Zhang H, Cooney D A, Sreenath A, Zhan Q, Agbaria R, Stowe E E, Fornace A J, Johns D G
Laboratory of Medicinal Chemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Mol Pharmacol. 1994 Dec;46(6):1063-9.
With increasing awareness of the mitochondrial toxicity associated with certain 2',3'-dideoxynucleosides used in anti-human immunodeficiency virus therapy, procedures for quantitative analyses of drug effects on mitochondrial DNA (mtDNA) have assumed enhanced importance. For this reason we have developed a method to measure the copy numbers of mtDNA in cultured MOLT-4 cells. First a hybrid competitive DNA template was synthesized by conventional polymerase chain reaction (PCR), using two custom-synthesized 40-mer composite primers incorporating mitochondrial displacement loop sequences linked by a non-mitochondrial cDNA template (a 76-base pair sequence from the tat/rev region of human immunodeficiency virus cDNA). For the competitive assay, increasing known copy numbers of the hybrid competitive template were added as an internal control to samples containing total cellular DNA. With this approach, two competitive PCR products were generated, 1) a mitochondrial displacement loop-derived fragment (182 base pairs) and 2) a competitive DNA template-derived fragment (156 base pairs). Absolute quantitation was achieved by radiometric comparison of the relative amounts of the two products. To test the versatility of this method, varying amounts of competitive template (6.6 x 10(4) to 6.6 x 10(9) copies) were used with a fixed quantity of total cellular DNA taken from cells cultured for 9 days in the presence or absence of selected pyrimidine and purine dideoxynucleosides. The results showed that the copy number of cellular mtDNA is 823 +/- 71 copies/cell in MOLT-4 cells. Little selective depletion of mtDNA, compared with total cellular DNA, was seen with the purine dideoxynucleosides examined; however, when the cells were exposed to the pyrimidine dideoxynucleoside 2',3'-dideoxycytidine (50 nM) for 9 days, mtDNA content was specifically depleted, although total cellular DNA decreased by only 10%. Thus, in addition to the presently used methods of assessing mitochondrial impairment, i.e., Southern blot analysis and electron microscopy, the competitive PCR method provides a third and convenient assay, with particular applicability to determination of mtDNA in very small numbers of cells.
随着人们对某些用于抗人类免疫缺陷病毒治疗的2'，3'-二脱氧核苷相关线粒体毒性的认识不断提高，定量分析药物对线粒体DNA（mtDNA）影响的方法变得愈发重要。因此，我们开发了一种测量培养的MOLT-4细胞中mtDNA拷贝数的方法。首先，通过常规聚合酶链反应（PCR）合成了一种杂交竞争DNA模板，使用了两种定制合成的40聚体复合引物，这些引物包含由非线粒体cDNA模板连接的线粒体置换环序列（来自人类免疫缺陷病毒cDNA的tat/rev区域的76个碱基对序列）。在竞争分析中，将已知拷贝数不断增加的杂交竞争模板作为内部对照添加到含有总细胞DNA的样品中。通过这种方法，产生了两种竞争PCR产物，1）线粒体置换环衍生片段（182个碱基对）和2）竞争DNA模板衍生片段（156个碱基对）。通过对两种产物相对量的放射性比较实现绝对定量。为了测试该方法的通用性，使用了不同量的竞争模板（6.6×10⁴至6.6×10⁹拷贝）与固定量的总细胞DNA，这些总细胞DNA取自于在存在或不存在选定的嘧啶和嘌呤二脱氧核苷的情况下培养9天的细胞。结果表明，MOLT-4细胞中细胞mtDNA的拷贝数为823±71拷贝/细胞。在所检测的嘌呤二脱氧核苷中，与总细胞DNA相比，mtDNA几乎没有选择性消耗；然而，当细胞暴露于嘧啶二脱氧核苷2'，3'-二脱氧胞苷（50 nM）9天时，mtDNA含量特异性降低，尽管总细胞DNA仅减少了10%。因此，除了目前用于评估线粒体损伤的方法，即Southern印迹分析和电子显微镜检查外，竞争PCR方法提供了第三种便捷的检测方法，特别适用于测定极少量细胞中的mtDNA。
