MONITORING OF INTRACELLULAR OXYGEN -CONCENTRATION DURING HYPOXIA INDUCIBLE FACTOR ACTIVATION USING OXYGEN PHOSPHORECSENCE QUENCHING

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MAMMALIAN CELLS ARE CRITICALLY DEPENDENT ON OXYGEN FOR SURVIVAL. THE MOLECULAR MECHANISMS BY WHICH CELLS SENSE AND RESPOND TO A REDUCTION IN OXYGEN SUPPLY ARE NOT KNOWN ALTHOUGH FINDINGS ON THE SIGNAL TRANSDUCTION PATHWAY HAVE BEEN REPORTED BY SEVERAL AUTHORS. A TYPICAL EXAMPLE IS THAT HYPOXIA STIMULATES TRANSCRIPTION OF A NUMBER OF GENES CRUCIAL TO SURVIVAL IN THE HYPOXIC STATE, AS WELL AS APOPTOSIS. THE ENHANCEMENT OF TRANSCRIPTION OF SOME GENES BY HYPOXIA IS MAINLY MEDIATED BY HYPOXIA-INDUCIBLE FACTOR-1 (HIF-1). HOWEVER, IT IS NOT CLEAR WHETHER THIS TRANSCRIPTION FACTOR ARE ACTIVATED BY HYPOXIA-INDUCED CHANGES IN ENERGY METABOLISM OR BY OXYGEN SHORTAGE DIRECTLY THROUGH AN OXYGEN SENSOR. TO ANSWER THIS, WE ATTEMPTED TO CORRELATE THE GENE-EXPRESSION AND INTRACELLULAR OXYGENATION STATE.GREEN FLUORESCENT PROTEIN (GFP) FROM AEQUOREA VICTORIA IS A POWERFUL FLUOROPHORE FORMED AUTOCATALYTICALLY BY INTERNAL CYCLIZATION AND OXIDATION OF THE SER-TYR-GLY SEQUENCE AT POSITIONS 65-67 WITHIN THE 2 … More 38 AMINO ACID. FOLLOWING THE CLONING OF THE CDNA FOR GFP, SEVERAL INVESTIGATORS TOOK A GREAT INTEREST IN THE EXPRESSION OF GFP AND GFP-FUSION PROTEINS AS A UNIQUE FLUOROPHORE TO MONITOR A RANGE OF INTRACELLULAR PROCESSES, ESPECIALLY THE LOCALIZATION OF PROTEINS. HIF- 1 IS THE TRANSCRIPTION REGULATORY PROTEIN AND TRANSLOCATE FROM THE CYTOPLASM TO THE NUCLEUS DURING HYPOXIA. WE OBSERVED THE NUCLEAR TRANSLAOCATION OF GFP LABELED HIF-1 BY COBALT TREATMENT WHICH MIMICS HYPOXIA.OXYGEN-DEPENDENT QUENCHING OF PHOSPHORESCENCE IS A FUNCTION OF THE FREQUENCY OF COLLISION BETWEEN THE PHOSPHOR AND MOLECULAR OXYGEN AND OF THE EFFICIENCY OF ENERGY TRANSFER DURING THESE COLLISMNS. WE DEVELOPED A DETECTION SYSYTE FOR PALLADIUM PORPHYRIN PHOSPHORESCENCE LIFETIME USING CONFOCAL MICROSCOPE. USING PHOSPHORESCENCE DECAY CONSTANT PREVIOUS REPORTED, THE OXYGEN CONCENTRATIONS IN THE MEDIUM WERE ESTIMATED AS 194 MICROMOLAR UNDER NORMOXIA AND 29 MICROMOLAR UNDER ANOXIA, RESPECTIVELY. WE PLAN TO DETERMINE OXYGEN GRADIENT BETWEEN CYTOSOL AND MEDIUM. Less