ALTERATION OF MITOCHONDRIAL GENE-EXPRESSION AND DISRUPTION OF RESPIRATORY-FUNCTION BY THE LIPOPHILIC ANTIFOLATE PYRIMETHAMINE IN MAMMALIAN-CELLS

To clone the mammalian gene(s) associated with a novel lipophilic anti folate resistance provoked by the antiparasitic drug pyrimethamine (As saraf, Y. G., and Slotky, J. I. (1993) J. Biol. Chem. 268, 4556-4566), differential screening of a cDNA library from pyrimethamine-resistant (Pyr(R100)) cells was used. This library was screened with total cDNA from wild-type and Pyr(R100) cells. Surprisingly, several differentia lly overexpressed cDNA clones were isolated from Pyr(R100) cells, many of which mapped to the mitochondrial genome. Several lines of evidenc e establish mitochondria as a new target for the cytotoxic activity of pyrimethamine. (a) At greater than or equal to 10 mu M, pyrimethamine inhibited mitochondrial respiration in viable wild-type cells. (b) El ectron microscopy revealed degenerated mitochondrial membrane cristae in Pyr(R100) cells. (c) Some mitochondrially encoded transcripts were prominently elevated, whereas the normally stable 12 S/16 S rRNA was d ecreased in Pyr(R100) cells. (d) Metabolic pulse chase labeling sugges ted an increased turnover rate of mitochondrially synthesized proteins in Pyr(R100) cells. (e) The specific activity of the key respiratory enzymatic complex cytochrome c oxidase was reduced by 6-fold in Pyr(R1 00) cells. (f) Consequently, the rate of respiration in intact Pyr(P10 0) cells was reduced by 3-fold. We conclude that pyrimethamine and pos sibly lipophilic analogues of methotrexate possess a folinic acid nonr escuable toxicity involving disruption of mitochondrial inner membrane structure and respiratory function, thereby establishing a new organe llar target for the cytotoxic effect elicited by lipid-soluble antifol ates.