RESUMO
We have identified a novel p53 regulated gene designated DDA3 through differential mRNA display on IW32 erythroleukemia cells containing a temperature sensitive p53 allele, tsp53val-135. DDA3 mRNA induction could be observed in all sublines expressing tsp53val-135 cultured at permissive temperature as well as in NIH3T3 cells undergoing DNA damage. Upregulation of DDA3 could be detected within 2 h after down-shifting the temperature to 32.5 degrees C; upon shifting back to 38.5 degrees C, DDA3 mRNA rapidly degraded with a half-life of less than 2 h. Actinomycin D, but not cycloheximide, inhibited the p53 dependent DDA3 induction, suggesting that the activation is through transcriptional regulation and does not require de novo protein synthesis. DDA3 was expressed in multiple mouse tissues including brain, spleen, lung, kidney and testis. Full-length DDA3 cDNA was cloned and it contained an open reading frame predicted to encode a proline rich protein of 329 amino acids. Overexpression of DDA3 in H1299 lung carcinoma cells suppressed colony formation. These results suggest that DDA3 is a p53-regulated gene that might participate in the p53-mediated growth suppression.
Assuntos
Regulação da Expressão Gênica , Fosfoproteínas/genética , RNA Mensageiro/genética , Células 3T3 , Sequência de Aminoácidos , Animais , Sequência de Bases , Carcinoma Pulmonar de Células não Pequenas , Núcleo Celular/metabolismo , Clonagem Molecular , Regulação Neoplásica da Expressão Gênica , Humanos , Leucemia Eritroblástica Aguda , Neoplasias Pulmonares , Masculino , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , Fosfoproteínas/biossíntese , Fosfoproteínas/química , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transcrição Gênica , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismoRESUMO
We investigated the mechanisms by which transforming growth factor (TGF)-beta2 inhibited prolactin mRNA expression in GH3 rat pituitary tumor cells. Maximal inhibition was observed with cells exposed to 5 ng/ml TGF-beta2 for 24 hr. Continuous presence of the hormone during the entire period was not necessary because exposure of cells to TGF-beta2 for 20 min was sufficient to trigger the same extent of prolactin mRNA inhibition at 24 hr as with its persistent presence. The action of TGF-beta2 could be abolished by cycloheximide or EGTA, suggesting the requirement of a newly synthesized protein and extracellular Ca2+. The response of prolactin mRNA to TGF-beta2 was inhibited by preincubation of cells with phorbol-12-myristate-13-acetate, which down-regulated protein kinase C (PKC). The activities of both the cytosolic and membrane PKC were significantly reduced at 20 min after TGF-beta2 addition, and inhibition continued to 24 hr, the last time point analyzed. However, the ratio of cytosolic to membrane PKC was not altered by TGF-beta2. Inhibition of PKC did not require the sustained presence of TGF-beta2. In vitro kinase assays of the immunoprecipitated PKC demonstrated that the activities of alpha, epsilon, mu, and zeta isozymes were significantly decreased in the TGF-beta2-treated cells, whereas that of PKClambda was not affected. Western blotting did not reveal any change in PKCepsilon steady state protein levels, suggesting TGF-beta2 inhibits PKC activity through a post-translational mechanism. Our results support that inhibition of PKC activity is an early event mediating TGF-beta2-inhibited prolactin mRNA expression in GH3 cells.
