Screening the p53 status of human cell lines using a yeast functional assay
Screening the p53 status of human cell lines using a yeast functional assay
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DOI：
10.1002/(sici)1098-2744(199708)19:4
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发表时间：
1997-08-01
影响因子：
4.6
通讯作者：
Kuroki, T
中科院分区：
文献类型：
--
作者：
Jia, LQ;Osada, M;Kuroki, T
我们已经筛选了156人细胞系的p53状态，包括142肿瘤细胞系从27个不同的肿瘤类型和14细胞系从正常组织通过使用分离的等位基因在酵母中的功能分析。该测定使我们能够基于表达的p53通过酿酒酵母中的p53响应性GAL 1启动子反式激活报告基因HIS 3的能力来对野生型p53表达进行评分。在142个肿瘤细胞系中，发现至少104个细胞系（73.2%）表达突变的p53基因：94个细胞系（66.2%）在两个等位基因中都突变，3个细胞系（2.1%）是杂合的，并且从7个细胞系（4.9%）扩增出nop 53 cDNA。在来自正常组织的14个细胞系中，所有转化或永生化细胞系均仅表达突变型p53。酵母细胞表达突变体p53来源于94细胞系进行了分析的温度敏感性生长。来自8个细胞系的p53 cDNA显示p53依赖性温度敏感性生长，在30 ℃生长，但在37 ℃不生长。分离到四种温度敏感性p53突变：密码子214处的CAT->CGT（H214 R）、密码子234处的TAC->TGC（Y234 C）、密码子272处的GTG->ATG（V272 M）和GAG->AAG（E285 K）。在38个肿瘤细胞系（26.8%）和所有二倍体成纤维细胞中检测到功能性野生型p53在早期和晚期群体倍增水平。这些结果有力地支持了以前的研究结果，即p53失活是发生在癌变和永生化过程中最常见的遗传事件之一。(C)1997 Wiley-Liss，Inc.
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 promoter 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 nop53 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. (C) 1997 Wiley-Liss, Inc.