RESUMO
Interferon alpha (IFN-alpha) inhibits growth, at least in part, through induction of apoptosis. However, the molecular mechanisms underlying IFN-alpha-induced apoptosis are not completely understood. In the present study, we found that IFN-alpha induced a sustained activation of c-Jun N-terminal kinase 1 (JNK1), but not extracellular kinases (ERKs), in Daudi B lymphoma cells, as assessed by Western blotting using phospho-specific antibodies. Several lines of evidence support the notion that the IFN-alpha-induced activation of JNK is responsible for IFN-alpha-induced apoptosis, at least in part, through upregulation of TNF-related apoptosis-inducing ligand (TRAIL). First, pretreatment of Daudi cells with a JNK inhibitor reduced IFN-alpha-induced upregulation of TRAIL and loss of mitochondrial membrane potential (DeltaPsim) and annexin-positive cells, which was assessed by flow cytometry. Second, a dominant-negative form of JNK1 (dnJNK1) also reduced these apoptotic events, while a constitutively active form of JNK1, MKK7-JNK1beta, enhanced them. Finally, treatment with IFN-alpha enhanced the promoter activity of the TRAIL gene, which was partially abrogated by either JNK inhibitor or dnJNK1, while it was moderately enhanced by MKK7-JNK1beta. These findings are useful for understanding molecular mechanisms of IFN-alpha-induced apoptosis and also for development of treatment modalities of some tumors with IFN-alpha.
Assuntos
Apoptose , Interferon-alfa/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/biossíntese , Linfoma de Células B/metabolismo , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genética , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Linhagem Celular Tumoral , Ativação Enzimática , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/efeitos dos fármacos , Linfoma de Células B/tratamento farmacológico , Proteínas de Transporte da Membrana Mitocondrial/fisiologia , Regiões Promotoras Genéticas , Ligante Indutor de Apoptose Relacionado a TNF , Células Tumorais Cultivadas , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
Type I interferon (IFN)-induced antitumor action is due in part to apoptosis, but the molecular mechanisms underlying IFN-induced apoptosis remain largely unresolved. In the present study, we demonstrate that IFN-beta induced apoptosis and the loss of mitochondrial membrane potential (delta psi m) in the murine CH31 B lymphoma cell line, and this was accompanied by the up-regulation of CD95, but not CD95-ligand (CD95-L), tumor necrosis factor (TNF), or TNF-related apoptosis-inducing ligand (TRAIL). Pretreatment with anti-CD95-L mAb partially prevented the IFN-beta-induced loss of delta psi m, suggesting that the interaction of IFN-beta-up-regulated CD95 with CD95-L plays a crucial role in the induction of fratricide. IFN-beta induced a sustained activation of c-Jun NH2-terminal kinase 1 (JNK1), but not extracellular signal-regulated kinases (ERKs). The IFN-beta-induced apoptosis and loss of delta psi m were substantially compromised in cells overexpressing a dominant-negative form of JNK1 (dnJNK1), and it was slightly enhanced in cells carrying a constitutively active JNK construct, MKK7-JNK1 fusion protein. The IFN-beta-induced up-regulation of CD95 together with caspase-8 activation was also abrogated in the dnJNK1 cells while it was further enhanced in the MKK7-JNK1 cells. The levels of cellular FLIP (c-FLIP), competitively interacting with caspase-8, were down-regulated by stimulation with IFN-beta but were reversed by the proteasome inhibitor lactacystin. Collectively, the IFN-beta-induced sustained activation of JNK mediates apoptosis, at least in part, through up-regulation of CD95 protein in combination with down-regulation of c-FLIP protein.
Assuntos
Apoptose/efeitos dos fármacos , Linfócitos B/efeitos dos fármacos , Interferon beta/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/efeitos dos fármacos , Linfoma de Células B/tratamento farmacológico , Receptor fas/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Citocinas/efeitos dos fármacos , Citocinas/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Proteína Ligante Fas , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Células Jurkat , Linfoma de Células B/imunologia , Linfoma de Células B/metabolismo , Glicoproteínas de Membrana/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mutação/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
CD40, a member of the tumor necrosis factor receptor (TNFR) family, promotes IgM, IgG, and IgA antibody (Ab) synthesis in combination with a variety of cytokines. Another TNFR family member, CD27, causes B cells to differentiate into antibody-forming cells, with marginal effects on proliferation. In the present study, we examined whether anti-CD27 monoclonal antibody (mAb) modulates the antibody production induced by anti-CD40 mAb immobilized on L cells expressing FcgammaRII (CDw32) in the presence of IL-2 and/or IL-10. The anti-CD40 mAb substantially enhanced IgM, IgG, and IgA production in combination with IL-2 and IL-10, whereas anti-CD27 mAb augmented it only marginally, as assessed by enzyme-linked immunosorbent assay. The addition of anti-CD27 mAb enhanced the anti-CD40-mediated IgM, IgG, and IgA antibody production only when both IL-2 and IL-10 were present in the culture. The CD27-positive B cell compartment generated synergistic antibody responses in response to four different stimulants, anti-CD27/anti-CD40 mAb and cytokines IL-2/IL-10, whereas the CD27-negative B cell compartment failed to do so. Kinetic analysis showed that anti-CD40 might function in the early phase of B cell activation, while anti-CD27 mAb functioned in the late stage. The addition of CD27(-) to CD27(+) B cells in various ratios did not have any effect on the antibody production, suggesting that CD27(+) to CD27(-) B cell interaction does not occur in this system. Our findings suggest that a member of the TNFR family, CD27, cooperates with CD40 to induce efficient antibody production in combination with cytokines IL-2 and IL-10.