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Rapid Analysis of Mitochondrial DNA Depletion by Fluorescence In Situ Hybridization and Immunocytochemistry : Potential Strategies for HIV Therapeutic Monitoring

Michael S. Janes, Bonnie J. Hanson, Dani M. Hill, Gayle M. Buller, Jakyoung Y. Agnew, Steven W. Sherwood, W. Gregory Cox, Kunihiro Yamagata and Roderick A. Capaldi

Molecular Probes, Inc. (MSJ,BJH,DMH,GMB,JYA,SWS,WGC), and Institute of Molecular Biology, University of Oregon, Eugene, Oregon (KY,RAC)

Correspondence to: Michael S. Janes, Molecular Probes, Inc., 29851 Willow Creek Road, Eugene, Oregon 97402. E-mail: mike.janes{at}probes.com

Nucleoside reverse transcriptase inhibitors (NRTIs) have been a mainstay in the treatment of human immunodeficiency virus since the introduction of azidothymidine (AZT) in 1987. However, none of the current therapies can completely eradicate the virus, necessitating long-term use of anti-retroviral drugs to prevent viral re-growth. One of the side effects associated with long-term use of NRTIs is mitochondrial toxicity stemming from inhibition of the mitochondrial DNA (mtDNA) polymerase , which leads to mtDNA depletion and consequently to mitochondrial dysfunction. Here we report the use of fluorescence in situ hybridization (FISH) and immunocytochemistry (ICC) to monitor mtDNA depletion in cultured fibroblasts treated with the NRTI 2',3'-dideoxycytidine (ddC). These techniques are amenable to both microscopy and flow cytometry, allowing analysis of populations of cells on a single-cell basis. We show that, as mtDNA depletion progresses, a mosaic population develops, with some cells being depleted of and others retaining mtDNA. These techniques could be useful as potential therapeutic monitors to indicate when NRTI therapy should be interrupted to prevent mitochondrial toxicity and could aid in the development of less toxic NRTIs by providing an assay suitable for pharmacodynamic evaluation of candidate molecules. (J Histochem Cytochem 52:1011–1018, 2004)

Key Words: mitochondria • oxidative phosphorylation • mitochondrial DNA • HIV • AIDS • mitochondrial disease • nucleoside reverse • transcriptase inhibitor • cytochrome c oxidase • immunocytochemistry • FISH

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