RESUMEN
Human GraB (hGraB) preferentially induces apoptosis via Bcl-2-regulated mitochondrial damage but can also directly cleave caspases and caspase substrates in cell-free systems. How hGraB kills cells when it is delivered by cytotoxic lymphocytes (CL) and the contribution of hGraB to CL-induced death is still not clear. We show that primary human natural killer (hNK) cells, which specifically used hGraB to induce target cell death, were able to induce apoptosis of cells whose mitochondria were protected by Bcl-2. Purified hGraB also induced apoptosis of Bcl-2-overexpressing targets but only when delivered at 5- to 10-fold the concentration required to kill cells expressing endogenous Bcl-2. Caspases were critical in this process as inhibition of caspase activity permitted clonogenic survival of Bcl-2-overexpressing cells treated with hGraB or hNK cells but did not protect cells that only expressed endogenous Bcl-2. Our data therefore show that hGraB triggers caspase activation via mitochondria-dependent and mitochondria-independent mechanisms that are activated in a hierarchical manner, and that the combined effects of Bcl-2 and direct caspase inhibition can block cell death induced by hGraB and primary hNK cells.
Asunto(s)
Apoptosis , Caspasas/metabolismo , Granzimas/metabolismo , Células Asesinas Naturales/enzimología , Mitocondrias/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Vesículas Secretoras/enzimología , Clorometilcetonas de Aminoácidos/farmacología , Apoptosis/efectos de los fármacos , Inhibidores de Caspasas , Técnicas de Cultivo de Célula , Células Cultivadas , Inhibidores de Cisteína Proteinasa/farmacología , Dipéptidos/farmacología , Activación Enzimática , Granzimas/antagonistas & inhibidores , Granzimas/genética , Células HeLa , Humanos , Células Asesinas Naturales/efectos de los fármacos , Mitocondrias/enzimología , Membranas Mitocondriales/metabolismo , Permeabilidad , Inhibidores de Proteasas/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Vesículas Secretoras/efectos de los fármacos , Factores de Tiempo , Transfección , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismoRESUMEN
IFI 16 is a member of the HIN-200 family of transcriptional regulators that suppress cell growth, modulate the cell cycle and have been linked to cellular differentiation. We hypothesized that the activity of IFI 16 depends on its level of expression and therefore studied the transcriptional activity of the IFI 16 promoter. A discrete sequence within the 5' untranslated region was required for constitutive activity of the promoter and the functional motif within this region was shown to be a consensus AP-1 site. Interestingly, this AP-1 site was also critical for IFN-induced activation of the promoter and consistent with these observations, treatment of cells with IFNgamma resulted in a rapid and robust induction of AP-1 activity that preceded expression of IFI 16. These experiments define the transcriptional mechanisms of IFI 16 gene regulation and provide evidence suggesting that AP-1 activation may be an important event in IFN signaling.