ANALYSIS OF P53 IN TUMOR CELL RESPONSE TO HYPOXIA

肿瘤细胞对缺氧反应中 P53 的分析

• 批准号: 6123754
• 负责人: L M NECKERS
• 金额: --
• 依托单位: DIVISION OF CLINICAL SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6123754
• 关键词: cobalt; deferoxamine; gene induction /repression; hypoxia; immunoprecipitation; molecular oncology; tissue /cell culture; transcription factor; transfection; tumor suppressor genes; tumor suppressor proteins

"Hypoxia-induced apoptosis of tumor cells in vivo requires wild type p53, and hypoxia is perhaps the most physiologic inducer of wtp53. However, the mechanism by which hypoxia induces p53 has remained elusive. Cells sense low oxygen tension via a heme-containing sensor protein, and the hypoxic state can thus be mimicked by both cobalt chloride and the iron chelator desferrioxamine. Like hypoxia, cobalt and DFX induce the novel transcription factor hypoxia-inducible factor 1 alpha (HIF-1alpha), which stimulates transcription of a number of hypoxia-related genes, including erythropoietin and vascular endothelial growth factor. We have shown in this study that cobalt and DFX both induce wtp53 via protein stabilization, without change in p53 mRNA level. Induction of p53 does not occur in a mutant hepatoma cell line unable to induce HIF-1alpha following exposure to hypoxic stimuli, nor in an embryonic stem cell line in which the HIF-1 gene has been knocked out. p53 immunoprecipitates from wtp53-expressing MCF7 cells exposed to DFX contain HIF-1alpha. Likewise, HA immunoprecipitates from p53-null PC3M cells co-transfected with HA-HIF-1alpha and wtp53 contain p53. These data suggest that HIF-1alpha and p53 proteins directly associate under hypoxic conditions. Finally, transfection of normoxic MCF7, but not PC3M, cells with HIF-1alpha alone stimulates the activity of a co-transfected p53-dependent reporter plasmid and elevates the steady-state level of endogenous p53 protein, supporting the hypothesis that hypoxic induction of wtp53 occurs via protein stabilization dependent on direct association with HIF-1alpha. Ongoing studies are examining mechanisms of HIF-1alpha stability in hypoxia and stimulation of nuclear entry of this protein. Additionally, a panel of p53 mutants are being studied to correlate inhibitory effects on HIF activity with p53 transcriptional activity and binding to HIF and/or p300. The mechanism by which hypoxia causes nuclear translocation of p53 is also being studied."

体内低氧诱导肿瘤细胞凋亡 需要野生型p53，而缺氧可能是最具生理学意义的 Wtp53的诱导物。然而，缺氧的机制 诱导P53基因的表达仍然难以捉摸。细胞感觉到低氧分压 通过一种含有血红素的感受器蛋白，缺氧状态可以 因此可以被氯化钴和铁络合剂所模仿 去铁胺。就像缺氧一样，钴和DFX导致了新的 转录因子缺氧诱导因子1α(HIF-1α)， 它刺激低氧相关的一些转录 促红细胞生成素等基因与血管内皮细胞生长 因素。我们在这项研究中已经表明，钴和DFX都 通过蛋白质稳定诱导wtP53，不改变P53 M RNA水平。突变体中未发生P53的诱导 暴露后不能诱导HIF-1α表达的肝癌细胞株 对低氧刺激，也不是在胚胎干细胞系中 HIF-1基因已被敲除。P53免疫沉淀来自于 经DFX处理的WtP53表达的MCF7细胞含有HIF-1α。 同样，HA免疫沉淀来自P53缺失的PC3M细胞 共转染HA-HIF-1α和wtP53的细胞中均含有P53。这些 数据表明，HIF-1α和P53蛋白直接相关 在低氧条件下。最后，转染常氧MCF7， 但不是PC3M，单独含有HIF-1α的细胞可以刺激这种活性 共转染的P53依赖的报告质粒，并提高 内源性P53蛋白的稳定水平，支持 低氧诱导wtp53是通过蛋白质发生的假说 稳定性依赖于与HIF-1α的直接联系。 正在进行的研究正在研究HIF-1α稳定性的机制 在缺氧和刺激下这种蛋白质的核进入。 此外，正在对一组p53突变进行研究，以确定其与 P53转录活性对HIF活性的抑制作用 并与HIF和/或P300结合。缺氧的机制 导致P53核转位的因素也在研究中。