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Effects of Thymidylate Synthase Inhibition on Thymidine Kinase Activity and Nucleoside Transporter Expression

Division of Experimental Therapeutics, The Ontario Cancer Institute, Toronto, Ontario M4X 1K9 Canada [J. P., B. M., C. E., D. W. H.], and Departments of Pharmacology [J. P., C. E.], Medical Biophysics [C. E., D. W. H.], and Medicine [C. E., D. W. H.], University of Toronto, Toronto, Ontario, Canada M5S 1A8

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 µM for 24-h exposures, and IC₅₀ and IC₉₀ for TS inhibition were 0.7 and 3.0 µM, 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 µM, 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.

¹ To whom requests for reprints should be addressed, at Ontario Cancer Institute/Princess Margaret Hospital, Room 499, 500 Sherbourne Street, Toronto, Ontario, M4X 1K9 Canada.

Received 9/27/94. Accepted 2/ 6/95.

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