RESUMEN

OBJECTIVES: Discrimination and bias in clinical training often take the form of microaggressions, which, albeit unintentional, are detrimental to the learning environment and well-being of students. Although there are a few reports of medical schools training students to respond to microaggressions, none have included a complementery student-led faculty training module. The aim of this study was to develop and evaluate a case-based approach to improving student resilience and increasing faculty awareness of microaggressions in the clinical setting. METHODS: We created four realistic cases of microaggressions and uncomfortable conversations, based on students' experiences on the wards, to implement training for incoming third-year students and their core faculty. Standardized patients were trained to effectively portray discriminatory faculty, residents, and patients. Institutional review board-approved surveys were administered and statistically analyzed to evaluate for efficacy. RESULTS: Students had greater mean confidence scores for responding to microaggressions immediately and at 6 months after the sessions (P < 0.05). Faculty showed improved mean confidence and understanding of the definition of a microaggression (P < 0.05). CONCLUSIONS: This approach had results similar to other studies, with the additional benefit of training faculty with the same scenarios. We believe that this method helped bridge the gap between students' notions of discrimination and faculty understanding of microaggressions.

Asunto(s)

Docentes , Microagresión , Comunicación , Humanos , Facultades de Medicina , Estudiantes

RESUMEN

The most common means of mobilizing autologous stem cells is G-CSF alone or combined with cyclophosphamide (CY) to obtain sufficient CD34+ cells for one to two transplants. There are few prospective, randomized studies investigating mobilization regimens in multiple myeloma (MM), especially after lenalidomide-based induction. We designed this prospective, randomized study to compare low-dose CY 2 g/m2 +G-CSF (arm A) and G-CSF alone (arm B) after lenalidomide-based up-front induction in MM. Of the 80 initially randomized patients, 69 patients were evaluable, 34 and 35 patients in arms A and B, respectively. The primary end point was the proportion of patients achieving a yield of ⩾3 × 10(6)/kg CD34+ cells with 1-2 aphereses, which was achieved in 94% and 77% in arms A and B, respectively (P=0.084). The median number of aphereses needed to reach the yield of ⩾3 × 10(6)/kg was lower in arm A than in arm B (1 vs. 2, P=0.035). Two patients needed plerixafor in arm A and five patients in arm B (P=0.428). Although CY-based mobilization was more effective, G-CSF alone was successful in a great majority of patients to reach the defined collection target after three cycles of lenalidomide-based induction.

Asunto(s)

Ciclofosfamida/administración & dosificación , Factor Estimulante de Colonias de Granulocitos/administración & dosificación , Movilización de Célula Madre Hematopoyética , Trasplante de Células Madre Hematopoyéticas , Talidomida/análogos & derivados , Adulto , Anciano , Autoinjertos , Ciclofosfamida/efectos adversos , Factor Estimulante de Colonias de Granulocitos/efectos adversos , Humanos , Quimioterapia de Inducción/efectos adversos , Quimioterapia de Inducción/métodos , Lenalidomida , Persona de Mediana Edad , Mieloma Múltiple , Talidomida/administración & dosificación , Talidomida/efectos adversos

RESUMEN

Apoptosis plays an important role in cancer biology. We investigated the expression of caspases 3 and 8 in malignant mesothelioma and malignant mesothelioma cell lines and putative changes in their ultrastructural expression prior and after exposure to epirubicin. Further studies were conducted to compare these changes to the localization and expression of the bcl-2 group of proteins bcl-X, bax and mcl-1, and Fas-Fas ligand in the same cells. In the histological samples, caspase 3 and 8 immunoreactivity was seen in 27/37 (73%) and 16/37 (43%) of the mesotheliomas. The immunostaining was cytoplasmic diffuse, granular, and occasionally nuclear. All six mesothelioma cell lines expressed caspases 3 and 8 by immunoblotting. After exposure to epirubicin the extent of apoptosis was increased in all cell lines investigated, being weakest in the most resistant M38K cell line. Immunoelectron microscopy revealed immunogold labeling for caspases 3 and 8 in the mitochondria with the accumulation of caspase 3 in the apoptotic bodies, while the mitochondrial localization of the bcl-2 proteins appeared to be very stable. Fas receptor could be detected by flow cytometry, whereas the most resistant cell line (M38K) lacked Fas ligand when assessed by RT-PCR. These results suggest the importance of caspase 3 during the apoptotic process of mesothelioma cells and indicate that epirubicin-induced apoptosis is independent of the mitochondrial pathway.

