Vol. 61, Issue 4, 879-884, April 2002

Down-Regulation of DNA Topoisomerase II in Human Colorectal Carcinoma Cells Resistant to a Protoberberine Alkaloid, Berberrubine

Mi Ran Kang and In Kwon Chung

Department of Biology, College of Science, and Protein Network Research Center, Yonsei University, Seoul, Korea

Berberrubine, a protoberberine alkaloid that exhibits antitumor activity in animal models, has been identified as a specific poison of DNA topoisomerase II in vitro. To better understand the mechanisms of cellular response to berberrubine, human colorectal carcinoma cells (AMC5) were selected for resistance to berberrubine. The resulting cell line (AMC5/B1) was 5.3-fold resistant to berberrubine in the absence of MDR1 overexpression. The AMC5/B1 line was cross-resistant to topoisomerase II-targeted drugs but showed no cross-resistance to other antitumor drugs. The patterns of cross-resistance to various drugs led us to examine the cellular contents of topoisomerase II. Topoisomerase II activity was ~2.8-fold lower in AMC5/B1 cells compared with parental cells. The AMC5/B1 line contained ~5-fold decrease in topoisomerase II protein level and ~2.5-fold decrease in topoisomerase II mRNA level. A comparison of the degradation kinetics of topoisomerase II mRNA demonstrated that there was no difference in mRNA stability between the two cell lines. Furthermore, the activity of topoisomerase II promoter in AMC5/B1 cells was about 25% of that in AMC5 parental cells when transient transfection experiments were performed with the promoter-luciferase reporter gene. These results indicate that down-regulation of topoisomerase II in AMC5/B1 cells occurs at the transcriptional level. Nucleotide sequencing of the topoisomerase II promoter regions revealed no mutations in AMC5/B1 cells. In summary, resistance to berberrubine in AMC5 cells is associated with decreased level of catalytically active topoisomerase II, suggesting that topoisomerase II is the cellular target of berberrubine in vivo.