Inhibition of p53-mediated transcriptional responses by mithramycin A
Inhibition of p53-mediated transcriptional responses by mithramycin A
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DOI：
10.1038/sj.onc.1208141
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发表时间：
2004-12-09
期刊：
影响因子：
8
通讯作者：
Kardassis, D
中科院分区：
文献类型：
--
作者：
Koutsodontis, G;Kardassis, D
在目前的工作中，我们证明了米曲霉素A，一种目前用于治疗骨佩吉特氏病和几种形式的癌症的药物，是人类肝癌细胞中肿瘤抑制蛋白P53的强烈激活剂。米曲霉素A激活P53的时间过程与已知的化疗化合物5-氟尿嘧啶(5-FU)相似。与5-FU不同，5-FU和米曲霉素A均可诱导P53丝氨酸15位特异性磷酸化，但不能激活P53靶基因，包括细胞周期抑制基因p21(Cip1)和促凋亡基因PUMA(P53上调的细胞凋亡介导物)和BAK(BCL2同源拮抗剂/杀手)，并阻断5-FU对上述基因的诱导作用。在Sp1缺陷细胞中的反式激活实验表明，米曲霉素A抑制了Sp1和P53对p21(Cip1)和PUMA启动子的转录激活。利用染色质免疫沉淀法和一种新的基于生物素化的蛋白质-蛋白质相互作用分析方法，我们证实了5-FU促进了溶液中P53-Sp1复合体的形成以及随后这两个因子对p21(Cip1)启动子的募集。米曲霉素A还促进P53对p21(Cip1)远端启动子的募集，但完全阻断Sp1对近端p21(Cip1)启动子的募集。我们的发现表明，米曲霉素A抑制Sp1与几个p53靶基因的启动子，如p21(Cip1)基因和某些促凋亡基因的结合，阻止了p53对这些基因的转录诱导，并提出了一种机制，可能解释了米特拉霉素A的某些肿瘤抑制和抗凋亡作用。
In the present work, we show that mithramycin A, a drug that is currently used for the treatment of patients with Paget's disease of the bone as well as with several forms of cancer, is a strong activator of the tumor suppressor p53 protein in human hepatoma cells. The time course of p53 activation by mithramycin A was similar to the known chemotherapeutic compound 5-fluorouracil (5-FU). Both 5-FU and mithramycin A induced site-specific phosphorylation of p53 at serine 15. However, in contrast to 5-FU, mithramycin A failed to activate p53 target genes including the cell cycle inhibitor p21(Cip1) gene as well as the proapoptotic genes PUMA (p53-upregulated mediator of apotosis) and BAK (bcl2-homologous antagonist/killer) and blocked the induction of the above genes by 5-FU. Using transactivation assays in Sp1-deficient cells, we showed that mithramycin A inhibited the transcriptional activation of the p21(Cip1) and PUMA promoters by Sp1 and p53. Using chromatin immunoprecipitation assays and a novel protein-protein interaction assay based on biotinylation in vivo, we established that 5-FU enhanced the formation of p53-Sp1 complexes in solution and the subsequent recruitment of both factors to the p21(Cip1) promoter. Mithramycin A also enhanced the recruitment of p53 to the distal p21(Cip1) promoter but totally blocked the recruitment of Sp1 to the proximal p21(Cip1) promoter. Our findings suggest that inhibition of Sp1 binding to the promoters of several p53 target genes, such as the p21(Cip1) gene as well as certain proapoptotic genes, by mithramycin A, prevents the transcriptional induction of these genes by p53 and propose a mechanism that could account for some of the tumor suppressing and antiapoptotic effects of mithramycin A.