Multiple mechanisms of resistance to polyglutamatable and lipophilic antifolates in mammalian cells: role of increased folylpolyglutamylation, expanded folate pools, and intralysosomal drug sequestration.
Chinese hamster ovary PyrR100 cells display more than 1000-fold resistance to pyrimethamine (Pyr), a lipophilic antifolate inhibitor of dihydrofolate reductase. PyrR100 cells had wild-type DHFR activity, lost folate exporter activity, and had a 4-fold increased activity of a low pH folic acid transporter. Here we report on the marked alterations identified in PyrR100 cells compared with parental cells: 1) approximately 100-fold decreased folic acid growth requirement; 2) a 25-fold higher glucose growth requirement in Pyr-containing medium; 3) a 2.5- to 4.1-fold increase in folylpolyglutamate synthetase activity; 4) a 3-fold increase in the accumulation of [3H]folic acid and a 3-fold expansion of the intracellular folate pools; 5) a 4-fold increase in the activity of the lysosomal marker beta-hexoseaminidase, suggesting an increased lysosome number/PyrR100 cell; and 6) a small reduction in the steady-state accumulation of [3H]Pyr and no evidence of catabolism or modification of cellular [3H]Pyr. Consequently, PyrR100 cells were markedly resistant to the lipophilic antifolates trimetrexate (40-fold) and AG377 (30-fold) and to the polyglutamatable antifolates 5,10-Dideaza-5,6,7,8-tetrahydrofolic acid (DDATHF) (26-fold) and AG2034 (14-fold). Resistance to these drugs was reversed in PyrR100 cells transferred into folate-depleted medium. In conclusion, these multiple resistance factors collectively result in a prominent increase in folate accumulation, an expansion of the intracellular folylpolyglutamate pool, and abolishment of the cytotoxic activity of polyglutamatable and lipophilic antifolates. The role of increased lysosome number per cell in sequestration of hydrophobic weak base drugs such as Pyr is also discussed as a novel mechanism of drug resistance.
Jansen
G
G
Department of Oncology, Section of Biochemical Pharmacology, University Hospital Vrije Universiteit, Amsterdam, The Netherlands.
Barr
H
H
Kathmann
I
I
Bunni
M A
MA
Priest
D G
DG
Noordhuis
P
P
Peters
G J
GJ
Assaraf
Y G
YG
D000818:Animals; D016466:CHO Cells; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D006224:Cricetinae; D004351:Drug Resistance; D005492:Folic Acid / Q000378:metabolism*; D005493:Folic Acid Antagonists / Q000378:metabolism / Q000494:pharmacology*; D005947:Glucose / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism*; D008727:Methotrexate / Q000378:metabolism / Q000494:pharmacology; D010453:Peptide Synthases / Q000378:metabolism; D011506:Proteins / Q000037:antagonists & inhibitors; D011624:Pteroylpolyglutamic Acids / Q000378:metabolism / Q000494:pharmacology; D011739:Pyrimethamine / Q000378:metabolism / Q000494:pharmacology; D014316:Tritium
D005493:Folic Acid Antagonists; D011506:Proteins; D011624:Pteroylpolyglutamic Acids; C018589:lysosomal proteins; D014316:Tritium; D005492:Folic Acid; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase; D005947:Glucose; D008727:Methotrexate; D011739:Pyrimethamine