Transactivational and DNA binding abilities of endogenous p53 in p53 mutant cell lines.
Transactivational and DNA binding abilities of endogenous p53 in p53 mutant cell lines.
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发表时间：
1994-07
期刊：
影响因子：
8
通讯作者：
Dorothy J. Park;H. Nakamura;A. Chumakov;J. Said;C. Miller;Dan-Lin Chen;H. Koeffler
中科院分区：
文献类型：
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作者：
Dorothy J. Park;H. Nakamura;A. Chumakov;J. Said;C. Miller;Dan-Lin Chen;H. Koeffler
具有p53基因的不同突变等位基因的细胞在体外具有不同的生物学和生化特性。越来越多的证据表明，p53是一种转录激活因子，最近，已经确定了p53的高亲和力DNA结合位点。本研究的目的是确定在体内，各种突变p53蛋白对它们的能力，介导的反式激活和特异性结合到DNA的效果。将p53应答或对照报告基因转染到18个人癌细胞系中，这些细胞系具有各种p53突变，有或没有野生型p53表达载体。CAT活性和DNA凝胶阻滞进行了研究，以测量这些内源性p53的反式激活和DNA结合。正如预期的那样，内源性产生的野生型p53与DNA结合序列结合，并且可以反式激活含有p53高亲和力DNA结合位点的报告构建体。在密码子273（273 His）处具有纯合p53突变的五个细胞系中的四个含有具有结合p53 DNA结合序列并反式激活的能力的p53。相反，在其他细胞系中存在的所有纯合的、非密码子273突变的p53（156 Pro，175 His，223 Leu，248 Gln，248 Trp，280 Lys）都没有反式激活能力。这些发现表明，在密码子273处突变的癌症的生物学可能不同于在其他位点具有p53突变的癌症。甲状腺癌细胞株WRO的p53在223位密码子（223 Leu）处发生突变，该突变株的p53能够结合p53 DNA识别序列，但不能反式激活。有趣的是，在一个外阴癌细胞系（A431）与p53突变的密码子273（273 His），p53不能反式激活，并给出了一个异常的凝胶阻滞带。CEM和SK-UT-1在密码子175/248（175 His/248 His）处具有复合杂合突变，产生可与DNA复合并具有反式激活作用的p53。相反，在具有纯合175 His或248 His p53突变的细胞系中，p53不能反式激活或结合p53反应元件。266 Glu p53突变杂合的细胞系（NPA）能够有效地反式激活含有p53 DNA结合位点的报告基因，因此没有证据表明内源性p53突变等位基因的显性负效应。总之，该体内研究进一步支持了不同的p53突变等位基因具有可能影响其功能的各种性质的想法。
Cells with divergent mutant alleles of the p53 gene have different biological and biochemical properties in vitro. Increasing evidence indicates that p53 is a transcriptional activator, and recently, high affinity DNA binding sites for p53 have been identified. The purpose of this study was to determine in vivo, the effect that various mutant p53 proteins have on their ability to mediate transactivation and to bind specifically to DNA. Either a p53 responsive or control reporter gene was transfected into 18 human carcinoma cell lines, having various p53 mutations, either with or without a wild-type p53 expression vector. The CAT activity and DNA gel retardation were studied to measure transactivation and DNA binding by these endogenous p53s. As expected, the endogenously produced wild-type p53 binds to DNA binding sequences and can transactivate a reporter construct containing a p53 high affinity DNA binding site. Four of five cell lines with homozygous p53 mutations at codon 273 (273His), contained p53 which had the ability to bind to p53 DNA binding sequences and transactivate. In contrast, all the homozygous, non-codon 273 mutant p53s (156Pro, 175His, 223Leu, 248Gln, 248Trp, 280Lys) present in the other cell lines had no transactivating ability. These findings suggest that the biology of cancers with mutations at codon 273 may be different than those with p53 mutations at other sites. The p53 from WRO, a thyroid carcinoma cell line with p53 mutation at codon 223 (223Leu), was able to bind p53 DNA recognition sequences, but was unable to transactivate. Interestingly, in a vulvar carcinoma cell line (A431) with a p53 mutation at codon 273 (273His), the p53 was unable to transactivate and gave an aberrant band on gel retardation. Both CEM and SK-UT-1, which have compound heterozygous mutations at codons 175/248 (175His/248His), produced p53 which can complex with DNA, as well as transactivate. In contrast, the p53 in cell lines with either homozygous 175His or 248His p53 mutations, were unable either to transactivate or bind to the p53 response element. A cell line (NPA) heterozygous for 266Glu p53 mutation, was able to efficiently transactivate a reporter containing a p53 DNA binding site, therefore showing no evidence of a dominant negative effect of the endogenous p53 mutant allele. In summary, this in vivo study further supports the idea that different p53 mutant alleles have various properties which may affect their function.