GENETIC ANALYSIS OF NEOPLASTIC TRANSFORMATION BY MYC

c-myc原癌基因与多种 人类肿瘤，包括淋巴瘤和白血病;此外， 体外转化活性已被清楚地证明。 尽管 令人信服的证据表明，c-myc蛋白参与细胞 增殖、有丝分裂和分化及其生化功能 在正常细胞生长和肿瘤转化中的作用 还有待确定。 我们的工作假设是，c-myc发挥其作用，通过 与其他尚未识别的细胞成分相互作用。 我们 建议通过隔离来定义这些组件的性质 非转化变体（回复突变体）的分子表征 从myc转化的细胞群体中。 这一战略基于我们的 预期至少在某些细胞“靶”上的遗传改变 通过逆转录病毒介导的插入突变诱导的基因将 削弱或抑制c-myc的体外转化能力。 我们 长期目标包括隔离和功能分析， 其改变（即激活或失活）负责 回复突变体表型。 我们的方法具有两个实验方案，旨在促进 分离这些回复突变体并鉴定负责的 改变了基因位点 首先，分离回复突变体， 将通过以下方法使c-myc基因的丢失或失活所引起的损伤最小化： 使用转化的大鼠成纤维细胞系， c-myc的整合（和功能）拷贝。 第二，我们将产生 通过逆转录病毒介导的插入诱变的回复突变体; 与“靶”基因相邻或在“靶”基因内的原病毒将作为一种物理载体， “标签”用于随后的基因分离。 一种富集稀有金属的方法 在含有大量过量的 转化的细胞将用于选择表型回复突变体（即， 表现出正常扁平形态的细胞）。 将选择回复突变体 根据若干标准进一步定性，包括 对c-myc再转化的抗性和产生转化的能力 病毒与复制能力的逆转录病毒重叠感染。 将进行体细胞杂交分析以确定 导致回复突变的遗传改变的性质 表型（即显性或隐性）。 我们将隔离 前病毒插入位点侧翼的细胞DNA序列， 回复突变体细胞系的分子克隆，并最终使用这些 序列作为探针，从表达中分离相关基因， 用于结构和功能表征的库。 我们预计，这种方法将开发新的试剂， 为了解决关于c-myc在以下方面的作用的几个基本问题， 肿瘤，假定的多步骤途径的组成， 致癌作用，更广泛地说，涉及的潜在机制 人类癌症

The c-myc proto-oncogene has been implicated in the genesis of a variety of human tumors, including lymphomas and leukemias; furthermore, its transforming activity in vitro has been clearly demonstrated. Despite compelling evidence for the involvement of the c-myc protein in cell proliferation, mitogenesis, and differentiation, its biochemical function and precise role in normal cellular growth and neoplastic transformation have yet to be established. Our working hypothesis is that c-myc exerts its effects through interaction with other, as yet unidentified, cellular components. We propose to define the nature of these components through the isolation and molecular characterization of non-transformed variants (revertants) from populations of myc-transformed cells. This strategy is based on our expectation that genetic alterations in at least some cellular "target" genes, induced by retroviral-mediated insertional mutagenesis, will impair or suppress the transforming capability of c-myc in vitro. Our long-term objectives involve the isolation and functional analysis of the gene(s) whose alteration (i.e. activation or inactivation) is responsible for the revertant phenotype. Our approach features two experimental schemes designed to facilitate the isolation of such revertants and the identification of the responsible altered genetic loci. First, the isolation of revertants resulting simply from loss or inactivation of the c-myc gene will be minimized by the use of a transformed rat fibroblast cell line containing two stably integrated (and functional) copies of c-myc. Second, we will generate revertants by retroviral-mediated insertional mutagenesis; integration of a provirus adjacent to or within a "target" gene will serve as a physical "tag" for subsequent gene isolation. A protocol for enrichment of rare non-transformed cells in a background containing a vast excess of transformed cells will be used to select phenotypic revertants (i.e. cells exhibiting normal flat morphology). Revertants will be selected for further characterization on the basis of several criteria, including resistance to retransformation by c-myc and ability to yield transforming virus upon superinfection with a replication-competent retrovirus. Somatic cell hybridization analyses will be performed to ascertain the nature of the genetic alterations responsible for the revertant phenotypes (i.e. dominance or recessiveness). We will isolate the cellular DNA sequences flanking the site of proviral insertion in revertant cell lines by molecular cloning, and, ultimately, use these sequences as probes to isolate the involved genes from expression libraries for structural and functional characterization. We anticipate that this approach will develop novel reagents with which to address several fundamental questions concerning the role of c-myc in neoplasia, the composition of the putative multistep pathway of carcinogenesis, and more broadly, the underlying mechanisms involved in human cancer.