RADIATION INDUCED SIGNAL TRANSDUCTION & GENE EXPRESSION

辐射感应信号传导

• 批准号: 2185718
• 负责人: STEVEN C MILLER
• 金额: $ 12.58万
• 依托单位: SRI INTERNATIONAL
• 依托单位国家: 美国
• 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
• 来源: https://reporter.nih.gov/project-details/2185718
• 关键词: DNA binding protein; DNA damage; biological signal transduction; cell fusion; clone cells; electroporation; flow cytometry; fluorescence microscopy; free radical oxygen; gene expression; genetic regulation; genetic transcription; heterokaryon; human genetic material tag; in situ hybridization; ionizing radiation; radiation dosage; radiation resistance; transfection; ultraviolet radiation

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.

辐射暴露会导致对许多人的直接和间接损害 细胞中的不同分子，辐射诱导的DNA损伤为 被认为是细胞死亡的原因。 哺乳动物细胞反应 通过特定的，可诱导的辐射引起的DNA损伤 通过不理理解的机制来调节的途径 这导致特定基因的激活和表达。 辐射的某些效果通过信号通路介导 由序列特异性DNA结合蛋白组成 参与反应基因的调节。 在我们的初步中 我们研究了诱导的作用 转录因子nf-（kappa）b介导a的表达 由辐射引起的启动子指导的报告基因 损坏：HIV1 LTR。 该提议的目的是进一步 定义活性氧中间体（ROI），DNA的作用 损坏和NF-（kappa）B DNA结合在调节中 辐射诱导的基因表达。 我们已经证明了小鼠L细胞激活的紫外线照射 NF-（KAPPA）B DNA结合和报告基因表达。 相比之下， 电离辐射的处理未能激活报告基因 表达。'诸如肿瘤坏死因子（TNF）和Phorbol之类的药物 产生ROI的酯（PMA）激活了NF-（kappa）B DNA结合 没有改变报告基因表达，表明 需要NF-（Kappa）B，但不足以激活基因 在此细胞系中的表达。 使用细胞融合到的策略 设计构造杂质者是为了在功能上测试 存在其他因素。 通过融合形成的异性恋 用含有小鼠L细胞的紫外线辐照的人类细胞含有 综合HIV1 LTR指导的LACZ基因直接证明 紫外线辐照激活了可以从 紫外线辐射的核并作用于未核实的靶基因 核。 DNA损伤或DNA构象中的失真 诱导基因不太可能涉及，因为记者 基因从未暴露于紫外线照射。 该系统将用于直接测试UV-和 在信号通路中的电离辐射损坏的DNA 没有暴露于辐射的细胞。 进一步探索 NF-（Kappa）B介导的基因调节与 对电离辐射的响应，我们表征了人类细胞 产生ROI的药物强烈诱导的线。 微孔介导的人类染色体转移将用于 评估与缺陷相互补充的人为因素的存在 在小鼠L细胞中的基因表达中。 从 这项调查将增加我们对细胞的理解 涉及辐射诱导的基因表达的机制。