contribute to the phenotype observed in CML.
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Diferenciación Celular/genética , División Celular/genética , Proteínas de Fusión bcr-abl/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Animales , Ciclo Celular , Células Clonales/efectos de los fármacos , Dexametasona/farmacología , Inhibidores de Crecimiento/farmacología , Interleucina-6/farmacología , Factor Inhibidor de Leucemia , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Linfocinas/farmacología , Macrófagos/citología , Ratones , Fenotipo , Proteínas Proto-Oncogénicas c-myc/genética , ARN Mensajero/genética , Células Tumorales CultivadasAsunto(s)
Apoptosis/fisiología , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Proteínas de la Membrana , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas , Animales , Proteínas Reguladoras de la Apoptosis , Proteína 11 Similar a Bcl2 , Proteínas Portadoras/metabolismo , Supervivencia Celular/fisiología , Homeostasis , Humanos , Interfase/fisiología , Leucocitos/citología , Leucocitos/metabolismo , Modelos Biológicos , Transducción de SeñalRESUMEN
D-type cyclins are induced in response to mitogens and are essential and rate-limiting for G1 phase progression in normal mammalian cells. Macrophages proliferating in response to colony-stimulating factor-1 (CSF-1) express cyclin D1 and to a lesser extent cyclin D2 but not cyclin D3. Previously we showed that the macrophage-activating agent lipopolysaccharide (LPS) blocks CSF-1-induced proliferation and cyclin D1 expression in macrophages. Here we report upon the effect of LPS on expression of cyclin D2 in normal mouse bone marrow-derived macrophages (BMM). Unexpectedly we found that this anti-mitogen raised levels of CSF-1-stimulated cyclin D2 mRNA and protein. Furthermore, LPS alone induced cyclin D2 but not cyclin D1. Inhibition of the MEK/ERK (MAPK/ERK kinase/extracellular signal-regulated kinase) mitogen-activated protein kinase pathway repressed LPS-induced cyclin D2 mRNA, whereas inhibition of the p38 mitogen-activated protein kinase enhanced expression. However, in contrast to cyclin D1, cyclin D2 in bone marrow-derived macrophages did not appear to be regulated by protein kinase A pathways. The present data (a) show elevation of a D-type cyclin in the absence of proliferation, (b) demonstrate inverse regulation of two distinct D-type cyclins under identical conditions, and (c) suggest that cyclin D2 plays a role in macrophage activation by LPS.
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Ciclina D1/biosíntesis , Ciclinas/biosíntesis , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , División Celular/efectos de los fármacos , AMP Cíclico/farmacología , Ciclina D2 , Sinergismo Farmacológico , Factor Estimulante de Colonias de Macrófagos/farmacología , Macrófagos/citología , Ratones , Transducción de SeñalRESUMEN
Coordinated interactions between cyclin-dependent kinases (Cdks), their target "pocket proteins" (the retinoblastoma protein [pRB], p107, and p130), the pocket protein binding E2F-DP complexes, and the Cdk inhibitors regulate orderly cell cycle progression. The cyclin D1 gene encodes a regulatory subunit of the Cdk holoenzymes, which phosphorylate the tumor suppressor pRB, leading to the release of free E2F-1. Overexpression of E2F-1 can induce apoptosis and may either promote or inhibit cellular proliferation, depending upon the cell type. In these studies overexpression of E2F-1 inhibited cyclin D1-dependent kinase activity, cyclin D1 protein levels, and promoter activity. The DNA binding domain, the pRB pocket binding region, and the amino-terminal Sp1 binding domain of E2F-1 were required for full repression of cyclin D1. Overexpression of pRB activated the cyclin D1 promoter, and a dominant interfering pRB mutant was defective in cyclin D1 promoter activation. Two regions of the cyclin D1 promoter were required for full E2F-1-dependent repression. The region proximal to the transcription initiation site at -127 bound Sp1, Sp3, and Sp4, and the distal region at -143 bound E2F-4-DP-1-p107. In contrast with E2F-1, E2F-4 induced cyclin D1 promoter activity. Differential regulation of the cyclin D1 promoter by E2F-1 and E2F-4 suggests that E2Fs may serve distinguishable functions during cell cycle progression. Inhibition of cyclin D1 abundance by E2F-1 may contribute to an autoregulatory feedback loop to reduce pRB phosphorylation and E2F-1 levels in the cell.
