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.

Characterization of a lipophilic antifolate resistance provoked by treatment of mammalian cells with the antiparasitic agent pyrimethamine.

We describe the characterization of an antitumor drug resistance following multiple step selection of hamster cells to the 2,4-diaminopyrimidines (DAP) metoprine, pyrimethamine (Pyr), and trimethoprim (Tmp). Pyr and Tmp are DAP lipophilic antifolates currently used as antiparasitic and antibacterial antibiotics, respectively. Dihydrofolate reductase (DHFR) from hamster cells bore a low or poor affinity to these DAP as compared to the hydrophilic folate antagonist methotrexate (MTX). Metoprine-resistant cells over-expressed DHFR enzyme and consequently displayed a high level of resistance to both hydrophilic and lipophilic antifolates including DAP but maintained wild type sensitivity to pleiotropic drugs involved in multi-drug resistance (MDR). In contrast, although Pyr- and Tmp-resistant cells expressed parental levels of wild type DHFR, they displayed a high degree of resistance to DAP and, surprisingly, to the lipophilic MTX analogs piritrexim (PTX) and trimetrexate (TMTX), while maintaining sensitivity to MTX. These drug-resistant cells maintained wild type mRNA levels of the MDR gene product P-glycoprotein and showed collateral hypersensitivity to pleiotropic drugs. To study the underlying mechanism of this apparently new resistance phenotype, we have employed fluorescein-methotrexate (F-MTX) labeling of cells and its displacement by different antifolates. Parental AA8 and Pyr-resistant cells showed a similar level of F-MTX labeling, however, while DAP, TMTX, and PTX showed an efficient competitive displacement of F-MTX from AA8 cells, Pyr-resistant cells displayed a persistent retention of F-MTX labeling in the presence of high concentrations of these lipophilic antifolates. Pyr-resistant cells showed a wild type displacement of F-MTX with MTX. This DAP resistance phenotype was unstable as it was rapidly lost upon growth under nonselective conditions. Furthermore, when the antifolate resistance levels of Pyr-resistant cells were plotted versus the ratios of the 50% F-MTX displacement values obtained with resistant and parental AA8 cells, a good correlation (r2 > 0.98) was obtained. We conclude that Pyr-resistant cells possess a novel phenotype that derives its resistance to lipophilic antifolates solely from a predominant decrease in the accumulation of DAP and lipid-soluble analogs of MTX.