Miller M S, Leone-Kabler S, Rollins L A, Wessner L L, Fan M, Schaeffer D O, McEntee M F, O'Sullivan M G
Department of Cancer Biology, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA.
Exp Lung Res. 1998 Jul-Aug;24(4):557-77. doi: 10.3109/01902149809087386.
Previous studies from this and other laboratories have shown that treatment of pregnant mice with 3-methylcholanthrene (MC) caused lung tumors in the offspring, the incidence of which correlated with fetal inducibility of Cyp1a1. Analysis of paraffin-embedded lung tissue for Ki-ras-2 mutations indicated that 79% of the lesions examined contained point mutations in codons 12 and 13 of the Ki-ras-2 gene locus, the majority of which (84%) were G-->T transversions. The mutational spectrum was dependent on the tumor stage, as both the incidence of mutation and type of mutation produced correlated with malignant progression of the tumor. Mutations occurred in 60% of the hyperplasias, 80% of the adenomas, and 100% of the adenocarcinomas. In the tumors with mutations, GLY12-->CYS12 transversions occurred in 100% of the hyperplasias, 42% of the adenomas, and 14% of the adenocarcinomas. GLY12-->VAL12 transversions were not observed in hyperplasias and occurred in 42% of the adenomas and 57% of the adenocarcinomas. The remaining ASP12 and ARG13 mutations occurred only in adenomas (17%) and adenocarcinomas (29%). The tumors were also analyzed for alterations in the structure or function of the tumor suppressor genes Rb, p53, and Cdkn2a. No mutations were observed in exons 5-8 of the p53 gene. SSCP analysis demonstrated that 2 of 15 lung tumors contained shifted bands at the Cdkn2a gene locus. Sequence analysis had identified these as mutations in exon 2, with a CAC-->TAC transition at base 301 (HIS74-->TYR74) in tumor 23-1 and GGG-->GAG transition at base 350 (GLY90-->GLU90) in tumor 36-1. Northern blot analysis of the larger tumors revealed that 14 of 14 of these large lung tumors exhibited markedly decreased expression of Rb gene transcripts. These results were confirmed by immunohistochemistry. The larger tumors, which exhibited features of adenocarcinomas, showed a marked reduction or almost complete absence of nuclear pRb staining compared with smaller adenomas and normal lung tissue. The results suggest that Ki-ras-2 mutations are an early and frequent event in lung tumorigenesis, and that the type of mutation produced by environmental chemicals can influence the carcinogenic potential of the tumor. The results obtained with the Cdkn2a and Rb genes suggest that alterations in the Rb regulatory axis may play a key role in the pathogenesis of the pulmonary tumors and appear to occur later in the neoplastic process. It appears from these experiments that the combination of mutated Ki-ras-2 and alterations in the Rb regulatory gene locus, which are frequent alterations in human lung tumors, may be the preferred pathway for lung tumor pathogenesis in mice exposed transplacentally to environmental carcinogens.
本实验室及其他实验室之前的研究表明，用3-甲基胆蒽（MC）处理怀孕小鼠会导致其后代发生肺肿瘤，肿瘤的发生率与Cyp1a1的胎儿诱导性相关。对石蜡包埋的肺组织进行Ki-ras-2突变分析表明，79%的检测病变在Ki-ras-2基因座的第12和13密码子处存在点突变，其中大多数（84%）是G→T颠换。突变谱取决于肿瘤分期，因为突变发生率和产生的突变类型均与肿瘤的恶性进展相关。增生性病变中60%发生突变，腺瘤中80%发生突变，腺癌中100%发生突变。在发生突变的肿瘤中，增生性病变中100%发生GLY12→CYS12颠换，腺瘤中42%发生该颠换，腺癌中14%发生该颠换。增生性病变中未观察到GLY12→VAL12颠换，腺瘤中42%发生该颠换，腺癌中57%发生该颠换。其余的ASP12和ARG13突变仅发生在腺瘤（17%）和腺癌（29%）中。还对肿瘤抑制基因Rb、p53和Cdkn2a的结构或功能改变进行了分析。在p53基因的第5 - 8外显子中未观察到突变。SSCP分析表明，15个肺肿瘤中有2个在Cdkn2a基因座出现条带迁移。序列分析确定这些为第2外显子中的突变，肿瘤23 - 1中第301位碱基发生CAC→TAC转换（HIS74→TYR74），肿瘤36 - 1中第350位碱基发生GGG→GAG转换（GLY90→GLU90）。对较大肿瘤进行Northern印迹分析显示，14个大的肺肿瘤中有14个Rb基因转录本表达明显降低。免疫组织化学证实了这些结果。与较小的腺瘤和正常肺组织相比，表现出腺癌特征的较大肿瘤显示核pRb染色明显减少或几乎完全缺失。这些结果表明，Ki-ras-2突变是肺肿瘤发生过程中早期且常见的事件，环境化学物质产生的突变类型可影响肿瘤的致癌潜能。对Cdkn2a和Rb基因的研究结果表明，Rb调节轴的改变可能在肺肿瘤发病机制中起关键作用，且似乎在肿瘤形成过程中出现得较晚。从这些实验看来，在经胎盘暴露于环境致癌物的小鼠中，突变的Ki-ras-2与Rb调节基因座改变的组合可能是肺肿瘤发病的首选途径，而这两种改变在人类肺肿瘤中也很常见。
