Mechanism and pharmacological specificity of dUTPase-mediated protection from DNA damage and cytotoxicity in human tumor cells
Mechanism and pharmacological specificity of dUTPase-mediated protection from DNA damage and cytotoxicity in human tumor cells
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DOI：
10.1007/s002800050829
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发表时间：
1998-10-01
影响因子：
3
通讯作者：
Maybaum, J
中科院分区：
文献类型：
--
作者：
Parsels, LA;Parsels, JD;Maybaum, J
目的：我们以前曾报道过E. coli dUTE（dutE）能保护HT 29细胞免受5-氟脱氧尿苷（FdUrd）诱导的DNA断裂和细胞毒性。在这里报道的研究中，我们进一步表征了一个HT 29克隆dutE 7中dutE表达改变FdUrd和其他胸苷酸合成酶（TS）抑制剂治疗效果的能力。此外，我们使用pLNCX-dutE逆转录病毒构建体开发了两个HuTu 80 dutE表达克隆，并测试了它们对FdUrd诱导的DNA片段化和细胞毒性的敏感性。研究方法：开发了dutE逆转录病毒表达系统和dutE抗体以促进dutE表达克隆的产生和筛选。用碱性洗脱或脉冲场凝胶电泳测试表达dutE的HT 29和HuTu 80克隆的药物诱导的DNA损伤和药物诱导的克隆形成性损失。结果如下：用100 μ M CB 3717或500 nM甲氨蝶呤（MTX）处理24小时后，dutE 7细胞对药物诱导的克隆形成性丧失的敏感性显著低于con 3细胞。DutE 7细胞在24 h时对CB 3717诱导的DNA片段化也具有抗性。然而，在用CB 3717或MTX处理48小时后，con 3和dutE 7细胞之间的存活率没有差异，即使在dutE 7细胞系中DNA损伤仍然大大减弱。此外，在两个HuTu 80克隆80 C和80 K中表达dutE不能保护这些细胞免受FdUrd诱导的DNA损伤或细胞毒性。结论：我们的结论是，尿嘧啶的错误掺入和随后的DNA损伤TS抑制剂诱导的细胞毒性，在HT 29细胞中的作用，是时间依赖性的，长期暴露于这些药物引起的细胞毒性在很大程度上是独立的DNA损伤，在这个细胞系。dutE不能保护HuTu 80细胞免受FdUrd的影响进一步表明，TS抑制导致的尿嘧啶错误掺入的重要性在细胞系中不同。
Purpose: We have reported previously that the expression of E. coli dUTPase (dutE) can protect HT29 cells from 5-fluorodeoxyuridine (FdUrd)-induced DNA fragmentation and cytotoxicity. In the study reported here, we further characterized the ability of dutE expression in one HT29 clone, dutE7, to alter the effects of treatment with FdUrd and other thymidylate synthase (TS) inhibitors. In addition, we developed two HuTu80 dutE-expressing clones using a pLNCX-dutE retroviral construct and tested their sensitivity to FdUrd-induced DNA fragmentation and cytotoxicity. Methods: Both a dutE retroviral expression system and a dutE antibody were developed to facilitate the generation and screening of dutE-expressing clones. HT29 and HuTu80 clones expressing dutE were tested for drug-induced DNA damage with either alkaline elution or pulsed field gel electrophoresis and drug-induced loss of clonogenicity. Results: Following a 24-h treatment with 100 mu M CB3717 or 500 nM methotrexate (MTX), dutE7 cells were significantly less sensitive to drug-induced loss of clonogenicity than con3 cells. DutE7 cells were also resistant to CB3717-induced DNA fragmentation at 24 h. However, following a 48-h treatment with CB3717 or MTX there was no difference in survival between con3 and dutE7 cells, even though DNA damage was still greatly attenuated in the dutE7 cell line. In addition, expression of dutE in two HuTu80 clones, 80 C and 80 K, did not protect these cells from FdUrd-induced DNA damage or cytotoxicity. Conclusions: We conclude that the role of uracil misincorporation and subsequent DNA damage in cytotoxicity induced by TS inhibitors, in HT29 cells, is time dependent, and that cytotoxicity caused by long-term exposure to these drugs is largely independent of resultant DNA damage, in this cell line. The inability of dutE to protect HuTu80 cells from FdUrd further suggests that the significance of uracil misincorporation resulting from TS inhibition varies among cell lines.