ONCOGENES AND THE RADIOSENSITIVITY OF DNA REPLICATION

目前的建议是基于新的观察， 暴露更持久地抑制DNA复制， 转染的原代大鼠胚胎成纤维细胞（REF）中的程度 癌基因H-ras + v-myc，与REF或获得的细胞系相比， 通过单独转染任一癌基因从REF中分离。 它旨在 研究ras和myc家族的癌基因以及 蛋白质磷酸化对这些晚期事件的调节DNA 在辐射细胞中复制。 我们测试的模型是基于 假设DNA损伤启动了一个转导途径， 将信号从myc传递到ras，ras反过来激活未知蛋白 降低p34 cdc 2激酶活性的激酶， DNA复制。 它假设该法规使用组件 被鉴定为DNA起始调节途径的一部分 复制在非辐照细胞，并推测，功能的 这些调节成分受激活和/或 原癌基因的过度表达。 有证据表明 ras和myc在转导起始中的协同作用 已知ras可激活不同的蛋白质家族， 激酶。 此外，最近的证据表明p34 cdc 2激酶是一个关键， 在DNA复制的起始事件的调节。 拟议 预计研究将提供关于以下方面贡献的信息： 癌基因激活到DNA复制的遗传控制。 它 提供了表征重要反应的第一步 p34 cdc 2激酶的作用 活动在过程中。 这项工作涉及研究的影响， ras和myc家族的癌基因单独或组合， 原代REF和永生化NIH 3 T3中DNA复制的调控 细胞 将使用这些癌基因的野生型和突变形式。 本构和条件向量将被用作测试工具 表达水平对辐射诱导的DNA抑制的影响 复制的 相关性将通过使用 表达反义RNA和核酶的载体， 这些致癌基因的转录本。 蛋白激酶的作用 将通过检查调制来测试所提出的机制 在与辐照细胞孵育后诱导DNA复制 增加或降低其活性的化合物。 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SV 40起源序列的细胞，或分离的细胞核， 用于试图确定DNA复制的近似效应 最终受到癌基因表达的影响。 丰富的 初步的实验证据提供支持我们的早期 这一建议所依据的意见和假设。 的 受辐射细胞中控制DNA复制的因子的存在 通过对共济失调毛细血管扩张症的细胞进行实验， (AT)患者 这些通路的激活可能使细胞 成功地应对环境的侮辱，损害其DNA， 以减少其不良后果。 DNA复制失调 是一种发生在正常细胞中的基本改变， 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.