Quantification of topoisomerase-DNA complexes in leukemia cells from patients undergoing therapy with a topoisomerase-directed agent.
Quantification of topoisomerase-DNA complexes in leukemia cells from patients undergoing therapy with a topoisomerase-directed agent.
复制标题
对接受拓扑异构酶靶向药物治疗的患者的白血病细胞中的拓扑异构酶-DNA 复合物进行定量。
DOI：
10.1007/bf00685085
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发表时间：
1994
影响因子：
3
通讯作者：
Zwelling,LA
中科院分区：
文献类型：
--
作者：
Ellis,AL;Nowak,B;Plunkett,W;Zwelling,LA
癌症化疗中使用的几种临床重要药物通过稳定DNA-拓扑异构酶复合物（拓扑异构酶-DNA交联）的形成来抑制I型（拓扑替康）或II型（安吖啶、依托泊苷）DNA拓扑异构酶。在各种细胞系中，药物诱导的DNA-蛋白质交联产生的幅度与药物诱导的细胞毒性的幅度相关。我们开发了一种简单的过滤结合试验，可测量直接从患者体内获得的白血病细胞中药物诱导的DNA-蛋白质交联，因为最广泛用于评估细胞中药物诱导的DNA-蛋白质交联的试验[十二烷基硫酸钠（SDS）/KCl沉淀和碱性洗脱]不容易适用于患者材料。将HL-60人白血病细胞或新鲜分离的患者白血病细胞与托泊替康、依托泊苷或安吖啶孵育;用SDS裂解;并在低盐缓冲液中应用于硝酸纤维素过滤器。只有当DNA与蛋白质共价结合时，它才能保留在过滤器上。通过与DNA序列的杂交，可以定量过滤器上保留的DNA量，DNA序列在人类基因组中无处不在。使用放射性标记的细胞，我们比较了直接与SDS/KCl沉淀法在检测足叶乙甙或安吖啶诱导的DNA-蛋白质交联HL-60细胞和安吖啶耐药HL-60/AMSA细胞的滤膜结合试验。SDS/KCl沉淀法和滤膜结合法均检测到依托泊苷诱导的HL-60和HL-60/AMSA细胞中的DNA-蛋白质交联，并检测到HL-60细胞中安吖啶诱导的DNA-蛋白质交联的频率高于HL-60/AMSA细胞。滤膜结合试验检测了体外暴露于托泊替康的新鲜分离白血病细胞中的DNA-蛋白质交联。来自白血病患者的Topotecan治疗的细胞与未治疗的细胞的DNA保留的比率范围为1.8至11.5。如果该试验可预测托泊替康的抗白血病作用，则检测到的交联的异质性与预期一致，托泊替康的抗白血病作用是可变的。这种新的过滤结合技术可能有助于预测个体患者肿瘤对抑制I型或II型DNA拓扑异构酶的药物的敏感性。
Several clinically important drugs utilized in cancer chemotherapy inhibit type I (Topotecan) or type II (amsacrine, etoposide) DNA topoisomerases by stabilizing the formation of DNA-topoisomerase complexes (topoisomerase-DNA cross-links). In various cell lines, the magnitude of drug-induced DNA-protein cross-link production correlates with the magnitude of cytotoxicity induced by the drugs. We developed a simple filter-binding assay that can measure drug-induced DNA-protein crosslinks in leukemia cells obtained directly from patients because the assays most widely used for assessment of drug-induced DNA-protein cross-links in cells [sodium dodecyl sulfate (SDS)/KCl precipitation and alkaline elution] are not readily applicable for use on patient material. HL-60 human leukemia cells or freshly isolated patients' leukemia cells were incubated with Topotecan, etoposide, or amsacrine; lysed with SDS; and applied to nitrocellulose filters in a low-salt buffer. DNA is retained on the filter only if it is covalently bound to protein. The amount of DNA retained on the filter is quantified by hibridization to thealusequence of DNA, which is distributed ubiquitously in the human genome. Using radiolabeled cells, we compared the filter-binding assay directly with the SDS/KCl precipitation assay in the detection of etoposide-or amsacrine-induced DNA-protein cross-links in HL-60 cells and amsacrine-resistant HL-60/AMSA cells. Both the SDS/KCl precipitation assay and the filter-binding assay detected etoposide-induced DNA-protein cross-links in HL-60 and HL-60/AMSA cells and detected a greater frequency of amsacrine-induced DNA-protein cross-links in HL-60 cells than in HL-60/AMSA cells. The filter-binding assay detected DNA-protein cross-links in freshly isolated leukemia cells exposed to Topotecan in vitro. The ratios of DNA retention for Topotecan-treated versus untreated cells from leukemia patients ranged from 1.8 to 11.5. The heterogeneity of this detected cross-linking was as might be expected if the assay were predictive of the antileukemic action of Topotecan, which is variable. This new filter-binding technique may be useful for predicting the sensitivity of individual patients' tumors to drugs that inhibit type I or type II DNA topoisomerases.