Asunto(s)

Apoptosis , Caspasas/análisis , Caspasas/metabolismo , Mesotelioma/enzimología , Antibióticos Antineoplásicos/farmacología , Caspasa 3 , Caspasa 8 , Activación Enzimática , Epirrubicina/farmacología , Proteína Ligando Fas , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Células Tumorales Cultivadas , Receptor fas/genética , Receptor fas/metabolismo

RESUMEN

It is generally accepted that the vascular endothelial growth factor (VEGF) signal system has no role in the maintenance of normal blood cell formation, although it obviously regulates the development of primitive hematopoiesis during an early stage of embryogenesis. The VEGF signaling pathway, however, might have some role in malignant hematopoiesis, since malignant hematopoietic cells, including acute myeloid leukemia (AML) cells, have been shown to express VEGF and its receptors. In endothelial cells, the VEGF/Flk-1/KDR signal system is a very important generator of nitric oxide (NO) through the activation of its downstream effectors phosphatidylinositol-3-OH-kinase (PI3-K), Akt kinase and endothelial NO synthase (eNOS). It is known that NO regulates hematopoiesis and modulates AML cell growth. The role of the VEGF signaling pathway in the control of AML cell growth through eNOS, however, has not been studied. By using the OCI/AML-2 cell line, which expresses VEGF receptor-2, ie Flk-1/KDR, eNOS and VEGF, as analyzed by flow cytometry, and produces VEGF into growth medium, as analyzed by ELISA, we showed that the Akt kinase and NOS activities in these cells were decreased by the inhibitors of VEGF, Flk-1/KDR and PI3-K, and NOS activity also by the direct inhibitor of NOS. The decreased NOS activity led to inhibition of clonogenic cell growth and, to some extent, induction of apoptosis. We also found that blast cells of bone marrow samples randomly taken from 14 AML patients uniformly expressed Flk-1/KDR and to varying degrees eNOS and VEGF, as analyzed by immunohistochemistry. We conclude that autocrine VEGF through Flk-1/KDR, by activating eNOS to produce NO through PI3-K/Akt kinase, maintains clonogenic cell growth in the OCI/AML-2 cell line. Since the patient samples did not express VEGF in all cases, it is possible that in vivo the regulatory connection between these two signal systems is also mediated via endocrine VEGF in addition to autocrine or paracrine VEGF.

Asunto(s)

Factores de Crecimiento Endotelial/fisiología , Leucemia Mieloide/enzimología , Linfocinas/fisiología , Óxido Nítrico Sintasa/metabolismo , Transducción de Señal , Enfermedad Aguda , Células HL-60 , Humanos , Inmunohistoquímica , Óxido Nítrico Sintasa de Tipo III , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular , Células Tumorales Cultivadas , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular

RESUMEN

AIMS: To investigate endothelial nitric oxide synthase (eNOS) expression in malignant mesothelioma and its association with expression of vascular endothelial growth factor (VEGF), its receptors FLK1 and FLT1, and vascular density. METHODS AND RESULTS: eNOS, VEGF, FLK1 and FLT1 were studied in 36 histological mesothelioma samples by immunohistochemistry. Two mesothelioma (M14K, M38K) and one non-neoplastic mesothelial cell line (MET-5A) were studied for eNOS mRNA expression by reverse transcriptase-polymerase chain reaction (RT-PCR). Vascular density was determined by staining the samples with an antibody to factor VIII. RT-PCR showed that mesothelioma cells synthesize eNOS in vitro. eNOS immunoreactivity was found in 32/36 (89%) tumours. VEGF, FLK1 and FLT1 expression was found in 17 (45%), 24 (69%) and 25 (71%) cases, respectively. FLK1 or FLT1 immunoreactivity was more often seen in epithelioid and biphasic mesotheliomas than in sarcomatoid ones (P=0.007 and P=0.011, respectively). There was a significant association between FLK1 and FLT1 immunoreactivity (P=0.032). No significant association was found between FLK1, FLT1, VEGF and eNOS immunoreactivity and vascular density. CONCLUSIONS: eNOS is strongly expressed in malignant mesothelioma. Since eNOS did not associate with VEGF, FLK1 or FLT1, its synthesis seems not to be regulated through VEGF in malignant mesothelioma as has been shown in non-neoplastic endothelial cells.

Asunto(s)

Vasos Sanguíneos/patología , Mesotelioma/patología , Óxido Nítrico Sintasa/análisis , Proteínas/análisis , Adulto , Anciano , Factores de Crecimiento Endotelial/análisis , Femenino , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Linfocinas/análisis , Masculino , Mesotelioma/genética , Mesotelioma/metabolismo , Persona de Mediana Edad , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo III , Proteínas Proto-Oncogénicas/análisis , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Proteínas Tirosina Quinasas Receptoras/análisis , Receptores de Factores de Crecimiento/análisis , Receptores de Factores de Crecimiento Endotelial Vascular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Supervivencia , Células Tumorales Cultivadas , Factor A de Crecimiento Endotelial Vascular , Receptor 1 de Factores de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular

