Belinsky S A, Middleton S K, Picksley S M, Hahn F F, Nikula K J
Inhalation Toxicology Research Institute, Albuquerque, New Mexico 87185, USA.
Radiat Res. 1996 Apr;145(4):449-56.
The risk from exposure to low-dose radiation in conjunction with cigarette smoking has not been estimated due in part to limited knowledge surrounding the molecular mechanisms underlying radiation-induced cancers. The purpose of this investigation was to determine the frequency for alterations in genes within the K-ras and p53 signal and cell cycle regulatory pathways, respectively, in X-ray-induced lung tumors in the F344/N rat. These tumors were examined for genetic alterations in the K-ras, c-raf-1, p53, mdm2 and cip1 genes. No K-ras mutations were detected by sequencing in 18 squamous cell carcinomas (SCCs) or 17 adenocarcinomas. However, using a K-ras codon 12 mutation selection assay, a codon 12 GGT --> GAT mutation was detected in one SCC, suggesting that activation of the K-ras proto-oncogene is both a rare and late event. Single-strand conformation polymorphism (SSCP) analysis of the kinase-binding domain of the c-raf-1 gene did not detect any polymorphisms. Three of 18 SCCs but none of the adenocarcinomas showed p53 nuclear immunoreactivity. Single-strand conformation polymorphism analysis of exons 4-9 of the p53 gene detected only an exon 9 mutation in one SCC. Mutations were not detected in the three SCCs with immunoreactive p53 protein. No amplification of the mdm2 gene was detected; however, nuclear mdm2 immunoreactivity was present in one of the three SCCs that stained positive for the p53 protein. Thus the increased level of p53 protein in one SCC may stem from stabilization by the mdm2 gene product. The complete cDNA of the rat cip1 gene comprising 810 bases was cloned and sequenced. Overall homology between the rat and human cip1 genes was 74%. Homology between the rat and mouse genes was 90%. The frequency of somatic mutations in exon 2 of the cip1 gene was determined by SSCP analysis. No alterations in electrophoretic mobility were detected. The results of this investigation indicate that alterations in the K-ras and p53 pathways do not play a major role in the genesis of X-ray-induced lung tumors in the rat.
由于对辐射诱发癌症的分子机制了解有限，目前尚未对低剂量辐射与吸烟共同作用产生的风险进行评估。本研究的目的是确定在F344/N大鼠的X射线诱发肺肿瘤中，K-ras和p53信号及细胞周期调控途径内基因改变的频率。对这些肿瘤进行了K-ras、c-raf-1、p53、mdm2和cip1基因的遗传改变检测。在18例鳞状细胞癌（SCC）或17例腺癌中，测序未检测到K-ras突变。然而，使用K-ras密码子12突变筛选试验，在1例SCC中检测到密码子12 GGT --> GAT突变，这表明K-ras原癌基因的激活既是罕见事件，也是晚期事件。对c-raf-1基因激酶结合域进行单链构象多态性（SSCP）分析，未检测到任何多态性。18例SCC中有3例显示p53核免疫反应性，但腺癌中均未显示。对p53基因第4至9外显子进行SSCP分析，仅在1例SCC中检测到第9外显子突变。在3例p53蛋白免疫反应阳性的SCC中未检测到突变。未检测到mdm2基因扩增；然而，在3例p53蛋白染色阳性的SCC中，有1例存在mdm2核免疫反应性。因此，1例SCC中p53蛋白水平升高可能源于mdm2基因产物的稳定作用。克隆并测序了包含810个碱基的大鼠cip1基因的完整cDNA。大鼠和人cip1基因之间的总体同源性为74%。大鼠和小鼠基因之间的同源性为90%。通过SSCP分析确定cip1基因第2外显子的体细胞突变频率。未检测到电泳迁移率改变。本研究结果表明，K-ras和p53途径的改变在大鼠X射线诱发肺肿瘤的发生中不发挥主要作用。
