Fukuda T, Kido A, Kajino K, Tsutsumi M, Miyauchi Y, Tsujiuchi T, Konishi Y, Hino O
Department of Experimental Pathology, Cancer Institute, 1-37-1 Kami-ikebukuro, Tokyo, Toshima-ku, 170-8455, Japan.
Biochem Biophys Res Commun. 1999 Jul 22;261(1):35-40. doi: 10.1006/bbrc.1999.0997.
To identify differentially expressed genes between highly and low metastatic rat transplantable osteosarcomas, we applied a modified AFLP (amplified fragment length polymorphisms) method for cDNA subtraction. The specific point of our modification is selective amplification using suppression PCR technique after restriction enzyme cutting. Our cDNA-AFLP gave high reproducibility (about 95%) in mRNA patterns and enabled us to clone four dominantly expressed genes in a highly metastatic tumor line. Three showed homology with known genes, encoding Ki-67, a proliferation-associated effective marker of malignancy, type IV collagen alpha-3, a major component of basement membrane, and KIAA77 for which the function is unknown. Although one fragment showed no database homology, we revealed a derivation from the rat homologue of the Drosophila melanogaster diaphanous gene (Dia) by cloning of longer cDNA. Dia genes, known to affect actin filament formation, are downstream effectors of Rho small GTPase. The results suggest that alterations in the expression of cytoskeletal protein, basement membrane elements, and proliferative markers may be important for metastasis of osteosarcomas.
为了鉴定高转移性和低转移性大鼠可移植骨肉瘤之间的差异表达基因，我们应用改良的AFLP（扩增片段长度多态性）方法进行cDNA消减。我们改良的具体要点是在限制性酶切后使用抑制性PCR技术进行选择性扩增。我们的cDNA-AFLP在mRNA模式中具有高重现性（约95%），并使我们能够在高转移性肿瘤细胞系中克隆四个优势表达基因。其中三个与已知基因具有同源性，分别编码Ki-67（一种与增殖相关的恶性有效标志物）、IV型胶原α-3（基底膜的主要成分）以及功能未知的KIAA77。尽管一个片段在数据库中无同源性，但通过克隆更长的cDNA，我们发现它源自果蝇透明基因（Dia）的大鼠同源物。已知Dia基因影响肌动蛋白丝的形成，是Rho小GTP酶的下游效应器。结果表明，细胞骨架蛋白、基底膜成分和增殖标志物表达的改变可能对骨肉瘤转移很重要。
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