Temple Mark D, Recabarren Patsy, McFadyen W David, Holmes R J, Denny W A, Murray Vincent
School of Biochemistry and Molecular Genetics, University of New South Wales, Sydney, NSW 2052, Australia.
Biochim Biophys Acta. 2002 Apr 12;1574(3):223-30. doi: 10.1016/s0167-4781(01)00365-7.
As part of an ongoing drug development programme, this paper describes the sequence specificity and time course of DNA adduct formation for a series of novel DNA-targeted analogues of cis-diaminedichloroplatinum(II) (cisplatin) (9-aminoacridine-4-carboxamide Pt complexes) in intact HeLa cells. The sequence specificity of DNA damage caused by cisplatin and analogues in human (HeLa) cells was studied using Taq DNA polymerase and a linear amplification/polymerase stop assay. Primer extension is inhibited by a Pt-DNA adduct, and hence the sites of these lesions can be analysed on DNA sequencing gels. The repetitive alphoid DNA sequence was used as the target DNA in human cells. The 9-aminoacridine-4-carboxamide Pt complexes exhibited a markedly different sequence specificity relative to cisplatin and other analogues. The sequence specificity of the 9-aminoacridine-4-carboxamide Pt complexes is shifted away from a preference for runs of guanines. The 9-aminoacridine-4-carboxamide Pt complexes have an enhanced preference for GA dinucleotides. This is the first occasion that an altered DNA sequence specificity has been demonstrated for a cisplatin analogue in human cells. A time course of DNA damage revealed that the DNA-targeted Pt complexes, consisting of four 9-aminoacridine-4-carboxamide Pt complexes and one acridine-4-carboxamide Pt complex, damaged DNA more rapidly compared to cisplatin and non-targeted analogues. A comparison of the time taken to reach half the maximum relative intensity indicated that the DNA-targeted Pt complexes reacted approximately 4-fold faster than cisplatin and the non-targeted analogues.
作为正在进行的药物研发项目的一部分，本文描述了一系列新型顺二氨基二氯铂（II）（顺铂）（9-氨基吖啶-4-甲酰胺铂配合物）的DNA靶向类似物在完整HeLa细胞中形成DNA加合物的序列特异性和时间进程。使用Taq DNA聚合酶和线性扩增/聚合酶终止试验研究了顺铂及其类似物在人（HeLa）细胞中引起的DNA损伤的序列特异性。引物延伸受到铂-DNA加合物的抑制，因此这些损伤位点可以在DNA测序凝胶上进行分析。重复的α卫星DNA序列被用作人细胞中的靶DNA。9-氨基吖啶-4-甲酰胺铂配合物相对于顺铂和其他类似物表现出明显不同的序列特异性。9-氨基吖啶-4-甲酰胺铂配合物的序列特异性从对鸟嘌呤序列的偏好发生了偏移。9-氨基吖啶-4-甲酰胺铂配合物对GA二核苷酸有增强的偏好。这是首次在人细胞中证明顺铂类似物的DNA序列特异性发生改变。DNA损伤的时间进程表明，由四个9-氨基吖啶-4-甲酰胺铂配合物和一个吖啶-4-甲酰胺铂配合物组成的DNA靶向铂配合物比顺铂和非靶向类似物更快地损伤DNA。达到最大相对强度一半所需时间的比较表明，DNA靶向铂配合物的反应速度比顺铂和非靶向类似物快约4倍。
