RESUMO
In the present study, we have investigated the mechanisms by which the restoration of wild-type (wt) p53 functions in p53 mutant cells increases their susceptibility to the cytotoxic action of tumor necrosis factor (TNF). Our data indicate that the resistance of p53-mutated cl.1001 cells to TNF-induced cell death was not due to a defect in the expression of TRADD and FADD, yet correlated with a reduced caspase-8 activation as well as a deficient mitochondrial membrane permeabilization. Moreover, cl.1001 cells failed to translocate the mitochondrial AIF and cytochrome c to the nucleus and to the cytosol, respectively, in response to TNF. Sensitization of these cells, following infection with a recombinant adenovirus encoding wtp53, to TNF-induced cytotoxicity resulted in the restoration of caspase-8 cleavage and the reestablishment of mitochondrial signs of apoptosis. These findings suggest that the cross-talk between p53 and TNF-induced cell death depends on mitochondria and that the combination of TNF and Adwtp53 may be a potential strategy to sensitize mutant p53 TNF-resistant tumors to the cytotoxic action of this cytokine.
Assuntos
Proteínas de Arabidopsis , Neoplasias da Mama/metabolismo , Caspases/metabolismo , Canais Iônicos/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Adenoviridae/genética , Fator de Indução de Apoptose , Western Blotting , Neoplasias da Mama/terapia , Caspase 8 , Caspase 9 , Sobrevivência Celular/efeitos dos fármacos , Grupo dos Citocromos c/metabolismo , Resistência a Medicamentos , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Flavoproteínas/metabolismo , Citometria de Fluxo , Genes Dominantes , Humanos , Potenciais da Membrana/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Microscopia Confocal , Microscopia de Fluorescência , Proteínas de Transporte da Membrana Mitocondrial , Poro de Transição de Permeabilidade Mitocondrial , Proteínas/genética , Proteínas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator 1 Associado a Receptor de TNF , Transfecção , Células Tumorais Cultivadas/citologia , Fator de Necrose Tumoral alfa/farmacologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/farmacologiaRESUMO
Inactivation of p53 has been implicated in many types of tumors particularly in non-small cell lung carcinoma, one of the most common cancers in which p53 mutation has been frequently identified. The aim of this study was to investigate the influence of p53 status on the regulation of tumor susceptibility to specific CTL-mediated cell death. For this purpose, we used a cytotoxic T lymphocyte clone, Heu127, able to lyse the human autologous lung carcinoma cell line, IGR-Heu, in a HLA-A2-restricted manner. Direct genomic DNA sequencing revealed that IGR-Heu expresses a mutated p53 at codon 132 of the exon 5 which results in the loss of p53 capacity to induce the expression of the p53-regulated gene product p21(waf/CIP1). Initial experiments demonstrated that IGR-Heu was resistant to Fas, TNF, and TRAIL apoptotic pathways. This correlated with the lack of p55 TNFRI, Fas, DR4, and DR5 expression. The effect of wild-type (wt) p53 restoration on the sensitization of IGR-Heu to autologous CTL clone lysis was investigated following infection of the tumor cell line with a recombinant adenovirus encoding the wt p53 (Adwtp53). We demonstrate that the restoration of wt p53 expression and function resulted in a significant potentiation of target cell susceptibility to CTL-mediated lysis. The wt p53-induced optimization of tumor cell killing by specific CTL involves at least in part Fas-mediated pathway via induction of CD95 expression by tumor cells but does not appear to interfere with granzyme B cytotoxic pathway.
Assuntos
Citotoxicidade Imunológica/genética , Inativação Gênica/imunologia , Proteínas de Neoplasias/fisiologia , Linfócitos T Citotóxicos/imunologia , Proteína Supressora de Tumor p53/fisiologia , Adenoviridae/genética , Apoptose/genética , Apoptose/imunologia , Células Clonais , Testes Imunológicos de Citotoxicidade , Vetores Genéticos , Granzimas , Humanos , Imunidade Inata/genética , Mutação , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Receptores do Fator de Necrose Tumoral/biossíntese , Serina Endopeptidases/fisiologia , Linfócitos T Citotóxicos/metabolismo , Linfócitos T Citotóxicos/patologia , Transativadores/genética , Transativadores/metabolismo , Transativadores/fisiologia , Ativação Transcricional/imunologia , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Receptor fas/biossíntese , Receptor fas/fisiologiaRESUMO
NO is a potent cellular mediator which has been shown to modulate several immune mechanisms. Using human T lymphocytes as responder cells in a primary mixed lymphocyte reaction, we demonstrated that, at the initiation of the culture, exogenously provided NO via sodium nitroprusside, in non-toxic concentrations, inhibited both allogeneic proliferative and primary cytotoxic responses in a dose-dependent manner. In contrast, it had no effect on the cytotoxic activity of established human TCR (alpha)beta and TCR (gamma)delta cytotoxic T lymphocyte (CTL) clones. The NO inhibitory effect on primary cytotoxic T cell response correlates with inhibition of T cell blastogenesis. Furthermore, under our stimulation conditions, NO induced an inhibition of IL-2 production, an alteration of IL-2R(alpha) expression, and a down-regulation of NF-AT translocation in CD4(+) and CD8(+)allostimulated T cells. Furthermore, we demonstrate that the inhibition of allospecific CTL activity by the NO donor was at least in part related to an inhibition of granzyme B and Fas ligand transcription as revealed respectively by RNase protection and RT-PCR analysis. These results suggest that NO may function to fine tune human CD3(+) T cell activation and subsequent CTL generation.