P-GLYCOPROTEIN EXPRESSION IN EHRLICH ASCITES TUMOR-CELLS AFTER IN-VITRO AND IN-VIVO SELECTION WITH DAUNORUBICIN

Fluctuation analysis experiments were performed to assess whether sele ction or induction determines expression of P-glycoprotein and resista nce in the murine Ehrlich ascites tumour cell line (EHR2) after exposu re to daunorubicin. Thirteen expanded populations of EHR2 cells were e xposed to daunorubicin 7.5 x 10(-9) M or 10(-8) hn for 2 weeks. Surviv ing clones were scored and propagated. Only clones exposed to daunorub icin 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 (m ean 3.6-fold). P-glycoprotein was significantly increased in 11 of 25 clones (44%). Analysis of variance supported the hypothesis that spont aneous mutations conferred drug resistance in EHR2 cells exposed to da unorubicin 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 gen eration. In contrast, induction seemed to determine resistance in EHR2 cells in vitro exposed to daunorubicin 10(-6) 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 d eveloped resistance to daunorubicin (12-fold), suggesting that in EHR2 /0.8/R the mdr1 gene was activated by induction. In conclusion, our st udy demonstrates that P-glycoprotein expression and daunorubicin resis tance are primarily acquired by selection of spontaneously arising mut ants. However, under certain conditions the mdr1 gene may be activated by induction.