Jia L Q, Osada M, Ishioka C, Gamo M, Ikawa S, Suzuki T, Shimodaira H, Niitani T, Kudo T, Akiyama M, Kimura N, Matsuo M, Mizusawa H, Tanaka N, Koyama H, Namba M, Kanamaru R, Kuroki T
Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan.
Mol Carcinog. 1997 Aug;19(4):243-53. doi: 10.1002/(sici)1098-2744(199708)19:4<243::aid-mc5>3.0.co;2-d.
We have screened the p53 status of 156 human cell lines, including 142 tumor cell lines from 27 different tumor types and 14 cell lines from normal tissues by using functional analysis of separated alleles in yeast. This assay enables us to score wild-type p53 expression on the basis of the ability of expressed p53 to transactivate the reporter gene HIS3 via the p53-responsive GAL1 promotor in Saccharomyces cerevisiae. Of 142 tumor cell lines, at least 104 lines (73.2%) were found to express the mutated p53 gene: 94 lines (66.2%) were mutated in both alleles, three lines (2.1%) were heterozygous, and no p53 cDNA was amplified from seven lines (4.9%). Of the 14 cell lines originating from normal tissues, all the transformed or immortalized cell lines expressed mutant p53 only. Yeast cells expressing mutant p53 derived from 94 cell lines were analyzed for temperature-sensitive growth. p53 cDNA from eight cell lines showed p53-dependent temperature-sensitive growth, growing at 30 degrees C but not at 37 degrees C. Four temperature-sensitive p53 mutations were isolated: CAT-->CGT at codon 214 (H214R), TAC-->TGC at codon 234 (Y234C), GTG-->ATG at codon 272 (V272M), and GAG-->AAG (E285K). Functionally wild-type p53 was detected in 38 tumor cell lines (26.8%) and all of the diploid fibroblasts at early and late population doubling levels. These results strongly support the previous findings that p53 inactivation is one of the most frequent genetic events that occurs during carcinogenesis and immortalization.
我们利用酵母中分离等位基因的功能分析，筛选了156个人类细胞系的p53状态，其中包括来自27种不同肿瘤类型的142个肿瘤细胞系和来自正常组织的14个细胞系。该检测方法使我们能够根据表达的p53通过酿酒酵母中p53反应性GAL1启动子反式激活报告基因HIS3的能力，对野生型p53表达进行评分。在142个肿瘤细胞系中，至少104个系（73.2%）被发现表达突变的p53基因：94个系（66.2%）两个等位基因均发生突变，3个系（2.1%）为杂合子，7个系（4.9%）未扩增出p53 cDNA。在14个来自正常组织的细胞系中，所有转化或永生化细胞系仅表达突变型p53。对来自94个细胞系的表达突变型p53的酵母细胞进行了温度敏感生长分析。来自8个细胞系的p53 cDNA显示出p53依赖性温度敏感生长，在30℃生长但在37℃不生长。分离出4个温度敏感型p53突变：密码子214处的CAT→CGT（H214R）、密码子234处的TAC→TGC（Y234C）、密码子272处的GTG→ATG（V272M）和GAG→AAG（E285K）。在38个肿瘤细胞系（26.8%）以及所有处于早期和晚期群体倍增水平的二倍体成纤维细胞中检测到功能上的野生型p53。这些结果有力地支持了先前的发现，即p53失活是致癌作用和永生化过程中最常见的遗传事件之一。