RESUMEN

We investigated the expression of thioredoxin (Trx) and thioredoxin reductase (TrxR) in 89 non-small cell lung carcinomas. Additionally, immortalized human bronchial epithelial cells (BEAS 2B) and four human lung carcinoma cells lines (A549, SK-MES-1, CALU-6, and A427) were studied by Western blot and reverse transcription-PCR for the synthesis of Trx and TrxR protein and mRNA expression in vitro. The histological samples were also studied for immunohistochemical p53 and proliferating cell nuclear antigen expression and apoptosis. In non-neoplastic lung, Trx and TrxR expression was seen in bronchial epithelial cells and alveolar macrophages, metaplastic alveolar epithelial cells, and chondrocytes of the bronchus. In non-small cell lung carcinomas, there was a widespread expression of Trx and TrxR with only three and eight cases negative, respectively. Trx and TrxR expression was located in both cytoplasmic and nuclear compartments of the cells. There was a statistical association between cytoplasmic and nuclear Trx or TrxR expression. Grade I-II tumors showed stronger cytoplasmic and nuclear Trx and TrxR immunoreactivity than grade III tumors. No association was found between p53 and proliferating cell nuclear antigen expression and Trx or TrxR immunoreactivity. However, apoptosis was inversely associated with nuclear Trx and TrxR positivity. In the cell lines studied, both non-neoplastic BEAS 2B cells and all of the carcinoma cell lines expressed Trx and TrxR proteins and mRNA. The results show that these redox-regulating proteins are highly expressed in lung carcinomas taking part in activation of transcriptional factors and regulation of apoptosis in non-small cell lung carcinoma. In high-grade tumors, Trx and TrxR expression is diminished, suggesting loss of redox regulation in tumors with low differentiation.

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas/enzimología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/metabolismo , Reductasa de Tiorredoxina-Disulfuro/biosíntesis , Tiorredoxinas/biosíntesis , Apoptosis , Western Blotting , Carcinoma/enzimología , Carcinoma/metabolismo , Diferenciación Celular , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Células Epiteliales/enzimología , Células Epiteliales/metabolismo , Femenino , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Masculino , Oxidación-Reducción , Antígeno Nuclear de Célula en Proliferación/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Fracciones Subcelulares , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/biosíntesis

RESUMEN

Thioredoxin (Trx) with a redoxactive dithiol together with NADPH and thioredoxin reductase (TrxR) is a major disulfide reductase regulating cellular redox state and cell proliferation and possibly contributing to the drug resistance of malignant cells. We assessed the Trx system in malignant pleural mesothelioma cell lines, in nonmalignant pleural mesothelium and in biopsies of malignant pleural mesothelioma. The mRNA and immunoreactive proteins of Trx and cytosolic and mitochondrial TrxR were positive in all four human mesothelioma cell lines investigated. Six cases of nonmalignant, histologically healthy pleural mesothelium showed no Trx or TrxR immunoreactivity, whereas immunohistochemistry on 26 biopsies of human malignant pleural mesothelioma showed positive Trx in all cases and positive TrxR in 23 (88%) of the cases. Moderate or strong immunoreactivity for Trx or TrxR was detected in 85% (22 cases) and 61% (14 cases) of the mesothelioma cases, respectively. Both Trx and TrxR staining patterns were mainly diffuse and cytoplasmic, but in 39% of the mesothelioma cases prominent nuclear staining could also be detected. Although staining for Trx and TrxR was seen in tumor cells, no significant association could be demonstrated between Trx or TrxR expression and tumor cell proliferation or apoptosis in the biopsies of mesothelioma. There was no significant association between the intensity of Trx or TrxR immunoreactivity and patient survival, which may possibly be related to moderate or intense Trx and TrxR reactivity in most of the cases. Although the Trx system may have an important role in the drug resistance of malignant mesothelioma, these studies also suggest that multiple factors contribute to the promotion, cell proliferation and apoptosis of malignant mesothelioma cells in vivo.

Asunto(s)

Mesotelioma/enzimología , Neoplasias Pleurales/enzimología , Reductasa de Tiorredoxina-Disulfuro/metabolismo , Tiorredoxinas/metabolismo , Adulto , Anciano , Femenino , Humanos , Inmunohistoquímica , Masculino , Mesotelioma/patología , Persona de Mediana Edad , Pleura/enzimología , Neoplasias Pleurales/patología , Células Tumorales Cultivadas , Regulación hacia Arriba

