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. 1996 Oct 1;319(Pt 1):307–313. doi: 10.1042/bj3190307

Transfection of human topoisomerase II alpha into etoposide-resistant cells: transient increase in sensitivity followed by down-regulation of the endogenous gene.

T Asano 1, T An 1, J Mayes 1, L A Zwelling 1, E S Kleinerman 1
PMCID: PMC1217769  PMID: 8870683

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.

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Selected References

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