ag-331 and Urinary-Bladder-Neoplasms

We have reported that noncytotoxic concentrations of 3'-azido-3'-deoxythymidine (AZT) increase the cytotoxicity of ICI D1694, a folate-based thymidylate synthase (TS) inhibitor, with increasing AZT incorporation into DNA. We postulated that the inhibition of TS by ICI D1694 would decrease 5'-deoxythymidine triphosphate (dTTP) pools, which compete with AZT triphosphate (AZT-TP) as a substrate for DNA polymerase. Furthermore, the inhibition of TS would increase the activity of both thymidine kinase (TK) and thymidylate kinase (TdK). Each of these consequences of TS inhibition would favor more incorporation of AZT into DNA. Thus, we reasoned that other TS inhibitors should also result in increased AZT incorporation into DNA and, perhaps, in increased cytotoxicity. N6-[4-(Morpholinosulfonyl)benzyl]-N6-methyl-2,6-diaminobenz[ cd]indole glucuronate (AG-331) differs from ICI D1694 in that it is a de novo designed lipophilic TS inhibitor, it does not require a specific carrier for cellular uptake, and it does not undergo intracellular polyglutamation. This potent TS inhibitor causes minimal cytotoxicity in MGH-U1 human bladder cancer cells. A 24-h exposure to 5 microM AG-331 causes almost complete TS inhibition but only 35% cell kill. The combination of AZT and AG-331 in MGH-U1 cells resulted in an enhanced antitumor effect relative to that of each agent alone; 50 microM AZT, noncytotoxic alone, increased the cell kill of induced by AG-331 from 35% to 50%. Biochemical studies of this combination revealed that simultaneous treatment with 5 microM AG-331 plus 1.8 microM [3H]-AZT produced as much as a 68% +/- 7% increase in AZT incorporation into DNA. This observation was associated with an increase in DNA single-strand breaks, measured as comet tail moment, of up to 6.6-fold. These studies support our original premise that TS inhibition favors increased incorporation of AZT into DNA and that the combination causes more cell kill than either drug alone in MGH-U1 cells.

Topics: Antimetabolites, Antineoplastic; Cell Division; DNA Damage; DNA-Directed DNA Polymerase; DNA, Single-Stranded; Drug Interactions; Drug Synergism; Humans; Image Processing, Computer-Assisted; Indoles; Substrate Specificity; Thymidylate Synthase; Thymine Nucleotides; Tumor Cells, Cultured; Urinary Bladder Neoplasms; Zidovudine

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.

Topics: Carrier Proteins; Humans; Indoles; Membrane Proteins; Nucleoside Transport Proteins; Quinazolines; Thiophenes; Thymidine Kinase; Thymidylate Synthase; Tumor Cells, Cultured; Urinary Bladder Neoplasms