RESUMEN

The interactions of the assembly factor P17 of bacteriophage PRD1 with liposomes were investigated by static light scattering, fluorescence spectroscopy, and differential scanning calorimetry. Our data show that P17 binds to positively charged large unilamellar vesicles composed of the zwitterionic 1-palmitoyl-2-oleoyl-phosphatidylcholine and sphingosine, whereas only a weak interaction is evident for 1-palmitoyl-2-oleoyl-phosphatidylcholine vesicles. P17 does not bind to negatively charged membranes composed of 1-palmitoyl-2-oleoyl-phosphatidylglycerol and 1-palmitoyl-2-oleoyl-phosphatidylcholine. Our differential scanning calorimetry results reveal that P17 slightly perturbs the phase behaviour of neutral phosphatidylcholine and negatively charged multilamellar vesicles. In contrast, the phase transition temperature of positively charged dimyristoylphosphatidylcholine/sphingosine multilamellar vesicles (molar ratio 9 : 1, respectively) is increased by approximately 2.4 degrees C and the half width of the enthalpy peak broadened from 1.9 to 5.6 degrees C in the presence of P17 (protein : lipid molar ratio 1 : 47). Moreover, the enthalpy peak is asymmetrical, suggesting that lipid phase separation is induced by P17. Based on the far-UV CD spectra, the alpha-helicity of P17 increases upon binding to positively charged micelles composed of Triton X-100 and sphingosine. We propose that P17 can interact with positively charged lipid membranes and that this binding induces a structural change on P17 to a more tightly packed and ordered structure.

Asunto(s)

Colifagos/metabolismo , Liposomas/química , Liposomas/metabolismo , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/metabolismo , Rastreo Diferencial de Calorimetría , Transferencia de Energía , Cinética , Leucina Zippers , Conformación Proteica , Dispersión de Radiación , Relación Estructura-Actividad , Termodinámica

RESUMEN

In this study we investigated the immunohistochemical expression of inducible nitric oxide synthase (iNOS) in a set of normal pleural mesothelial tissues, malignant mesotheliomas, mesothelioma cell lines and metastatic pleural adenocarcinomas. Furthermore, the expression of mRNA was assessed in four malignant mesothelioma cell lines in culture. Apoptosis and vascular density in malignant mesotheliomas was assessed by the TUNEL method and by immunohistochemistry with an antibody against FVIII-related antigen. Immunohistochemically mesothelial cells in non-neoplastic healthy pleural tissues were mostly negative for iNOS. Positivity for iNOS was observed in 28/38 (74%) and 24/25 (96%) of malignant mesotheliomas and metastatic pleural adenocarcinomas, respectively. Epithelial and mixed mesotheliomas expressed more often strong iNOS immunoreactivity compared to the sarcomatoid subtype (P = 0.023). Moreover, metastatic adenocarcinomas expressed more often iNOS positivity than mesotheliomas (P = 0.021). Experiments with the cell lines confirmed that malignant mesothelioma cells are capable of synthesizing iNOS. No significant association was found between iNOS expression and apoptosis or vascular density in malignant mesotheliomas. The higher expression of iNOS in the epithelial subtype of mesothelioma and pleural metastatic adenocarcinoma might be due to an increased sensitivity of these cell types to cytokine-mediated iNOS upregulation. The strong expression of iNOS suggests a putative role for NO in the growth and progression of these tumours.

Asunto(s)

Mesotelioma/enzimología , Óxido Nítrico Sintasa/biosíntesis , Pleura/enzimología , Neoplasias Pleurales/enzimología , Adenocarcinoma/irrigación sanguínea , Adenocarcinoma/enzimología , Adenocarcinoma/secundario , Anticuerpos , Apoptosis/fisiología , Línea Celular Transformada , Epitelio/enzimología , Factor VIII/inmunología , Femenino , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Masculino , Mesotelioma/irrigación sanguínea , Mesotelioma/patología , Persona de Mediana Edad , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa/inmunología , Óxido Nítrico Sintasa de Tipo II , Neoplasias Pleurales/irrigación sanguínea , Neoplasias Pleurales/secundario , Pleuresia/enzimología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Células Tumorales Cultivadas

RESUMEN

The role of the p53 pathway in apoptosis induced by all-trans-retinoic acid (ATRA) was studied in 5 human acute myeloid leukaemia (AML) cell lines, OU-AML-3, -4, -5, -7 and -8, previously established and characterized by the authors. Although all the cell lines have a wild-type (wt) p53 gene, the protein is in a mutant conformation detectable by the anti-p53 antibody PAb 240. Exposure of the cell lines to 1.0 microM ATRA for 72 h caused induction of apoptosis detectable by morphology and the annexin V assay. The number of apoptotic cells according to the annexin V assay varied from 16 +/- 8% (OU-AML-7) to 61 +/- 4% (OU-AML-3) in ATRA-treated cells, while it was 7 +/- 6% in control cells. Western blotting and flow cytometry showed down-regulation of the p53 protein by ATRA. The conformation of p53 remained unchanged, being detectable in flow cytometry by PAb 240, but not by PAb 1620 (an antibody which only detects p53 in wt conformation). At the same time bcl-2 was down-regulated as shown by Western blotting and flow cytometry, while no induction of bax was observed by ATRA. On the basis of these results, ATRA-induced apoptosis in these AML cell lines is independent of the p53 pathway, although it is associated with the down-regulation of bcl-2.

