The thymidylate synthase inhibitor, ICI D1694, overcomes translational detainment of the enzyme.
We have investigated the mechanism of inactivation of thymidylate synthase (TS) by ICI D1694 (a folate-based quinazoline) in normal versus tumor-derived human mammary epithelial cells. ICI D1694 is a very potent cytotoxic agent against these cells with IC50 values of 1-2 nM. Its growth inhibitory activity was completely reversed by the addition of thymidine, confirming that TS is its sole target in these cells. Remarkably, TS protein levels rose by 10-40-fold following treatment with ICI D1694, depending on cell type, while TS mRNA levels remained constant. The mechanism appears to be a release of "detainment" of TS translation, since addition of cycloheximide, a translational inhibitor, blocked the TS protein levels from rising. But coadministration of 5,6-dichlorobenzimidazole, a transcriptional inhibitor, did not overcome protein accumulation, nor did thymidine which overcomes growth inhibition by ICI D1694. 5,10-Methylenetetrahydrofolate (via folinic acid), however, did block the effects of ICI D1694, showing that the drug has its effect upon both detainment and enzyme inhibition by binding to the folate substrate site of TS. In addition, in the presence of ICI D1694, TS protein was no longer cell cycle-regulated as evident by its constitutive expression in synchronized cells. This accumulation and constitutive expression of TS induced by D1694 should increase drug resistance under a clinical setting. We suggest that an ideal inhibitor of TS would target the TS allosteric site that binds to TS mRNA, responsible for specific translation of the protein, thereby complimenting inactivation of the enzyme.
Keyomarsi
K
K
Division of Cell Growth and Regulation, Dana Farber Cancer Institute, Boston, Massachusetts 02115.
Samet
J
J
Molnar
G
G
Pardee
A B
AB
D002453:Cell Cycle / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D004795:Enzyme Stability; D006801:Humans; D014176:Protein Biosynthesis; D011799:Quinazolines / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D013876:Thiophenes / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D013936:Thymidine / Q000378:metabolism; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured
D011799:Quinazolines; D012333:RNA, Messenger; D013876:Thiophenes; D003513:Cycloheximide; D013940:Thymidylate Synthase; C068874:raltitrexed; D013936:Thymidine