RESUMO
The expression of genes involved in p53-mediated apoptosis was studied using cDNA microarray after treating isogenic cell lines with either ionizing radiation or doxorubicin. Most of the known p53 transcriptional activation target genes clustered in a functional category defined by early and p53-dependent induction, regardless of the type of stress. Apoptotic protease activating factor-1 (APAF-1) emerged from this analysis as a novel p53 target gene. Genomic sequences upstream of the APAF-1 transcription start site contain a classic p53-responsive element that bound to p53. Consistently, p53 directly induced APAF-1 gene expression. Furthermore, DNA damage-mediated induction of APAF-1 mRNA and protein expression, accompanied by apoptosis, were strictly dependent on wild-type p53 function. These data are consistent with the hypothesis that APAF-1 is an essential downstream effector of p53-mediated apoptosis.
Assuntos
Apoptose/genética , Dano ao DNA , Proteínas/genética , Proteína Supressora de Tumor p53/genética , Fator Apoptótico 1 Ativador de Proteases , Linhagem Celular , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Perfilação da Expressão Gênica , Humanos , Linfócitos/efeitos dos fármacos , Linfócitos/fisiologia , Linfócitos/efeitos da radiação , Família Multigênica , Análise de Sequência com Séries de Oligonucleotídeos , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genética , Ativação Transcricional/efeitos da radiação , Células Tumorais CultivadasRESUMO
The Bloom syndrome (BS) protein, BLM, is a member of the RecQ DNA helicase family that also includes the Werner syndrome protein, WRN. Inherited mutations in these proteins are associated with cancer predisposition of these patients. We recently discovered that cells from Werner syndrome patients displayed a deficiency in p53-mediated apoptosis and WRN binds to p53. Here, we report that analogous to WRN, BLM also binds to p53 in vivo and in vitro, and the C-terminal domain of p53 is responsible for the interaction. p53-mediated apoptosis is defective in BS fibroblasts and can be rescued by expression of the normal BLM gene. Moreover, lymphoblastoid cell lines (LCLs) derived from BS donors are resistant to both gamma-radiation and doxorubicin-induced cell killing, and sensitivity can be restored by the stable expression of normal BLM. In contrast, BS cells have a normal Fas-mediated apoptosis, and in response to DNA damage normal accumulation of p53, normal induction of p53 responsive genes, and normal G(1)-S and G(2)-M cell cycle arrest. BLM localizes to nuclear foci referred to as PML nuclear bodies (NBs). Cells from Li-Fraumeni syndrome patients carrying p53 germline mutations and LCLs lacking a functional p53 have a decreased accumulation of BLM in NBs, whereas isogenic lines with functional p53 exhibit normal accumulation. Certain BLM mutants (C1055S or Delta133-237) that have a reduced ability to localize to the NBs when expressed in normal cells can impair the localization of wild type BLM to NBs and block p53-mediated apoptosis, suggesting a dominant-negative effect. Taken together, our results indicate both a novel mechanism of p53 function by which p53 mediates nuclear trafficking of BLM to NBs and the cooperation of p53 and BLM to induce apoptosis.
