3′-azido-3′-deoxythymidine cytotoxicity and metabolism in the human colon tumor cell line HCT-8

Abstract

We have reported that 3′-azido-3′-deoxythymidine (AZT) possesses significant cytotoxicity in human tumor models when combined with agents that inhibit de novo thymidylate (dTMP) synthesis, such as 5-fluorouracil (FUra) and methotrexate (MTX). To aid in the further development of these and related cancer chemotherapeutic regimens, this study was undertaken to identify the biochemical processes relevant to the induction of AZT cytotoxicity in the model human colon tumor cell line HCT-8. The ic 50 of AZT in this cell line after a 5-day exposure was 55 μM. In cells incubated for 5 days with various concentrations of [ 3H]AZT alone, both [ 3H]AZT nucleotide pools and [ 3H]AZT incorporation into DNA increased as the concentration of AZT in the medium increased. In addition, a 5-day exposure to AZT, at medium concentrations ⩽ 100 μM, resulted in a reduction in dTMP synthase (EC 2.1.1.45; methylene tetrahydrofolate:deoxyuridine-5′-monophosphate C methyltransferase) and dThd kinase (EC 2.7.1.27; ATP: thymidine phosphotransferase) activities, compared with cells incubated without drug. The ic 50 of AZT was unchanged when the medium concentration of dThd was increased from 0.1 to 50 μM. Increasing the concentration of dThd to 50 μM also did not affect intracellular pools of [ 3H]AZTDP and [ 3H]AZTTP or the degree to which [ 3H]AZT was incorporated into cellular DNA, but did reduce intracellular [ 3H]AZTMP by ∼ 75%. The degree to which 3′-amino-3′-deoxythymidine (AMT) was generated from AZT and incorporated into DNA also was not affected by varying the medium concentration of dThd. However, the amount of [ 3H]-AMT detected in DNA, ⩽ 3 pmol/10 6 cells at medium concentrations of [ 3H]AZT ⩽ 100 μM, was below that associated with significant cytotoxicity in these cells. These data support the notion that, in this model, AZT cytotoxicity is determined by the relative size of AZTTP pools and its utilization in DNA synthesis. Studies to verify this relationship assessed the effect of alterations in the concentration of dTTP and [ 3H]AZTTP on [ 3H]AZT incorporation into newly synthesized DNA in vitro, using DNA polymerases isolated from HCT-8 cells. The results of these studies confirmed that alterations in the concentration of either dTTP or AZTTP to reduce the dTTP/AZTTP ratio resulted in an increase in AZT incorporation into DNA. These findings are discussed in light of their biochemical implications and relevance to ongoing clinical trials.