FRET-based Biosensors to Monitor Redox in Cell Cycle Regulation

描述（由申请人提供）：癌症可以被视为细胞增殖和细胞死亡之间的平衡异常地有利于前者的状态。我们和其他人已经发现，细胞内氧化还原环境对调节增殖和细胞死亡之间平衡的正常细胞过程产生深远影响，包括DNA合成、酶激活、细胞周期进程、增殖、分化和凋亡。事实上，可以认为氧化还原稳态是细胞命运管理的核心。不幸的是，介导细胞内氧化还原敏感性和调节的分子机制仍然不清楚。目前改变细胞内氧化还原状态的药理学方法受到以下限制：（i）它们不能独立于整体生物化学改变和细胞毒性而操作，以及（ii）需要显著操纵扰乱细胞内稳态的培养条件。我们的遗传构建体克服了这些限制，因为它们能够在不破坏细胞的情况下实时和扩展地评估细胞内氧化还原的改变。这些构建体使用荧光共振能量转移（FRET），供体和受体荧光团之间的距离和取向依赖性能量转移过程。在这些生物传感器中，氧化还原的变化诱导连接供体和受体的氧化还原敏感开关的构象变化，改变它们的距离，这反过来又导致FRET效率的可检测变化。在这里，我们建议进一步定义我们的FRET生物传感器相对于细胞内氧化还原环境的变化，似乎决定细胞命运的灵敏度和动态范围。该方法的优点包括：（1）量化氧化还原状态变化的能力;（2）传感器浓度的独立性;和（3）通过交换构建体中的开关或荧光团模块来精确调节氧化还原灵敏度和范围的能力。 目标1：通过比较非转化成纤维细胞和同基因猪肿瘤细胞系是否存在接触抑制，确定基因工程FRET氧化还原生物传感器在增殖过程中的灵敏度和动态范围。具体而言，提出了在细胞生长的连续阶段期间检测生理相关的变化。 目标二：确定FRET生物传感器对用化疗药物氟尿嘧啶和多柔比星处理的等基因HCT 116 p53+/+和p53-/-细胞的细胞内氧化还原环境的变化以及谷胱甘肽体内平衡的扰动的敏感程度。具体地，细胞内氧化还原环境将响应于与调节谷胱甘肽的生物合成或代谢的试剂组合的常见化疗药物而可视化。 目的3：建立第二代FRET生物传感器，允许视觉监测和解剖的细胞器内局部氧化还原电位。具体而言，我们打算量化的亚细胞器内的氧化还原电位的差异，在一个非平衡稳态相对于彼此在活细胞。 总之，拟议的工作将提供新的分子工具，使在细胞内和细胞器内水平的氧化还原信号在癌症发展中的作用的深入检查。 公共卫生相关性：本计画追求新颖的分子工具-氧化还原敏感生物感测器-以深入探讨氧化还原讯号在与癌症发展相关的细胞过程中所扮演的角色。这些生物传感器的优化将使氧化还原电位的局部变化可视化，这可能会调节细胞周期的进展，并介导细胞生长的接触依赖性抑制，细胞生长的破坏是癌症的关键标志。最终，这些工具将增强对癌细胞在多大程度上失去了伴随正常细胞生长的氧化还原电位变化的能力以及对这些变化的敏感性的理解。

DESCRIPTION (provided by applicant): Cancer can be viewed as a state in which the balance between cell proliferation and cell death aberrantly favors the former. We and others have discovered that the intracellular redox environment exerts a profound influence on the normal cellular processes that regulate the balance between proliferation and cell death, including DNA synthesis, enzyme activation, cell cycle progression, proliferation, differentiation, and apoptosis. In fact, it could be argued that redox homeostasis is central to the governance of cell fate. Unfortunately, molecular mechanisms mediating redox sensitivity and regulation within cells are still poorly defined. Current pharmacological methods to alter intracellular redox state are limited by (i) their inability to operate independent of global biochemical alterations and cellular toxicity, and (ii) the required significant manipulation of culture conditions that perturb intracellular homeostasis. Our genetic constructs overcome these limitations as they enable real-time and extended assessment of alterations in intracellular redox without cellular disruption. These constructs use fluorescence resonance energy transfer (FRET), a distance- and orientation- dependent energy transfer process between donor and acceptor fluorophores. In these biosensors a change in redox induces a conformational change in the redox-sensitive switch that links the donor and acceptor, changing their distance, which in turn causes a detectable change in FRET efficiency. Here we propose to further define the sensitivity and dynamic range of our FRET biosensors relative to changes in the intracellular redox environment that appear to dictate cell fate. Advantages of this approach include: (1) the ability to quantify the change in redox state; (2) independence of sensor concentration; and (3) the ability to precisely tune the redox sensitivity and range by exchange of the switch or the fluorophore modules in the construct. Aim 1: Define the sensitivity and dynamic range of genetically engineered FRET redox biosensors during proliferation by comparison of nontransformed fibroblasts and isogenic porcine tumor cell lines with respect to the presence or absence of contact inhibition. Specifically, detection of physiologically relevant changes during successive stages of cell growth is proposed. Aim 2: Determine the extent to which the FRET biosensors are sensitive to changes in the intracellular redox environment of isogenic HCT116 p53+/+ and p53-/- cells treated with the chemotherapeutic drugs fluorouracil and doxorubicin in combination with perturbations in glutathione homeostasis. Specifically, the intracellular redox environment will be visualized in response to common chemotherapeutic drugs in combination with agents that modulate biosynthesis or metabolism of glutathione. Aim 3: Create second generation FRET biosensors that permit visual monitoring and dissection of intraorganellar local redox potentials. Specifically, we intend to quantify differences in redox potentials within subcellular organelles that are at a nonequilibrium steady-state with respect to each other in living cells. In sum, the proposed work will provide novel molecular tools that enable in depth examination of the role of redox signaling at the intracellular and intraorganellar level in cancer development. PUBLIC HEALTH RELEVANCE: This project pursues novel molecular tools-redox-sensitive biosensors-that will enable in depth examination of the role of redox signaling in cellular processes related to cancer development. Optimization of these biosensors will enable visualization of local changes in redox potential that might regulate progression through the cell cycle and mediate contact-dependent inhibition of cell growth, the disruption of which is a key hallmark of cancer. Ultimately, the tools will enhance understanding of the extent to which cancerous cells have lost the ability to mount changes in redox potential that accompany normal cell growth versus their sensitivity to these changes.