Effect of transfection of a Drosophila topoisomerase II gene into a human brain tumour cell line intrinsically resistant to etoposide

Abstract

The human brain tumour cell line HBT20 is intrinsically resistant to etoposide 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 etoposide. A D-topo II construct in a pMAMneo vector under the control of a mouse mammary tumour virus (MMTV) promoter was transfected into HBT20 cells. The gene is inducible by dexamethasone (Dex). The growth rate of the transfected cells and percentage of the cells in G1, S and G2M was no different than the parental cells. Survival after etoposide exposure (10 microM x 2 h) was measured by colony formation. Parental cells and cells transfected by pMAMneo vector alone showed no enhanced etoposide sensitivity after 24 h of Dex stimulation. By contrast, D-topo II transfected cells were sensitised 3-fold when etoposide treatment was preceded by 24 h Dex stimulation. Northern blotting and Western blotting confirmed that Dex had induced D-topo II expression in the sensitised cells. However, in D-topo II-transfected cells increasing the 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 than 24 h. At these distal times, the total cellular topo II levels (endogenous + 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 resistant HBT20 cells to etoposide but that the time frame of that sensitisation is limited.