GENETIC ANALYSIS OF NEOPLASTIC TRANSFORMATION BY MYC

原癌基因c-myc与多种肿瘤的发生有关。 人类肿瘤，包括淋巴瘤和白血病；此外，其 体外转化活性已被清楚地证明。尽管 C-myc蛋白参与细胞内作用的有力证据 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精确作用 都还没有确定。 我们的工作假设是c-myc通过 与其他尚未确定的细胞成分的相互作用。我们 建议通过隔离来定义这些组件的性质 和未转化变异体(回复变异体)的分子特征 从myc转化的细胞群体中。这一战略基于我们的 预期至少某些细胞“靶点”中的基因改变 由逆转录病毒介导的插入突变诱导的基因将 体外抑制或削弱c-myc的转化能力。我们的 长期目标包括分离和功能分析 基因(S)，其改变(即激活或失活)是负责的 对于突变的表型。 我们的方法具有两个试验性方案，旨在促进 这类返回体的分离和责任人的鉴定 遗传基因发生了改变。首先，导致的回复菌株的分离 仅由c-myc基因丢失或失活造成的风险将降至最低 转化的大鼠成纤维细胞系的应用 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突变获得的返回体；整合 与“靶标”基因相邻或位于“靶标”基因内的前病毒将作为物质 “标签”，用于后续的基因分离。一种稀土元素的浓缩方案 背景中的未转换单元格包含大量过剩的 转化的细胞将被用来选择表型逆转株(即 表现出正常扁平形态的细胞)。改革者将被选中 根据几个标准进一步定性，包括 对c-myc再转化的抗性和高产转化能力 当病毒与复制能力强的逆转录病毒重叠感染时。 将进行体细胞杂交分析以确定 导致突变的基因改变的性质 表型(即显性或隐性)。我们将分离出 前病毒插入部位两侧的细胞DNA序列 通过分子克隆恢复细胞系，并最终使用这些 以序列为探针从表达中分离相关基因 用于结构和功能表征的文库。 我们预计，这种方法将开发出新的试剂， 解决有关c-myc在细胞周期中作用的几个基本问题 肿瘤，推测的多步途径的组成 致癌，以及更广泛地说，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.