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Proteínas Portadoras , Ciclina D1/genética , Quinasas Ciclina-Dependientes/metabolismo , Regiones Promotoras Genéticas , Factor de Transcripción Sp1/metabolismo , Factores de Transcripción/metabolismo , Células 3T3 , Animales , Proteínas de Ciclo Celular/metabolismo , Ciclina D1/metabolismo , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Factor de Transcripción E2F4 , Citometría de Flujo , Humanos , Ratones , Proteína de Retinoblastoma/metabolismo , Proteína 1 de Unión a Retinoblastoma , Factor de Transcripción DP1 , Trofoblastos/enzimología , Células Tumorales CultivadasRESUMEN
GATA-1 is a tissue-specific DNA-binding protein containing two zinc-finger-like domains. It is expressed predominantly in erythrocytes. Consensus binding sites for GATA-1 have been found in the regulatory elements of all erythroid-specific genes examined. GATA-1 protein is required for erythroid differentiation beyond the proerythroblast stage. In this paper, we demonstrate that the overexpression of GATA-1 in murine erythroleukaemia (MEL) cells alleviates DMSO-induced terminal erythroid differentiation. Hence, there is no induction of globin gene transcription and the cells do not arrest in the G1 phase of the cell cycle. Furthermore, we demonstrate that expression of GATA-1 in non-transformed erythroid precursors also affects their proliferative capacity and terminal differentiation, as assayed by adult globin gene transcription. To gain insight into the mechanism of this effect, we studied the levels and activities of regulators of cell-cycle progression during DMSO-induced differentiation. A decrease in cyclin D-dependent kinase activity was observed during the induction of both control and GATA-1-overexpressing MEL cells. However, cyclin E-dependent kinase activity decreased more than 20-fold in control but less than 2-fold in GATA-1-overexpressing MEL cells upon induction. Thus GATA-1 may exert its effects by regulating cyclin E-dependent kinase activity. We also show that GATA-1 binds to the retinoblastoma protein in vitro, but not to the related protein p107, which may indicate that GATA-1 interacts directly with specific members of the cell-cycle machinery in vivo. We conclude that GATA-1 regulates cell fate, in terms of differentiation or proliferation, by affecting the cell-cycle apparatus.
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Ciclo Celular/genética , Proteínas de Unión al ADN/fisiología , Eritroblastos/citología , Factores de Transcripción/fisiología , Animales , Diferenciación Celular , Quinasas Ciclina-Dependientes/metabolismo , Proteínas de Unión al ADN/genética , Dimetilsulfóxido , Eritroblastos/química , Factores de Unión al ADN Específico de las Células Eritroides , Factor de Transcripción GATA1 , Regulación del Desarrollo de la Expresión Génica , Globinas/genética , Humanos , Leucemia Experimental , Ratones , Proteínas Nucleares/metabolismo , Fosforilación , Unión Proteica , Proteínas Recombinantes de Fusión , Proteína de Retinoblastoma/metabolismo , Proteína p107 Similar a la del Retinoblastoma , Factores de Transcripción/genética , Transcripción Genética , Células Tumorales CultivadasRESUMEN
Myeloid maturation appears to require exit from the cell cycle and leads to activation of apoptosis in the differentiated cells. The level of Bcl-2, which is known to promote cell survival, is shown here to influence both these critical steps. Bcl-2 function during myelomonocytic differentiation was investigated by introducing a deregulated bcl-2 gene into HL60 promyelocytic leukemia cells, which can be induced to exit the cell cycle and differentiate into granulocytes or monocytes. Deregulated Bcl-2 expression did not itself promote differentiation but extended the lifespan of mature cells elicited by granulocytic or monocytic inducers. Unexpectedly, in response to induction, Bcl-2 overexpression markedly potentiated and hastened cell cycle withdrawal into G(0). Enhanced survival cannot account for the elevated numbers of G(0) cells, because they arose under induction conditions that did not kill control cells. Since the cell cycle status and growth of uninduced cells was not affected by Bcl-2-overexpression, its cell cycle inhibitory activity must require an induction signal. While cell cycle withdrawal may be necessary for maturation, it was not sufficient, implicating a requirement for specific differentiative signals. These results identify, for the first time, a function for the bcl-2 proto-oncogene that is separable from its enhancement of cell survival.
