title: Characterization of a lipophilic antifolate resistance provoked by treatment of mammalian cells with the antiparasitic agent pyrimethamine.
abstract: 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.
authors.affiliation: Department of Biology, Technion-Israel Institute of Technology, Haifa.
authors.forename: Y G
authors.initials: YG
authors.lastname: Assaraf
authors.forename: J I
authors.initials: JI
authors.lastname: Slotky
mesh_terms: D000818:Animals; D016466:CHO Cells; D002470:Cell Survival / Q000187:drug effects; D006224:Cricetinae; D004351:Drug Resistance; D005434:Flow Cytometry; D005493:Folic Acid Antagonists / Q000494:pharmacology*; D002955:Leucovorin / Q000494:pharmacology; D011739:Pyrimethamine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D014295:Trimethoprim / Q000378:metabolism / Q000494:pharmacology
chemical_list: D005493:Folic Acid Antagonists; D012333:RNA, Messenger; C006308:metoprine; D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase; D002955:Leucovorin; D011739:Pyrimethamine