EFFECT OF TRANSFECTION OF A DROSOPHILA TOPOISOMERASE-II GENE INTO A HUMAN BRAIN-TUMOR CELL-LINE INTRINSICALLY RESISTANT TO ETOPOSIDE

The human brain tumour cell line HBT20 is intrinsically resistant to e toposide and does not express mdr-1 mRNA, These studies were conducted to determine whether transfecting a Drosophila (D) topoisomerase II ( topo II) gene into HBT20 cells could increase their sensitivity to eto poside. A D-topo II construct in a pMAMneo vector under the control of a mouse mammary tumour virus (MMTV) promoter was transfected into HBT 20 cells. The gene is inducible by dexamethasone (Dex). The growth rat e of the transfected cells and percentage of the cells in G(1), S and G(2)M was no different than the parental cells. Survival after etoposi de exposure (10 mu M x 2 h) was measured by colony formation. Parental cells and cells transfected by pMAMneo vector alone showed no enhance d etoposide sensitivity after 24 h of Dex stimulation. By contrast, D- topo II transfected cells were sensitised 3-fold when etoposide treatm ent was preceded by 24 h Dex stimulation. Northern blotting and Wester n blotting confirmed that Dex had induced D-topo II expression in the sensitised cells. However, in D-topo II-transfected cells increasing t he duration of Dex stimulation to 48 h eliminated the sensitisation to etoposide although increased MMTV promoter activity and expression of the D-topo II gene persisted. Measurement of endogenous human topo-II mRNA and protein revealed a decrease after Dex exposure of greater th an 24 h. At these distal times, the total cellular topo II levels (end ogenous + exogenous) may be decreased, which may explain why increased sensitivity to etoposide could no longer be demonstrated. This model suggests that D-topo II gene transfection can sensitise de novo resist ant HBT20 cells to etoposide but that the time frame of that sensitisa tion is limited.