RESUMO
By analyzing the gene expression profile between tumor cells and revertant counterparts that have a suppressed malignant phenotype, we previously reported a significant down-regulation of translationally controlled tumor protein (TCTP) in the revertants. In the present study, we derived, by using the H1 parvovirus as a selective agent, revertants from three major solid cancers: colon, lung, and melanoma cell lines. These cells have a strongly suppressed malignant phenotype both in vitro and in vivo. The level of TCTP is decreased in most of the revertants. To verify whether inhibition of TCTP expression induces changes in the malignant phenotype, in the classical, well established model of "flat reversion," v-src-transformed NIH3T3 cells were transfected with antisense TCTP. By inhibiting the expression of TCTP, the number of revertant cells was raised to 30%, instead of the reported rate for spontaneous flat revertants of 10(-6). Because TCTP encodes for a histamine-releasing factor, we tested the hypothesis that inhibitors of the histaminic pathway could be effective against tumor cells. We show that some antihistaminic compounds (hydroxyzine and promethazine) and other pharmacological compounds with a related structure (including thioridazine and sertraline) kill tumor cells and significantly decrease the level of TCTP. All together, these data suggest that, with tumor reversion used as a working model, TCTP was identified as a target and drugs were selected that decrease its expression and kill tumor cells.
Assuntos
Proteínas de Neoplasias/genética , Neoplasias/patologia , Biossíntese de Proteínas , Animais , Sequência de Bases , Linhagem Celular Transformada , Linhagem Celular Tumoral , Primers do DNA , Humanos , Camundongos , Células NIH 3T3 , Fenótipo , Proteína Tumoral 1 Controlada por TraduçãoRESUMO
Siah proteins are E3 ubiquitin ligases. They are homologues of the Drosophila seven in absentia (Sina), a protein required for the R7 photoreceptor development. We have previously found that the expression of human siah-1 and its mouse homologue siah-1b are induced by p53 during apoptosis and tumor reversion. So far, no evidence that the siah-1b gene is a direct transcriptional target of p53 has been provided. In the present study we investigate this issue. Northern blot analysis with a specific probe demonstrates an increase in siah-1b transcription on activation of endogenous and inducible exogenous p53. To explore whether this effect is directly mediated by p53 we analyzed 20 kb of chromosome X DNA, containing the siah-1b locus. A p53-binding site was identified in the siah-1b promoter, located at nucleotides -2155/-2103 relative to the translational start site. This site is composed of two half-sites, conforming to the p53-binding consensus sequence but separated by a nonclassical 33-bp spacer. In luciferase assays, p53 induces a substantial increase in siah-1b promoter activity. Gel shift and DNase-I-footprinting studies, combined with mutational analysis and chromatin immunoprecipitation, indicate that p53 effectively binds the siah-1b promoter in vitro and in vivo. Thus, the siah-1b gene is a direct transcriptional target of p53.
Assuntos
Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Proteína Supressora de Tumor p53/metabolismo , Animais , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Sequência Consenso , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Técnicas In Vitro , Íntrons , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ativação Transcricional , Ubiquitina-Proteína LigasesRESUMO
The translationally controlled tumor protein (TCTP), also known as histamine-releasing factor (HRF), is encoded by a gene (Tpt1) that is highly conserved throughout phylogeny. TCTP is implicated in cell growth, acute allergic response, and apoptosis. In the present study, seven putative Tpt1 genes with different chromosomal localizations were identified in the mouse genome. In six of them, analysis of the 5' and 3' untranslated regions revealed the presence of flanking direct repeats and residual poly(A) tails typical of pseudogenes. Only three of the seven genes can produce a protein of the expected molecular weight. We isolated the genomic DNA of these three genes to analyze their sequence, genomic organization, and in vitro promoter activity. We found that mouse Tpt1 is localized on chromosome 14 with a canonical intron-exon organization, a functional promoter, and only one transcript that is ubiquitously expressed in all tissues.
