Rapid analysis of mitochondrial DNA depletion by fluorescence in situ hybridization and immunocytochemistry: Potential strategies for HIV therapeutic monitoring
Rapid analysis of mitochondrial DNA depletion by fluorescence in situ hybridization and immunocytochemistry: Potential strategies for HIV therapeutic monitoring
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DOI：
10.1369/jhc.3a6209.2004
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发表时间：
2004-08-01
影响因子：
3.2
通讯作者：
Capaldi, RA
中科院分区：
文献类型：
--
作者：
Janes, MS;Hanson, BJ;Capaldi, RA
自1987年叠氮胸苷（AZT）问世以来，核苷类逆转录酶抑制剂（NRTI）一直是治疗人类免疫缺陷病毒的主要药物。然而，目前没有一种疗法可以完全根除病毒，因此需要长期使用抗逆转录病毒药物来防止病毒再生长。与长期使用NRTI相关的副作用之一是线粒体毒性，其源于线粒体DNA（mtDNA）聚合酶γ的抑制，导致mtDNA耗竭，从而导致线粒体功能障碍。在这里，我们报告了使用荧光原位杂交（FISH）和免疫细胞化学（ICC）监测培养的成纤维细胞与NRTI 2 '，3'-双脱氧胞苷（ddC）处理的mtDNA消耗。这些技术适用于显微镜和流式细胞术，允许在单细胞基础上分析细胞群。我们发现，随着mtDNA耗竭的进展，嵌合体群体的发展，一些细胞被耗尽，其他细胞保留mtDNA。这些技术可以作为潜在的治疗监测器，以指示何时应中断NRTI治疗以防止线粒体毒性，并且可以通过提供适合于候选分子的药效学评价的测定来帮助开发毒性较小的NRTI。
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.