Asunto(s)

Apoptosis/efectos de los fármacos , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Genes p53 , Leucemia Mieloide/patología , Proteínas de Neoplasias/fisiología , Tretinoina/farmacología , Proteína p53 Supresora de Tumor/fisiología , Enfermedad Aguda , Apoptosis/genética , Western Blotting , Citometría de Flujo , Genes bcl-2 , Humanos , Leucemia Mieloide/genética , Proteínas de Neoplasias/biosíntesis , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/patología , Proteína p53 Supresora de Tumor/biosíntesis , Proteína X Asociada a bcl-2

RESUMEN

Proliferation and apoptotic indices of tumour cells may have important prognostic significance. Manganese superoxide dismutase (MnSOD), an important anti-oxidant enzyme, has been shown to decrease proliferation of malignant cells transfected with the MnSOD gene. The aim of the present study was to investigate the indices of cell proliferation and apoptosis and their prognostic significance in human mesothelioma and to assess the effect of MnSOD on the proliferation and apoptosis of the mesothelioma cells expressing high constitutive MnSOD activity. Tissue sections from 35 subjects with malignant pleural mesothelioma were studied for cell proliferation by Ki-67 immunohistochemistry and for apoptosis by the TUNEL assay. In additional experiments, 2 mesothelioma cell lines expressing either low (M14K) or high (M38K) MnSOD levels were assessed for proliferative and apoptotic responses to epirubicin. The median proliferation and apoptotic indices of the mesothelioma tissue were 8.2% and 0.75%, respectively. Patients with a high proliferation (>8%) or apoptotic index (>0.75%) showed a worse prognosis (p < 0.001). MnSOD expression was inversely correlated with cell proliferation (p = 0.02). Our cell line experiments indicated that cells expressing high MnSOD levels were more resistant to apoptosis and showed lower proliferation when exposed to epirubicin in vitro. These findings show that high proliferation and apoptosis are associated with a poor prognosis of mesothelioma and that a high MnSOD level is associated with low proliferation of tumour cells. Furthermore, experiments with cultured mesothelioma cells suggest the importance of MnSOD in the proliferation and apoptosis caused by drug exposure.

Asunto(s)

Apoptosis/fisiología , Mesotelioma/enzimología , Mesotelioma/patología , Neoplasias Pleurales/enzimología , Neoplasias Pleurales/patología , Superóxido Dismutasa/metabolismo , Adulto , Anciano , Biopsia , División Celular/fisiología , Femenino , Humanos , Inmunohistoquímica , Antígeno Ki-67/metabolismo , Masculino , Persona de Mediana Edad , Necrosis , Pronóstico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Superóxido Dismutasa/biosíntesis , Células Tumorales Cultivadas

RESUMEN

We investigated the possible roles of mitochondrial manganese superoxide dismutase (MnSOD) and bcl-2 in etoposide-induced cell death in acute myeloblastic leukaemia (AML) using two subclones of the OCI/AML-2 cell line, the etoposide-sensitive (ES) and the etoposide-resistant (ER), as models. Cell death after 24 h exposure to 10 micromol/l etoposide was about 60% and 70% in the ES subclone and about 20% and 25% in the ER subclone, when analysed by trypan blue and annexin V respectively. Cytochrome c efflux from mitochondria to cytosol was observed after 4 h of exposure in both subclones, whereas the activation of caspase-3 was not detectable until after 12 h of exposure in the ES subclone and 24 h of exposure in the ER subclone, using Western blotting. The decrease in mitochondrial membrane potential, when analysed by the JC-1 probe fluorocytometrically, also appeared to take place later in the ER than in the ES subclone. Both subclones showed evident basal expression of MnSOD and bcl-2 by Western blotting. Etoposide caused a potent induction of MnSOD, more than 400% at 12 h, in the ER but not in the ES subclone. No significant change in bcl-2 expression could be observed in either of the subclones during exposure to etoposide when analysed by Western blotting or flow cytometry. In conclusion, we suggest that MnSOD might have a special role in the protection of AML cells against etoposide-induced cell death. Although unable to influence the cytochrome c efflux to cytosol, MnSOD might prevent the disruption of mitochondrial membrane potential, which evidently leads to cell death by releasing various activators of apoptosis.

Asunto(s)

Antineoplásicos Fitogénicos/uso terapéutico , Etopósido/uso terapéutico , Leucemia Mieloide Aguda/enzimología , Mitocondrias/enzimología , Superóxido Dismutasa/biosíntesis , Apoptosis/efectos de los fármacos , Western Blotting , Caspasa 3 , Caspasas/metabolismo , Grupo Citocromo c/metabolismo , Resistencia a Antineoplásicos , Inducción Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/uso terapéutico , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Mitocondrias/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Superóxido Dismutasa/metabolismo , Células Tumorales Cultivadas

