Mirski S E, Cole S P
Cancer Research Laboratories, Queen's University, Kingston, Ontario, Canada.
Cancer Res. 1995 May 15;55(10):2129-34.
Many clinically important antineoplastic agents exert their cytotoxicity through interaction with the M(r) 170,000 topoisomerase II alpha, an essential nuclear enzyme. Resistance to these agents has been associated frequently with either a decrease in the levels of topoisomerase II alpha or a qualitative change that alters the interaction of this enzyme with a drug or DNA. Using a VP-16-selected lung cancer cell line, H209/V6, we have identified a third resistance mechanism which involves an aberrant subcellular location of the topoisomerase II alpha isoenzyme. We have shown previously that H209/V6 cells express two topoisomerase II alpha mRNAs (6.1 and 4.8 kilobases) but only a single catalytically active protein which has a M(r) of 160,000 and is located primarily in the cytoplasm (Mirski et al., Cancer Res., 53: 4866-4873, 1993; Feldhoff et al., Cancer Res., 54: 756-762, 1994). In the present study we have determined that this mutant M(r) 160,000 topoisomerase II alpha is encoded by the shorter 4.8-kilobase mRNA. The sequencing of reverse transcriptase-PCR products from H209/V6 cells and subsequent Northern blot analyses showed that a sequence of 988 nucleotides from the 3'-coding and 3'-noncoding region of the normal topoisomerase II alpha is absent from the 4.8-kilobase mRNA. This shorter mRNA is predicted to encode a topoisomerase II alpha protein that no longer contains the 109 COOH-terminal amino acids of the normal enzyme but instead contains 34 new amino acids encoded by a sequence that was previously in the 3'-noncoding region of the mRNA. Confirmation that the COOH terminus of topoisomerase II alpha is no longer present in the M(r) 160,000 protein in H209/V6 cells was obtained by immunoblot analysis. Sequence analyses indicate that 3 putative bipartite nuclear localization signals in the M(r) 160,000 protein are disrupted or lost. Our results suggest that sequences within the COOH-proximal domain of human topoisomerase II alpha serve an important nuclear localization function.
许多具有临床重要性的抗肿瘤药物通过与分子量为170,000的拓扑异构酶IIα相互作用发挥其细胞毒性，拓扑异构酶IIα是一种重要的核酶。对这些药物的耐药性通常与拓扑异构酶IIα水平的降低或改变该酶与药物或DNA相互作用的定性变化有关。利用一种经依托泊苷筛选的肺癌细胞系H209/V6，我们发现了第三种耐药机制，该机制涉及拓扑异构酶IIα同工酶异常的亚细胞定位。我们之前已表明，H209/V6细胞表达两种拓扑异构酶IIα mRNA（6.1和4.8千碱基），但仅有一种催化活性蛋白，其分子量为160,000，主要位于细胞质中（米尔斯基等人，《癌症研究》，53: 4866 - 4873, 1993；费尔德霍夫等人，《癌症研究》，54: 756 - 762, 1994）。在本研究中，我们确定这种分子量为160,000的突变型拓扑异构酶IIα由较短的4.8千碱基mRNA编码。对H209/V6细胞逆转录酶 - PCR产物的测序及随后的Northern印迹分析表明，正常拓扑异构酶IIα 3'编码区和3'非编码区的988个核苷酸序列在4.8千碱基mRNA中缺失。预计这种较短的mRNA编码一种拓扑异构酶IIα蛋白，该蛋白不再包含正常酶的109个COOH末端氨基酸，而是包含由先前位于mRNA 3'非编码区的序列编码的34个新氨基酸。通过免疫印迹分析证实H209/V6细胞中分子量为160,000的蛋白中拓扑异构酶IIα的COOH末端不再存在。序列分析表明，分子量为160,000的蛋白中的3个假定的双分型核定位信号被破坏或丢失。我们的结果表明，人拓扑异构酶IIα的COOH近端结构域内的序列具有重要的核定位功能。
