Quantitation of mitochondrial DNA in human lymphoblasts by a competitive polymerase chain reaction method: application to the study of inhibitors of mitochondrial DNA content.

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.

Characterization of 2',3'-dideoxycytidine diphosphocholine and 2',3'-dideoxycytidine diphosphoethanolamine. Prominent phosphodiester metabolites of the anti-HIV nucleoside 2',3'-dideoxycytidine.

2',3'-Dideoxycytidine (ddCyd) is among the most potent of the anti-human immunodeficiency virus (HIV) agents of the dideoxynucleoside class. Its pharmacologically active metabolite 2',3'-dideoxycytidine 5'-triphosphate (ddCTP) is an effective inhibitor of HIV reverse transcriptase and thus of HIV replication. ddCyd differs, however, from other dideoxynucleoside agents such as 3'-azido-3'-deoxythymidine and 2',3'-dideoxyinosine in its capacity to generate phosphodiester metabolites (i.e. ddCDP choline and ddCDP ethanolamine). We have synthesized and characterized these two diesters, and established their identity with the metabolites formed in ddCyd-treated Molt-4 cells. Toward this end, the biologically generated metabolites have been isolated on a preparative scale and compared with the synthetic compounds mass spectroscopically, chromatographically, and enzymatically (i.e. their relative susceptibility to the catabolic enzymes alkaline phosphatase and venom phosphodiesterase). The concentration reached by each of these two phosphodiesters within cells can, under certain conditions, equal or exceed that of ddCTP, and their half-times of disappearance are long, indicating that they may serve as depot forms of ddCyd. The possible role of these phosphodiesters in contributing to the unusual toxicity of ddCyd is discussed.