RESUMEN

BACKGROUND AND OBJECTIVE: All-trans retinoic acid (ATRA) induces growth arrest and apoptosis in acute myeloblastic leukemia (AML) cells. Since cellular redox state regulates these events, we were interested in studying whether it has any role in the responsiveness of AML cells to ATRA. DESIGN AND METHODS: Two human AML cell lines, the ATRA-sensitive OU-AML-3, and the ATRA-resistant OU-AML-7, were used as models. Clonogenic cell culture assay, annexin V method, and measurement of mitochondrial membrane potential were used for the determination of cell growth and apoptosis. Peroxide formation was analyzed by flow cytometry, glutathione and g-glutamylcysteine synthetase (g-GCS) activity was determined spectrophotometrically, and the expression of manganese superoxide dismutase (MnSOD) by Western blotting. RESULTS: ATRA inhibited clonogenic cell growth and induced apoptosis particularly in OU-AML-3 cells. The OU-AML-7 cells had a higher basal level of glutathione and g-GCS activity than the OU-AML-3 cells. ATRA enhanced the generation of peroxides after 24h exposure, which was more prominent in the sensitive than the resistant cell line and was not preventable by N-acetyl-L-cysteine. ATRA also increased the activity of g-GCS, which was associated with increased intracellular glutathione in the resistant cell line, while the glutathione level was maintained in the sensitive cell line. During ATRA exposure, MnSOD was induced in the sensitive cell line, but not until after 72 h. Buthionine sulfoximine significantly increased the inhibitory effect of ATRA on colony formation in both cell lines, but only marginally enhanced the effect of ATRA on the induction of apoptosis. INTERPRETATION AND CONCLUSIONS: The balance between oxidative and antioxidative actions of ATRA, as well as the basal redox state of the cells seem to have a definite influence on the responsiveness of AML cells to ATRA.

Asunto(s)

Apoptosis/efectos de los fármacos , División Celular/efectos de los fármacos , Leucemia Mieloide Aguda/patología , Oxidantes/farmacología , Tretinoina/farmacología , Células Clonales/efectos de los fármacos , Células Clonales/fisiología , Glutatión/efectos de los fármacos , Glutatión/metabolismo , Humanos , Leucemia Mieloide Aguda/fisiopatología , Oxidantes/fisiología , Oxidación-Reducción/efectos de los fármacos , Peróxidos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Especies Reactivas de Oxígeno/fisiología , Superóxido Dismutasa/efectos de los fármacos , Tretinoina/fisiología , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/fisiología , gamma-Glutamil Hidrolasa/efectos de los fármacos , gamma-Glutamil Hidrolasa/metabolismo

RESUMEN

Two subclones of the OCI/AML-2 cell line, etoposide-sensitive (ES) and etoposide-resistant (ER), established by the authors, were used as models. We investigated whether the Fas pathway is involved in etoposide-induced apoptosis in acute myeloblastic leukemia (AML). Both of the studied subclones expressed the Fas receptor (FasR), but only the ER cell line expressed the Fas ligand (FasL). Etoposide caused an increase in the mean fluorescence intensity of FasR in both subclones, and an induction of FasL in the ES subclone. However, no change in the numbers of apoptotic cells induced by etoposide was observed when FasR was blocked by an antagonist anti-Fas antibody, nor was an agonist anti-Fas antibody alone cytotoxic to the subclones or enhanced the cytotoxic effect of etoposide. The Fas-resistant phenotype of the AML cells was converted to a Fas-sensitive one by cycloheximide (CHX) suggesting the presence of an inhibitory protein of the Fas pathway in the cells. In etoposide-induced apoptosis, the effect of CHX was different, apoptosis-preventing. In conclusion, etoposide-induced apoptosis is not mediated by the Fas pathway in AML.

Asunto(s)

Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Etopósido/farmacología , Leucemia Mieloide Aguda/tratamiento farmacológico , Receptor fas/fisiología , Cicloheximida/farmacología , Proteína Ligando Fas , Humanos , Leucemia Mieloide Aguda/patología , Glicoproteínas de Membrana/análisis , Células Tumorales Cultivadas , Receptor fas/análisis

RESUMEN

The present study investigated whether all-trans retinoic acid (ATRA)-induced apoptosis in acute myeloblastic leukaemia (AML) is related to changes in mitochondrial function. Two human AML cell lines, OU-AML-3 and OU-AML-7, known to be inducible to time-dependent apoptosis of varying degrees by ATRA, were used. Apoptosis induced by ATRA was shown to be a slow event. It was detected by the DNA electrophoretic method and cytofluorimetrical annexin V assay after 48 h exposure, and by morphology and polyADPribose polymerase (PARP) cleavage after 72 h exposure of AML cells to ATRA. The efflux of mitochondrial cytochrome c to cytosol was notable in Western blotting after 48 h exposure of the cells to ATRA and was observed before the drop in the mitochondrial membrane potential, which only took place after 72 h exposure, when measured by flow cytometry and a JC-1 probe. The apoptotic events in mitochondria were more evident in the OU-AML-3 than the OU-AML-7 cell line. This might relate to the different bcl-2 contents of the cell lines: the basic bcl-2 levels of the OU-AML-7 cell line were almost twofold compared to that of the OU-AML-3 cell line, as analysed by the ELISA method. However, both of the cell lines showed progressive down-regulation of bcl-2, which began after 12-24 h exposure of the cells to ATRA as determined by ELISA, Western blotting and flow cytometry. The present results show that mitochondria have a role in ATRA-induced apoptosis in AML cells and down-regulation of bcl-2 is related to it. In view of the previously published studies, the present results underline the fact that the timing of apoptotic events, such as fragmentation of DNA, externalization of phosphatidylserine, cytochrome c efflux, change in mitochondrial membrane potential and cleavage of PARP, are, to a notable extent, cell type and inducer-dependent.

