Devereux Theodora R, Holliday Wanda, Anna Colleen, Ress Nancy, Roycroft Joseph, Sills Robert C
Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, PO Box 12233, National Institutes of Health, Research Triangle Park, NC 27709, USA.
Carcinogenesis. 2002 Oct;23(10):1737-43. doi: 10.1093/carcin/23.10.1737.
Previous work showed a correlation between K-ras mutation and loss of heterozygosity (LOH) on chromosome 6 in the region of K-ras in lung carcinomas from B6C3F1 mice. We hypothesized that mitogen-activated protein kinase (MAPK) would be activated only in those lung neoplasms with both K-ras mutation and LOH. As MAPK activity can be correlated directly with signal detection using antibodies to phosphorylated MAPK, we were able to analyze lung carcinomas from B6C3F1 mice for the presence or absence of MAPK activity by western analysis. Vanadium pentoxide-induced mouse lung carcinomas, which had been shown to have a high frequency of K-ras mutations and LOH on chromosome 6 and for which frozen tumor tissue was available, were used for this study. Total MAPK expression levels were similar between normal lung and lung carcinomas. Phospho-MAPK was elevated in five of six lung carcinoma samples examined in which K-ras mutations and chromosome 6 LOH were identified and in four of five carcinomas with K-ras mutations that lacked LOH. Phospho-MAPK was undetectable or weakly expressed in seven carcinomas examined without K-ras mutations and in normal lung. By immunohistochemistry three K-ras positive/LOH negative samples exhibited multifocal areas of nuclear and cytoplasmic staining for phospho-MAPK. Large amounts of non-staining fibroblasts, lymphocytes and macrophages were also observed in these tumors. Two of these lung carcinomas were microdissected and chromosome 6 LOH was detected in regions of phospho-MAPK positive cells. These results suggest that MAPK is activated during vanadium pentoxide-induced B6C3F1 mouse lung tumorigenesis following K-ras mutation and loss of the wild-type K-ras allele.
先前的研究表明，在B6C3F1小鼠的肺癌中，K-ras突变与6号染色体上K-ras区域的杂合性缺失（LOH）之间存在关联。我们推测，丝裂原活化蛋白激酶（MAPK）仅在那些同时具有K-ras突变和LOH的肺肿瘤中被激活。由于MAPK活性可以通过使用抗磷酸化MAPK抗体直接与信号检测相关联，我们能够通过蛋白质印迹分析来检测B6C3F1小鼠肺癌中MAPK活性的有无。本研究使用了五氧化二钒诱导的小鼠肺癌，这些肺癌已被证明具有高频率的K-ras突变和6号染色体上的LOH，并且有冷冻肿瘤组织可用。正常肺组织和肺癌组织中总的MAPK表达水平相似。在检测的六个肺癌样本中有五个样本中磷酸化MAPK水平升高，其中鉴定出K-ras突变和6号染色体LOH；在五个具有K-ras突变但缺乏LOH的肺癌样本中有四个样本中磷酸化MAPK水平升高。在检测的七个没有K-ras突变的肺癌样本以及正常肺组织中，未检测到磷酸化MAPK或其表达较弱。通过免疫组织化学，三个K-ras阳性/LOH阴性样本显示出磷酸化MAPK在细胞核和细胞质中的多灶性染色区域。在这些肿瘤中还观察到大量无染色的成纤维细胞、淋巴细胞和巨噬细胞。对其中两个肺癌进行显微切割，并在磷酸化MAPK阳性细胞区域检测到6号染色体LOH。这些结果表明，在五氧化二钒诱导的B6C3F1小鼠肺癌发生过程中，K-ras突变和野生型K-ras等位基因缺失后MAPK被激活。
