Engineered Platforms to Manipulate Intracellular Redox

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中，化学和分子生物学正在走到一起，为理解和操纵复杂生物医学问题（例如，肿瘤发生）。与此同时，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然不清楚。目前改变细胞内氧化还原电位的药理学方法需要显著操纵扰乱细胞内稳态的培养条件。为了克服这一问题，并回答有关细胞内氧化还原和细胞生长的基本问题，该提案侧重于创建工程电化学平台，这将使细胞内氧化还原和新的遗传结构的精确操纵，这将使实时和扩展的评估细胞内氧化还原的变化，而不破坏细胞。配备细胞研究平台和生物传感器进行可视化（SA 1和2），我们将解决主要生物医学相关的中心细胞生物学问题，即细胞内氧化还原和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：设计和验证工程电化学（EEC）平台，用于细胞研究，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）的比例并使用荧光显微镜使用GSH和ROS标记物以及新型基因构建体来测量细胞内和细胞器内氧化还原状态与外部施加电位的函数关系。具体目标2：开发和验证FRET生物传感器，允许细胞内和intraorganellar氧化还原电位的视觉监测。所设想的编码基于FRET的氧化还原传感器的遗传构建体将被稳定地转染到靶细胞中，从而允许实时监测活细胞中的细胞内氧化还原电位。阐明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生物传感器来确定如何可逆地操纵细胞内氧化还原状态影响非转化和转化的人成纤维细胞系中的细胞生长。非转化细胞变得越来越氧化的密度依赖性接触抑制的同时。因此，我们推测，突变的氧化还原调节信号通路，使细胞无法启动接触抑制可能有助于肿瘤的发生。将使用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（不显示接触抑制）的细胞内氧化还原状态来解决该假设，以确定细胞内环境的进行性氧化是否以及如何有助于密度依赖性接触抑制。

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.