TRANSFECTION OF HUMAN TOPOISOMERASE II-ALPHA INTO ETOPOSIDE-RESISTANTCELLS - TRANSIENT INCREASE IN SENSITIVITY FOLLOWED BY DOWN-REGULATIONOF THE ENDOGENOUS GENE

We have investigated the possibility of overcoming the resistance of h uman brain tumour cells (HBT20) to etoposide by transferring the norma l human topoisomerase II alpha ((H-topo II) gene into these cells. H-t opo II in a mammalian expression vector containing a glucocorticoid-in ducible mouse mammary tumour virus (MMTV) promoter was transfected int o etoposide-resistant HBT20 cells (HBT20-hTOP2MAM). HBT20 cells transf ected with pMAMneo vector alone served as control cells (HBT20-MAM). T hese were stable transfections. Following a 2 h dexamethasone treatmen t, H-topo II mRNA expression, protein production, etoposide-induced DN A-protein complex formation and sensitivity to etoposide were increase d in HBT20-hTOP2MAM cells compared with control HBT20-MAM cells and wi th HBT20-hTOP2MAM cells not treated with dexamethasone. However, mRNA and protein levels and cell sensitivity returned to baseline when incu bation with dexamethasone was continued for 24 h. This decrease from t he 2 h values could not be explained by a loss of the MMTV promoter re sponse to dexamethasone, (H-topo II alpha promoter)-(chloramphenicol a cetyltransferase) constructs containing regions -559-0 and -2400-0 wer e 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 n ot altered compared with that in control cells. Our data indicate that the exogenously produced H-topo II may have a negative-feedback effec t on the endogenous topoisomerase II promoter, causing down-regulation of the endogenous gene.