RESUMEN
Virus-induced activation of nuclear factor-kappa B (NF-kappaB) is required for Type 3 (T3) reovirus-induced apoptosis. We now show that NF-kappaB is also activated by the prototypic Type 1 reovirus strain Lang (T1L), which induces significantly less apoptosis than T3 viruses, indicating that NF-kappaB activation alone is not sufficient for apoptosis in reovirus-infected cells. A second phase of virus-induced NF-kappaB regulation, where NF-kappaB activation is inhibited at later times following infection with T3 Abney (T3A), is absent in T1L-infected cells. This suggests that inhibition of NF-kappaB activation at later times post infection also contributes to reovirus-induced apoptosis. Reovirus-induced inhibition of stimulus-induced activation of NF-kappaB is significantly associated with apoptosis following infection of HEK293 cells with reassortant reoviruses and is determined by the T3 S1 gene segment, which is also the primary determinant of reovirus-induced apoptosis. Inhibition of stimulus-induced activation of NF-kappaB also occurs following infection of primary cardiac myocytes with apoptotic (8B) but not non-apoptotic (T1L) reoviruses. Expression levels of the NF-kappaB-regulated cellular FLICE inhibitory protein (cFLIP) reflect NF-kappaB activation in reovirus-infected cells. Further, inhibition of NF-kappaB activity and cFLIP expression promote T1L-induced apoptosis. These results demonstrate that inhibition of stimulus-induced activation of NF-kappaB and the resulting decrease in cFLIP expression promote reovirus-induced apoptosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Orthoreovirus Mamífero 3/fisiología , FN-kappa B/antagonistas & inhibidores , Orthoreovirus de los Mamíferos/fisiología , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD , Línea Celular , Regulación de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Miocitos Cardíacos/virología , Virus Reordenados/fisiología , Infecciones por Reoviridae/fisiopatologíaRESUMEN
TRAIL (TNF-related apoptosis-inducing ligand) induces apoptosis in susceptible cells by binding to death receptors 4 (DR4) and 5 (DR5). TRAIL preferentially induces apoptosis in transformed cells and the identification of mechanisms by which TRAIL-induced apoptosis can be enhanced may lead to novel cancer chemotherapeutic strategies. Here we show that reovirus infection induces apoptosis in cancer cell lines derived from human breast, lung and cervical cancers. Reovirus-induced apoptosis is mediated by TRAIL and is associated with the release of TRAIL from infected cells. Reovirus infection synergistically and specifically sensitizes cancer cell lines to killing by exogenous TRAIL. This sensitization both enhances the susceptibility of previously resistant cell lines to TRAIL-induced apoptosis and reduces the amount of TRAIL needed to kill already sensitive lines. Sensitization is not associated with a detectable change in the expression of TRAIL receptors in reovirus-infected cells. Sensitization is associated with an increase in the activity of the death receptor-associated initiator caspase, caspase 8, and is inhibited by the peptide IETD-fmk, suggesting that reovirus sensitizes cancer cells to TRAIL-induced apoptosis in a caspase 8-dependent manner. Reovirus-induced sensitization of cells to TRAIL is also associated with increased cleavage of PARP, a substrate of the effector caspases 3 and 7.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis , Caspasas/fisiología , Glicoproteínas de Membrana/farmacología , Neoplasias/patología , Neoplasias/virología , Orthoreovirus de los Mamíferos/fisiología , Factor de Necrosis Tumoral alfa/farmacología , Proteínas Reguladoras de la Apoptosis , Caspasa 8 , Caspasa 9 , Doxorrubicina/farmacología , Células HeLa , Humanos , Glicoproteínas de Membrana/fisiología , Neoplasias/tratamiento farmacológico , Poli(ADP-Ribosa) Polimerasas/metabolismo , ARN Neoplásico/biosíntesis , Receptores del Factor de Necrosis Tumoral/biosíntesis , Receptores del Factor de Necrosis Tumoral/genética , Ligando Inductor de Apoptosis Relacionado con TNF , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
Viral infection often perturbs host cell signaling pathways including those involving mitogen-activated protein kinases (MAPKs). We now show that reovirus infection results in the selective activation of c-Jun N-terminal kinase (JNK). Reovirus-induced JNK activation is associated with an increase in the phosphorylation of the JNK-dependent transcription factor c-Jun. Reovirus serotype 3 prototype strains Abney (T3A) and Dearing (T3D) induce significantly more JNK activation and c-Jun phosphorylation than does the serotype 1 prototypic strain Lang (T1L). T3D and T3A also induce more apoptosis in infected cells than T1L, and there was a significant correlation between the ability of these viruses to phosphorylate c-Jun and induce apoptosis. However, reovirus-induced apoptosis, but not reovirus-induced c-Jun phosphorylation, is inhibited by blocking TRAIL/receptor binding, suggesting that apoptosis and c-Jun phosphorylation involve parallel rather than identical pathways. Strain-specific differences in JNK activation are determined by the reovirus S1 and M2 gene segments, which encode viral outer capsid proteins (sigma1 and mu1c) involved in receptor binding and host cell membrane penetration. These same gene segments also determine differences in the capacity of reovirus strains to induce apoptosis, and again a significant correlation between the capacity of T1L x T3D reassortant reoviruses to both activate JNK and phosphorylate c-Jun and to induce apoptosis was shown. The extracellular signal-related kinase (ERK) is also activated in a strain-specific manner following reovirus infection. Unlike JNK activation, ERK activation could not be mapped to specific reovirus gene segments, suggesting that ERK activation and JNK activation are triggered by different events during virus-host cell interaction.
Asunto(s)
Proteínas de la Cápside , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Reoviridae/fisiología , Animales , Apoptosis/fisiología , Cápside/genética , Línea Celular , Activación Enzimática , Hemaglutininas , Proteínas Quinasas JNK Activadas por Mitógenos , Ratones , Receptores del Factor de Necrosis Tumoral/fisiología , Reoviridae/genética , Transducción de Señal , Proteínas Virales/genéticaRESUMEN
Members of the tumor necrosis factor (TNF) receptor superfamily and their activating ligands transmit apoptotic signals in a variety of systems. We now show that the binding of TNF-related, apoptosis-inducing ligand (TRAIL) to its cellular receptors DR5 (TRAILR2) and DR4 (TRAILR1) mediates reovirus-induced apoptosis. Anti-TRAIL antibody and soluble TRAIL receptors block reovirus-induced apoptosis by preventing TRAIL-receptor binding. In addition, reovirus induces both TRAIL release and an increase in the expression of DR5 and DR4 in infected cells. Reovirus-induced apoptosis is also blocked following inhibition of the death receptor-associated, apoptosis-inducing molecules FADD (for FAS-associated death domain) and caspase 8. We propose that reovirus infection promotes apoptosis via the expression of DR5 and the release of TRAIL from infected cells. Virus-induced regulation of the TRAIL apoptotic pathway defines a novel mechanism for virus-induced apoptosis.