RESUMO
The cdc25C phosphatase dephosphorylates cdc2 kinase which then in complex with cyclin B can catalyse transition from the G(2) phase to mitosis. We demonstrate that transcription of cdc25C is repressed by p53 in a dose-dependent manner. In stably transfected DLD-1 colorectal adenocarcinoma cells, cdc25C expression is down-regulated when p53 is induced from a (tet)-off-regulated system. In contrast to p53, its homologue p73 is not able to down-modulate cdc25C expression. A previously identified site in the cdc25C promoter can bind p53 in vitro and, when placed in a heterologous construct, is able to activate transcription. However, transcriptional repression by p53 is not mediated through this site but is dependent on a segment containing three CCAAT-boxes. In general down-regulation of cdc25C transcription by reducing the levels of active cdc2 kinase contributes to G(2) arrest and G(2)/M checkpoint control. This reveals functional differences between p73 and p53 in regulating cell division.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/fisiologia , Proteínas Nucleares/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Fosfatases cdc25/genética , Animais , Fator de Ligação a CCAAT/fisiologia , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ligação a DNA/genética , Regulação para Baixo , Genes Supressores de Tumor , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , Elementos de Resposta , Deleção de Sequência , Transfecção , Células Tumorais Cultivadas , Proteína Tumoral p73 , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor , Fosfatases cdc25/biossínteseRESUMO
During normal cell cycles, the function of mitotic cyclin-cdk1 complexes, as well as of cdc25C phosphatase, is required for G2 phase progression. Accordingly, the G2 arrest induced by DNA damage is associated with a down-regulation of mitotic cyclins, cdk1, and cdc25C phosphatase expression. We found that the promoter activity of these genes is repressed in the G2 arrest induced by DNA damage. We asked whether the CCAAT-binding NF-Y modulates mitotic cyclins, cdk1, and cdc25C gene transcription during this type of G2 arrest. In our experimental conditions, the integrity of the CCAAT boxes of cyclin B1, cyclin B2, and cdc25C promoters, as well as the presence of a functional NF-Y complex, is strictly required for the transcriptional inhibition of these promoters. Furthermore, a dominant-negative p53 protein, impairing doxorubicin-induced G2 arrest, prevents transcriptional down-regulation of the mitotic cyclins, cdk1, and cdc25C genes. We conclude that, as already demonstrated for cdk1, NF-Y mediates the transcriptional inhibition of the mitotic cyclins and the cdc25C genes during p53-dependent G2 arrest induced by DNA damage. These data suggest a transcriptional regulatory role of NF-Y in the G2 checkpoint after DNA damage.
Assuntos
Fator de Ligação a CCAAT/metabolismo , Proteínas de Ciclo Celular/genética , Ciclina B/genética , Fase G2/fisiologia , Regiões Promotoras Genéticas/genética , Fosfatases cdc25/genética , Fator de Ligação a CCAAT/genética , Células Cultivadas , Ciclina B1 , Dano ao DNA , Regulação para Baixo , Doxorrubicina/farmacologia , Mitose , Músculo Esquelético/citologia , Transcrição Gênica , Proteína Supressora de Tumor p53/genéticaRESUMO
The tumour suppressor protein p53 has functions in controlling the G(1)/S and G(2)/M transitions. Central regulators for progression from G(2) to mitosis are B-type cyclins complexed with cdc2 kinase. In mammals two cyclin B proteins are found, cyclin B1 and B2. We show that upon treatment of HepG2 cells with 5-fluorouracil or methotrexate, p53 levels increase while concentrations of cyclin B2 mRNA, measured by RT-PCR with the LightCycler system, are reduced. In DLD-1 colorectal adenocarcinoma cells (DLD-1-tet-off-p53) cyclin B1 and B2 mRNA levels drop after expression of wild-type p53 but not after induction of a DNA binding-deficient mutant of p53. Analysis of the cyclin B2 promoter reveals specific repression of this gene by p53. Transfection of wild-type p53 into SaOS-2 cells shuts off transcription from a cyclin B2 promoter-luciferase construct whereas a p53 mutant protein does not. The cyclin B2 promoter does not contain a consensus p53 binding site. Most of the p53-dependent transcriptional responsiveness resides in its 226 bp core promoter. Taken together with earlier observations on p53-dependent transcription of cyclin B1, our results suggest that one way of regulating G(2) arrest may be a reduction in cyclin B levels through p53-dependent transcriptional repression.
Assuntos
Ciclina B/genética , Proteína Supressora de Tumor p53/fisiologia , Ciclina B1 , Ciclina B2 , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Luciferases/genética , Luciferases/metabolismo , Metotrexato/farmacologia , Mutação , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transcrição Gênica , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genéticaRESUMO
Reduced expression of the cyclin-dependent kinase inhibitor p27 has previously been correlated with fatal clinical outcome in some tumors, including gastric, breast, and prostate cancers. For hepatocellular carcinoma, the findings are equivocal. In situ hybridization and immunohistochemistry were performed on a series of 203 curatively (R0) resected hepatocellular carcinomas and in corresponding non-cancerous liver tissue to detect p27. Patients receiving liver transplantation were excluded. The results were correlated with histopathological stage according to the UICC system, Edmondson grade, several other histopathological factors of possible prognostic significance, and finally patient survival. Whereas p27 mRNA was expressed homogeneously in all carcinomas examined, the p27 protein was found in various amounts. The labeling index of p27 protein was significantly lower in advanced stages of the disease (P < 0.001, chi(2) = 28.1). We observed decreased p27 protein in higher pT categories (P < 0.001, chi(2) = 24.7) and in multiple tumor nodules (P < 0.001, chi(2) = 9.3). Multivariate Cox survival analysis identified age, co-existing cirrhosis, and Edmondson grade as independent prognostic factors. We conclude that evaluation of p27 in hepatocellular carcinoma is useful to predict stage of disease and may have clinical significance, e.g., in predicting optimal therapeutic regimes.
