An Attempt to Clone Amplified Genes which are Overexpressed

对人食管癌细胞株进行基因扩增分析。C-myc基因扩增在高分化型中明显高于其他型，并伴有其他基因的扩增。发现细胞周期蛋白D1 （CCND1）基因扩增而不表达的细胞系，CCND2基因高表达。这些结果表明，不同的遗传改变导致了不同组织学亚型的食管癌，CCND1基因不一定是染色体11q13扩增的靶基因，CCND家族基因在食管癌发生中的作用是互补的。为了克隆扩增基因，我们尝试了一种结合凝胶内再生和基于RT-PCR的cDNA文库的新方法。在PCR扩增过程中，扩增片段的偏倚会干扰每个基因表达的大小。然而，我们建立了RT-PCR定量基因表达的方法。比较CCND1基因表达细胞和未表达细胞的启动子区域。目前在1.3 kbp区域未发现差异，但扩增子结构复杂，被认为是由结构相似但不相似的扩增单元串联重复组成。建立了一种改良的凝胶内再生法来克隆扩增的序列。这种方法使我们能够克隆一些重复的DNA序列。其克隆扩增序列的改进正在进行中。

Gene amplification was analyzed in human esophageal carcinoma cell lines. c-myc gene amplification was significantly more detected in well-differentiated type compared with other types and was accompanied with amplification of other genes. A cell line with cyclin D1 (CCND1) gene amplification without its expression was found and they showed hyperexpression of the CCND2 gene. These suggest that distinctively different genetic changes contribute to histologically different subtypes of esophageal carcinomas, that CCND1 gene is not necessary a target gene of chromosome 11q13 amplification and that CCND family genes contribute to esophageal carcinogenesis complimentarily each other. In order to clone amplified gene we tried a novel method which is combination of In-gel renaturation and RT-PCR based cDNA library. Bias of amplified fragments during PCR amplification interferes magnitudes of each gene expression. However, we established quantification of gene expression by RT-PCR.The promoter region of the amplified CCND1 gene was compared between cells with CCND1gene expression and those without expression. So far no difference was found in 1.3 kbp region, however, structure of amplicon is complicated and is thought to be composed of tandem repeat of amplified units of which structures are alike but not similar.A modified version of In-gel renaturation method was developed to clone amplified sequences. This method allowed us to clone some repeated DNA sequences. Its refinements to clone amplified sequences are underway.