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Ciclo Celular/genética , Supervivencia Celular/genética , Genes bcl-2/fisiología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Dimetilsulfóxido/farmacología , Regulación hacia Abajo , Genes bcl-2/efectos de los fármacos , Humanos , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/patología , Proto-Oncogenes Mas , Fase de Descanso del Ciclo Celular/genética , Tretinoina/farmacología , Células Tumorales CultivadasRESUMEN
There is currently much interest in the mechanisms of action of antiproliferative agents and their effects on cell cycle machinery. In the present study we examined the mechanisms of action of four unrelated agents known to inhibit proliferation of CSF-1-stimulated bone marrow-derived macrophages (BMM). We report that 8-bromo-cAMP (8Br-cAMP) and lipopolysaccharide (LPS) potently reduced CSF-1-stimulated cyclin D1 protein, and cyclin-dependent kinase (cdk) 4 mRNA and protein levels, while the inhibitory effects of the Na+/ H+ antiport inhibitor 5-(N',N'-dimethyl) amiloride (DMA) and interferon gamma (IFN gamma ) were only weak. All agents repressed CSF-1-stimulated retinoblastoma protein phosphorylation. Furthermore, 8Br-cAMP and to a lesser extent IFN gamma, also reduced CSF-1-stimulated levels of E2F DNA binding activity in a macrophage cell line, BAC1.2F5. An explanation for the different effects of the agents is that 8Br-cAMP and LPS were found to arrest BMM in early/mid-G1, while IFN gamma and DMA arrested cells in late G1 or early S phase. These data indicate that (1) different antiproliferative agents can arrest the same cell type at distinct checkpoints in G1 and (2) effects of antiproliferative agents on cell cycle machinery is linked to the position at which they arrest cells in G1.
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Antineoplásicos/farmacología , Proteínas Portadoras , Proteínas de Ciclo Celular , Proteínas de Unión al ADN , Fase G1/efectos de los fármacos , Macrófagos/citología , Macrófagos/efectos de los fármacos , Proteínas Proto-Oncogénicas , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Amilorida/análogos & derivados , Amilorida/farmacología , Animales , Secuencia de Bases , Ciclo Celular/efectos de los fármacos , Ciclo Celular/fisiología , División Celular/efectos de los fármacos , Ciclina D1 , Quinasa 4 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/farmacología , Ciclinas/metabolismo , ADN/metabolismo , Factores de Transcripción E2F , Fase G1/fisiología , Interferón gamma/farmacología , Lipopolisacáridos/farmacología , Macrófagos/metabolismo , Ratones , Datos de Secuencia Molecular , Proteínas Oncogénicas/metabolismo , Fosforilación/efectos de los fármacos , Proteína de Retinoblastoma/metabolismo , Proteína 1 de Unión a Retinoblastoma , Factor de Transcripción DP1 , Factores de Transcripción/metabolismoRESUMEN
Induction of c-myc gene expression is an essential response to growth promoting agents, including colony-stimulating factor 1 (CSF-1). Down regulation of c-myc expression occurs in response to a variety of negative growth regulators in many cell types. However, for many of these systems the causal link between c-myc down regulation and growth arrest remains to be established. Here we show for CSF-1-dependent BAC1.2F5 mouse macrophages that interferon-gamma (IFN gamma) results in a midlate G1 phase decrease of CSF-1-dependent c-myc mRNA and subsequent cell cycle arrest. Introduction of a deregulated c-myc gene into these cells, which prevents the IFN gamma-mediated decrease in c-myc expression, overrides the cell cycle arrest and restores CSF-1-dependent growth in the presence of the cytokine. This result contrasts with the macrophage growth arrest induced by cAMP elevation, which also suppresses c-myc expression, but is not overcome by a deregulated c-myc gene. These results show that inhibition of c-myc expression is an essential component in IFN gamma-mediated cell cycle arrest and demonstrates that distinct mechanisms contribute to IFN gamma- and cAMP-mediated growth arrest in macrophages.
