Lagergren J, Reichard P
Biochem Pharmacol. 1987 Sep 15;36(18):2985-91. doi: 10.1016/0006-2952(87)90213-9.
Inhibition of cell growth and DNA synthesis by hydroxyurea is thought to occur via an effect on the enzyme ribonucleotide reductase leading to a block of deoxyribonucleotide synthesis. Earlier attempts to bypass such a block by delivering deoxyribonucleosides to the medium of cultured cells have given equivocal results. Complications arise in such experiments from the specificity of the phosphorylating enzymes since 3 of the 4 deoxyribonucleosides are substrates for the same enzyme, with widely differing Km values, and from allosteric effects exerted by deoxyribonucleotides. We simplify this situation by using a mutant hamster V79 line that lacks the enzyme dCMP deaminase. The cells contain a 20-fold enlarged dCTP pool and require thymidine for optimal growth. Concentrations of hydroxyurea (50 or 100 microM) that in short-term experiments inhibited DNA synthesis depleted the dATP pool without seriously affecting pyrimidine deoxyribonucleotide pools. The dATP pool could be restored by addition of deoxyadenosine but this depleted the dGTP pool. This depletion could be counteracted by the simultaneous addition of deoxyguanosine but then critically depended on the relative concentrations of the two purine deoxyribonucleosides, with optimal results at 1 microM deoxyadenosine + 100 microM deoxyguanosine. Under those conditions the inhibition of DNA synthesis by hydroxyurea was partially reversed.
羟基脲对细胞生长和DNA合成的抑制作用被认为是通过影响核糖核苷酸还原酶，导致脱氧核糖核苷酸合成受阻而发生的。早期试图通过向培养细胞培养基中添加脱氧核苷来绕过这种阻断的尝试，结果并不明确。在这类实验中会出现复杂情况，因为4种脱氧核苷中的3种是同一种磷酸化酶的底物，其Km值差异很大，而且脱氧核苷酸会产生变构效应。我们通过使用缺乏dCMP脱氨酶的仓鼠V79突变细胞系来简化这种情况。这些细胞的dCTP池扩大了20倍，并且需要胸腺嘧啶核苷才能实现最佳生长。在短期实验中抑制DNA合成的羟基脲浓度（50或100 microM）会耗尽dATP池，而不会严重影响嘧啶脱氧核苷酸池。添加脱氧腺苷可以恢复dATP池，但这会耗尽dGTP池。同时添加脱氧鸟苷可以抵消这种耗尽，但这关键取决于两种嘌呤脱氧核苷的相对浓度，在1 microM脱氧腺苷 + 100 microM脱氧鸟苷时效果最佳。在这些条件下，羟基脲对DNA合成的抑制作用会部分逆转。
