Bank B B, Kanganis D, Liebes L F, Silber R
Department of Medicine, New York University Medical Center, New York 10016.
Cancer Res. 1989 Feb 1;49(3):554-9.
Chlorambucil (CLB) uptake by chronic lymphocytic leukemia lymphocytes was studied using a radiometric and a newly developed high-performance liquid chromatography assay. CLB labeled with 14C in either the chloroethyl group or phenyl ring was used with identical results. Drug accumulation by the cells was found to peak at 30 s, was independent of temperature, and was proportional to medium CLB concentration over a wide range. Efflux from cells loaded with CLB and resuspended in drug-free medium was nearly complete at 30 s. The metabolic inhibitors 2-deoxyglucose and NaN3, the nitrogen mustard transport inhibitor hemicholinium-3, and another alkylating agent, melphalan, had no effect on drug uptake. We conclude that CLB enters and exits chronic lymphocytic leukemia lymphocytes by simple diffusion. Cells from 17 patients with all stages of chronic lymphocytic leukemia were studied including three with CLB-resistant disease, and no heterogeneity was found in the peak cell-associated CLB content or in metabolite pattern on high-performance liquid chromatography. These findings make it unlikely that transport or cellular drug metabolism are factors in drug resistance. Drug-DNA binding was found to be temperature-sensitive and increased with time of incubation. Gel filtration of DNA before and after enzymatic digestion indicated the presence of drug-DNA adducts. High-performance liquid chromatography analysis of digested DNA and DNA treated by neutral thermal hydrolysis suggested the presence of multiple adducts. Most of the radioactivity was found as purine adducts. Studies with CLB labeled at two different sites revealed the presence of the phenyl group and ethyl chains in the adducts. A survey of patients showed increased drug-DNA binding in cells from patients with clinical CLB resistance.
使用放射性测定法和新开发的高效液相色谱分析法研究了苯丁酸氮芥（CLB）被慢性淋巴细胞白血病淋巴细胞摄取的情况。使用在氯乙基或苯环上标记有¹⁴C的CLB，结果相同。发现细胞对药物的积累在30秒时达到峰值，与温度无关，并且在很宽的范围内与培养基中CLB的浓度成正比。在不含药物的培养基中重悬的、已加载CLB的细胞在30秒时几乎完全排出药物。代谢抑制剂2-脱氧葡萄糖和NaN₃、氮芥转运抑制剂hemicholinium-3以及另一种烷化剂美法仑对药物摄取没有影响。我们得出结论，CLB通过简单扩散进入和离开慢性淋巴细胞白血病淋巴细胞。研究了17例处于慢性淋巴细胞白血病各阶段患者的细胞，包括3例对CLB耐药的患者，在细胞相关CLB含量峰值或高效液相色谱分析的代谢物模式方面未发现异质性。这些发现使得转运或细胞药物代谢不太可能是耐药的因素。发现药物与DNA的结合对温度敏感，并随孵育时间增加。酶消化前后DNA的凝胶过滤表明存在药物-DNA加合物。对消化后的DNA和经中性热水解处理的DNA进行高效液相色谱分析表明存在多种加合物。大部分放射性被发现为嘌呤加合物。对在两个不同位点标记的CLB进行的研究表明加合物中存在苯基和乙基链。对患者的一项调查显示，临床CLB耐药患者细胞中的药物与DNA结合增加。
