CYTOTOXIC AND BIOCHEMICAL IMPLICATIONS COMBINING AZT AND AG-331

We have reported that noncytotoxic concentrations of 3'-azido-3'-deoxy thymidine (AZT) increase the cytotoxicity of ICI D1694, a folate-based thymidylate synthase (TS) inhibitor, with increasing AZT incorporatio n into DNA. We postulated that the inhibition of TS by ICI D1694 would decrease 5'-deoxythymidine triphosphate (dTTP) pools, which compete w ith AZT triphosphate (AZT-TP) as a substrate for DNA polymerase. Furth ermore, the inhibition of TS would increase the activity of both thymi dine kinase (TK) and thymidylate kinase (TdK). Each of these consequen ces of TS inhibition would favor more incorporation of AZT into DNA. T hus, we reasoned that other TS inhibitors should also result in increa sed AZT incorporation into DNA and, perhaps, in increased cytotoxicity . fonyl)benzyl]-N-6-methyl-2,6-diaminobenz[cd]indole glucuronate (AG-3 31) differs from ICI D1694 in that it is a de novo designed lipophilic TS inhibitor, it does not require a specific carrier for cellular upt ake, and it does not undergo intracellular polyglutamation. This poten t TS inhibitor causes minimal cytotoxicity in MGH-U1 human bladder can cer cells. A 24-h exposure to 5 mu M AG-331 causes almost complete TS inhibition but only 35% cell kill. The combination of AZT and AG-331 i n MGH-U1 cells resulted in an enhanced antitumor effect relative to th at of each agent alone; 50 mu M AZT, noncytotoxic alone, increased the cell kill of induced by AG-331 from 35% to 50%. Biochemical studies o f this combination revealed that simultaneous treatment with 5 mu M AG -331 plus 1.8 mu M [H-3]-AZT produced as much as a 68% +/- 7% increase in AZT incorporation into DNA. This observation was associated with a n 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 th e combination causes more cell kill than either drug alone in MGH-U1 c ells.