RESUMO
Tumor suppressor p53 has been implicated in cell stress response and determines cell fate of either growth arrest or apoptosis. Heat shock proteins (Hsps) expressed under stress usually confer survival protection to the cell or interruption in the apoptotic pathways. Although Hsp90 can physically interact with p53, whether or not the hsp90 gene is influenced downstream of p53 in UV irradiation-induced apoptosis remains unclear. We have found that the level of p53 is elevated with the decline of Hsp90 in UV-irradiated cells and that malfunction of Hsp90, as inhibited by geldanamycin, enhances the p53-involved UV irradiation-induced apoptosis. In addition, the expression of the hsp90beta gene was reduced in both UV-irradiated and wild type p53-transfected cells. These results suggest a negative correlation between the trans factor p53 and a chaperone gene hsp90beta in apoptotic cells. Mutation analysis demonstrated that the p53 binding site in the first exon was indispensable for p53 regulation on the hsp90beta gene. In addition, with p53 bound at the promoter of the hsp90beta gene, mSin3a and p300 were differentially recruited in UV irradiation-treated or untreated Jurkat cells in vivo. The evidence of p53-repressed hsp90beta gene expression in UV-irradiated cells shed light on a novel pathway of Hsp90 in the survival control of the stressed cells.
Assuntos
Proteínas de Choque Térmico HSP90/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Antibióticos Antineoplásicos/farmacologia , Apoptose , Sequência de Bases , Benzoquinonas , Sítios de Ligação , Western Blotting , Núcleo Celular/metabolismo , Separação Celular , Sobrevivência Celular , Cloranfenicol O-Acetiltransferase/metabolismo , DNA/metabolismo , Análise Mutacional de DNA , Relação Dose-Resposta a Droga , Éxons , Citometria de Fluxo , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Humanos , Imunoprecipitação , Células Jurkat , Lactamas Macrocíclicas , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Plasmídeos/metabolismo , Mutação Puntual , Regiões Promotoras Genéticas , Ligação Proteica , Quinonas/farmacologia , RNA Mensageiro/metabolismo , Fatores de Tempo , Transfecção , Raios UltravioletaRESUMO
OBJECTIVE: To establish a RT-PCR system for detecting mRNA expression level of 4 hsp genes in human cells. METHODS: RT-PCR system was established with gene cloning, gene recombination, in vitro transcription and RT-PCR techniques; The detection of expression level of 4 hsp genes in SW13 cell was carried out with this system. RESULTS: In SW13 cell, hsp70 and hsp90 alpha were typical heat shock induced genes, while hsp60 and hsp90 beta were efficiently expressed and further induced by heat-shock to various extent. CONCLUSIONS: In our hands novel RT-PCR system can be used to detect mRNA expression level of 4 human hsp genes.
