RADIATION-INDUCED SIGNAL TRANSDUCTION & GENE EXPRESSION

辐射感应信号传导

基本信息

批准号：
3468945
负责人：
STEVEN C MILLER
金额：
$ 12.53万
依托单位：
SRI INTERNATIONAL
依托单位国家：
美国
项目类别：
财政年份：
1992
资助国家：
美国
起止时间：
1992-08-12 至 1997-07-31
项目状态：
已结题

项目摘要

Radiation exposure results in direct and indirect damage to many different molecules in cells, and radiation-induced DNA damage is believed to be the cause of cell death. Mammalian cells respond to radiation-induced DNA damage through specific, inducible pathways that are regulated through poorly understood mechanisms that result in the activation and expression of specific genes. Some effects of radiation are mediated through signaling pathways composed of sequence-specific DNA binding proteins that are involved in the regulation of responsive genes. In our preliminary studies we have investigated the role of the inducible transcription factor NF-(kappa)B in mediating the expression of a reporter gene directed by a promoter that is induced by radiation damage: the HIV1 LTR. The objective of this proposal is to further define the role of reactive oxygen intermediates (ROIs), DNA damage, and NF-(kappa)B DNA binding in the regulation of radiation-induced gene expression. We have demonstrated that UV irradiation of mouse L cells activated NF-(kappa)B DNA binding and reporter gene expression. In contrast, treatment with ionizing radiation failed to activate reporter gene expression.' Agents such as tumor necrosis factor (TNF) and phorbol ester (PMA), which generate ROIs, activated NF-(kappa)B DNA binding without altering reporter gene expression, suggesting that NF-(kappa)B was required but was not sufficient to activate gene expression in this cell line. A strategy of using cell fusion to construct heterokaryons was devised to functionally test for the presence of additional factors. Heterokaryons formed by fusing UV-irradiated human cells with mouse L cells containing an integrated HIV1 LTR-directed lacZ gene directly demonstrated that UV irradiation activated a trans-acting pathway that can pass from a UV-irradiated nucleus and act on a target gene in a noniiradiated nucleus. DNA damage or distortion in the DNA conformation around the inducible gene is unlikely to be involved, because the reporter gene was never exposed to UV irradiation. This system will be used to directly test the role of UV- and ionizing radiation-damaged DNA in the signaling pathway in the absence of exposing cells to radiation. To further explore the relationship between NF-(kappa)B-mediated gene regulation and the response to ionizing radiation, we have characterized a human cell line that is strongly induced by agents that generate ROIs. Microcell-mediated human chromosome transfer will be used to evaluate the existence of human factors that complement the defect in gene expression in mouse L cells. The information derived from this investigation will increase our understanding of cellular mechanisms involved in radiation-induced gene expression.

辐射暴露会对许多人造成直接和间接的损害细胞中的不同分子，辐射引起的 DNA 损伤是被认为是细胞死亡的原因。哺乳动物细胞做出反应通过特定的诱导性DNA损伤来应对辐射引起的DNA损伤通过知之甚少的机制调节的途径导致特定基因的激活和表达。辐射的一些影响是通过信号通路介导的由序列特异性 DNA 结合蛋白组成参与反应基因的调节。在我们的初步我们研究了诱导物的作用转录因子 NF-(kappa)B 介导 a 表达由辐射诱导的启动子指导的报告基因损害：HIV1 LTR。该提案的目的是进一步定义活性氧中间体 (ROI)、DNA 的作用损伤和 NF-(kappa)B DNA 结合在调节中的作用辐射诱导的基因表达。我们已经证明，紫外线照射小鼠 L 细胞会激活 NF-(kappa)B DNA 结合和报告基因表达。相比之下，电离辐射治疗未能激活报告基因表达。'肿瘤坏死因子 (TNF) 和佛波醇等药物酯 (PMA)，产生 ROI，激活 NF-(kappa)B DNA 结合不改变报告基因表达，表明需要 NF-(kappa)B，但不足以激活基因在此细胞系中表达。利用细胞融合的策略构建异核体的目的是为了功能测试额外因素的存在。融合形成的异核体紫外线照射的人类细胞与小鼠 L 细胞含有整合的 HIV1 LTR 导向的 lacZ 基因直接证明紫外线照射激活了反式作用途径，可以从紫外线照射的细胞核并作用于未照射的细胞核中的靶基因核。 DNA 损伤或周围 DNA 构象扭曲诱导基因不太可能参与其中，因为报告者基因从未暴露于紫外线照射。该系统将用于直接测试紫外线和电离辐射损伤的DNA在信号通路中没有将细胞暴露于辐射。为进一步探索 NF-(kappa)B 介导的基因调控与对电离辐射的反应，我们已经表征了人体细胞由生成 ROI 的代理强烈诱导的线。微细胞介导的人类染色体转移将用于评估补充缺陷的人为因素的存在小鼠 L 细胞的基因表达。信息来源于这项研究将增加我们对细胞的了解辐射诱导基因表达的机制。