title: Altered expression of topoisomerase IIalpha contributes to cross-resistant to etoposide K562/MX2 cell line by aberrant methylation.
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) IIalpha protein levels in K562/MX2 cells were lower of those in K562/P cells on immunoblot analysis and decreased expression of Topo IIalpha 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 IIalpha 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 IIalpha 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 IIalpha gene and increased methylation, and that 5AZ could prove to be a novel treatment for etoposide-resistant cell lines, such as K562/MX2.
authors.affiliation: Department of Pediatrics, Nippon Medical School, Tokyo, Japan. VFF13540@nifty.ne.jp
authors.forename: T
authors.initials: T
authors.lastname: Asano
authors.forename: K
authors.initials: K
authors.lastname: Nakamura
authors.forename: H
authors.initials: H
authors.lastname: Fujii
authors.forename: N
authors.initials: N
authors.lastname: Horichi
authors.forename: T
authors.initials: T
authors.lastname: Ohmori
authors.forename: K
authors.initials: K
authors.lastname: Hasegawa
authors.forename: T
authors.initials: T
authors.lastname: Isoe
authors.forename: M
authors.initials: M
authors.lastname: Adachi
authors.forename: N
authors.initials: N
authors.lastname: Otake
authors.forename: Y
authors.initials: Y
authors.lastname: Fukunaga
mesh_terms: D000951:Antigens, Neoplasm / Q000096:biosynthesis* / Q000187:drug effects; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D015152:Blotting, Northern; D002360:Carubicin / Q000031:analogs & derivatives* / Q000494:pharmacology*; D045744:Cell Line, Tumor; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation / Q000187:drug effects; D004250:DNA Topoisomerases, Type II / Q000096:biosynthesis* / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000187:drug effects; D000077209:Decitabine; D019008:Drug Resistance, Neoplasm / Q000187:drug effects / Q000235:genetics; D005047:Etoposide / Q000494:pharmacology*; D005434:Flow Cytometry; D006801:Humans; D015151:Immunoblotting; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D000951:Antigens, Neoplasm; D000970:Antineoplastic Agents; D004268:DNA-Binding Proteins; D012333:RNA, Messenger; C053537:morpholinoanthracycline MX2; D005047:Etoposide; D000077209:Decitabine; D002360:Carubicin; D004250:DNA Topoisomerases, Type II; D001374:Azacitidine