Darnowski J W, Goulette F A
Department of Medicine, Brown University, Providence, RI.
Biochem Pharmacol. 1994 Nov 1;48(9):1797-805. doi: 10.1016/0006-2952(94)90466-9.
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 IC50 of AZT in this cell line after a 5-day exposure was 55 microM. 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 < or = 100 microM, 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 IC50 of AZT was unchanged when the medium concentration of dThd was increased from 0.1 to 50 microM. Increasing the concentration of dThd to 50 microM 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 approximately 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, < or = 3 pmol/10(6) cells at medium concentrations of [3H]AZT < or = 100 microM, 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.
我们曾报道，3'-叠氮-3'-脱氧胸苷（AZT）与抑制胸腺嘧啶脱氧核苷酸（dTMP）从头合成的药物（如5-氟尿嘧啶（FUra）和甲氨蝶呤（MTX））联合使用时，在人类肿瘤模型中具有显著的细胞毒性。为了帮助进一步开发这些及相关的癌症化疗方案，开展了本研究以确定在模型人结肠肿瘤细胞系HCT-8中与AZT细胞毒性诱导相关的生化过程。该细胞系经5天暴露后AZT的半数抑制浓度（IC50）为55微摩尔。在单独用不同浓度的[3H]AZT孵育5天的细胞中，随着培养基中AZT浓度的增加，[3H]AZT核苷酸池以及[3H]AZT掺入DNA的量均增加。此外，与未用药物孵育的细胞相比，在培养基浓度≤100微摩尔时暴露于AZT 5天，导致胸苷酸合成酶（EC 2.1.1.45；亚甲基四氢叶酸：脱氧尿苷-5'-单磷酸C甲基转移酶）和脱氧胸苷激酶（EC 2.7.1.27；ATP：胸苷磷酸转移酶）活性降低。当培养基中脱氧胸苷（dThd）的浓度从0.1微摩尔增加到50微摩尔时，AZT的IC50未改变。将dThd浓度增加到50微摩尔也不影响细胞内[3H]AZTDP和[3H]AZTTP池，以及[3H]AZT掺入细胞DNA的程度，但确实使细胞内[3H]AZTMP减少了约75%。从AZT生成并掺入DNA的3'-氨基-3'-脱氧胸苷（AMT）的程度也不受dThd培养基浓度变化的影响。然而，在[3H]AZT培养基浓度≤100微摩尔时，在DNA中检测到的[3H]-AMT量≤3皮摩尔/10^6个细胞，低于这些细胞中与显著细胞毒性相关的量。这些数据支持这样一种观点，即在该模型中，AZT细胞毒性由AZTTP池的相对大小及其在DNA合成中的利用情况决定。为验证这种关系而进行的研究评估了dTTP和[3H]AZTTP浓度变化对使用从HCT-8细胞分离的DNA聚合酶在体外将[3H]AZT掺入新合成DNA的影响。这些研究结果证实，改变dTTP或AZTTP的浓度以降低dTTP/AZTTP比率会导致AZT掺入DNA增加。根据其生化意义及其与正在进行的临床试验的相关性对这些发现进行了讨论。
