Mello J A, Lippard S J, Essigmann J M
Department of Chemistry, Whitaker College of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge 02139, USA.
Biochemistry. 1995 Nov 14;34(45):14783-91. doi: 10.1021/bi00045a020.
The effects of cis-diamminedichloroplatinum(II) (cis-DDP) and trans-DDP adducts on mammalian transcription in vivo have been investigated. A plasmid containing the beta-galactosidase (beta-gal) reporter gene was modified with either of the two platinum compounds and transfected into human or hamster cell lines. A 2-3 fold higher level of transcription was observed in both cell lines from plasmids containing trans-DDP adducts as compared to plasmids modified by cis-DDP. This difference in transcriptional activity was not decreased in human and rodent nucleotide excision repair deficient cell lines, indicating that more efficient excision repair of the trans-DDP adducts was not the cause of its lower ability to block transcription in this assay. For this conclusion to be valid, it is assumed that trans-DDP adducts are repaired primarily by the nucleotide excision repair pathway, as is the case with the adducts of cis-DDP. The possibility that trans-DDP adducts are preferentially bypassed by RNA polymerase was examined by monitoring the elongation of beta-gal mRNA on damaged templates in vivo. Nascent beta-gal mRNA transcripts were recovered from excision repair deficient xeroderma pigmentosum A cells transfected with platinated plasmids, and the extent of RNA synthesis was measured by using ribonuclease protection. Fourfold more trans-DDP than cis-DDP adducts were required to inhibit transcription elongation by 63%. RNA polymerase II bypassed cis- and trans-DDP DNA adducts with efficiencies of 0-16% and 60-70%, respectively. These data provide insight into the differential toxicity of the two platinum isomers.
已研究了顺二氯二氨合铂（II）（顺铂）和反式二氯二氨合铂加合物对哺乳动物体内转录的影响。将含有β-半乳糖苷酶（β-gal）报告基因的质粒用这两种铂化合物之一进行修饰，然后转染到人或仓鼠细胞系中。与用顺铂修饰的质粒相比，在含有反式二氯二氨合铂加合物的质粒转染的两种细胞系中均观察到转录水平提高了2 - 3倍。在人和啮齿动物核苷酸切除修复缺陷细胞系中，这种转录活性差异并未降低，这表明反式二氯二氨合铂加合物更有效的切除修复并非其在该实验中阻断转录能力较低的原因。为使该结论有效，假定反式二氯二氨合铂加合物主要通过核苷酸切除修复途径进行修复，顺铂加合物的情况也是如此。通过监测体内受损模板上β-gal mRNA的延伸，研究了RNA聚合酶优先绕过反式二氯二氨合铂加合物的可能性。从用铂化质粒转染的切除修复缺陷的着色性干皮病A细胞中回收新生的β-gal mRNA转录本，并使用核糖核酸酶保护法测量RNA合成的程度。抑制转录延伸63%所需的反式二氯二氨合铂加合物比顺铂加合物多四倍。RNA聚合酶II绕过顺铂和反式二氯二氨合铂DNA加合物的效率分别为0 - 16%和60 - 70%。这些数据为这两种铂异构体的差异毒性提供了深入了解。
