Role of substrate depletion in the inhibition of thymidylate biosynthesis by the dihydrofolate reductase inhibitor trimetrexate in cultured hepatoma cells.
The effects of the lipid-soluble dihydrofolate reductase inhibitor, trimetrexate, on the inhibition of thymidylate biosynthesis as a result of perturbation in cellular folate pools in H35 hepatoma cells in vitro has been investigated. Exposure of the cultures to increasing concentrations of trimetrexate between 2 and 20 nM causes a marked reduction in de novo thymidylate biosynthesis and a concomitant decrease in (6R)5,10-methylenetetrahydropteroylpolyglutamate (5,10-CH2H4PteGlun) from 2.0-0.2 microM, respectively. This is accompanied by an increase in H2PteGlun from 1.2 microM in control cultures to 4.7 microM in cultures exposed to 20 nM trimetrexate. The dependency of de novo thymidylate biosynthesis on intracellular 5,10-CH2H4PteGlun in trimetrexate-treated cells is compared with (a) the relationship of thymidylate biosynthesis on intracellular levels of 5,10-CH2H4PteGlun in folate-depleted cells supplemented with increments of folic acid and (b) the substrate (5,10-CH2H4PteGlun) dependence of purified thymidylate synthase from the same source. All three results are nearly identical demonstrating that trimetrexate-dependent inhibition of de novo thymidylate biosynthesis is primarily a result of substrate depletion. These results coupled with the weak inhibitory properties of H2PteGlun for thymidylate synthase Ki = 5.0 microM) suggest that H2PteGlun accumulation is not the major determinant in inhibiting thymidylate synthase following trimetrexate inhibition but under certain conditions has the potential to enhance the inhibition caused by substrate depletion.
Rhee
M S
MS
Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany 12201-0509.
Balinska
M
M
Bunni
M
M
Priest
D G
DG
Maley
G F
GF
Maley
F
F
Galivan
J
J
D006528:Carcinoma, Hepatocellular / Q000201:enzymology / Q000378:metabolism*; D005492:Folic Acid / Q000031:analogs & derivatives*; D008113:Liver Neoplasms / Q000201:enzymology / Q000378:metabolism*; D011624:Pteroylpolyglutamic Acids / Q000378:metabolism*; D011799:Quinazolines / Q000494:pharmacology*; D013938:Thymidine Monophosphate / Q000097:blood*; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013942:Thymine Nucleotides / Q000097:blood*; D016597:Trimetrexate; D014407:Tumor Cells, Cultured / Q000378:metabolism
D011624:Pteroylpolyglutamic Acids; D011799:Quinazolines; D013942:Thymine Nucleotides; C024897:dihydropteroylpolyglutamate; C024898:methylenetetrahydropteroylpolyglutamate; D013938:Thymidine Monophosphate; D005492:Folic Acid; D013940:Thymidylate Synthase; D016597:Trimetrexate