To clone the mammalian gene(s) associated with a novel lipophilic antifolate resistance provoked by the antiparasitic drug pyrimethamine (Assaraf, 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 differentially overexpressed cDNA clones were isolated from Pyr(R100) cells, many of which mapped to the mitochondrial genome. Several lines of evidence 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) Electron 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 decreased in Pyr(R100) cells. (d) Metabolic pulse chase labeling suggested 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(R100) cells. (f) Consequently, the rate of respiration in intact Pyr(P100) cells was reduced by 3-fold. We conclude that pyrimethamine and possibly lipophilic analogues of methotrexate possess a folinic acid nonrescuable toxicity involving disruption of mitochondrial inner membrane structure and respiratory function, thereby establishing a new organellar target for the cytotoxic effect elicited by lipid-soluble antifolates.
