Rapid analysis of mitochondrial DNA depletion by fluorescence in situ hybridization and immunocytochemistry: potential strategies for HIV therapeutic monitoring.
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 gamma, 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.
Janes
Michael S
MS
Molecular Probes, Inc., 29851 Willow Creek Road, Eugene, Oregon 97402, USA. mike.janes@probes.com
Hanson
Bonnie J
BJ
Hill
Dani M
DM
Buller
Gayle M
GM
Agnew
Jakyoung Y
JY
Sherwood
Steven W
SW
Cox
W Gregory
WG
Yamagata
Kunihiro
K
Capaldi
Roderick A
RA
D002460:Cell Line; D004272:DNA, Mitochondrial / Q000096:biosynthesis*; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence; D016133:Polymerase Chain Reaction; D018894:Reverse Transcriptase Inhibitors / Q000009:adverse effects*; D016047:Zalcitabine / Q000009:adverse effects*
D004272:DNA, Mitochondrial; D018894:Reverse Transcriptase Inhibitors; D016047:Zalcitabine