Rand A, Glenn K S, Alvares C P, White M B, Thibodeau S M, Karnes W E
Mayo Foundation, Rochester, MN 55905, USA.
Cancer Lett. 1996 Jan 2;98(2):183-91.
We have sequenced p53 in three colon cancer cell lines capable of autonomous proliferation. SNU-C1 and SNU-C4 cells, whose autonomous growth is dependent upon autocrine stimulation of epidermal growth factor receptor (EGFR), had wildtype p53 sequence of exons 4-9. In contrast, an EGFR ligand-independent cell line, SNU-C5, had heterozygous missense mutations affecting codons 218 (valine to leucine) and 248 (arginine to tryptophan) of p53. Bacterial cloning of p53 from SNU-C5 cells showed that the 248trp and 218leu mutants were both expressed and on separate alleles. 248trp is a common 'hot spot' mutant of p53 with variable dominant negative activity depending on the celullar context. Valine 218, in contrast, is rarely affected by mutation in cancers and is located in a region of the hydrophobic core domain away from 'hot spot' DNA contact sights. However, valine 218 is completely conserved across species, prompting us to investigate the function of 218leu in SNU-C5 cells. SNU-C5 cells exhibited complete loss of normal p53 function as evidenced by over-expression of p53 protein and by failure to show induction of p53, waf-1, mdm-2 or G1/S arrest in response to the DNA damaging agent, bleomycin. In a yeast p53 functional assay (FASAY), 50% of the clones were unable to transactivate a p53-specific promoter required for yeast colony expansion at 25, 30 or 37 degrees C. Sequencing of the p53 insert from several randomly selected wild-type and mutant yeast clones revealed that 218leu-bearing clones retained their ability to transactivate the p53-specific promoter. As expected, the 248trp-bearing clones lost this function. These data indicate that although 218leu retains normal transactivation activity on a p53 promoter in yeast at physiological temperatures, it is not capable of normal p53 function in the presence of a 248trp allele in SNU-C5 cells. It remains unclear whether the strong dominant negative activity of 248trp in SNU-C5 cells is related to the cellular context or to an unresolved abnormality of 218leu function.
我们对三种能够自主增殖的结肠癌细胞系中的p53进行了测序。SNU - C1和SNU - C4细胞的自主生长依赖于表皮生长因子受体（EGFR）的自分泌刺激，其外显子4 - 9的p53序列为野生型。相比之下，一种不依赖EGFR配体的细胞系SNU - C5，其p53存在杂合错义突变，影响p53的第218位密码子（缬氨酸突变为亮氨酸）和第248位密码子（精氨酸突变为色氨酸）。从SNU - C5细胞中对p53进行细菌克隆显示，248trp和218leu突变体均有表达且位于不同等位基因上。248trp是p53常见的“热点”突变体，其显性负性活性因细胞环境而异。相比之下，缬氨酸218在癌症中很少发生突变，位于疏水核心结构域中远离“热点”DNA接触位点的区域。然而，缬氨酸218在所有物种中完全保守，这促使我们研究SNU - C5细胞中218leu的功能。SNU - C5细胞表现出正常p53功能的完全丧失，这通过p53蛋白的过表达以及在DNA损伤剂博来霉素作用下未能诱导p53、waf - 1、mdm - 2或G1/S期阻滞得以证明。在酵母p53功能测定（FASAY）中，50%的克隆在25、30或37摄氏度下无法激活酵母菌落扩增所需的p53特异性启动子。对几个随机选择的野生型和突变型酵母克隆的p53插入片段进行测序发现，携带218leu的克隆保留了激活p53特异性启动子的能力。正如预期的那样，携带248trp的克隆失去了该功能。这些数据表明，尽管218leu在生理温度下在酵母中对p53启动子保留正常的反式激活活性，但在SNU - C5细胞中存在248trp等位基因的情况下，它不能发挥正常的p53功能。目前尚不清楚248trp在SNU - C5细胞中的强显性负性活性是与细胞环境有关还是与218leu功能未解决的异常有关。
