Kameda H, Risinger J I, Han B B, Baek S J, Barrett J C, Abe T, Takeuchi T, Glasgow W C, Eling T E
Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Mol Cell Biol. 2001 Oct;21(20):6895-905. doi: 10.1128/MCB.21.20.6895-6905.2001.
An in vitro transformation system of carcinogen-treated Syrian hamster embryo (SHE) cell cultures represents multistep genetic and nongenetic changes that develop during the neoplastic progression of normal cells to tumor cells in vivo. During this neoplastic progression, SHE cells demonstrate an altered response to epidermal growth factor (EGF). In the present report, we examined the role of the adapter protein Gab1 (Grb2-associated binder-1) in the neoplastic progression of SHE cells. We used two asbestos-transformed SHE cell clones in different neoplastic stages: a 10W+8 clone, which is immortal and retains the ability to suppress the tumorigenicity of tumor cells in cell-cell hybrid experiments, and a 10W-1 clone, which has lost this tumor suppressor ability. 10W+8 cells expressed full-length 100-kDa Gab1 and associated 5.2-kb mRNA. Upon repeated cell passaging, 10W-1 cells showed increasing expression of a novel 87-kDa form of Gab1 as well as 4.6-kb mRNA with diminishing expression of the original 100-kDa Gab1. cDNA encoding the 87-kDa Gab1 predicts a form of Gab1 lacking the amino-terminal 103 amino acids (Gab1(Delta1-103)), which corresponds to loss of most of the pleckstrin homology (PH) domain. Gab1(Delta1-103) retains the ability to be phosphorylated in an EGF-dependent manner and to associate with the EGF receptor and SHP-2 upon EGF stimulation. The endogenous expression of Gab1(Delta1-103) in 10W-1 cells appeared closely related to EGF-dependent colony formation in soft agar. Moreover, transfection and expression of Gab1(Delta1-103), but not Gab1, in 10W+8 cells enhanced their EGF-dependent colony formation in soft agar. These results demonstrate that Gab1 is a target of carcinogen-induced transformation of SHE cells and that the expression of a Gab1 variant lacking most of the PH domain plays a specific role in the neoplastic progression of SHE cells.
致癌物处理的叙利亚仓鼠胚胎（SHE）细胞培养物的体外转化系统代表了正常细胞在体内向肿瘤细胞的肿瘤进展过程中发生的多步骤遗传和非遗传变化。在这个肿瘤进展过程中，SHE细胞对表皮生长因子（EGF）的反应发生了改变。在本报告中，我们研究了衔接蛋白Gab1（Grb2相关结合蛋白-1）在SHE细胞肿瘤进展中的作用。我们使用了处于不同肿瘤阶段的两个石棉转化的SHE细胞克隆：一个10W + 8克隆，它是永生的，并且在细胞 - 细胞杂交实验中保留抑制肿瘤细胞致瘤性的能力；还有一个10W - 1克隆，它已经失去了这种肿瘤抑制能力。10W + 8细胞表达全长100 kDa的Gab1和相关的5.2 kb mRNA。在细胞反复传代后，10W - 1细胞显示出一种新的87 kDa形式的Gab1以及4.6 kb mRNA的表达增加，而原来的100 kDa Gab1的表达减少。编码87 kDa Gab1的cDNA预测了一种缺少氨基末端103个氨基酸的Gab1形式（Gab1(Delta1 - 103)），这对应于大部分普列克底物蛋白同源（PH）结构域的缺失。Gab1(Delta1 - 103)保留了以EGF依赖方式被磷酸化以及在EGF刺激下与EGF受体和SHP - 2结合的能力。10W - 1细胞中Gab1(Delta1 - 103)的内源性表达似乎与软琼脂中EGF依赖的集落形成密切相关。此外，在10W + 8细胞中转染并表达Gab1(Delta1 - 103)，而不是Gab1，增强了它们在软琼脂中EGF依赖的集落形成。这些结果表明Gab1是致癌物诱导的SHE细胞转化的靶点，并且缺少大部分PH结构域的Gab1变体的表达在SHE细胞的肿瘤进展中起特定作用。
