Monti Paola, Campomenosi Paola, Ciribilli Yari, Iannone Raffaella, Inga Alberto, Abbondandolo Angelo, Resnick Michael A, Fronza Gilberto
Mutagenesis-Laboratory, National Cancer Research Institute (IST), Largo R. Benzi, 10, 16132-Genova, Italy.
Oncogene. 2002 Mar 7;21(11):1641-8. doi: 10.1038/sj.onc.1205250.
The tumour suppressor gene p53 is frequently mutated in human cancer. Tumour derived p53 mutants are usually transcriptionally inactive, but some mutants retain the ability to transactivate a subset of p53 target genes. In addition to simple loss of function, some p53 mutants may be carcinogenic through a dominant negative mechanism. Aiming at a more general classification of p53 mutants into predictive functional categories it is important to determine (i) which p53 mutants are dominant, (ii) what features characterize dominant mutants and (iii) whether dominance is target gene specific. The ability of 71 p53 mutants to inhibit wild type p53 was determined using a simple yeast transcriptional assay. Approximately 30% of the mutants were dominant. They preferentially affect highly conserved amino acids (P<0.005), which are frequently mutated in tumours (P<0.005), and usually located near the DNA binding surface of the protein (P<0.001). Different tumour-derived amino acid substitutions at the same codon usually have the same dominance phenotype. To determine whether the ability of p53 mutants to inhibit wild type p53 is target gene specific, the dominance towards p21, bax, and PIG3 binding sites was examined. Approximately 40% of the 45 mutants examined were dominant for the p21 (17/45) or PIG3 (20/45) responsive elements and 71% (32/45) were dominant for the bax responsive element. These differences are statistically significant (p21 vs bax, P<0.003; bax vs PIG3, P<0.02, Fisher's exact test) and defined a hierarchy of dominance. Finally, we extended the analysis to a group of mutants isolated in BRCA-associated tumours, some of which retained wild type level of transcription in yeast as well as in human cells, but show gain of function in transformation assays. Since transformation assays require transdominant inhibition of the endogenous wild type allele, one possible explanation for the behaviour of the BRCA-associated mutants is that they adopt conformations able to bind DNA alone but not in mixed tetramers with wild type p53. The yeast data do not support this explanation, because all BRCA-associated mutants that behaved as wild type in transcription assay were recessive in dominance assays.
肿瘤抑制基因p53在人类癌症中经常发生突变。肿瘤衍生的p53突变体通常转录无活性，但一些突变体保留了反式激活p53靶基因子集的能力。除了简单的功能丧失外，一些p53突变体可能通过显性负性机制致癌。为了将p53突变体更普遍地分类为预测性功能类别，确定以下几点很重要：（i）哪些p53突变体是显性的；（ii）显性突变体的特征是什么；（iii）显性是否具有靶基因特异性。使用简单的酵母转录测定法确定了71个p53突变体抑制野生型p53的能力。大约30%的突变体是显性的。它们优先影响高度保守的氨基酸（P<0.005），这些氨基酸在肿瘤中经常发生突变（P<0.005），并且通常位于蛋白质的DNA结合表面附近（P<0.001）。同一密码子处不同的肿瘤衍生氨基酸取代通常具有相同的显性表型。为了确定p53突变体抑制野生型p53的能力是否具有靶基因特异性，检测了对p21、bax和PIG3结合位点的显性。在检测的45个突变体中，约40%（17/45）对p21反应元件或PIG3反应元件（20/45）是显性的，71%（32/45）对bax反应元件是显性的。这些差异具有统计学意义（p21与bax，P<0.003；bax与PIG3，P<0.02，Fisher精确检验），并定义了显性层次。最后，我们将分析扩展到一组在BRCA相关肿瘤中分离的突变体，其中一些在酵母以及人类细胞中保留了野生型转录水平，但在转化试验中显示出功能获得。由于转化试验需要对内源性野生型等位基因进行反式显性抑制，BRCA相关突变体行为的一种可能解释是，它们采用的构象能够单独结合DNA，但不能与野生型p53形成混合四聚体。酵母数据不支持这一解释，因为在转录试验中表现为野生型的所有BRCA相关突变体在显性试验中都是隐性的。
