P-glycoprotein expression in Ehrlich ascites tumour cells after in vitro and in vivo selection with daunorubicin

Abstract

Fluctuation analysis experiments were performed to assess whether selection or induction determines expression of P-glycoprotein and resistance in the murine Ehrlich ascites tumour cell line (EHR2) after exposure to daunorubicin. Thirteen expanded populations of EHR2 cells were exposed to daunorubicin 7.5 x 10(-9) M or 10(-8) M for 2 weeks. Surviving clones were scored and propagated. Only clones exposed to daunorubicin 7.5 x 10(-9) M could be expanded for investigation. Drug resistance was assessed by the tetrazolium dye (MTT) cytotoxicity assay. Western blot was used for determination of P-glycoprotein. Compared with EHR2, the variant cells were 2.5- to 5.2-fold resistant to daunorubicin (mean 3.6-fold). P-glycoprotein was significantly increased in 11 of 25 clones (44%). Analysis of variance supported the hypothesis that spontaneous mutations conferred drug resistance in EHR2 cells exposed to daunorubicin 7.5 x 10(-9) M. At this level (5 log cell killing) of drug exposure, the mutation rate was estimated at 4.1 x 10(-6) per cell generation. In contrast, induction seemed to determine resistance in EHR2 cells in vitro exposed to daunorubicin 10(-8) M. The revertant EHR2/0.8/R was treated in vivo with daunorubicin for 24 h. After treatment, P-glycoprotein increased in EHR2/0.8/R (sevenfold) and the cell line developed resistance to daunorubicin (12-fold), suggesting that in EHR2/0.8/R the mdr1 gene was activated by induction. In conclusion, our study demonstrates that P-glycoprotein expression and daunorubicin resistance are primarily acquired by selection of spontaneously arising mutants. However, under certain conditions the mdr1 gene may be activated by induction.