The role of DNA synthesis inhibition in the cytotoxicity of 2',2'-difluoro-2'-deoxycytidine.
The role of DNA synthesis inhibition in the cytotoxicity of 2',2'-difluoro-2'-deoxycytidine.
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DNA 合成抑制在 2,2-二氟-2-脱氧胞苷的细胞毒性中的作用。
DOI：
10.1007/s00280-003-0661-5
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发表时间：
2003
期刊：
影响因子：
--
通讯作者：
Shewach,DonnaS
中科院分区：
文献类型：
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作者：
Ostruszka,LeoJ;Shewach,DonnaS
目的抗癌药物 2',2'-二氟-2'-脱氧胞苷 (dFdCyd) 的细胞毒性与其掺入 DNA 相关。然而，细胞毒性也可能来自 DNA 合成的抑制，原因是 (1) dFdCyd 二磷酸介导的核糖核苷酸还原酶抑制，或 (2) dFdCyd 的 5'-三磷酸 (dFdCTP) 对 DNA 聚合酶的直接抑制。为了阐明 DNA 合成抑制在 dFdCyd 细胞毒性中的作用，我们在 U251 和 D54 人胶质母细胞瘤细胞系中将 dFdCyd 与羟基脲 (HU)（一种核糖核苷酸还原酶抑制剂）和 aphidicolin（一种 DNA 聚合酶抑制剂）进行比较。 方法使用集落形成测定法测定对 dFdCyd、HU 和 aphidicolin 的敏感性。通过双参数流式细胞仪测量这些药物对 DNA 合成的影响，并通过高效液相色谱分析对核苷酸池水平的影响。结果HU 和 aphidicolin 引起的细胞毒性明显低于 dFdCyd 的多对数杀伤作用。当以等毒浓度（24小时IC50值）使用时，dFdCyd和HU主要减少嘌呤dNTP池，但dFdCyd的效果不如HU。 4-12 小时后，dFdCyd 使 dATP 减少约 80%，并且需要 8-24 小时才能使 DNA 合成减少 50%。相比之下，HU 在 2 小时内迅速耗尽 dATP > 98%，从而导致 DNA 合成抑制 > 90%。 Aphidicolin 的浓度与其 DNA 聚合酶的 Ki 值 (1 μM) 相似，可在 2 小时内使 DNA 合成减少 > 70%。然而，这仅使细胞存活率降低了 10%（U251 细胞）和 40%（D54 细胞）。结论这些结果表明，HU 和 aphidicolin 比 dFdCyd 产生更快速、更深刻的 DNA 合成抑制作用，但细胞毒性显着降低。这表明，DNA 合成的抑制导致了用 dFdCyd 观察到的多对数细胞毒性的不到一个对数，而 dFdCTP 掺入 DNA 是更致命的事件。
PurposeCytotoxicity from the anticancer drug 2′,2′-difluoro-2′-deoxycytidine (dFdCyd) has been correlated with its incorporation into DNA. However, cytotoxicity may also result from inhibition of DNA synthesis, due to either (1) dFdCyd diphosphate-mediated inhibition of ribonucleotide reductase, or (2) direct inhibition of DNA polymerases by the 5′-triphosphate of dFdCyd (dFdCTP). To elucidate the role of DNA synthesis inhibition in the cytotoxicity of dFdCyd, we compared dFdCyd to hydroxyurea (HU), a ribonucleotide reductase inhibitor, and aphidicolin, an inhibitor of DNA polymerases, in the U251 and D54 human glioblastoma cell lines.MethodsSensitivity to dFdCyd, HU, and aphidicolin were determined using a colony formation assay. The effects of these drugs on DNA synthesis were measured by dual parameter flow cytometry, while the effects on nucleotide pool levels were analyzed by high-performance liquid chromatography.ResultsHU and aphidicolin elicited substantially less cytotoxicity than the multi-log killing with dFdCyd. When used at equitoxic concentrations (24-h IC50values), dFdCyd and HU decreased purine dNTP pools primarily, but dFdCyd was less effective than HU. dFdCyd had decreased dATP by about 80% after 4–12 h, and required 8–24 h to decrease DNA synthesis by 50%. In contrast, HU rapidly depleted dATP by >98% within 2 h, which resulted in >90% inhibition of DNA synthesis. Aphidicolin at a concentration similar to its Kivalues for DNA polymerases (1 μM) decreased DNA synthesis by >70% within 2 h. However, this decreased cell survival by only 10% (U251 cells) and 40% (D54 cells).ConclusionsThese results demonstrate that HU and aphidicolin produced a more rapid and profound inhibition of DNA synthesis than dFdCyd, but resulted in significantly less cytotoxicity. This suggests that inhibition of DNA synthesis accounted for less than one log of the multi-log cytotoxicity observed with dFdCyd, whereas incorporation of dFdCTP into DNA is a more lethal event.