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

1)将高转移性黑色素瘤细胞(A375黑色素瘤细胞)的DNA导入非转移性小鼠细胞系(AH8对照)，建立了一个克隆。2)将人乳腺癌细胞株MDA435多次移植到裸鼠体内，建立了肺转移克隆，建立了MDA4A4和未转移克隆MDA2C5，利用基因差异显示技术分离了4个克隆，经基因库同源检索，证实为新分子。Northern杂交结果显示，这些克隆的条带大小分别为2.5kb和4.5kb左右。这些克隆仅在4A4克隆中表达，而在2C5中未检测到表达，提示这些分子与肿瘤转移密切相关。接下来，我们将4A4和2C5细胞移植到裸鼠体内，提取每种细胞的RNA，进行Northern印迹分析。有趣的是，这些克隆在转移细胞中并不一定高表达，这表明这些分子的表达水平会随着环境的变化而变化。此外，我们尝试用基因芯片系统检测4A4和2C5克隆的基因表达谱，目前有20000个克隆被列为转移相关基因的候选分子，包括之前分离的4个DNA克隆，目前正在进行密切的分析。

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.