DNA repair initiated in chronic lymphocytic leukemia lymphocytes by 4-hydroperoxycyclophosphamide is inhibited by fludarabine and clofarabine.
DNA repair initiated in chronic lymphocytic leukemia lymphocytes by 4-hydroperoxycyclophosphamide is inhibited by fludarabine and clofarabine.
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发表时间：
2001-11
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通讯作者：
T. Yamauchi;B. Nowak;M. Keating;W. Plunkett
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作者：
T. Yamauchi;B. Nowak;M. Keating;W. Plunkett
慢性淋巴细胞性白血病（CLL）淋巴细胞对DNA烷基化的反应是切除修复，随着细胞对烷基化试剂产生抗性，修复程度增加。由于将核苷酸类似物掺入修复补丁中会在静止细胞中产生死亡信号，因此在烷化剂抗性细胞中切除修复的能力增加可以促进核苷酸类似物的掺入。我们假设核苷类似物与烷化剂的基于机制的相互作用可以引起大于CLL细胞的相加杀伤。实验设计来自50名对烷化剂不敏感的CLL患者的淋巴细胞在体外用4-过氧化氢环磷酰胺（4-HC）处理，有或没有预先与氟达拉滨核苷（F-ara-A）或氯法拉滨（Cl-F-ara-A）孵育。DNA损伤修复动力学通过单细胞凝胶电泳（彗星）测定来确定。结果CLL淋巴细胞迅速启动并完成切除修复反应4-HC。用10 μ M F-ara-A或10 μ M Cl-F-ara-A预孵育2小时可抑制4-HC引发的修复，在细胞内浓度为50 μ M F-ara-ATP或5 μ M Cl-F-ara-ATP时抑制作用达到峰值。将4-HC与F-ara-A或Cl-F-ara-A组合产生的凋亡细胞死亡多于每种单独组合的总和。细胞毒性的增加与DNA切口的初始大小和核苷类似物的修复抑制程度成比例，表明修复抑制和诱导细胞死亡之间存在密切相关性。结论：CLL淋巴细胞中活跃的DNA修复可能是促进核苷类似物掺入并增加其细胞毒性的生物学靶点。因此，与抗性疾病相关的增加的修复能力可以被操纵以获得治疗优势。
PURPOSE Chronic lymphocytic leukemia (CLL) lymphocytes respond to DNA alkylation by excision repair, with the extent of repair increasing as the cells acquire resistance to alkylating agents. Because incorporation of nucleotide analogues into the repair patches elicits death signals in quiescent cells, the increased capacity for excision repair in alkylator-resistant cells could facilitate incorporation of nucleotide analogues. We hypothesized that the mechanism-based interaction of nucleoside analogues with alkylating agents could elicit greater than additive killing of CLL cells. EXPERIMENTAL DESIGN Lymphocytes from 50 patients with CLL that were not refractory to alkylators were treated in vitro with 4-hydroperoxycyclophosphamide (4-HC) with or without prior incubation with fludarabine nucleoside (F-ara-A) or with clofarabine (Cl-F-ara-A). DNA damage repair kinetics were determined by the single-cell gel electrophoresis (comet) assay. Cytotoxicity was assessed by staining with annexin V. RESULTS CLL lymphocytes promptly initiated and completed excision repair in response to 4-HC. A 2-h preincubation with 10 microM F-ara-A or 10 microM Cl-F-ara-A inhibited the repair initiated by 4-HC, with inhibition peaking at the intracellular concentrations of 50 microM F-ara-ATP or 5 microM Cl-F-ara-ATP. Combining 4-HC with either F-ara-A or Cl-F-ara-A produced more than additive apoptotic cell death than the sum of each alone. The increase in cytotoxicity was proportional to the initial magnitude of the DNA incision and to the extent of repair inhibition by the nucleoside analogues, suggesting close correlation between the repair inhibition and induction of cell death. CONCLUSIONS DNA repair, which is active in CLL lymphocytes, may be a biological target for facilitating the incorporation of nucleoside analogues and increasing their cytotoxicity. Thus, the increased repair capacity associated with resistant disease may be manipulated to therapeutic advantage.