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 wit h certain 2',3'-dideoxynucleosides used in anti-human immunodeficiency virus therapy, procedures for quantitative analyses of drug effects o n mitochondrial DNA (mtDNA) have assumed enhanced importance. For this reason we have developed a method to measure the copy numbers of mtDN A in cultured MOLT-4 cells. First a hybrid competitive DNA template wa s synthesized by conventional polymerase chain reaction (PCR), using t wo custom-synthesized 40-mer composite primers incorporating mitochond rial displacement loop sequences linked by a non-mitochondrial cDNA te mplate (a 76-base pair sequence from the tat/rev region of human immun odeficiency virus cDNA). For the competitive assay, increasing known c opy numbers of the hybrid competitive template were added as an intern al control to samples containing total cellular DNA. With this approac h, two competitive PGR products were generated, 1) a mitochondrial dis placement 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 purin e dideoxynucleosides. The results showed that the copy number of cellu lar mtDNA is 823 +/- 71 copies/cell in MOLT-4 cells. Little selective depletion of mtDNA, compared with total cellular DNA, was seen with th e purine dideoxynucleosides examined; however, when the cells were exp osed to the pyrimidine dideoxynucleoside 2',3'-dideoxycytidine (50 nM) for 9 days, mtDNA content was specifically depleted, although total c ellular DNA decreased by only 10%. Thus, in addition to the presently used methods of assessing mitochondrial impairment, i.e., Southern blo t analysis and electron microscopy, the competitive PCR method provide s a third and convenient assay, with particular applicability to deter mination of mtDNA in very small numbers of cells.