Withoff S, Smit E F, Meersma G J, van den Berg A, Timmer-Bosscha H, Kok K, Postmus P E, Mulder N H, de Vries E G, Buys C H
Department of Internal Medicine, University Hospital Groningen, The Netherlands.
Lab Invest. 1994 Jul;71(1):61-6.
We have modified a polymerase chain reaction (PCR)-aided transcript titration assay (1) in order to allow quantitation of low amounts of DNA topoisomerase II alpha mRNA in small RNA samples.
The titration assay was used to quantitate the amount of DNA topoisomerase II alpha mRNA in a human small cell lung carcinoma cell line, GLC4 and its drug-resistant sublines, GLC4/ADR and GLC4/CDDP. These cell lines show differences in DNA topoisomerase II alpha protein level and DNA topoisomerase II enzyme activity. To validate the titration assay, the results were compared with the results of a DNA topoisomerase II enzyme activity assay and DNA topoisomerase II alpha northern and western blotting assays.
Using the titration assay, we were able to quantitate DNA topoisomerase II alpha mRNA on a picogram level starting with less than 1 micrograms of total RNA/cell line. GLC4/ADR showed a markedly decreased DNA topoisomerase II alpha mRNA level that seemed to be unchanged in GLC4/CDDP when compared with the parental cell line. The results obtained with this assay are confirmed by the western blot data and are not in contradiction with the northern blot results obtained for the three cell lines.
The DNA topoisomerase II alpha titration assay is a highly sensitive new technique to study the role of DNA topoisomerase II alpha in drug resistance and may help to identify cancer types and patients most likely to respond to DNA topoisomerase II targeted drugs. The decrease in DNA topoisomerase II alpha protein level and DNA topoisomerase II activity in GLC4/ADR may result from transcriptional down regulation of DNA topoisomerase II alpha.
我们对聚合酶链反应（PCR）辅助的转录本滴定分析方法（1）进行了改进，以便能够对小RNA样本中少量的DNA拓扑异构酶IIα mRNA进行定量分析。
采用滴定分析方法对人小细胞肺癌细胞系GLC4及其耐药亚系GLC4/ADR和GLC4/CDDP中的DNA拓扑异构酶IIα mRNA含量进行定量分析。这些细胞系在DNA拓扑异构酶IIα蛋白水平和DNA拓扑异构酶II酶活性方面存在差异。为验证滴定分析方法，将结果与DNA拓扑异构酶II酶活性分析、DNA拓扑异构酶IIα Northern印迹和Western印迹分析结果进行比较。
使用滴定分析方法，我们能够从每个细胞系少于1微克的总RNA开始，在皮克水平上对DNA拓扑异构酶IIα mRNA进行定量分析。与亲代细胞系相比，GLC4/ADR显示出DNA拓扑异构酶IIα mRNA水平显著降低，而GLC4/CDDP中的该水平似乎未发生变化。该分析方法获得的结果得到了Western印迹数据的证实，并且与三个细胞系的Northern印迹结果不矛盾。
DNA拓扑异构酶IIα滴定分析是一种高度灵敏的新技术，可用于研究DNA拓扑异构酶IIα在耐药性中的作用，并可能有助于识别最有可能对DNA拓扑异构酶II靶向药物产生反应的癌症类型和患者。GLC4/ADR中DNA拓扑异构酶IIα蛋白水平和DNA拓扑异构酶II活性的降低可能是由于DNA拓扑异构酶IIα的转录下调所致。
