title: Imaging in real-time with FRET the redox response of tumorigenic cells to glutathione perturbations in a microscale flow.
abstract: Despite the potential benefits of selective redox-modulating strategies for cancer therapy, an efficacious methodology for testing therapies remains elusive because of the difficulty in measuring intracellular redox potentials over time. In this report, we have incorporated a new FRET-based biosensor to follow in real time redox-sensitive processes in cells transformed to be tumorigenic and cultured in a microfluidic channel. A microfluidic network was used to control micro-scale flow near the cells and at the same time deliver drugs exogenously. Subsequently, the response of a redox homeostasis circuit was tested, namely reduced glutathione (GSH)/oxidized glutathione(GSSG), to diamide, a thiol oxidant, and two drugs used for cancer therapies: BSO (L-buthionine-[SR]-sulfoximine) and BCNU (carmustine). The main outcome from these experiments is a comparison of the temporal depletion and recovery of GSH in single living cells in real-time. These data demonstrate that mammalian cells are capable of restoring a reduced intracellular redox environment in minutes after an acute oxidative insult is removed. This recovery is significantly delayed by (i) the inhibition of GSH biosynthesis by BSO; (ii) the inactivation of glutathione reductase by BCNU; and (iii) in tumorigenic cells relative to an isogenic non-tumorigenic control cell line.
authors.affiliation: University of Notre Dame, 316 Stinson-Remick Hall, South Bend, IN 46556. clin7@nd.edu
authors.forename: Chunchen
authors.initials: C
authors.lastname: Lin
authors.forename: Vladimir L
authors.initials: VL
authors.lastname: Kolossov
authors.forename: Gene
authors.initials: G
authors.lastname: Tsvid
authors.forename: Lisa
authors.initials: L
authors.lastname: Trump
authors.forename: Jennifer Jo
authors.initials: JJ
authors.lastname: Henry
authors.forename: Jerrod L
authors.initials: JL
authors.lastname: Henderson
authors.forename: Laurie A
authors.initials: LA
authors.lastname: Rund
authors.forename: Paul J A
authors.initials: PJ
authors.lastname: Kenis
authors.forename: Lawrence B
authors.initials: LB
authors.lastname: Schook
authors.forename: H Rex
authors.initials: HR
authors.lastname: Gaskins
authors.forename: Gregory
authors.initials: G
authors.lastname: Timp
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D015374:Biosensing Techniques / Q000379:methods*; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D016466:CHO Cells; D002330:Carmustine / Q000494:pharmacology; D002461:Cell Line, Transformed; D058948:Cell Tracking / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D003958:Diamide / Q000378:metabolism / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005978:Glutathione / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019803:Glutathione Disulfide / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence / Q000379:methods; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013552:Swine; D014162:Transfection
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; D011994:Recombinant Proteins; C527701:enhanced cyan fluorescent protein; C054562:yellow fluorescent protein, Bacteria; D003958:Diamide; D049452:Green Fluorescent Proteins; D019328:Buthionine Sulfoximine; D005978:Glutathione; D002330:Carmustine; D019803:Glutathione Disulfide