Son Y S, Suh J M, Ahn S H, Kim J C, Yi J Y, Hur K C, Hong W S, Muller M T, Chung I K
Laboratory of Cell Biology, Korea Cancer Center Hospital, Seoul.
Cancer Chemother Pharmacol. 1998;41(5):353-60. doi: 10.1007/s002800050751.
A human stomach-adenocarcinoma cell line (MKN-45) was selected for resistance to Adriamycin by stepwise exposure to increasing concentrations of this agent. The resulting cell line (MKN/ADR) exhibited a high level of cross-resistance to topoisomerase II (topo II)-targeted drugs such as Adriamycin, mitoxantrone, and etoposide but showed no cross-resistance to other chemotherapeutic agents such as cisplatin, carboplatin, 5-fluorouracil, or mitomycin-C. P-glycoprotein encoded by the mdr-1 gene was not overexpressed in the MKN/ADR cell line. The doubling time of the MKN/ADR cell line (2.1 days) increased only slightly as compared with that of the MKN cell line (1.7 days). The patterns of cross-resistance to various chemotherapeutic agents led us to examine the cellular contents of topo II in both the drug-sensitive and the drug-resistant cells. Extractable topo II enzyme activity was 3-fold lower in MKN/ADR cells as compared with the parental MKN cells. Levels of topoisomerase I (topo I) catalytic activity were similar in both wild-type MKN and drug-resistant MKN/ADR cells. Southern-blot analysis of genomic DNA probed with topo IIalpha or IIbeta showed no sign of either gene rearrangement or hypermethylation. Northern-blot analysis revealed that both topo IIalpha and topo IIbeta mRNA transcripts were essentially identical in the MKN and MKN/ADR cells. In contrast, Western-blot analysis revealed an approximately 20-fold lower level of topo IIalpha in drug-resistant cells as compared with drug-sensitive cells, whereas topo IIbeta levels were similar in both lines. Moreover, the amount of in vivo topo IIalpha-DNA covalent complexes formed in the presence of etoposide was also approximately 20-fold lower in drug-resistant cells. No mutation was detected in the promoter region of the topo IIalpha gene in resistant cells as compared with sensitive cells. Thus, low levels of topo IIalpha polypeptide cannot be ascribed to changes in the mRNA levels. Collectively, the data suggest that a quantitative reduction in topo IIalpha may contribute to the resistance of MKN cells to Adriamycin and other topo II-targeted drugs.
通过逐步暴露于浓度不断增加的阿霉素，筛选出一株人胃腺癌细胞系（MKN - 45）使其对阿霉素产生耐药性。所得细胞系（MKN/ADR）对拓扑异构酶II（topo II）靶向药物如阿霉素、米托蒽醌和依托泊苷表现出高度的交叉耐药性，但对其他化疗药物如顺铂、卡铂、5 - 氟尿嘧啶或丝裂霉素 - C没有交叉耐药性。mdr - 1基因编码的P - 糖蛋白在MKN/ADR细胞系中未过度表达。MKN/ADR细胞系的倍增时间（2.1天）与MKN细胞系（1.7天）相比仅略有增加。对各种化疗药物的交叉耐药模式促使我们检测药物敏感细胞和耐药细胞中topo II的细胞含量。与亲代MKN细胞相比，MKN/ADR细胞中可提取的topo II酶活性低3倍。拓扑异构酶I（topo I）催化活性水平在野生型MKN和耐药MKN/ADR细胞中相似。用topo IIα或IIβ探针进行基因组DNA的Southern印迹分析未显示基因重排或高甲基化的迹象。Northern印迹分析表明，topo IIα和topo IIβ mRNA转录本在MKN和MKN/ADR细胞中基本相同。相比之下，Western印迹分析显示，与药物敏感细胞相比，耐药细胞中topo IIα水平低约20倍，而topo IIβ水平在两株细胞中相似。此外，在依托泊苷存在下形成的体内topo IIα - DNA共价复合物的量在耐药细胞中也低约20倍。与敏感细胞相比，耐药细胞中topo IIα基因启动子区域未检测到突变。因此，topo IIα多肽水平低不能归因于mRNA水平的变化。总体而言，数据表明topo IIα的定量减少可能导致MKN细胞对阿霉素和其他topo II靶向药物产生耐药性。
