Kubota N, Kanzawa F, Nishio K, Takeda Y, Ohmori T, Fujiwara Y, Terashima Y, Saijo N
Pharmacology Division, National Cancer Center Research Institute, Tokyo, Japan.
Biochem Biophys Res Commun. 1992 Oct 30;188(2):571-7. doi: 10.1016/0006-291x(92)91094-7.
CPT-11, a recently developed topoisomerase I (Topo I) inhibitor, attracts the attention not only of basic researchers but also of clinicians because of its high antitumor activity. The CPT-11 resistant human lung cancer cell line, PC-7/CPT, showed 10-fold resistance compared to parental cell line, PC-7. The total activity of Topo I in the resistant cell line was one fourth that of the parental sensitive cell line. The Topo I from the resistant cells was also 5-fold more resistant to the inhibitory effect of CPT-11 than that of the parental cells. We speculated that the alteration of the Topo I gene may be responsible for the change in topoisomerase activity of the CPT-11 resistant cell line. Therefore, we analyzed the mutation of Topo I gene using the method of single strand conformation polymorphism of polymerase chain reaction and the reverse transcriptase. We divided Topo I cDNA into ten fragments which overlapped each other and covered whole coding sequences of the Topo I cDNA. We observed mobility shift of two fragments in the PC-7/CPT, suggesting the presence of some mutations in these fragments. We performed the direct-sequencing of these portions by the dideoxy chain termination method and observed an altered sequence having a G to A base change in PC-7/CPT. This base substitution results in replacement of the conserved threonine at 729 position with alanine. These results suggest that the point mutation of Topo I gene is related to the decreases of Topo I activity and the sensitivity to Topo I inhibitor in PC-7/CPT cells.
CPT-11是一种最近研发的拓扑异构酶I（Topo I）抑制剂，因其高抗肿瘤活性不仅吸引了基础研究人员，也引起了临床医生的关注。CPT-11耐药的人肺癌细胞系PC-7/CPT与亲代细胞系PC-7相比，显示出10倍的耐药性。耐药细胞系中Topo I的总活性是亲代敏感细胞系的四分之一。耐药细胞中的Topo I对CPT-11抑制作用的耐药性也比亲代细胞高5倍。我们推测Topo I基因的改变可能是CPT-11耐药细胞系拓扑异构酶活性变化的原因。因此，我们使用聚合酶链反应的单链构象多态性方法和逆转录酶分析了Topo I基因的突变。我们将Topo I cDNA分成十个相互重叠的片段，覆盖了Topo I cDNA的整个编码序列。我们在PC-7/CPT中观察到两个片段的迁移率变化，表明这些片段中存在一些突变。我们通过双脱氧链终止法对这些部分进行直接测序，在PC-7/CPT中观察到一个改变的序列，有一个从G到A的碱基变化。这种碱基替换导致729位保守的苏氨酸被丙氨酸取代。这些结果表明，Topo I基因的点突变与PC-7/CPT细胞中Topo I活性的降低和对Topo I抑制剂的敏感性有关。
