Molecular cloning of the cancer metastasis associated gene and its clinical applications.

我们已经建立了两种引起下述转移的人细胞系。1）通过用来自高转移性黑素瘤细胞（A375黑素瘤细胞）的DNA转染非转移性小鼠细胞系（AH8对照）来建立一个克隆。2）将人乳腺癌细胞系MDA 435多次移植于裸鼠体内，建立了肺转移克隆MDA 4A4和非肺转移克隆MDA 2C5。经北方印迹分析，这两个克隆的大小分别为2.5kb和4.5kb左右。仅在4A 4克隆中检测到这些克隆的表达，而在2C 5中未检测到这些分子，表明这些分子与癌症转移密切相关。我们接下来将4A 4和2C 5细胞移植到裸小鼠体内，提取每个细胞的RNA并进行北方印迹分析。有趣的是，这些克隆在转移性细胞中不一定高度表达，这表明这些分子的表达水平根据环境条件而变化。我们尝试用20000个人mRNA克隆，通过DNA微阵列系统检测4A4和2C5克隆的mRNA表达谱，目前有200多个克隆被列为转移相关基因的候选分子包括先前分离的4个DNA克隆在内的密切分析正在进行中。

We have established two human cell lines which cause metastasis written below.1) One clone was established by transfecting with DNA from highly metastatic melanoma cells (A375 melanoma cell) into non-metastatic mouse cell line (AH8 control). This cells highly metastasize to lung when transplanted to the nude mouse.2) Human breast cancer cell line, MDA 435, was transplanted repeatedly to nude mice and metastatic clones to the lung, MDA 4A4 and non-metastatic clone, MDA 2C5 were established.We have previously isolated 4 clones by cDNA differential display method and these clones were revealed as new molecules after homology search by the gene bank. The sizes of these clones by Northern blot were about 2.5kb and 4.5kb as a single band, respectedly. The expressions of the clones were detected only in 4A4 clones and not detected in 2C5 suggesting that these molecules are closely related to cancer metastasis.We next transplanted 4A4 and 2C5 cells into nude mice and extracted RNA from each cells and Northern blot analysis was performed. Interestingly, these clones were not necessarily highly expressed in metastatic cells suggesting that the expression levels of these molecules changes according to the conditions of the circumstances.Moreover, we have tried to detect the mRNA expression profiles of 4A4 and 2C5 clones by DNA microarray system using 20000 human mRNA clones and now more than 200 clones are listed as candidate molecule as metastasis related gene and close analysis including the 4 DNA clones which were isolated previously, are on going now.