抗肿瘤药物顺式二氨二氯铂（II）与DNA的加合物：形成、鉴定及定量分析

Adducts of the antitumor drug cis-diamminedichloroplatinum(II) with DNA: formation, identification, and quantitation.

作者信息

Fichtinger-Schepman A M, van der Veer J L, den Hartog J H, Lohman P H, Reedijk J

出版信息

Biochemistry. 1985 Jan 29;24(3):707-13. doi: 10.1021/bi00324a025.

Abstract

Salmon sperm DNA, treated with the antitumor agent cis-diamminedichloroplatinum(II) (cis-DDP), was enzymatically degraded to (oligo)nucleotides. Four Pt-containing products were identified by 1H NMR after preparative chromatography on a diethylaminoethyl-Sephacel column at pH 8.8. In all identified adducts, comprising approximately 90% of the total Pt in the DNA, Pt was linked to the N7 atoms of the nucleobases guanine and adenine. The two major adducts were cis-Pt(NH3)2d(pGpG) and cis-Pt-(NH3)2d(pApG), both derived from intrastrand cross-links of cis-DDP on neighboring nucleobases. Only the d(pApG) but not the d(pGpA) adduct could be detected. Two minor adducts were Pt(NH3)3dGMP, resulting from monofunctionally bound cis-DDP to guanine, and cis-Pt(NH3)2d(GMP)2, originating from interstrand cross-links on two guanines as well as from intrastrand cross-links on two guanines separated by one or more bases. For analytical purposes we developed an improved method to determine cis-DDP adducts. Routinely, 40-micrograms samples of enzymatically degraded cis-DDP-treated DNA are now analyzed by separation of the mononucleotides and Pt-containing (oligo)nucleotides on the anion-exchange column Mono Q (FPLC) at pH 8.8 (completed within 14 min) and subsequent determination of the Pt content in the collected fractions by atomic absorption spectroscopy. The method was used to optimize the digestion conditions for cis-DDP-treated DNA. In kinetic studies on the formation of the various adducts, a clear preference of the Pt compound to react with guanines occurring in the base sequence d(pGpG) was established.

摘要

用抗肿瘤药物顺二氯二氨铂（II）（顺铂）处理过的鲑鱼精DNA被酶解为（寡）核苷酸。在pH 8.8条件下，于二乙氨基乙基-葡聚糖凝胶柱上进行制备色谱分离后，通过1H NMR鉴定出四种含铂产物。在所有鉴定出的加合物中，铂与DNA中约90%的总铂相连，铂与碱基鸟嘌呤和腺嘌呤的N7原子相连。两种主要加合物是顺铂（NH3）2d（pGpG）和顺铂（NH3）2d（pApG），两者均源自顺铂在相邻碱基上的链内交联。只能检测到d（pApG）加合物，而检测不到d（pGpA）加合物。两种次要加合物是Pt（NH3）3dGMP，它是由顺铂单功能结合到鸟嘌呤产生的，以及顺铂（NH3）2d（GMP）2，它源自两个鸟嘌呤之间的链间交联以及由一个或多个碱基隔开的两个鸟嘌呤之间的链内交联。为了分析目的，我们开发了一种改进的方法来测定顺铂加合物。通常，现在通过在pH 8.8的阴离子交换柱Mono Q（FPLC）上分离单核苷酸和含铂（寡）核苷酸（14分钟内完成），然后通过原子吸收光谱法测定收集馏分中的铂含量，来分析40微克经酶解的顺铂处理过的DNA样品。该方法用于优化顺铂处理过的DNA的消化条件。在各种加合物形成的动力学研究中，确定了铂化合物明显倾向于与碱基序列d（pGpG）中出现的鸟嘌呤反应。