Asunto(s)
Genes myc/fisiología , Interferón gamma/fisiología , Macrófagos/citología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Animales , División Celular/efectos de los fármacos , División Celular/genética , Línea Celular , AMP Cíclico/metabolismo , ADN/biosíntesis , Regulación hacia Abajo , Regulación de la Expresión Génica , Genes myc/efectos de los fármacos , Interferón gamma/farmacología , Factor Estimulante de Colonias de Macrófagos/fisiología , Ratones , ARN Mensajero/metabolismoRESUMEN
Little is known of the mechanisms controlling the G0/G1 transition of the cell cycle. The induction of immediate early gene expression, thought to be important for this process, suggests that the key factors controlling this transition preexist in quiescent cells. The E2F family of transcription factors likely play an important role in this process, because E2F DNA-binding activity exists in quiescent cells, and the induction of at least some immediate early genes requires intact E2F regulatory promoter sites. Here, we show that the major G0 E2F activity of primary human T cells, E2F-4, is stably bound to the p130 pocket protein in association with a DP heterodimerization partner. p130-E2F-4 binding has functional implications because p130 effectively suppressed E2F-4-mediated trans-activation, and coexpression of E2F4 overcame p130-mediated G1 arrest more efficiently than RB-induced G1 blockade. Conversely, E2F-1 overrode an RB-induced G1 block more efficiently than E2F-4. Thus, p130 and RB appear to induce cell cycle arrest via biochemically distinct mechanisms that involve different E2F family members.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Inhibidores de Crecimiento/metabolismo , Fosfoproteínas , Proteínas/metabolismo , Proteína de Retinoblastoma/metabolismo , Factores de Transcripción/metabolismo , Factor de Transcripción E2F4 , Fase G1/fisiología , Humanos , Unión Proteica , Fase de Descanso del Ciclo Celular/fisiología , Proteína p130 Similar a la del Retinoblastoma , Linfocitos T/citología , Linfocitos T/metabolismo , Activación Transcripcional , Células Tumorales CultivadasRESUMEN
The E2F family of transcription factors has been implicated in the regulation of cell proliferation, and E2F-binding sites are present in the promoters of several growth-regulating genes. E2F family members are functionally regulated, in part, by complex formation with one or more members of the nuclear pocket protein family, RB, p107, and p130. Pocket protein regulation of E2F likely contributes to normal cellular growth control. While the three cloned species of E2F, E2F-1, E2F-2, and E2F-3, are known to be targets of RB interaction, no E2F species has yet been shown to be a specific p107 or p130 target. Here, we describe the cloning of a new member of the E2F family, E2F-4, which forms heterodimers with a member(s) of the DP family and, unlike some family members, is present throughout the cell cycle and appears to be a differentially phosphorylated p107-binding partner. p107 binding not only can be linked to the regulation of E2F-4 transcriptional activity, but also to suppression of the ability of E2F-4 to transform an immortalized rodent cell line.
Asunto(s)
Proteínas Portadoras , Proteínas de Ciclo Celular , Proteínas de Unión al ADN/química , Proteínas Nucleares/química , Factores de Transcripción/química , Proteínas E2 de Adenovirus/genética , Secuencia de Aminoácidos , Secuencia de Bases , Ciclo Celular , Mapeo Cromosómico , Cromosomas Humanos Par 16 , Clonación Molecular , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Factor de Transcripción E2F2 , Factor de Transcripción E2F3 , Factor de Transcripción E2F4 , Regulación de la Expresión Génica , Humanos , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular , Polimorfismo Genético , Regiones Promotoras Genéticas , Proteína 1 de Unión a Retinoblastoma , Proteína p107 Similar a la del Retinoblastoma , Factor de Transcripción DP1 , Activación Transcripcional , TransfecciónRESUMEN
Although increased Na+/H+ exchange (antiport) activity has been shown to precede both granulocytic and monocytic differentiation of HL60 cells, its requirement for this process remains controversial. We show here that, in the absence of HCO3-, the Na+/H+ exchanger is the predominant mechanism involved in rapid recovery of pHi from cytoplasmic acidosis; however, in the presence of HCO3-, other mechanisms also contribute to pHi homeostasis. In the absence of HCO3-, dimethyl sulfoxide and 4 beta-phorbol 12-myristate 13 alpha-acetate result in an acute activation of antiport activity, whereas all-trans-retinoic acid, 1,25-dihydroxyvitamin D3, gamma-interferon, and tumor necrosis factor alpha do not. Furthermore, whether or not HCO3- is present, the amiloride analogue, 5-N,N-dimethylamiloride, a potent and more specific antiport inhibitor than the parent compound, failed to suppress HL60 cell proliferation or the decreased proliferation in response to dimethyl sulfoxide, all-trans-retinoic acid, 1,25-dihydroxyvitamin D3, gamma-interferon, or tumor necrosis factor alpha. In the absence of HCO3-, 5-N,N-dimethylamiloride also had little effect on the ability of these agents to induce functional maturation as assessed by acquisition of respiratory burst activity. Inhibition of antiport activity also did not prevent 4 beta-phorbol 12-myristate 13 alpha-acetate-induced expression of c-fos mRNA or cell adherence. The results suggest that, even under conditions in which the Na+/H+ exchanger is the predominant operable pHi regulator, antiport activity is not required for HL60 proliferation or differentiation.