Characterisation of multidrug-resistant Ehrlich ascites tumour cells selected in vivo for resistance to etoposide

An Ehrlich ascites tumour cell line (EHR2) was selected for resistance to e toposide (VP16) by in vivo exposure to this agent. The resulting cell line (EHR2/VP16) was 114.3-, 5.7-, and 4.0-fold resistant to VP16, daunorubicin, and vincristine, respectively. The amount of salt-extractable immunoreacti ve topoisomerase II alpha and beta in EHR2/VP16 was reduced by 30-40% relat ive to that in EHR2. The multidrug resistance-associated protein (MRP) mRNA was increased 20-fold in EHR2/VP16 as compared with EHR2, whereas the expr ession of P-glycoprotein was unchanged. In EHR2/VP16, the steady-state accu mulation of [H-3]VP16 and daunorubicin was reduced by 64% and 17%, respecti vely, as compared with EHR2. Deprivation of energy by addition of sodium az ide increased the accumulation of both drugs to the level of sensitive cell s. When glycolysis was restored by the addition of glucose to EHR2/VP16 cel ls loaded with drug in the presence of sodium azide, extrusion of [H-3]VP16 and daunorubicin was induced. Addition of verapamil (25 mu M) decreased th e efflux of daunorubicin to the level of sensitive cells, but had only a mo derate effect on the efflux of [H-3]VP16. The resistant cells showed modera te sensitisation to VP16 on treatment with verapamil, whereas cyclosporin A had no effect. Compared with that of sensitive cells, the ATPase activity of plasma membrane vesicles prepared from EHR2/VP16 cells was very low. Van adate inhibited the ATPase activity of EHR2IVP16 microsomes with a K-i valu e of 30 mu M. ATPase activity was slightly stimulated by daunorubicin, wher eas vinblastine, verapamil, and cyclosporin A had no effect. In conclusion, development of resistance to VP16 in EHR2 is accompanied by a significant reduction in topoisomerase II (alpha and beta) and by increased expression of MRP mRNA (20-fold). MRP displays several points of resemblance to P-glyc oprotein in its mode of action: 1) like P-glycoprotein, MRP causes resistan ce to a range of hydrophobic drugs; 2) MRP decreases drug accumulation in t he cells and this decrease is abolished by omission of energy; and 3) MRP i ncreases efflux of drug from cells. However, compared with that of P-glycop rotein-positive cells, the ATPase activity of MRP-positive cells is found t o be low and not able to be stimulated by verapamil. BIOCHEM PHARMACOL 60;3 :353-361, 2000. (C) 2000 Elsevier Science Inc.