FRET-based Biosensors to Monitor Redox in Cell Cycle Regulation

基于 FRET 的生物传感器可监测细胞周期调节中的氧化还原

• 批准号: 8305728
• 负责人: Rex Gaskins
• 金额: $ 26.84万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8305728
• 关键词: Anabolism; Apoptosis; Area; Biochemical; Biochemical Phenomena; Biological; Biological Models; Biosensor; Cancerous; Cell Cycle; Cell Cycle Progression; Cell Cycle Regulation; Cell Death; Cell Proliferation; Cell physiology; Cells; Cloning; Contact Inhibition; Coupling; Cultured Cells; DNA biosynthesis; Detection; Development; Dissection; Doxorubicin; Electron Transport; Energy Transfer; Engineering; Environment; Enzyme Activation; Equilibrium; Family suidae; Fibroblasts; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorouracil; Gases; Gene Expression; Generations; Genetic; Glutathione; Glutathione Disulfide; Glutathione Metabolism Pathway; HCT116 Cells; Homeostasis; Human; Imagery; Life; Link; Malignant Neoplasms; Measurement; Mediating; Methods; Modification; Molecular; Molecular Analysis; Monitor; Normal Cell; Organelles; Oxidation-Reduction; Performance; Pharmaceutical Preparations; Process; Protein Chemistry; Regulation; Relative (related person); Role; Signal Transduction; Staging; Sum; TP53 gene; Techniques; Technology; Time; Toxic effect; Tumor Cell Line; Visual; Work; base; cell growth; colon cancer cell line; cytotoxic; design; fluorophore; innovation; novel; oxidation; physical process; programs; public health relevance; ratiometric; response; sensor; tool; tumor

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.

描述（由申请人提供）：癌症可以被视为一种细胞增殖和细胞死亡之间的平衡异常有利于前者的状态。我们和其他人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的影响，包括DNA合成、酶激活、细胞周期进程、增殖、分化和凋亡。事实上，可以说氧化还原稳态对于细胞命运的调控至关重要。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)它们无法独立于整体生化改变和细胞毒性而发挥作用，以及(ii)需要对扰乱细胞内稳态的培养条件进行显着操作。我们的基因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变化。这些构建体使用荧光共振能量转移（FRET），这是供体和受体荧光团之间依赖于距离和方向的能量转移过程。在这些生物传感器中，氧化还原的变化会引起连接供体和受体的氧化还原敏感开关的构象变化，从而改变它们的距离，进而导致 FRET 效率发生可检测的变化。在这里，我们建议进一步定义我们的 FRET 生物传感器相对于似乎决定细胞命运的细胞内氧化还原环境变化的灵敏度和动态范围。这种方法的优点包括：（1）能够量化氧化还原态的变化； (2) 与传感器浓度无关； (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：通过比较未转化的成纤维细胞和同基因猪肿瘤细胞系是否存在接触抑制，确定增殖过程中基因工程 FRET 氧化还原生物传感器的灵敏度和动态范围。具体来说，建议检测细胞生长的连续阶段期间的生理相关变化。 目标 2：确定 FRET 生物传感器对用化疗药物氟尿嘧啶和阿霉素处理的同基因 HCT116 p53+/+ 和 p53-/- 细胞的细胞内氧化还原环境变化以及谷胱甘肽稳态扰动的敏感程度。具体来说，细胞内氧化还原环境将响应常见化疗药物与调节谷胱甘肽生物合成或代谢的药物的组合而显现。 目标 3：创建第二代 FRET 生物传感器，允许视觉监测和解剖细胞器内局部氧化还原电位。具体来说，我们打算量化活细胞中彼此处于非平衡稳态的亚细胞细胞器内氧化还原电位的差异。 总之，拟议的工作将提供新颖的分子工具，能够深入研究氧化还原信号在细胞内和细胞器内水平在癌症发展中的作用。 公共健康相关性：该项目追求新型分子工具——氧化还原敏感的生物传感器——这将能够深入研究氧化还原信号在与癌症发展相关的细胞过程中的作用。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，而细胞生长的破坏是癌症的一个关键标志。最终，这些工具将增强人们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它们对这些变化的敏感性的了解。

项目成果

Distinct responses of compartmentalized glutathione redox potentials to pharmacologic quinones targeting NQO1.

区室化谷胱甘肽氧化还原电位对靶向 NQO1 的药理学醌的独特反应。

• DOI: 10.1016/j.bbrc.2016.12.082
• 发表时间: 2017
• 期刊: Biochemical and biophysical research communications
• 影响因子: 3.1
• 作者: Kolossov,VladimirL;Ponnuraj,Nagendraprabhu;Beaudoin,JessicaN;Leslie,MatthewT;Kenis,PaulJ;Gaskins,HRex
• 通讯作者: Gaskins,HRex

Development of a high-dynamic range, GFP-based FRET probe sensitive to oxidative microenvironments.

• DOI: 10.1258/ebm.2011.011009
• 发表时间: 2011-06-01
• 期刊: Experimental biology and medicine (Maywood, N.J.)
• 作者: Kolossov VL;Spring BQ;Clegg RM;Henry JJ;Sokolowski A;Kenis PJ;Gaskins HR
• 通讯作者: Gaskins HR

Imaging in real-time with FRET the redox response of tumorigenic cells to glutathione perturbations in a microscale flow.

• DOI: 10.1039/c0ib00071j
• 发表时间: 2011-03
• 期刊: Integrative biology : quantitative biosciences from nano to macro
• 作者: Lin C;Kolossov VL;Tsvid G;Trump L;Henry JJ;Henderson JL;Rund LA;Kenis PJ;Schook LB;Gaskins HR;Timp G
• 通讯作者: Timp G