Rapid Analysis of Mitochondrial DNA Depletion by Fluorescence In Situ Hybridization and Immunocytochemistry: Potential Strategies for HIV Therapeutic Monitoring
| Autor: | W. Gregory Cox, Roderick A. Capaldi, Jakyoung Y. Agnew, Dani Hill, Steven W. Sherwood, Gayle Buller, Kunihiro Yamagata, Michael S. Janes, Bonnie J. Hanson |
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| Rok vydání: | 2004 |
| Předmět: |
0301 basic medicine
Mitochondrial DNA Histology Mitochondrial disease Population Mitochondrion Biology DNA Mitochondrial Polymerase Chain Reaction Virus Cell Line Nucleoside Reverse Transcriptase Inhibitor 03 medical and health sciences immune system diseases medicine Humans education In Situ Hybridization Fluorescence education.field_of_study 030102 biochemistry & molecular biology medicine.diagnostic_test Zalcitabine Fibroblasts medicine.disease Immunohistochemistry Virology Molecular biology Mitochondrial toxicity 030104 developmental biology Reverse Transcriptase Inhibitors Anatomy Fluorescence in situ hybridization |
| Zdroj: | Journal of Histochemistry & Cytochemistry. 52:1011-1018 |
| ISSN: | 1551-5044 0022-1554 |
| DOI: | 10.1369/jhc.3a6209.2004 |
| Popis: | 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 γ, 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. |
| Databáze: | OpenAIRE |
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