Isolation and functional analysis of the genes involved in suppression of transformation in primary cells

DRS基因最初是从大鼠原代胚胎成纤维细胞cDNA文库中分离出来的，是一种抑制v-src转化的基因。DRS蛋白有一个跨膜结构域，在C末端有一个较短的胞内结构域，以及三个共识重复(寿司基序)。我们已经发现，在各种人类癌细胞系和恶性组织中，包括结肠、膀胱、卵巢、肺和前列腺组织中，DRS mRNA的表达显著降低。此外，通过逆转录病毒载体将DRS基因导入这些癌细胞系，可抑制非锚定生长和致瘤性。这些结果表明，在人类肿瘤的发生发展过程中，DRS基因的表达下调与恶性表型的表达密切相关。对DRS基因缺失突变体的分析表明，跨膜区内的C末端区域和N末端区域的三个共同重复(CR)对于抑制细胞的锚定非依赖性生长是必不可少的。DRS基因对人癌细胞的锚定非依赖性生长的抑制与细胞周期蛋白A基因非依赖于Rb的下调有关。我们还分离到了小鼠DRS的一个新的变异体(MDRS-2)，它除了含有三个CRS的小鼠同源物(MDRS-1)外，还含有两个CRS。两种类型的DRS基因在正常小鼠组织中均有表达。MDRS-1有能力抑制这些细胞的贴壁非依赖性生长，而MDRS-2没有，这表明缺乏一个CR是抑制贴壁非依赖性生长的关键。此外，我们还分离了一个小鼠基因组克隆用于基因打靶，DRS基因敲除小鼠的构建正在进行中。

The drs gene was originally isolated as a suppressor gene against v-src transformation from rat primary embryo fibroblast cDNA library. The Drs protein has one transmembrane domain, a short intracellular domain in the C terminus, and three consensus repeats (Sushi motifs). We have shown that expression of drs mRNA was markedly reduced in a variety of human cancer cell lines and malignant tissues, including those of the colon, bladder, ovary, lung, and prostate. Furthermore, introduction of drs cDNA by retrovirus vector into these cancer cell lines caused suppression of anchorage-independent growth and tumorigenicity. These findings indicate that downregulation of drs mRNA is closely correlated with expression of malignant phenotypes in development of human cancers. Analyses with deletion mutants of the drs gene revealed that both the C-terminal region inside the transmembrane domain and three consensus repeats (CR) in the N-terminal region are essential for the suppression of anchorage-independent growth of the cells. An Rb-independent downregulation of cyclin A mRNA was involved in the suppression of anchorage-independent growth by the drs gene in human cancer cells. We also isolated a novel variant cDNA of mouse drs (mDRS-2) which contains two CRs in addition to a mouse homologue of drs (mDRS-1) which contains three CRs. Both types of drs mRNA were expressed in normal mouse tissues. The mDRS-1 had the ability to suppress anchorage-independent growth of these cells whereas mDRS-2 did not, indicating that the lack of one CR is critical for suppression of anchorage-independent growth. Furthermore, we isolatede a mouse genomic clone for gene targetting and construction of drs-knockout mouse is in progress.