MECHANISTIC STUDY OF APOPTOTIC CELL DEATH AND INVASION POTENTIAL USING SIGNAL TRANSDUCTION INHIBITORS IN THE ONCOGENE-TRANSFECTED HUMAN CELLS

使用信号转导抑制剂对转染癌基因的人类细胞进行凋亡细胞死亡和侵袭潜力的机制研究

基本信息

批准号：
10670415
负责人：
NAKANO Shuji
金额：
$ 2.11万
依托单位：
KYUSHU UNIVERSITY
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 1999
项目状态：
已结题

项目摘要

Although src or ras oncogene induces transformation, their precise roles in the apoptotic machinery and invasion remain to be clarified. We examined the role of these oncogenes on the detachment-induced apoptosis termed anoikis and invasion potential, using HAG-1 human epithelial cell lines transfected with v-src or activated ras. Non-tumorigenic parental, mock-transfected, and ras-transfected HAG-1 cells underwent anoikis. By contrast, tumorigenic, v-src-transformed cells did not exhibit such apoptotic features. pp125^<FAK> focal adhesion kinase (FAK), was constitutively activated only in the v-src-transformed cells. Moreover, herbimycin A, a Src tyrosine kinase inhibitor, reduced tyrosyl phosphorylation of pp125^<FAK>, and reversed resistance to anoikis. These data suggest that pp60^<v-src> may substitute for integrin in the activation of pp125^<FAK>, thereby evading anoikis, and that the association of pp60^<src> with FAK might be required for acquisition of anchorage-independence. … More The functional role of ras in the acquisition of fully neoplastic potentials by v-src-transformed human gallbladder HAG/src3-1 cells, were investigated by introducing adenoviral vector containing dominant negative Ras (DN/Ras) into these cells. The anchorage-independent growth of the HAG/src3-1 was inhibted by DN/Ras by 93%. The HAG/src3-1 cells transfected with DN/ras failed to form tumors. Thus, v-src might requires a Ras-MAP kinase signaling cascade in the acquisition of tumorigenic potential. To examine the potential role of Rac, a small GTPase binding protein which acts downstream of Ras, experiments using adenovirus vectors containing DN/Ras or dominant negative Rac (DN/Rac), were performed. The data indicated that both DN/Ras and DN/Rac reduced the invasive potential of src-transfected cells by 60% and 99%, respectively, as compared to AdCALacZ containing β-gal gene as a control. Moreover, DN/Rac suppressed completely the tumorigenic potentials of src-transfected cells in nude mice. These results suggest that Src, Ras, Rac, all appeared to participate in the invasion processes, and that Rac may act downstream of these signaling pathways of invasion and tumorigenicity. Less

尽管SRC或RAS癌基因诱导转化，但它们在凋亡机制和入侵中的精确作用仍有待阐明。我们使用用V-SRC或激活的Ras翻译的HAG-1人类上皮细胞系检查了这些癌基因在脱离诱导的凋亡中的作用，称为Anoikis和侵袭潜能。非肿瘤父母，模拟转染和RAS转染的HAG-1细胞发生了Anoikis。相比之下，肿瘤性，V-SRC转化的细胞不存在这样的凋亡特征。 PP125^<fak>焦点粘附激酶（FAK）仅在V-SRC转换的细胞中始终激活。此外，Herbimycin A，一种SRC酪氨酸激酶抑制剂，降低了PP125^<fak>的酪酶磷酸化，并反向对Anoikis的抗性。这些数据表明，pp60^<v-src>可能在PP125^<fak>的激活中代替整联蛋白，从而逃避了Anoikis，并且可能需要PP60^<src>与FAK的关联来获取锚定独立性。 …通过将含有主要的负RAS（DN/RAS）主要的腺病毒载体引入腺病毒载体（DN/RAS）引入腺病毒载体，研究了RAS在获得完全肿瘤电位中的功能作用。 DN/RAS抑制了hag/src3-1的锚定非依赖性生长93％。用DN/RAS翻译的hag/src3-1细胞未能形成肿瘤。这就是V-SRC在获得肿瘤潜能时可能需要RAS-MAP激酶信号传导级联。为了检查RAC的潜在作用，RAC的潜在作用是一种小的GTPase结合蛋白，其作用于RAS的下游，进行了使用含有DN/RAS或显性阴性RAC（DN/RAC）的腺病毒载体的实验。数据表明，与含有β-gal基因的ADCALACZ相比，DN/RAS和DN/RAS分别将SRC转染细胞的侵入性降低了60％和99％。此外，DN/RAC完全抑制了裸鼠中SRC转染细胞的致瘤潜力。这些结果表明，SRC，RAS，RAC似乎都参与了入侵过程，RAC可能在这些入侵和肿瘤性的信号传导途径的下游作用。较少的