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

原代细胞转化抑制相关基因的分离和功能分析

• 批准号: 10670205
• 负责人: INOUE Hirokazu
• 金额: $ 1.98万
• 依托单位: Shiga University of Medical Science (2000)Osaka University (1998-1999)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10670205/
• 关键词: Tumor suppressor gene; Transformation; Drs gene; 足場非依存性増殖; 初代培養細胞; がん遺伝子

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.

drs基因最初是作为v-src转化的抑制基因从大鼠原代胚胎成纤维细胞cDNA文库中分离得到的。Drs蛋白具有一个跨膜结构域、在C末端的短胞内结构域和三个共有重复序列（Sushi基序）。我们已经发现，在多种人类癌细胞系和恶性组织中，包括结肠、膀胱、卵巢、肺和前列腺中，drs mRNA的表达显著降低。此外，通过逆转录病毒载体将drs cDNA导入这些癌细胞系中引起非锚定生长和致瘤性的抑制。这些发现表明，在人类癌症的发展中，drs mRNA的下调与恶性表型的表达密切相关。与drs基因的缺失突变体的分析表明，跨膜结构域内的C-末端区域和N-末端区域中的三个共识重复序列（CR）是必不可少的抑制锚定非依赖性生长的细胞。Rb的非依赖性下调细胞周期蛋白A的mRNA参与抑制锚定非依赖性生长的drs基因在人类癌细胞。我们还分离了小鼠drs的一种新型变体cDNA（mDRS-2），除了含有三个CR的小鼠drs同源物（mDRS-1）之外，还含有两个CR。两种类型的drs mRNA在正常小鼠组织中均有表达。mDRS-1具有抑制这些细胞的锚定非依赖性生长的能力，而mDRS-2则没有，这表明缺乏一个CR对于抑制锚定非依赖性生长至关重要。此外，我们还分离了一个小鼠基因组克隆用于基因打靶，并正在构建drs基因敲除小鼠。

项目成果

N.Yoshioka et al.: "Isolation of transformation suppressor genes by cDNA subtraction: Lumican suppresses transformation by v-src and v-K-ras"J.Virol.. 74. 1008-1013 (2000)

N.Yoshioka 等：“通过 cDNA 消减法分离转化抑制基因：Lumican 通过 v-src 和 v-K-ras 抑制转化”J.Virol.. 74. 1008-1013 (2000)

H.Inoue et al.: "Suppression of v-Src transformation by the drs gene." J.Virol.72. 2532-2537 (1998)

H.Inoue 等人：“drs 基因抑制 v-Src 转化。”

A.Yamashita et al.: "Suppression of anchorage-independent growth of human cancer cell lines by the drs gene."Oncogene. 18. 4777-4787 (1999)

A.Yamashita 等人：“drs 基因抑制人类癌细胞系的锚定非依赖性生长。”Oncogene。

Shimakage, M., Kawahara, K., Kikkawa, N., Sasagawa, T., Yutsudo, M., and Inoue, H.: "Downregulation of drs mRNA in human colon adenocarcinoma."Int.J.Cancer. 87. 5-11 (2000)

Shimakage, M.、Kawahara, K.、Kikkawa, N.、Sasakawa, T.、Yutsudo, M. 和 Inoue, H.：“人类结肠腺癌中 drs mRNA 的下调。”Int.J.Cancer。

S.Kyo etal.: "Expression of human telomerase subunits in uvariain maliginant,borderline and benisn tumors." Int.J.Cancer. in press. (1999)

S.Kyo 等人：“人类端粒酶亚基在卵巢恶性肿瘤、交界性肿瘤和贝尼森肿瘤中的表达。”