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 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 (PyrR100) cells was used. This library was screened with total cDNA from wild-type and PyrR100 cells. Surprisingly, several differentially overexpressed cDNA clones were isolated from PyrR100 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 > or = 10 microM, pyrimethamine inhibited mitochondrial respiration in viable wild-type cells. (b) Electron microscopy revealed degenerated mitochondrial membrane cristae in PyrR100 cells. (c) Some mitochondrially encoded transcripts were prominently elevated, whereas the normally stable 12 S/16 S rRNA was decreased in PyrR100 cells. (d) Metabolic pulse-chase labeling suggested an increased turnover rate of mitochondrially synthesized proteins in PyrR100 cells. (e) The specific activity of the key respiratory enzymatic complex cytochrome c oxidase was reduced by 6-fold in PyrR100 cells. (f) Consequently, the rate of respiration in intact PyrR100 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.
Sprecher
H
H
Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel.
Barr
H M
HM
Slotky
J I
JI
Tzukerman
M
M
Eytan
G D
GD
Assaraf
Y G
YG
D000251:Adenosine Triphosphatases / Q000096:biosynthesis; D000818:Animals; D015139:Blotting, Southern; D016466:CHO Cells; D002999:Clone Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary; D004272:DNA, Mitochondrial / Q000302:isolation & purification / Q000378:metabolism*; D004351:Drug Resistance / Q000235:genetics; D003576:Electron Transport Complex IV / Q000096:biosynthesis*; D005293:Ferritins / Q000096:biosynthesis; D005493:Folic Acid Antagonists / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects*; D015723:Gene Library; D008322:Mammals; D008854:Microscopy, Electron; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D009245:NADH Dehydrogenase / Q000096:biosynthesis*; D010101:Oxygen Consumption / Q000187:drug effects* / Q000235:genetics; D011739:Pyrimethamine / Q000494:pharmacology*; D015183:Restriction Mapping; D014158:Transcription, Genetic / Q000187:drug effects
D018076:DNA, Complementary; D004272:DNA, Mitochondrial; D005493:Folic Acid Antagonists; D005293:Ferritins; D009245:NADH Dehydrogenase; D003576:Electron Transport Complex IV; D000251:Adenosine Triphosphatases; D011739:Pyrimethamine