COOPERATIVE EFFECTS OF VIRAL AND CELLULAR ONCOGENES

正常细胞向完全恶性肿瘤的发展是一个多步骤的过程。 涉及外部诱变或生长之间相互作用的过程 促进刺激和激活一个或多个不同的成员 一组称为癌基因的细胞基因。癌基因介导的多步法 转化已经在原代细胞培养中得到证实，但并不是 已建立的细胞系的一般特性。C3H1OT1/2细胞系是一种 异常，其行为类似于原代细胞。 人H-ras癌基因和MC29病毒myc癌基因。RAS/MYC合作 在C3H1OT1/2成纤维细胞中，病灶形成增加和 高度转化的细胞形态，反映了 在软琼脂中锚定独立生长。以评估其贡献 各癌基因对ras/myc转化表型C3H1OT1/2细胞的作用 表达ras、myc或两者的癌基因将被检测其水平 癌基因在裸鼠体内的表达及致瘤性检测。这个 共转染C3H1OT1/2细胞后病灶形成的增加 与ras和myc结合，结合MC29 Gag-1基因的定点突变。 Myc基因，将用于鉴定对ras/myc重要的myc序列。 合作变换，并且还识别指定 Gag-myc蛋白的核定位。时间关系 协同转化中ras表达与myc表达的关系 将在使用可诱导的c-myc C3H1OT1/2的时间进程研究中进行探索 细胞系。为了进一步剖析分子中的中间体 在C3H1OT1/2细胞中ras/myc协同作用的途径，我们将制备 C3H1OT1/2和C3H1OT1/2 Myc细胞文库的构建及差异筛选 消息库上调或下调以响应 MYC过度表达。将对鉴定出的DNA进行检测，以确定 Myc相关基因在不同细胞系中的适当表达模式。饱满 将长度的cDNA亚克隆到表达载体中，并转染到 用于功能分析的C3H1OT1/2细胞。这篇文章中概述的实验 提案旨在增加我们对myc功能的了解 哺乳动物细胞，并鉴定其产物是 对文化的多步骤转变和进步很重要 体内的肿瘤形成。

The progression of a normal cell to a fully malignant tumor is a multistep process involving interactions between external mutagenic or growth promoting stimuli and the activation of one or more members of a diverse set of cellular genes called oncogenes. Oncogene-mediated multistep transformation has been demonstrated in primary cell cultures but is not a general property of established cell lines. The C3H1OT1/2 cell line is an exception and behaves similarly to primary cells when transfected with the human H-ras oncogene and the MC29 viral myc oncogene. Ras/myc cooperation in C3H1OT1/2 fibroblasts is marked by an increase in focus formation and a highly transformed cell morphology that reflects an increased potential for anchorage independent growth in soft agarose. To assess the contribution of each oncogene to the ras/myc transformed phenotype, C3H1OT1/2 cell lines expressing ras, myc or both oncogenes will be examined for levels of oncogene expression and assayed for tumorigenicity in nude mice. The increase in focus formation following co-transfection of C3H1OT1/2 cells with ras and myc, coupled with site-directed mutagenesis of the MC29 gag- myc gene, will be used to identify the myc sequences important to ras/myc cooperative transformation and also to identify the sequences specifying nuclear localization of the gag-myc protein. The temporal relationship between ras expression and myc expression in cooperative transformation will be explored in time-course studies using an inducible c-myc C3H1OT1/2 cell line. In an effort to dissect further the molecular intermediates in the pathway of ras/myc cooperation in C3H1OT1/2 cells, we will prepare cDNA libraries from C3H1OT1/2 and C3H1OT1/2 myc cells and differentially screen the libraries for messages up-regulated or down-regulated in response to myc over-expression. The cDNAs identified will be examined for the appropriate pattern of myc-related expression in various cell lines. Full length cDNAs will be subcloned into expression vectors and transfected into C3H1OT1/2 cells for functional assays. The experiments outlined in this proposal are designed to increase our knowledge of myc function in mammalian cells and to identify additional genes whose products are important to multistep transformation in culture and the progression of tumor formation in vivo.