RESUMEN
The temporal gene expression profile during the entire process of apoptosis and cell cycle progression in response to p53 in human ovarian cancer cells was explored with cDNA microarrays representing 33 615 individual human genes. A total of 1501 genes (4.4%) were found to respond to p53 (approximately 80% of these were repressed by p53) using 2.5-fold change as a cutoff. It was anticipated that most of p53 responsive genes resulted from the secondary effect of p53 expression at late stage of apoptosis. To delineate potential p53 direct and indirect target genes during the process of apoptosis and cell cycle progression, microarray data were combined with global p53 DNA-binding site analysis. Here we showed that 361 out of 1501 p53 responsive genes contained p53 consensus DNA-binding sequence(s) in their regulatory region, approximately 80% of which were repressed by p53. This is the first time that a large number of p53-repressed genes have been identified to contain p53 consensus DNA-binding sequence(s) in their regulatory region. Hierarchical cluster analysis of these genes revealed distinct temporal expression patterns of transcriptional activation and repression by p53. More genes were activated at early time points, while more repressed genes were found after the onset of apoptosis. A small-scale quantitative chromatin immunoprecipitation analysis indicated that in vivo p53-DNA interaction was detected in eight out of 10 genes, most of which were repressed by p53 at the early onset of apoptosis, suggesting that a portion of p53 target genes in the human genome could be negatively regulated by p53 via sequence-specific DNA binding. The approaches and genes described here should aid the understanding of global gene regulatory network of p53.
Asunto(s)
Apoptosis/genética , Ciclo Celular/genética , Neoplasias Ováricas/genética , Transcripción Genética , Proteína p53 Supresora de Tumor/genética , Adenoviridae/genética , Secuencia de Aminoácidos , Sitios de Unión , Cromatina/metabolismo , Análisis por Conglomerados , Secuencia Conservada , Femenino , Perfilación de la Expresión Génica , Genoma Humano , Humanos , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Neoplasias Ováricas/patología , Pruebas de Precipitina , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transfección/métodos , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Survivin is an inhibitor of apoptosis protein, which is over-expressed in most tumors. Aberrant expression of survivin and loss of wild-type p53 in many tumors prompted us to investigate a possible link between these two events. Here we show that wild-type p53 represses survivin expression at both mRNA and protein levels. Transient transfection analyses revealed that the expression of wild-type p53, but not mutant p53, was associated with strong repression of the survivin promoter in various cell types. The over-expression of exogenous survivin protein rescues cells from p53-induced apoptosis in a dose-dependent manner, suggesting that loss of survivin mediates, at least, in part the p53-dependent apoptotic pathway. In spite of the presence of two putative p53-binding sites in the survivin promoter, deletion and mutation analyses suggested that neither site is required for transcriptional repression of survivin expression. This was confirmed by chromatin immunoprecipitation assays. Further analyses suggested that the modification of chromatin within the survivin promoter could be a molecular explanation for silencing of survivin gene transcription by p53.
Asunto(s)
Apoptosis , Proteínas Cromosómicas no Histona/genética , Inhibidores de Cisteína Proteinasa/genética , Proteínas Asociadas a Microtúbulos , Proteína p53 Supresora de Tumor/genética , Adenoviridae , Antibióticos Antineoplásicos/farmacología , Secuencia de Bases , Western Blotting , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Inhibidores de Cisteína Proteinasa/metabolismo , Cartilla de ADN/química , Doxorrubicina/farmacología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas Inhibidoras de la Apoptosis , Luciferasas/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Datos de Secuencia Molecular , Proteínas de Neoplasias , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Pruebas de Precipitina , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , ARN Neoplásico/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Survivin , Transcripción Genética , Transfección , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
In this study we used adenovirus vector-mediated transduction of either the p53 gene (rAd-p53) or the p21(WAF1/CIP1) gene (rAd-p21) to mimic both p53-dependent and -independent up-regulation of p21(WAF1/CIP1) within a human ovarian cancer cell line, 2774, and the derivative cell lines, 2774qw1 and 2774qw2. We observed that rAd-p53 can induce apoptosis in both 2774 and 2774qw1 cells but not in 2774qw2 cells. Surprisingly, overexpression of p21(WAF1/CIP1) also triggered apoptosis within these two cell lines. Quantitative reverse transcription-PCR analysis revealed that the differential expression of BAX, BCL2, and caspase 3 genes, specific in rAd-p53-induced apoptotic cells, was not altered in rAd-p21-induced apoptotic cells, suggesting p21(WAF1/CIP1)-induced apoptosis through a pathway distinguishable from p53-induced apoptosis. Expression analysis of 2774qw1 cells infected with rAd-p21 on 60,000 cDNA microarrays identified 159 genes in response to p21(WAF1/CIP1) expression in at least one time point with 2.5-fold change as a cutoff. Integration of the data with the parallel microarray experiments with rAd-p53 infection allowed us to extract 66 genes downstream of both p53 and p21(WAF1/CIP1) and 93 genes in response to p21(WAF1/CIP1) expression in a p53-independent pathway. The genes in the former set may play a dual role in both p53-dependent and p53-independent pathways, and the genes in the latter set gave a mechanistic molecular explanation for p53-independent p21(WAF1/CIP1)-induced apoptosis. Furthermore, promoter sequence analysis suggested that transcription factor E2F family is partially responsible for the differential expression of genes following p21(WAF1/CIP1). This study has profound significance toward understanding the role of p21(WAF1/CIP1) in p53-independent apoptosis.