1. ¹Department of Pediatrics, Nippon Medical School, Tokyo, Japan
2. ²Pharmaceutical Research Laboratory, Kirin Brewery Co., Ltd, Gunma, Japan
3. ³The 1st Department of Internal Medicine, Showa University School of Medicine, Tokyo, Japan
4. ⁴Department of Medical Oncology, Tochigi Cancer Center Hospital, Tochigi, Japan
5. ⁵Pharmacology Division, National Cancer Center Institute, Tokyo, Japan
6. ⁶School of Science and Engineering, Teikyo University, Tochigi, Japan

Correspondence: Dr T Asano, Department of Pediatrics, Nippon Medical School, Chiba-Hokusoh Hospital, 1715 Kamakari, Inba-mura, Inba-gun, Chiba Prefecture 270-1894, Japan. E-mail: VFF13540@nifty.ne.jp

⁷These authors contributed equally to this work

⁸These authors share senior authorship for this work

Received 12 October 2004; Accepted 8 February 2005; Published online 29 March 2005.

Abstract

KRN 8602 (MX2) is a novel morpholino anthracycline derivative having the chemical structure 3'-deamino-3'-morpholino-13-deoxo-10-hydroxycarminomycin hydrochloride. To investigate the mechanisms of resistance to MX2, we established an MX2-resistant phenotype (K562/MX2) of the human myelogeneous leukaemia cell line (K562/P), by continuously exposing a suspension culture to increasing concentrations of MX2. K562/MX2 cells were more resistant to MX2 than the parent cells, and also showed cross-resistance to etoposide and doxorubicin. Topoisomerase (Topo) II protein levels in K562/MX2 cells were lower of those in K562/P cells on immunoblot analysis and decreased expression of Topo II mRNA was seen in K562/MX2 cells. Topoisomerase II catalytic activity was also reduced in the nuclear extracts from K562/MX2 cells when compared with K562/P cells. Aberrant methylated CpG of Topo II gene was observed in K562/MX2 cells when compared with the parent line on methylation-specific restriction enzyme analysis. To overcome the drug resistance to MX2 and etoposide, we investigated treatment with 5-Aza-2'-deoxycytidine (5AZ), which is a demethylating agent, in K562/MX2 cells. 5-Aza-2'-deoxycytidine treatment increased Topo II mRNA expression in K562/MX2 cells, but not in K562/P cells, and increased the cytotoxicity of MX2 and etoposide. Methylated CpG was decreased in K562/MX2 cells after 5AZ treatment. We concluded that the mechanism of drug resistance to MX2 and etoposide in K562/MX2 cells might be the combination of decreased expression of Topo II gene and increased methylation, and that 5AZ could prove to be a novel treatment for etoposide-resistant cell lines, such as K562/MX2.