CP-31398 restores DNA-binding activity to mutant p53 in vitro but does not affect p53 homologs p63 and p73
CP-31398 restores DNA-binding activity to mutant p53 in vitro but does not affect p53 homologs p63 and p73
复制标题
DOI：
10.1074/jbc.m401854200
复制
发表时间：
2004-10-29
影响因子：
4.8
通讯作者：
Dasmahapatra, B
中科院分区：
文献类型：
--
作者：
Demma, MJ;Wong, S;Dasmahapatra, B
p53蛋白在维持哺乳动物细胞的基因组稳定性中起主要作用。p53突变发生在超过50%的所有癌症中，并且指示难以治疗的高度侵袭性癌症。最近，人们对恢复突变型p53的生长抑制功能的治疗方法产生了高度兴趣。已经报道了几种化合物恢复突变型p53的野生型功能。其中一种化合物CP-31398已被证明在体内有效，但问题是它是否真的影响p53。在这里，我们表明，突变型p53，分离与CP-31398处理的细胞，是能够结合p53反应元件在体外。我们还表明，该化合物恢复DNA结合活性的突变p53细胞染色质免疫沉淀测定。此外，使用纯化的p53核心结构域从两个不同的热点突变体（R273 H和R249 S），我们表明，CP-31398可以恢复DNA结合活性的剂量依赖性方式。使用定量DNA结合试验，我们还表明CP-31398显著增加与同源DNA结合的突变型p53的量（B-max）及其对DNA的亲和力（K-d）。然而，该化合物不影响野生型p53对DNA的亲和力（K-d值），并且仅略微增加Bmax。在类似的测定中，PRIMA 1对p53核心DNA结合活性没有任何影响。我们还表明，CP-31398对p53同源物p63和p73的DNA结合活性没有影响。
The p53 protein plays a major role in the maintenance of genome stability in mammalian cells. Mutations of p53 occur in over 50% of all cancers and are indicative of highly aggressive cancers that are hard to treat. Recently, there has been a high degree of interest in therapeutic approaches to restore growth suppression functions to mutant p53. Several compounds have been reported to restore wild type function to mutant p53. One such compound, CP-31398, has been shown effective in vivo, but questions have arisen to whether it actually affects p53. Here we show that mutant p53, isolated from cells treated with CP-31398, is capable of binding to p53 response elements in vitro. We also show the compound restores DNA-binding activity to mutant p53 in cells as determined by a chromatin immunoprecipitation assay. In addition, using purified p53 core domain from two different hotspot mutants (R273H and R249S), we show that CP-31398 can restore DNA-binding activity in a dose-dependent manner. Using a quantitative DNA binding assay, we also show that CP-31398 increases significantly the amount of mutant p53 that binds to cognate DNA (B-max) and its affinity (K-d) for DNA. The compound, however, does not affect the affinity (K-d value) of wild type p53 for DNA and only increases Bmax slightly. In a similar assay PRIMA1 does not have any effect on p53 core DNA-binding activity. We also show that CP-31398 had no effect on the DNA-binding activity of p53 homologs p63 and p73.