Characterisation of Non-P-Glycoprotein multidrug-resistant Ehrlich ascites tumour cells selected for resistance to mitoxantrone

An Ehrlich ascites tumour cell line (EHR2) was selected in vivo for resistance to mitoxantrone (MITOX). The resistant cell line (EHR2/MITOX) was 6123-, 33-, and 30-fold-resistant to mitoxantrone, daunorubicin, and etoposide, respectively, but retained sensitivity to vincristine. The resistant cells showed moderate sensitisation to mitoxantrone on treatment with verapamil or cyclosporin A. Compared with EHR2, the multidrug resistance-associated protein mRNA was increased 13-fold in EHR2/MITOX. Western blot analysis showed an unchanged, weak expression of P-glycoprotein. Topoisomerase IIα was reduced to one-third in EHR2/MITOX relative to EHR2 cells, whereas topoisomerase IIβ was present in EHR2 but could not be detected in EHR2/MITOX. In the resistant subline, net accumulation of MITOX (120 min) and daunorubicin (60 min) was reduced by 43% and 27%, respectively, as compared with EHR2. The efflux of daunorubicin from preloaded EHR2/MITOX cells was significantly increased. EHR2/MITOX microsomes had a significant basal unstimulated ATPase activity. The apparent K i value for vanadate inhibition of the ATPase activity in EHR2/MITOX microsomes was not significantly different from the K i value for P-glycoprotein-positive cells. However, whereas verapamil (50 μM) inhibited the ATPase activity of EHR2/MITOX microsomes, it stimulated the ATPase activity of microsomes derived from P-glycoprotein-positive cells. In conclusion, the resistance in EHR2/MITOX was multifactorial and appeared to be associated with: 1) a quantitative reduction in topoisomerase IIα and β protein; 2) reduced drug accumulation, probably as a result of increased expression of a novel transport protein with ATPase activity; and 3) increased expression of MRP mRNA.