title: Transfection of human topoisomerase II alpha into etoposide-resistant cells: transient increase in sensitivity followed by down-regulation of the endogenous gene.
abstract: We have investigated the possibility of overcoming the resistance of human brain tumour cells (HBT20) to etoposide by transferring the normal human topoisomerase II alpha (H-topo II) gene into these cells. H-topo II in a mammalian expression vector containing a glucocorticoid-inducible mouse mammary tumour virus (MMTV) promoter was transfected into etoposide-resistant HBT20 cells (HBT20-hTOP2MAM). HBT20 cells transfected with pMAMneo vector alone served as control cells (HBT20-MAM). These were stable transfections. Following a 2 h dexamethasone treatment, H-topo II mRNA expression, protein production, etoposide-induced DNA-protein complex formation and sensitivity to etoposide were increased in HBT20-hTOP2MAM cells compared with control HBT20-MAM cells and with HBT20-hTOP2MAM cells not treated with dexamethasone. However, mRNA and protein levels and cell sensitivity returned to baseline when incubation with dexamethasone was continued for 24 h. This decrease from the 2 h values could not be explained by a loss of the MMTV promoter response to dexamethasone. (H-topo II alpha promoter)-(chloramphenicol acetyltransferase) constructs containing regions -559-0 and -2400-0 were significantly down-regulated in HBT20-hTOP2MAM cells treated for 24 h with dexamethasone compared with dexamethasone-treated control cells. H-topo II mRNA stability after 24 h of dexamethasone treatment was not altered compared with that in control cells. Our data indicate that the exogenously produced H-topo II may have a negative-feedback effect on the endogenous topoisomerase II promoter, causing down-regulation of the endogenous gene.
authors.affiliation: Department of Cell Biology, University of Texas M.D. Anderson Cancer Center, Houston 77030, USA.
authors.forename: T
authors.initials: T
authors.lastname: Asano
authors.forename: T
authors.initials: T
authors.lastname: An
authors.forename: J
authors.initials: J
authors.lastname: Mayes
authors.forename: L A
authors.initials: LA
authors.lastname: Zwelling
authors.forename: E S
authors.initials: ES
authors.lastname: Kleinerman
mesh_terms: D000818:Animals; D000951:Antigens, Neoplasm; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D015152:Blotting, Northern; D001932:Brain Neoplasms / Q000188:drug therapy / Q000201:enzymology / Q000235:genetics; D004247:DNA / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000235:genetics*; D004268:DNA-Binding Proteins; D003907:Dexamethasone / Q000494:pharmacology; D015536:Down-Regulation; D019008:Drug Resistance, Neoplasm; D005047:Etoposide / Q000494:pharmacology*; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007527:Isoenzymes / Q000235:genetics*; D051379:Mice; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D015183:Restriction Mapping; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D000951:Antigens, Neoplasm; D000972:Antineoplastic Agents, Phytogenic; D004268:DNA-Binding Proteins; D007527:Isoenzymes; D012333:RNA, Messenger; D005047:Etoposide; D003907:Dexamethasone; D004247:DNA; D004250:DNA Topoisomerases, Type II