Campomenosi P, Monti P, Aprile A, Abbondandolo A, Frebourg T, Gold B, Crook T, Inga A, Resnick M A, Iggo R, Fronza G
Mutagenesis-Laboratory, National Cancer Research Institute (IST), Largo R. Benzi, 10, 16132-Genova, Italy.
Oncogene. 2001 Jun 14;20(27):3573-9. doi: 10.1038/sj.onc.1204468.
The human p53 protein acts mainly as a stress inducible transcription factor transactivating several genes involved in cell cycle arrest (e.g. p21) or apoptosis (e.g. Bax, PIG3). Roughly half of all human tumours contains p53 missense mutations. Virtually all tumour-derived p53 mutants are unable to activate Bax transcription but some retain the ability to activate p21 transcription. Identification of these mutants may have valuable clinical implications. We have determined the transactivation ability of 77 p53 mutants using reporter yeast strains containing a p53-regulated ADE2 gene whose promoter is regulated by p53 responsive elements derived from the regulatory region of the p21, Bax and PIG3 genes. We also assessed the influence of temperature on transactivation. Our results indicate that a significant proportion of mutants [16/77 (21%); 10/64 (16%) considering only tumour-derived mutants] are transcriptionally active, especially with the p21 promoter. Discriminant mutants preferentially affect less conserved (P<0.04, Fisher's exact test), more rarely mutated (P<0.006, Fisher's exact test) amino acids. Temperature sensitivity is frequently observed, but is more common among discriminant than non-discriminant mutants (P<0.003, Fisher's exact test). Finally, we extended the analysis to a group of mutants isolated in BRCA-associated tumours that surprisingly were indistinguishable from wild type in standard transcription, growth suppression and apoptosis assays in human cells, but showed gain of function in transformation assays. The incidence of transcriptionally active mutations among this group was significantly higher than in the panel of mutants studied previously (P<0.001, Fisher's exact test). Since it is not possible to predict the behaviour of a mutant from first principles, we propose that the yeast assay be used to compile a functional p53 database and fill the gap between the biophysical, pharmacological and clinical fields.
人类p53蛋白主要作为一种应激诱导转录因子，可激活多个参与细胞周期阻滞（如p21）或细胞凋亡（如Bax、PIG3）的基因。大约一半的人类肿瘤含有p53错义突变。几乎所有肿瘤来源的p53突变体都无法激活Bax转录，但有些仍保留激活p21转录的能力。鉴定这些突变体可能具有重要的临床意义。我们使用含有p53调控的ADE2基因的报告酵母菌株，测定了77个p53突变体的转录激活能力，该基因的启动子由源自p21、Bax和PIG3基因调控区域的p53反应元件调控。我们还评估了温度对转录激活的影响。我们的结果表明，相当一部分突变体[16/77（21%）；仅考虑肿瘤来源的突变体时为10/64（16%）]具有转录活性，尤其是对p21启动子。有鉴别能力的突变体优先影响保守性较低（P<0.04，Fisher精确检验）、突变较少（P<0.006，Fisher精确检验）的氨基酸。经常观察到温度敏感性，但在有鉴别能力的突变体中比无鉴别能力的突变体更常见（P<0.003，Fisher精确检验）。最后，我们将分析扩展到一组在BRCA相关肿瘤中分离出的突变体，令人惊讶的是，这些突变体在人类细胞的标准转录、生长抑制和细胞凋亡检测中与野生型无法区分，但在转化检测中显示出功能获得。这组突变体中转录活性突变的发生率明显高于之前研究的突变体组（P<0.001，Fisher精确检验）。由于无法从第一原理预测突变体的行为，我们建议使用酵母检测来编制功能性p53数据库，填补生物物理、药理学和临床领域之间的空白。
