Characterisation of non-P-glycoprotein multidrug-resistant Ehrlich ascitestumour cells selected for resistance to mitoxantrone

An Ehrlich ascites tumour cell line (EHR2) was selected in vivo for resista nce 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 o r cyclosporin A. Compared with EHR2, the multidrug resistance-associated pr otein mRNA was increased 13-fold in EHR2/MITOX. Western blot analysis showe d an unchanged, weak expression of P-glycoprotein. Topoisomerase II alpha w as reduced to one-third in EHR2/MITOX relative to EHR2 cells, whereas topoi somerase II beta was present in EHR2 but could not be detected in EHR2/MITO X. In the resistant subline, net accumulation of MITOX (120 min) and daunor ubicin (60 min) was reduced by 43% and 27%, respectively, as compared with EHR2. The efflux of daunorubicin from preloaded EHR2/MITOX cells was signif icantly increased. EHR2/MITOX microsomes had a significant basal unstimulat ed ATPase activity. The apparent K-i value for vanadate inhibition of the A TPase activity in EHR2/MITOX microsomes was not significantly different fro m the K-i value for P-glycoprotein-positive cells. However, whereas verapam il (50 mu M) inhibited the ATPase activity of EHR2/MITOX microsomes, it sti mulated the ATPase activity of microsomes derived from P-glycoprotein-posit ive cells. In conclusion, the resistance in EHR2/MITOX was multifactorial a nd appeared to be associated with: 1) a quantitative reduction in topoisome rase II alpha and beta protein; 2) reduced drug accumulation, probably as a result of increased expression of a novel transport protein with ATPase ac tivity; and 3) increased expression of MRP mRNA. BIOCHEM PHARMACOL 60;3:363 -370, 2000. (C) 2000 Elsevier Science Inc.