RESUMEN
We have compared the cytotoxic/cytostatic responses of the SKW6.4 lymphoblastoid B-cells to the alkylating agent chlorambucil, the purine analog fludarabine, the non-genotoxic activator of the p53 pathway, Nutlin-3, used alone or in association with the death-inducing ligand recombinant TRAIL. Exposure to chlorambucil, fludarabine, and Nutlin-3 induced p53 accumulation and variably affected cell cycle progression in SKW6.4 lymphoblastoid cells. In particular, chlorambucil induced cell cycle accumulation at the G2/M checkpoint; Nutlin-3 induced early cell cycle arrest at the G1/S checkpoint, while fludarabine showed an intermediate behavior. On the other hand, recombinant TRAIL alone did not affect cell cycle progression but induced a rapid increase of apoptosis. Analysis of the gene expression profile of the p53-transcriptional targets showed distinct features between chlorambucil, Nutlin-3 and fludarabine, which likely account for their differential effect on cell cycle in SKW6.4 cells. In particular, chlorambucil upregulated the steady-state mRNA expression of SFN/14-3-3sigma, a gene involved in G2/M cell cycle arrest. Of note, all agonists upregulated TRAIL-R2 expression in SKW6.4 cells both at the mRNA and protein levels. Consistently, pretreatment with chlorambucil, fludarabine and Nutlin-3 enhanced SKW6.4 sensitivity to TRAIL-mediated apoptosis.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Imidazoles/farmacología , Leucemia Linfocítica Crónica de Células B/patología , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Linfocitos B/patología , Crisis Blástica/patología , Línea Celular Tumoral , Clorambucilo/farmacología , Humanos , Regulación hacia Arriba/genética , Vidarabina/análogos & derivados , Vidarabina/farmacologíaRESUMEN
UNLABELLED: Exposure of human pre-osteoclasts to the MDM2 antagonist Nutlin-3 activated the p53 pathway and significantly decreased the entry of pre-osteoclasts in the S phase in response to RANKL. Moreover, repeated exposure to Nutlin-3 suppressed osteoclastic differentiation, without affecting cell survival at any culture time. INTRODUCTION: The p53 oncosuppressor coordinates an intracellular network involved in protection from malignant transformation and cell cycle control; its activation is tightly regulated by the murine double minute 2 (MDM2) gene and p53-MDM2 interaction can be disrupted by selective small molecule inhibitors, the Nutlins. Although the ability of Nutlins to suppress the growth of wildtype p53 tumors has been clearly established, their biological activity in normal cells and tissues has not been extensively studied. MATERIALS AND METHODS: Peripheral blood mononuclear cell pre-osteoclasts were cultured with macrophage-colony stimulating factor (M-CSF) + RANKL or co-cultured with SaOS-2 osteosarcoma cells in the presence of IL-1beta to induce osteoclastic differentiation. Cell cycle was analyzed by BrdU incorporation. The degree of osteoclastic differentiation was monitored at different culture times by TRACP and DAPI staining, as well as by TRACP-5b ELISA. Finally, the role of p53 in mediating the biological activity of Nutlin-3 was studied using specific siRNA. RESULTS: Exposure of human pre-osteoclasts to RANKL induced an early (24 h) increase in the percentage of cells in the S phase, followed by the exit from the cell cycle at later time-points. The simultaneous addition of Nutlin-3 and RANKL dose-dependently decreased the percentage of pre-osteoclasts in the S phase and induced a rapid accumulation of p53 protein coupled with the induction of p53 target genes. Unexpectedly, the administration of Nutlin-3 to pre-osteoclasts at early culture times significantly suppressed the final output of osteoclasts at day 14 of culture. The role of p53 in mediating this biological activity of Nutlin-3 was underscored by gene knockdown experiments, in which the anti-osteoclastic activity of Nutlin-3 was significantly counteracted by siRNA specific for p53. Nutlin-3 also significantly decreased the formation of osteoclasts in a co-culture system of SaOS-2 osteosarcoma and pre-osteoclastic cells. CONCLUSIONS: These findings indicate that Nutlin-3 abrogates both pre-osteoclastic proliferation and differentiation through a p53-dependent pathway and may have therapeutic implications for those neoplastic diseases characterized by an abnormal osteoclastic activity.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Imidazoles/farmacología , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Adhesión Celular/efectos de los fármacos , Linaje de la Célula/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Humanos , Factor Estimulante de Colonias de Macrófagos/farmacología , Osteoclastos/citología , Osteosarcoma/patología , Ligando RANK/metabolismo , Factores de Tiempo , Transcripción Genética/genética , Proteína p53 Supresora de Tumor/genética , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Despite accumulating evidence showing that TNF-related apoptosis inducing ligand (TRAIL) plays a role in vascular biology and that its decoy receptor osteoprotegerin (OPG) is expressed in the vessel wall, modulation of these TNF and TNF-R family members in the early phases of diabetes mellitus has not been investigated. The expression of TRAIL and of OPG was examined both at the mRNA and protein levels in control and streptozotocin (SZT)-induced diabetic rats at early time points after the induction of diabetes mellitus. No differences in the steady-state mRNA levels of TRAIL were noticed by quantitative RT-PCR among the two groups of animals. On the other hand, diabetic rats showed a rapid and significant increase of the steady-state mRNA levels of OPG in the aortic wall of diabetic animals with respect to vehicle-treated (control) animals. These findings were confirmed at the protein level by analysing the amount of TRAIL and OPG proteins in aortic lysates by either Western blot or immunohistochemistry. Thus, an abnormal elevation of the OPG/TRAIL ratio in the vessel wall characterizes the early onset of diabetes mellitus and might represent a molecular mechanism involved in the vascular dysfunction characterizing diabetes mellitus.
