RESUMO
Using a novel cell-based assay to profile transcriptional pathway targeting, we have identified a new functional class of thalidomide analogs with distinct and selective antileukemic activity. These agents activate nuclear factor of activated T cells (NFAT) transcriptional pathways while simultaneously repressing nuclear factor-kappaB (NF-kappaB) via a rapid intracellular amplification of reactive oxygen species (ROS). The elevated ROS is associated with increased intracellular free calcium, rapid dissipation of the mitochondrial membrane potential, disrupted mitochondrial structure, and caspase-independent cell death. This cytotoxicity is highly selective for transformed lymphoid cells, is reversed by free radical scavengers, synergizes with the antileukemic activity of other redox-directed compounds, and preferentially targets cells in the S phase of the cell cycle. Live-cell imaging reveals a rapid drug-induced burst of ROS originating in the endoplasmic reticulum and associated mitochondria just prior to spreading throughout the cell. As members of a novel functional class of "redoxreactive" thalidomides, these compounds provide a new tool through which selective cellular properties of redox status and intracellular bioactivation can be leveraged by rational combinatorial therapeutic strategies and appropriate drug design to exploit cell-specific vulnerabilities for maximum drug efficacy.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Imunossupressores/farmacologia , Leucemia/imunologia , Talidomida/farmacologia , Sinalização do Cálcio/imunologia , Morte Celular/efeitos dos fármacos , Morte Celular/imunologia , Linhagem Celular Transformada , Avaliação Pré-Clínica de Medicamentos/métodos , Retículo Endoplasmático/imunologia , Humanos , Imunidade Celular/efeitos dos fármacos , Imunidade Celular/imunologia , Imunossupressores/uso terapêutico , Leucemia/tratamento farmacológico , Mitocôndrias/imunologia , NF-kappa B/imunologia , Fatores de Transcrição NFATC/imunologia , Oxirredução/efeitos dos fármacos , Espécies Reativas de Oxigênio/imunologia , Talidomida/análogos & derivados , Talidomida/uso terapêuticoRESUMO
Although extensive homology exists between related genes p53 and p73, recent data suggest that the family members have divergent roles. We demonstrate that the differential regulatory roles of p53 family member p73 are highly cell-context and promoter-specific. Full-length p73 expressed in the transformed leukemia cell line Jurkat behaves as a specific dominant negative transcriptional repressor of the cell cycle inhibitor gene p21 and blocks p53-mediated apoptosis. These findings provide evidence for a new mechanism in oncogenesis through which the functional properties of p73 can be altered in an inheritable and cell-specific fashion independent of transcriptional coding.
Assuntos
Apoptose , Proteínas de Ligação a DNA/fisiologia , Genes Dominantes , Leucemia/metabolismo , Proteínas Nucleares/fisiologia , Caspase 3 , Caspases/metabolismo , Linhagem Celular , Linhagem Celular Transformada , Núcleo Celular/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Genes Supressores de Tumor , Humanos , Marcação In Situ das Extremidades Cortadas , Células Jurkat , Luciferases/metabolismo , Microscopia de Fluorescência , Proteínas Nucleares/metabolismo , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Transcrição Gênica , Ativação Transcricional , Transfecção , Proteína Tumoral p73 , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de TumorRESUMO
In this study, we demonstrate that p53 directly inhibits expression of the T cell growth factor (IL-2) in activated T cells. This repression is independent of the intrinsic transcriptional activity of p53 and is mediated by the Tax-responsive CD28RE-3'-12-O-tetradecanoylphorbol-13-acetate response element (AP1) element of the IL-2 promoter. Coexpression of the Tax oncogene causes full reversal of this repression through coordinate targeting of p300, CREB, and the NF-kappaB pathways. Paradoxically, IL-2 repression by p53 is not reversed by mdm2. Instead, mdm2 represses the IL-2 promoter by a mechanism that is synergistic with p53 and resistant to Tax reversal. The p300 structure-function studies show that these effects are linked to competitive associations among p53, Tax, and mdm2 with multiple domains of p300. The functional outcome of these antagonistic associations is revealed further by the observation that Tax and p53 induce apoptosis in activated T cells through separate and mutually exclusive pathways. Interestingly, both pathways are abrogated by mdm2. These results provide evidence that a dynamic interplay, between Tax and specific elements of the p53 network, mediates growth factor expression and programmed cell death in activated T cells.