Engineered Platforms to Manipulate Intracellular Redox

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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.