Engineered Platforms to Manipulate Intracellular Redox

操纵细胞内氧化还原的工程平台

• 批准号: 7097553
• 负责人: Paul J. A. Kenis
• 金额: $ 20.88万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7097553
• 关键词: contact inhibition; intracellular; oxidation reduction reaction

DESCRIPTION (provided by applicant): In redox systems research domains that traditionally belong to the physical sciences, chemistry and molecular biology are coming together to offer new synergistic opportunities for understanding and manipulating basic cellular processes that underlie complex biomedical problems (e.g., tumorigenesis). Parallel with this recognition emerges that intracellular redox status exerts influence on the normal cellular processes of DNA synthesis, selective gene expression, cell cycle progression, proliferation, differentiation, and apoptosis. However, molecular mechanisms mediating redox sensitivity are still poorly defined. Current pharmacological methods to alter intracellular redox potential require significant manipulation of culture conditions that perturb intracellular homeostasis. To overcome this problem and to answer fundamental questions concerning intracellular redox and cell growth, this proposal focuses on the creation of engineered electrochemical platforms that will enable precise manipulation of intracellular redox and novel genetic constructs that will enable real-time and extended assessment of alterations in intracellular redox without cellular disruption. Equipped with cell study platforms and biosensors for visualization (SA 1 & 2), we will address a central cell biological question of primary biomedical relevance that being the relationship between intracellular redox and density-dependent contact inhibition of cell growth (SA 3). The proposed research will thus aid public health by aiming to unravel the role of intracellular redox in uncontrolled cell growth (i.e. tumorigenesis). Specific Aim 1: Design and validate engineered electrochemical (EEC) platforms for cell studies that permit precise control of the intracellular redox environment. We will measure intracellular and intraorganellar redox state as a function of externally applied potential by monitoring the ratios of redox-active species (GSH/GSSG) and with fluorescence microscopy using markers for GSH and ROS, as well as novel gene constructs. Specific Aim 2: Develop and validate FRET biosensors that permit visual monitoring of intracellular and intraorganellar redox potentials. The envisioned genetic constructs encoding FRET-based redox sensors will be stably transfected into target cells allowing real-time monitoring of intracellular redox potentials in live cells. Incorporating organelle-specific targeting sequences will permit the monitoring of intraorganellar redox potential. Specific Aim 3: Use EEC platforms and FRET biosensors to determine how reversibly manipulating intracellular redox status affects cell growth in non-transformed and transformed human fibroblast cell lines. Nontransformed cells become increasingly oxidized concurrent with density-dependent contact inhibition. Thus, we hypothesize that mutations in redox-regulated signaling pathways that render cells unable to initiate contact inhibition may contribute to tumorigenesis. This hypothesis will be addressed with the EEC platforms (and FRET Biosensors for visualization) by reversibly manipulating the intracellular redox status of nontransformed IMR-90 human fibroblasts versus HT-1080 human fibrosarcoma cells (do not exhibit contact inhibition) to determine whether and how progressive oxidation of the intracellular environment contributes to density-dependent contact inhibition.

描述（由申请人提供）：在传统上属于物理科学的氧化还原系统研究领域，化学和分子生物学正在结合在一起，为理解和操纵复杂生物医学问题（例如肿瘤发生）背后的基本细胞过程提供新的协同机会。与这一认识并行的是，细胞内氧化还原状态对 DNA 合成、选择性基因表达、细胞周期进程、增殖、分化和凋亡的正常细胞过程产生影响。然而，介导氧化还原敏感性的分子机制仍不清楚。目前改变细胞内氧化还原电位的药理学方法需要对扰乱细胞内稳态的培养条件进行大量操作。为了克服这个问题并回答有关细胞内氧化还原和细胞生长的基本问题，该提案重点关注创建工程电化学平台，该平台将能够精确操纵细胞内氧化还原和新颖的遗传结构，从而能够实时和扩展地评估细胞内的变化。细胞内氧化还原而不破坏细胞。配备细胞研究平台和可视化生物传感器（SA 1 和 2），我们将解决主要生物医学相关性的核心细胞生物学问题，即细胞内氧化还原与细胞生长的密度依赖性接触抑制之间的关系（SA 3）。因此，拟议的研究将通过旨在揭示细胞内氧化还原在不受控制的细胞生长（即肿瘤发生）中的作用来帮助公众健康。具体目标 1：设计和验证用于细胞研究的工程电化学 (EEC) 平台，允许精确控制细胞内氧化还原环境。我们将通过监测氧化还原活性物质 (GSH/GSSG) 的比率并使用 GSH 和 ROS 标记以及新的基因构建体通过荧光显微镜来测量细胞内和细胞器内氧化还原状态作为外部施加电位的函数。具体目标 2：开发并验证 FRET 生物传感器，允许视觉监测细胞内和细胞器内氧化还原电位。设想的编码基于 FRET 的氧化还原传感器的基因构建体将被稳定转染到靶细胞中，从而能够实时监测活细胞中的细胞内氧化还原电位。合并细胞器特异性靶向序列将允许监测细胞器内氧化还原电位。具体目标 3：使用 EEC 平台和 FRET 生物传感器来确定可逆地操纵细胞内氧化还原状态如何影响非转化和转化的人成纤维细胞系中的细胞生长。未转化的细胞变得越来越氧化，同时发生密度依赖性接触抑制。因此，我们假设氧化还原调节信号通路中的突变使细胞无法启动接触抑制，这可能有助于肿瘤发生。这一假设将通过 EEC 平台（和用于可视化的 FRET 生物传感器）通过可逆地操纵未转化的 IMR-90 人成纤维细胞与 HT-1080 人纤维肉瘤细胞（不表现出接触抑制）的细胞内氧化还原状态来解决，以确定是否以及如何进展。细胞内环境的氧化有助于密度依赖性接触抑制。