RESUMEN
p53-induced death domain protein (PIDD) facilitates p53-dependent apoptosis through the interaction with components of the death receptor signaling pathways. However, the role of PIDD in hepatocellular carcinoma (HCC) development remains unknown. In this study, we investigated the expression pattern of PIDD in clinical HCC samples and adjacent non-cancerous tissues using immunohistochemistrical and Western blot analyses. The results showed that PIDD was lowly expressed in HCC tissues and HCC cell lines, compared with the adjacent non-tumorous tissues and LO2 normal hepatocytes. In addition, clinicopathological analysis showed that the expression of PIDD was closely related with multiple clinicopathological variables, such as American Joint Committee on Cancer (AJCC) stage, AFP, and poor prognosis of HCC. Univariate and multivariate survival analyses demonstrated that PIDD could serve as an independent prognostic factor to predict the survival of HCC patients. We used serum starvation-refeeding experiment to explore the involvement of PIDD in HCC cell cycle regulation. We found that PIDD was accumulated in growth-arrested HCC cells and was progressively decreased when cells entered into S phase. Moreover, flow cytometry and cell counting kit-8 (CCK-8) assays indicated that depleting the expression of PIDD could facilitate cell cycle progression and accelerate cell proliferation in HepG2 cells, while overexpression of PIDD could result in cell cycle arrest at G1 phase and hinder the cell proliferation in Hep3B cells. Finally, flow cytometry revealed that overexpression of PIDD slightly increased the apoptosis of HCC cells. Taken together, we concluded that PIDD may be a valuable prognostic marker and promising therapeutic target of HCC.
Asunto(s)
Carcinoma Hepatocelular/patología , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/fisiología , Neoplasias Hepáticas/patología , Proteínas de Neoplasias/fisiología , Adulto , Anciano , Apoptosis , Carcinoma Hepatocelular/metabolismo , Ciclo Celular , División Celular , Línea Celular Tumoral , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/análisis , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/antagonistas & inhibidores , Femenino , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/análisis , Proteínas de Neoplasias/antagonistas & inhibidores , Pronóstico , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Adulto JovenRESUMEN
This protocol describes activation, isolation, and analysis of the CD95 (APO-1/Fas) death-inducing signaling complex (DISC) using affinity purification. Activation is achieved using a biotin-labeled anti-CD95 antibody and the native DISC complex is captured using streptavidin beads. This approach minimizes both the number of steps involved and any potential nonspecific interactions or cross-reactivity of antibodies commonly seen in immunoprecipitations using unlabeled antibodies and protein A/G beads. Composition of the isolated complex is analyzed via western blot to identify known DISC components, and dimerization-induced autocatalytic processing of procaspase-8 at the DISC can be confirmed by detection of caspase-8 cleavage products. The potential for DISC-associated caspase-8 to activate the caspase cascade can be determined by measuring caspase-8-dependent cleavage of the fluorigenic substrate Ac-IETD.AFC, or by performing a bioassay using exogenous protein substrates.
Asunto(s)
Apoptosis , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/análisis , Complejos Multienzimáticos/química , Complejos Multienzimáticos/aislamiento & purificación , Transducción de Señal , Receptor fas/química , Receptor fas/aislamiento & purificación , Autoanticuerpos/metabolismo , Biotina/metabolismo , Western Blotting , Cromatografía de Afinidad , Humanos , Células Jurkat , Microesferas , Complejos Multienzimáticos/metabolismo , Coloración y Etiquetado , Estreptavidina/metabolismo , Receptor fas/metabolismoRESUMEN
We previously reported that the small GTPase Rab27 and its effectors regulate isoproterenol (IPR)-stimulated amylase release from rat parotid acinar cells. Although activation of Rab27 by a specific guanine nucleotide exchange factor (GEF) is thought to be required for amylase release, its activation mechanism is poorly understood, because GEF for Rab27 has not been reported in parotid acinar cells. In the present study, we investigated the possible involvement of MADD/DENN/Rab3GEP, which was recently described as a Rab27-GEF in melanocytes, in amylase release from rat parotid acinar cells. Reverse transcription-PCR analyses indicated that mRNA of DENND family members, including MADD, was expressed in parotid acinar cells. MADD protein was also expressed in the cytosolic fraction of parotid acinar cells. Incubation of an antibody against the C-terminal 150 amino acids of MADD (anti-MADD-C antibody) with streptolysin O-permeabilized parotid acinar cells caused not only inhibition of IPR-induced amylase release but also reduction in the amount of GTP-Rab27. Our findings indicated that MADD functions as a GEF for Rab27 in parotid acinar cells and that its GEF activity for Rab27, i.e., GDP/GTP cycling, is required for IPR-induced amylase release.
Asunto(s)
Células Acinares/citología , Amilasas/metabolismo , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/metabolismo , Exocitosis , Factores de Intercambio de Guanina Nucleótido/metabolismo , Glándula Parótida/citología , Proteínas de Unión al GTP rab/metabolismo , Células Acinares/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/análisis , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/genética , Expresión Génica , Factores de Intercambio de Guanina Nucleótido/análisis , Factores de Intercambio de Guanina Nucleótido/genética , Guanosina Trifosfato/metabolismo , Humanos , Masculino , ARN Mensajero/análisis , ARN Mensajero/genética , Ratas , Ratas Wistar , Estreptolisinas/metabolismo , Proteínas rab27 de Unión a GTPRESUMEN
The extrinsic apoptosis pathway is activated when certain members of the tumor necrosis factor (TNF) receptor superfamily (TNFRSF) are oligomerized by their cognate ligands that are members of the TNF superfamily (TNFSF). The apoptosis-inducing capacity of a member of the TNFRSF relies on the presence of a death domain (DD) in the intracellular portion of the receptor protein. Such receptors are also referred to as death receptors. Binding of a TNFSF ligand to a TNFRSF receptor that is expressed on the surface of a cell results in the formation of a receptor proximal protein complex. This protein complex is the platform for further signaling events within the cell. In case of death receptors like TNF-related apoptosis-inducing ligand receptor 1 (TRAIL-R1/DR4), TRAIL-R2 (KILLER/APO-2/DR5/TRICK), CD95 (Fas, APO-1), or TNF receptor 1 (TNF-R1), this complex is termed death-inducing signaling complex (DISC). The compositions of the various DISCs have been intensively studied in the last 12 years. For the CD95 and the TRAIL-R1/R2 DISCs, it is now clear that the adaptor protein Fas-associated DD protein (FADD) forms part of these complexes and is necessary for recruitment of the proapoptotic signaling molecules caspase-8 and caspase-10. Recruitment of these proteases allows for their activation at the DISC and subsequent induction of apoptosis. The caspase-8 homologous cellular FLICE-like inhibitory protein (cFLIP) can also be recruited to the DISC. cFLIP acts as an anti-apoptotic regulator by interfering with activation of caspases 8 and 10 at the DISC. Interestingly, treatment of TRAIL-resistant tumor cells with conventional chemotherapeutic drugs or with proteasome inhibitors renders these cells sensitive for TRAIL-induced apoptosis. By applying the methodology of the biochemical analysis of the TRAIL DISC described here, we were able to show that this sensitization is mainly due to changes in the biochemical composition of the DISC as the apoptosis-initiating protein complex of the extrinsic pathway.