COOPERATIVE EFFECTS OF VIRAL AND CELLULAR ONCOGENES

正常细胞发展为完全恶性肿瘤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成员的多样性 一组叫做癌基因的细胞基因。 癌基因介导的多步 转化已经在原代细胞培养物中得到证实，但不是一种有效的方法。 建立的细胞系的一般性质。 C3 H1 OT 1/2细胞系是一种 异常，并且当转染时与原代细胞的行为相似。 人H-ras癌基因和MC 29病毒myc癌基因。 Ras/Myc协同作用 在C3 H1 OT 1/2成纤维细胞中，以病灶形成增加和 高度转化的细胞形态，反映了 在软琼脂糖中的锚定独立生长。 评估贡献 每个癌基因与ras/myc转化表型的C3 H1 OT 1/2细胞系 将检测表达ras、myc或这两种癌基因的患者的 癌基因表达并测定裸鼠中的致瘤性。 的 C3 H1 OT 1/2细胞共转染后病灶形成增加 与ras和myc，再加上定点突变的MC 29 gag- myc基因，将用于鉴定对ras/myc重要的myc序列 合作变换，并确定指定的序列 gag-myc蛋白的核定位。 的时间关系 协同转化中ras和myc基因表达之间的关系 将在使用诱导型c-myc C3 H1 OT 1/2的时程研究中探索 细胞系 为了进一步剖析 为研究ras/myc在C3 H1 OT 1/2细胞中协同作用途径，我们将制备cDNA 从C3 H1 OT 1/2和C3 H1 OT 1/2 myc细胞的文库和差异筛选 用于响应于 myc过表达。 将检查鉴定的cDNA的 在各种细胞系中myc相关表达的适当模式。 充分 长度cDNA将被亚克隆到表达载体中并转染到 用于功能测定的C3 H1 OT 1/2细胞。 本文中概述的实验 这些建议旨在增加我们对myc功能的了解， 哺乳动物细胞，并确定其产物是 重要的是在文化和文化的进展多步转化 体内肿瘤形成。

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.