Li L, Liu X, Glassman A B, Keating M J, Stros M, Plunkett W, Yang L Y
Division of Laboratory Medicine, The University of Texas M. D. Anderson Cancer Center, Houston 77030, USA.
Cancer Res. 1997 Apr 15;57(8):1487-94.
Fludarabine (9-beta-arabinofuranosyl-2-fluoroadenine-5'-monophosphate) is clinically active against chronic lymphocytic leukemia and low-grade lymphomas. We reported previously that fludarabine nucleoside synergistically enhanced cisplatin (CDDP)-induced cytotoxicity in vitro, and that the synergism was concomitant with inhibition of removal of cellular CDDP-induced DNA interstrand cross-links, which are presumably repaired by homologous recombinational repair. To extend our work, we investigated whether fludarabine inhibits nucleotide excision repair (NER) of CDDP-induced DNA intrastrand adducts. The effect of fludarabine on NER was determined using a cell-free system in which a plasmid containing the DNA adducts served as the substrate for repair enzymes in whole-cell extracts from repair-competent cells. To prevent the cell-bound high mobility group box-containing proteins from interfering with repair, cell extracts were depleted with high mobility group box proteins by immunoprecipitation prior to the assay. Repair synthesis, measured by the incorporation of [(32)P]dATP or [(32)P]dCTP, was inhibited by 50% at 26 or 43 microM fludarabine triphosphate, respectively; the effect was dose dependent and may have resulted from the termination of repair-patch elongation. These results were consistent with those from pulse-chase experiments demonstrating the conversion of nicked circular plasmid to the closed circular form by cell extracts filling the repair gaps. When proliferating cell nuclear antigen-depleted cell extracts were used and aphidicolin was added in the repair assay to arrest NER at the incision/excision stage, 100 microM fludarabine triphosphate inhibited about 55% of the conversion of nicked plasmids from the closed circular damaged plasmid substrate; the inhibition was dose dependent. We conclude that fludarabine triphosphate inhibited NER at the steps of incision and repair synthesis. These results suggest that fludarabine may serve as a potential repair modulator to improve the antitumor efficacies of combination regimens containing agents that induce NER.
氟达拉滨（9-β-阿拉伯呋喃糖基-2-氟腺嘌呤-5'-单磷酸）对慢性淋巴细胞白血病和低度淋巴瘤具有临床活性。我们之前报道过，氟达拉滨核苷在体外可协同增强顺铂（CDDP）诱导的细胞毒性，且这种协同作用与抑制细胞内CDDP诱导的DNA链间交联的去除有关，这些交联大概是通过同源重组修复来修复的。为了拓展我们的研究工作，我们研究了氟达拉滨是否抑制CDDP诱导的DNA链内加合物的核苷酸切除修复（NER）。使用无细胞系统来确定氟达拉滨对NER的影响，在该系统中，含有DNA加合物的质粒作为有修复能力细胞的全细胞提取物中修复酶的底物。为防止细胞结合的含高迁移率族框蛋白干扰修复，在测定前通过免疫沉淀用高迁移率族框蛋白使细胞提取物耗尽。通过掺入[(32)P]dATP或[(32)P]dCTP来测量的修复合成，在26或43 microM氟达拉滨三磷酸时分别被抑制50%；该作用呈剂量依赖性，可能是由于修复补丁延伸的终止所致。这些结果与脉冲追踪实验的结果一致，该实验表明有修复能力细胞的提取物通过填补修复缺口将带切口的环状质粒转化为闭环形式。当使用增殖细胞核抗原耗尽的细胞提取物并在修复测定中加入阿非科林以将NER阻滞在切口/切除阶段时，100 microM氟达拉滨三磷酸抑制了约55%的从闭环受损质粒底物的带切口质粒的转化；该抑制呈剂量依赖性。我们得出结论，氟达拉滨三磷酸在切口和修复合成步骤抑制NER。这些结果表明，氟达拉滨可能作为一种潜在的修复调节剂，以提高包含诱导NER的药物的联合治疗方案的抗肿瘤疗效。
