title: Effects of thymidylate synthase inhibition on thymidine kinase activity and nucleoside transporter expression.
abstract: The effects of de novo dTMP inhibition by N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N- methylamino]-2- thenoyl)-L-glutamic acid (D1694) or N6-[4-(morpholinosulfonyl)benz]-N6-diaminobenz[cd]indole glucuronate (AG-331) on clonogenic survival, thymidylate synthase (TS) and thymidine kinase (TK) activity, and expression of S-(p-nitrobenzyl)-6-thioinosine-sensitive nucleoside transporter (NT) sites were addressed in the human bladder cancer cell line, MGH-U1. These two TS inhibitors are structurally diverse. D1694 is a folate-based TS inhibitor, whereas AG-331 is a novel agent that inhibits the cofactor binding site of the enzyme. They also differ with respect to their cytotoxic effects in this cell line; D1694 cytotoxic 50% inhibitory concentration (IC50) and IC90 were 6.0 and 9.0 nM, respectively and IC50 and IC90 for TS inhibition were 2.5 and 4.8 nM, respectively. In contrast, AG-331 cytotoxic IC50 could not be achieved even at concentrations of up to 20 microM for 24-h exposures, and IC50 and IC90 for TS inhibition were 0.7 and 3.0 microM, respectively. Similar effects for D1694 and AG-331 were observed in their modulation of TK activity and NT expression. 5-(SAENTA-x8)-Fluorescein, a highly modified form of adenosine incorporating a fluorescein molecule which binds with a 1:1 stoichiometry to S-(p-nitrobenzyl)-6-thioinosine-sensitive NT sites, was used to investigate the expression of NT following exposure of cells to D1694 and AG-331. TK activity was addressed by the metabolism of [3H]thymidine to [3H]TMP by cellular extracted protein and by an alternative flow cytometric method using a modified form of thymidine incorporating a fluorescent molecule, dansyl-5-amino-2-deoxyuridine. Results obtained by both methods were comparable. At concentrations of 5 and 10 nM, D1694 increased TK activity 2.3-4.5-fold and NT expression 34-39-fold. AG-331, at concentrations of 5 and 10 microM, increased TK activity 1.8-2.5-fold and NT expression 22-31-fold, respectively. These data suggest that TK activity and NT expression have a common regulatory mechanism which is sensitive to endogenous dTTP pools and that the salvage pathway is a complex system of kinases coordinated with transport of nucleosides.
authors.affiliation: Division of Experimental Therapeutics, Ontario Cancer Institute, Toronto, Canada.
authors.forename: J
authors.initials: J
authors.lastname: Pressacco
authors.forename: B
authors.initials: B
authors.lastname: Mitrovski
authors.forename: C
authors.initials: C
authors.lastname: Erlichman
authors.forename: D W
authors.initials: DW
authors.lastname: Hedley
mesh_terms: D002352:Carrier Proteins / Q000378:metabolism*; D006801:Humans; D007211:Indoles / Q000494:pharmacology*; D008565:Membrane Proteins / Q000378:metabolism*; D033703:Nucleoside Transport Proteins; D011799:Quinazolines / Q000494:pharmacology*; D013876:Thiophenes / Q000494:pharmacology*; D013937:Thymidine Kinase / Q000096:biosynthesis*; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors*; D014407:Tumor Cells, Cultured; D001749:Urinary Bladder Neoplasms / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D007211:Indoles; D008565:Membrane Proteins; D033703:Nucleoside Transport Proteins; D011799:Quinazolines; D013876:Thiophenes; C087748:AG 331; D013940:Thymidylate Synthase; D013937:Thymidine Kinase; C068874:raltitrexed