ONCOGENES AND THE RADIOSENSITIVITY OF DNA REPLICATION

目前的提议是基于新的观察，即辐射 暴露会更持久地、更大范围地抑制 DNA 复制 转染原代大鼠胚胎成纤维细胞（REF）的程度 癌基因 H-ras 加 v-myc，与 REF 或获得的细胞系相比 通过单独用任一癌基因转染从 REF 中分离。 它的设计目的是 研究 ras 和 myc 家族癌基因的参与以及 蛋白质磷酸化对 DNA 调控的这些晚期事件的影响 在受辐射的细胞中复制。 我们测试的模型基于 DNA 损伤启动转导途径的假设 将信号从 myc 传输至 ras，从而激活未知蛋白质 降低 p34cdc2 激酶活性的激酶，并因此启动 DNA 复制。 它假设该法规利用组件 被鉴定为 DNA 起始调控途径的一部分 在未受辐射的细胞中复制，并推测其功能 这些调节成分受到激活和/或的影响 原癌基因的过度表达。 有证据表明 ras 和 myc 在转导启动中的合作 途径，并且 ras 已知可激活不同的蛋白质家族 激酶。 此外，最近的证据表明 p34cdc2 激酶是一个关键 DNA 复制起始事件的调节因子。 拟议的 研究预计将提供有关贡献的信息 癌基因激活对DNA复制的遗传控制。 它 提供了表征重要响应的第一步 受辐射细胞中的通路并检查 p34cdc2 激酶的作用 过程中的活动。 这项工作涉及研究影响 ras 和 myc 家族的癌基因单独或组合， 初级 REF 和永生化 NIH 3T3 中 DNA 复制的调节 细胞。 将使用这些致癌基因的野生型和突变体形式。 本构向量和条件向量将用作测试工具 表达水平对辐射诱导的 DNA 抑制的影响 复制。 相关性将通过使用进一步测试 表达反义RNA和核酶的载体 这些癌基因的转录本。 蛋白激酶的贡献 将通过检查调制来测试拟议的机制 受辐射细胞孵育后诱导 DNA 复制 增加或减少其活性的化合物。 最后，两个 利用细胞提取物和质粒进行 DNA 复制的模型系统 携带猿猴病毒SV40起源序列，或分离的细胞核将 用于尝试识别 DNA 复制的直接影响 最终受到癌基因表达的影响。 丰富的 提供了初步的实验证据支持我们之前的 该提案所依据的观察结果和假设。 的 受辐射细胞中控制 DNA 复制的因素的存在 共济失调毛细血管扩张细胞的实验表明 （AT）患者。 此类途径的激活可能允许细胞 成功应对损害其 DNA 的环境侵害，并且 以减少其不良后果。 DNA 复制的放松管制 是正常细胞在以下过程中发生的基本改变 它转化为恶性细胞。

The present proposal is based on the novel observation that radiation exposure inhibits DNA replication more persistently and to a larger extent in primary rat embryo fibroblasts (REF) transfected with the oncogenes H-ras plus v-myc, as compared to REF or, to cell lines obtained from REF by transfection with either oncogene alone. It is designed to study the involvement of oncogenes of the ras and myc families and of protein phosphorylation on these late events f regulation of DNA replication in irradiated cells. The model we test is based on the hypothesis that DNA damage initiates a transduction pathway that transmits signals from myc to ras which in turn activates unknown protein kinases that reduce p34cdc2 kinase activity, and as a result initiation of DNA replication. It assumes that this regulation utilizes components identified as parts of the regulatory pathway for initiation of DNA replication in non-irradiated cells, and speculates that the function of these regulatory components is affected by activation and/or overexpression of proto-oncogenes. There is evidence suggesting cooperation between ras and myc in the initiation of transduction pathways, and ras is known to activate different families of protein kinases. Furthermore, recent evidence points to p34cdc2 kinase as a key regulator in the initiation events of DNA replication. The proposed research is expected to provide information on the contribution of oncogene activation to the genetic control of DNA replication. It provides the first steps towards characterization of important response pathways in irradiated cells and examines the role of p34cdc2 kinase activity in the process. The work involves the study of the effect of oncogenes of the families ras and myc alone or in combination, on the regulation of DNA replication in primary REF and immortalized NIH 3T3 cells. Wild type and mutant forms of these oncogenes will be used. Constitutive and conditional vectors will be employed as tools to test the effect of expression level on radiation induced inhibition of DNA replication. The correlation will be further tested by the use of vectors expressing antisense RNA and ribozymes directed against the transcripts of these oncogenes. The contribution of protein kinases in the proposed mechanism will be tested by examining the modulations induced in DNA replication after incubation of irradiated cells with compounds that either increase or decrease their activity. Finally, two model systems for DNA replication utilizing cell extracts and plasmids carrying the simian virus SV40 origin sequences, or isolated nuclei will be used in a attempt to identify proximate effects of DNA replication ultimately affected by the expression of oncogenes. A wealth of preliminary experimental evidence is provided supporting our earlier observations and the hypotheses on which this proposal rests. The existence e of factors that control DNA replication in irradiated cells has been suggested by experiments with cells from ataxia telangiectasia (AT) patients. Probably activation of such pathways allows the cell to successfully respond to environmental insults that damage its DNA, and to reduce their adverse consequences. Deregulation of DNA replication is a fundamental alteration occurring in a normal cell in the process of its conversion to a malignant cell.