Asunto(s)

Apoptosis/efectos de los fármacos , Leucemia Mieloide Aguda/patología , Mitocondrias/fisiología , Tretinoina/farmacología , División Celular , Grupo Citocromo c/metabolismo , Regulación hacia Abajo , Expresión Génica , Genes bcl-2/fisiología , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/fisiopatología , Células Tumorales Cultivadas

RESUMEN

As interleukin-6 (IL-6) has been shown to have diverse effects on blast cell growth in acute myeloblastic leukemia (AML), and as a soluble (s) form of IL-6 receptor (IL-6R) agonizes IL-6 effects in many cell types, we investigated whether sIL-6R was able to modulate clonogenic blast cell growth in AML. The proliferation responses of eight autonomously growing AML cell lines and eight primary AML blast cell samples were compared with their IL-6 and sIL-6R expression. Only three of the 16 AML samples were influenced by IL-6, two of them being stimulated and one inhibited by it. The sIL-6R-induced responses were more frequent, however, and, in contrast to those by IL-6, always stimulatory: clonogenic cell growth in six of the 16 AML samples was stimulated by sIL-6R treatment. All the cell lines and four of the seven primary blast cell samples analyzed expressed IL-6, and the expression was associated with unresponsiveness to exogenous IL-6. sIL-6R was also frequently expressed by AML cells: only one of the samples was negative for it. However, there was no correlation between sIL-6R expression and the responsiveness of cells to exogenous sIL-6R. The work presented here shows that sIL-6R is able to stimulate blast cell growth in AML. As AML blast cells are provided by exogenous IL-6 and sIL-6R in a bone marrow environment, and as many of them also express IL-6 and sIL-6R themselves in vitro, it is possible that signaling through the IL-6/sIL-6R system plays a role in maintaining their growth also in vivo.

Asunto(s)

Leucemia Mieloide Aguda/patología , Receptores de Interleucina-6/fisiología , Adolescente , Adulto , Anciano , Crisis Blástica/tratamiento farmacológico , Crisis Blástica/genética , División Celular/efectos de los fármacos , Línea Celular , Femenino , Expresión Génica , Humanos , Interleucina-6/genética , Interleucina-6/farmacología , Masculino , Persona de Mediana Edad , ARN Mensajero/metabolismo , Receptores de Interleucina-6/genética , Solubilidad , Ensayo de Tumor de Célula Madre

RESUMEN

We analysed the status of the p53 gene and protein in eight newly established acute myeloid leukaemia (AML) cell lines representing blast cells of either de novo leukaemia patients in first remission or patients with relapsed and chemotherapy-resistant disease causing their death. There were no mutations in the p53 gene in any of the cell lines as analysed by single-strand conformation polymorphism of amplified exons 5-8. However, the p53 protein was clearly and consistently expressed in all of these cell lines, as shown by immunohistochemistry, Western blotting and flow cytometry. The consistently expressed p53 protein was located in both the nucleus and the cytoplasm of all the cell lines and, as shown by flow cytometry, it was mostly in a conformation typical of the mutated protein. These AML cell lines offer a tool for studying the production and function of the p53 protein and its possible role in cell cycle regulation and chemoresistance as well as in the regulation of apoptosis in AML.

Asunto(s)

ADN de Neoplasias/genética , Regulación Neoplásica de la Expresión Génica , Genes p53/genética , Leucemia Mieloide Aguda/genética , Adulto , Anciano , Apoptosis , Western Blotting , Ciclo Celular/genética , Análisis Mutacional de ADN , ADN de Neoplasias/análisis , Resistencia a Antineoplásicos/genética , Femenino , Citometría de Flujo , Humanos , Masculino , Persona de Mediana Edad , Células Tumorales Cultivadas

