RESUMEN
It has been reported that mitochondria-independent or mitochondria-dependent (type I/II) Fas signaling pathways in leukemia cells depend on the amount of active caspase-8. However, Bid molecules, which could not be cleaved in type I cells, could be effectively cleaved by recombinant active caspase-8 in vitro. The cleavage of recombinant Bid by recombinant active caspase-8 could be blocked by anti-p10 and anti-p18 specific antibodies. Fas receptors could be similarly internalized into cytoplasm in type I and type II cells. Interestingly, p10 subunit of active caspase-8 could be detected in both type I and II cells, while p18 subunit of active caspase-8 could be detected only in type II cells but not in type I cells. These results demonstrated that p18 subunit was necessary for Bid cleavage and the mitochondria pathway might be dependent on the release of p18 subunit from active caspase-8.
Asunto(s)
Caspasa 8/metabolismo , Transducción de Señal , Receptor fas/metabolismo , Anticuerpos/farmacología , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Caspasa 8/química , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Endocitosis/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Humanos , Leucemia/metabolismo , Leucemia/patología , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
OBJECTIVE: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor superfamily, which induces apoptosis in cancer cells but not in normal cells. Akt/protein kinase B, when phosphorylated to its active form, promotes cell survival and blocks apoptosis. The aim of this study was to investigate the role of Akt pathway in acquired TRAIL resistance of acute lymphoblastic leukemia cells. MATERIALS AND METHODS: MB-IT and NALM-24 cells that developed resistance to TRAIL, i.e., TRAIL-resistant cells (MB-IT R and NALM-24 R) were established from TRAIL-sensitive acute lymphoblastic leukemia cell lines (MB-IT S and NALM-24 S), respectively, through application of TRAIL and repetitive limiting dilution. Apoptosis was measured by flow cytometry using propidium iodide/Annexin-V fluorescein isothiocyanate staining. TRAIL receptor cell surface expression of MB-IT and NALM-24 were analyzed by flow cytometry. Protein levels were analyzed by Western blot analysis. RESULTS: The obtained resistant cell lines presented the same pattern of receptor expression as sensitive parent cells, and the internalization of DR5 after TRAIL treatment was similar. Caspase-8/3, FLIP, BID, XIAP were cleaved/downregulated in sensitive cells after treatment with TRAIL, but not in the resistant cells. We also observed that phosphoinositide-3-kinase (PI3K)/Akt pathway was constitutively active in resistant clones, and was not downregulated upon TRAIL treatment. Phosphate and tensin homologue deleted on chromosome 10 (PTEN) level was the same in both sensitive cells and resistant cells, but was quickly downregulated in sensitive cells after TRAIL treatment. Also, resistant cells expressed a high level of phosphorylated inactive form of PTEN than the sensitive cells. Expression levels of PH domain leucine-rich repeat protein phosphatase were slightly higher in sensitive than resistant cells. When resistant cells were treated with LY 294002 (a PI3K inhibitor), the expression level of phosphorylated Akt was distinctly downregulated, and there was induction of apoptosis when these cells were treated with a combination of TRAIL and LY 294002. When MB-IT-sensitive cells were treated with okadaic acid, a phosphatase inhibitor, TRAIL-induced apoptosis was significantly reduced. CONCLUSION: These results suggest that cellular resistance to TRAIL could be developed through phosphorylation (activation) of Akt and phosphorylation (inactivation) of PTEN.
Asunto(s)
Apoptosis/fisiología , Fosfohidrolasa PTEN/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/fisiología , Antígenos CD/genética , Línea Celular Tumoral , Resistencia a Antineoplásicos , Femenino , Humanos , Inmunofenotipificación , Cariotipificación , Microscopía Confocal , Fosfohidrolasa PTEN/genética , Fosforilación , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Proto-Oncogénicas c-akt/genética , Ligando Inductor de Apoptosis Relacionado con TNF/genéticaAsunto(s)
Quilotórax/cirugía , Enfermedades Pulmonares/diagnóstico por imagen , Linfangiectasia/diagnóstico por imagen , Sistema Linfático/anomalías , Derrame Pericárdico/cirugía , Complicaciones Posoperatorias/diagnóstico por imagen , Quilotórax/etiología , Disnea/etiología , Insuficiencia de Crecimiento/etiología , Resultado Fatal , Femenino , Neoplasias de Cabeza y Cuello , Pérdida Auditiva Bilateral , Hemangioma Capilar , Humanos , Lactante , Enfermedades Pulmonares/complicaciones , Linfangiectasia/complicaciones , Derrame Pericárdico/etiología , Radiografía , Insuficiencia Respiratoria/etiologíaRESUMEN
The relationship between apoptosis and the cell cycle remains unclear. In the present study we have investigated the relationship between cell cycle progression and the activation of caspases (caspase-3 and caspase-8) in Fas (CD95)-mediated apoptosis in asynchronously growing leukemia cells. We found that cells expressing the active form of caspase-3 were cyclin A/B1 and Ki-67 negative but cyclin E positive, whereas expression of the active form of caspase-8 was detected in cyclin A/B1/E-negative and Ki-67-negative cells. In addition, both the activation of caspases and Fas-mediated apoptosis were completely abolished when leukemia cells were arrested in early G1 phase. Using post-sorting western blot analysis, we demonstrated that caspase-3 and caspase-8 were activated in p27-negative cells. These results suggest that caspase-3 would be activated in cells entering into late G1 or early S phase, and caspase-8 would be activated in middle or late G1 phase. The speed of cell cycle progression from G1 to S phase might be influential in the speed of caspase activation and induction of Fas-mediated apoptosis.