Asunto(s)
Aorta/metabolismo , Diabetes Mellitus/metabolismo , Regulación de la Expresión Génica , Osteoprotegerina/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Animales , Aorta/patología , Glucemia/metabolismo , Diabetes Mellitus/genética , Diabetes Mellitus/patología , Modelos Animales de Enfermedad , Masculino , Osteoprotegerina/genética , ARN Mensajero/genética , Ratas , Ratas Wistar , Ligando Inductor de Apoptosis Relacionado con TNF/genéticaAsunto(s)
Diabetes Mellitus/fisiopatología , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Vasodilatadores/farmacología , Animales , Aorta/efectos de los fármacos , Aorta/fisiología , Diabetes Mellitus/inducido químicamente , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Masculino , Ratas , Ratas Wistar , Proteínas Recombinantes/farmacología , Vasodilatación/efectos de los fármacosRESUMEN
It has been clearly established that receptor activator of nuclear factor kappa B ligand (RANKL) is a key cytokine involved in the differentiation of osteoclastic precursors of the monocytic/macrophagic lineage. However, relatively little information is available on the ability of RANKL to modulate the expression of genes controlling cell survival/apoptosis and proliferation in human osteoclastic cells in comparison to macrophages. For this purpose, CD14+ human peripheral blood mononuclear cells, which express the cognate high affinity receptor activator of nuclear factor kappa B (RANK), were differentiated along the macrophagic or osteoclastic lineage by adding macrophage-colony stimulating factor (M-CSF) or M-CSF plus RANKL in culture for 12 days. RANKL up-regulated the expression of the chemokine MIP1alpha, which potentiates osteoclastic differentiation and simultaneously activated both anti-apoptotic (Bcl-2) and pro-apoptotic (CIDEB, PYCARD, and BAK-1) genes. Moreover, RANKL markedly up-regulated cylin D2, while it significantly decreased the levels of cyclin A, cyclin-dependent kinase 2, and other cyclin-dependent kinases, in keeping with the notion that end-stage osteoclasts are nondividing cells. Finally, a long-term exposure of RANKL up-regulated the adaptor protein TRAF3 but not TRAF6.
Asunto(s)
Apoptosis/genética , Ciclo Celular/genética , Expresión Génica , Leucocitos Mononucleares/metabolismo , Factor Estimulante de Colonias de Macrófagos/metabolismo , Macrófagos/metabolismo , Osteoclastos/metabolismo , Ligando RANK/metabolismo , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Antígeno CD11b/metabolismo , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular/genética , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Expresión Génica/efectos de los fármacos , Perfilación de la Expresión Génica , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Receptores de Lipopolisacáridos/metabolismo , Factor Estimulante de Colonias de Macrófagos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Análisis de Secuencia por Matrices de Oligonucleótidos , Osteoclastos/inmunología , Ligando RANK/farmacología , ARN Mensajero/metabolismo , Transducción de Señal/genéticaRESUMEN
The small-molecule inhibitor of murine double minute (MDM-2), Nutlin-3, induced variable apoptosis in primary acute myeloid leukemia (AML) blasts and promoted myeloid maturation of surviving cells, as demonstrated by analysis of CD11b and CD14 surface antigens and by morphologic examination. Although the best-characterized activity of Nutlin-3 is activation of the p53 pathway, Nutlin-3 induced maturation also in one AML sample characterized by p53 deletion, as well as in the p53(-/-) human myeloblastic HL-60 cell line. At the molecular level, the maturational activity of Nutlin-3 in HL-60 cells was accompanied by the induction of E2F1 transcription factor, and it was significantly counteracted by specific gene knockdown with small interfering RNA for E2F1. Moreover, Nutlin-3, as well as tumor necrosis factor (TNF) alpha, potentiated the maturational activity of recombinant TNF-related apoptosis-inducing ligand (TRAIL) in HL-60 cells. However, although TNF-alpha significantly counteracted the proapoptotic activity of TRAIL, Nutlin-3 did not interfere with the proapoptotic activity of TRAIL. Taken together, these data disclose a novel, potentially relevant therapeutic role for Nutlin-3 in the treatment of both p53 wild-type and p53(-/-) AML, possibly in association with recombinant TRAIL.