RESUMEN

The role of the interleukin-6/interleukin-6 receptor (IL-6/IL-6R) system in regulating blast cell growth in 8 acute myeloblastic leukemia (AML) patient-derived cell lines was investigated. As they all expressed IL-6R and as none of them responded to exogenous IL-6 under conventional serum-supplemented culture conditions, we investigated whether signaling through IL-6R plays any role in maintaining their spontaneous colony growth. This was done by treating the cells with monoclonal antibodies made against the ligand-specific IL-6R alpha-chain or the signal transducer gp130. In serum-supplemented cultures inhibition of gp130 function did not affect the cell line growth, whereas anti-IL-6R alpha-chain antibody reduced colony growth. While some of the cell lines also showed similar growth characteristics in a serum-free environment, some others changed their growth pattern and stopped responding to anti-IL-6R alpha-chain treatment. At the same time, these cell lines also began to respond to exogenously added IL-6 and, interestingly, were stimulated by anti-gp130 antibody. Hence, in some of the blast cells, clonogenic cell growth seemed to be also negatively controlled by an endogenously produced growth-depressing cytokine or cytokines that utilize gp130. All the cell lines, whether cultured in the presence or absence of serum expressed IL-6 both at mRNA and protein level. The current results indicate that AML cells can use IL-6 as a growth stimulating factor, supplied either paracrinely or autocrinely. This could implicate the use of anti-IL-6R alpha-chain antagonists in AML treatment, not IL-6.

Asunto(s)

Interleucina-6/farmacología , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Células Madre Neoplásicas/patología , Receptores de Interleucina-6/metabolismo , Transducción de Señal , Adulto , Anciano , División Celular/efectos de los fármacos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Receptores de Interleucina-6/agonistas , Células Tumorales Cultivadas

RESUMEN

As interleukin (IL-6) ahs been reported to have diverse effects on acute myeloblastic leukemia (AML) blast cell growth, we investigated whether the level of IL-6 receptor (IL-6R) expression by blast cells is associated with their susceptibility to proliferate in response to exogenous IL-6. For absolute quantification of IL-6R transcript numbers, we established a quantitative IL-6R reverse transcription polymerase chain reaction (RT-PCR) method with an internal RNA standard. In the present work, two types of AML blast cells were investigated, namely autonomously growing cell line cells (n = 8) and non cultured native blast cells (n = 20), including those from which the cell lines originate. The native blast cells expressed an average of 2.8 x 10(7) +/- 1.9 x 10(7) IL-6R transcripts in one microgram of total cellular RNA, whereas the expression by the cell line cells was significantly more abundant, the value being 8.3 x 10(7) +/- 2.8 X 10(7) (P < 0.001). The proliferation responses were evaluated by exposing the cells to IL-6 (1000 U/ml) in a clonogenic cell culture assay and, in the case of the cell line cells, in a long-term suspension culture assay as well. None of the autonomously growing cell lines responded to exogenous IL-6, whereas the native blast cell showed either stimulatory, inhibitory or neutral responses. Thus, the IL-6R expression level did not predict whether the cells proliferated in response to exogenous IL-6, which shows that IL-6R quantification cannot be used as a screening test prior to possibly applying this cytokine to clinical use in AML therapy.

Asunto(s)

Regulación Neoplásica de la Expresión Génica/fisiología , Interleucina-6/farmacología , Leucemia Mieloide Aguda/genética , Receptores de Interleucina-6/genética , Adolescente , Adulto , Anciano , Humanos , Leucemia Mieloide Aguda/patología , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/análisis , ARN Neoplásico/análisis , Receptores de Interleucina-6/análisis , Células Tumorales Cultivadas

RESUMEN

The aim of the present work was to investigate whether acute myeloblastic leukaemia (AML) blast cells express a soluble (s) form of interleukin 6 (IL-6) receptor (R), and if they do, what is the mechanism of production. Eight AML patient cell lines and 25 primary AML blast cell samples were investigated. The cell lines secreted high quantities of sIL-6R into their culture medium when examined by enzyme-linked immunosorbent assay (ELISA). To determine whether sIL-6R is synthesized by a mechanism of alternative splicing, RNA was analysed from all the AML blast cell samples by using reverse transcription polymerase chain reaction. In this method, primer sites flanking the transmembrane domain were utilized and the alternatively spliced IL-6R mRNA was distinguished from the non-spliced transcript form by size. All the cell lines and 64% of the primary blast cell samples expressed the alternatively spliced IL-6R mRNA. To confirm the phenomenon of alternative splicing at protein level, cytoplasmic protein fractions of the cell lines were investigated by using a sensitive adaptation of the Western blot method. All the cell lines expressed two IL-6R proteins sized 80 and 50 kDa and corresponding to the membraneous and soluble forms of IL-6R, respectively. In conclusion, the results obtained at both mRNA and protein levels strongly support alternative splicing as a mechanism of sIL-6R production in AML. Because sIL-6R modulates the effects of IL-6 on target cells, differences in sIL-6R expression levels may partially explain the previously observed diversity in IL-6-induced growth responses in AML

Asunto(s)

Empalme Alternativo , Interleucina-6/biosíntesis , Interleucina-6/genética , Leucemia Mieloide Aguda/metabolismo , Adolescente , Adulto , Anciano , Femenino , Humanos , Leucemia Mieloide Aguda/genética , Masculino , Persona de Mediana Edad , ARN Mensajero/genética , Células Tumorales Cultivadas