ONCOGENES AND THE RADIOSENSITIVITY OF DNA REPLICATION

目前的建议是基于一种新的观察结果，即辐射 暴露对DNA复制的抑制更持久，影响更大 体外培养的大鼠胚胎成纤维细胞(REF) 癌基因H-ras加v-myc与REF或，与获得的细胞系的比较 通过单独用两种癌基因中的任何一种来转染REF。它的设计目的是 研究ras和myc家族癌基因的参与以及 DNA调控中这些晚期事件的蛋白质磷酸化 在受辐射的细胞中复制。我们测试的模型基于 假设DNA损伤启动了一条转导途径， 从myc向ras传递信号，ras进而激活未知蛋白质。 降低p34cdc2激酶活性的激酶，从而启动 DNA复制。它假定该法规使用组件 被确认为启动DNA的调控途径的一部分 在未受辐射的细胞中复制，并推测 这些监管组件受激活和/或 原癌基因的过度表达。有证据表明 Ras和myc在启动转导过程中的合作 通路，而ras已知可以激活不同的蛋白质家族。 激活剂。此外，最近的证据表明，p34cdc2激酶是一个关键 DNA复制启动事件中的调节因子。建议数 预计研究将提供关于以下贡献的信息 癌基因激活对DNA复制的遗传控制。它 为确定重要响应的特征提供了第一步 辐射后细胞内的信号转导途径及p34cdc2激酶的作用 过程中的活动。这项工作涉及到对 Ras和myc家族的癌基因单独或组合在 初级REF和永生化NIH3T3中DNA复制的调控 细胞。这些癌基因的野生型和突变型将被使用。 将使用本构向量和条件向量作为测试工具 基因表达水平对辐射抑制DNA的影响 复制。这种相关性将通过使用 靶向反义RNA和核酶的表达载体 这些癌基因的转录本。蛋白激酶在细胞周期中的作用 建议的机制将通过检查调制来进行测试 照射后细胞孵育后DNA复制诱导 增强或降低其活性的化合物。最后，两个 利用细胞提取液和质粒进行DNA复制的模型系统 携带猴病毒SV40起源序列或分离的核将 被用来试图确定DNA复制的直接影响 最终受癌基因表达的影响。一大笔钱 提供了初步的实验证据，支持我们早期的 这项建议所依据的观察和假设。这个 辐射细胞中是否存在控制DNA复制的因素 是通过对共济失调毛细血管扩张症细胞的实验提出的 病人。这种通路的激活很可能允许细胞 成功应对破坏其DNA的环境侮辱，以及 以减少其不良后果。放松对DNA复制的管制 是发生在正常细胞的过程中的根本改变 它会转化为恶性细胞。

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.