Mirski S E, Voskoglou-Nomikos T, Young L C, Deeley R G, Campling B G, Gerlach J H, Cole S P
Cancer Research Laboratories, Queen's University, Kingston, Ontario, Canada.
Lab Invest. 2000 Jun;80(6):787-95. doi: 10.1038/labinvest.3780083.
Certain drugs used in the treatment of lung cancer and other human malignancies are cytotoxic because of their ability to interact with the two isoforms of topoisomerase II (topo II), topo IIalpha and topo IIbeta. As part of an effort to evaluate the contribution of topo II alterations to drug sensitivity and resistance in lung cancer, we have developed a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay to measure levels of topo II alpha and beta mRNAs simultaneously using a single pair of primers with sequences common to both isoforms. The PCR products derived from the topo II alpha and beta mRNAs are both 446 bp but have different electrophoretic mobilities in a nondenaturing polyacrylamide gel, allowing sensitive, rapid quantitation when the products are radiolabeled with [35S]-dATP. Using this RT-PCR method, poly(A+) RNA from 13 non-small cell lung cancer (NSCLC) cell lines was analyzed. The results obtained indicated that the cell lines express a wide range of topo II alpha mRNA levels (12-fold) and topo IIbeta mRNA levels (5.5-fold). Tumor and normal lung tissues from 25 patients with NSCLC were also examined. In the tumor samples, the levels of the topo II alpha and beta mRNAs were similar. However, mean topo IIalpha mRNA levels in the tumors were approximately 7-fold higher than those of the paired normal lung tissues. In contrast, topo IIbeta mRNA levels were similar in both tumor and normal lung. Topo II alpha and beta mRNA levels were both significantly lower in the squamous cell tumors than in the adenocarcinoma samples. Topo IIbeta mRNA levels in the squamous cell tumors were also significantly lower than those in paired normal lung tissue. The RT-PCR method described is reliable and convenient, and for the first time, makes the rapid simultaneous direct comparison of topo IIalpha and topo IIbeta mRNA levels feasible in large numbers of clinical samples.
某些用于治疗肺癌和其他人类恶性肿瘤的药物具有细胞毒性，因为它们能够与拓扑异构酶II（topo II）的两种同工型，即拓扑异构酶IIα和拓扑异构酶IIβ相互作用。作为评估拓扑异构酶II改变对肺癌药物敏感性和耐药性影响的一部分工作，我们开发了一种半定量逆转录聚合酶链反应（RT-PCR）检测方法，使用一对对两种同工型都通用的引物同时测量拓扑异构酶IIα和β mRNA的水平。来自拓扑异构酶IIα和β mRNA的PCR产物均为446 bp，但在非变性聚丙烯酰胺凝胶中具有不同的电泳迁移率，当产物用[35S]-dATP进行放射性标记时，可实现灵敏、快速的定量分析。使用这种RT-PCR方法，对13种非小细胞肺癌（NSCLC）细胞系的聚腺苷酸（poly(A+)）RNA进行了分析。所得结果表明，这些细胞系表达的拓扑异构酶IIα mRNA水平范围很广（12倍），拓扑异构酶IIβ mRNA水平范围也很广（5.5倍）。还检测了25例NSCLC患者的肿瘤组织和正常肺组织。在肿瘤样本中，拓扑异构酶IIα和β mRNA的水平相似。然而，肿瘤中拓扑异构酶IIα mRNA的平均水平比配对的正常肺组织高约7倍。相比之下，肿瘤和正常肺组织中拓扑异构酶IIβ mRNA的水平相似。鳞状细胞肿瘤中拓扑异构酶IIα和β mRNA的水平均显著低于腺癌样本。鳞状细胞肿瘤中拓扑异构酶IIβ mRNA的水平也显著低于配对的正常肺组织。所述的RT-PCR方法可靠且方便，首次使得在大量临床样本中快速同时直接比较拓扑异构酶IIα和拓扑异构酶IIβ mRNA水平成为可能。
