DIFFERENTIAL OVER-EXPRESSION OF MDR1 GENES IN MULTIDRUG-RESISTANT RAT GLIOBLASTOMA CELL-LINES SELECTED WITH DOXORUBICIN OR VINCRISTINE

We have compared the pharmacological and molecular characteristics of 2 cell lines derived from the C6 rat glioblastoma, and selected for resistance either to doxorubicin (C6 0.5 line) or to vincristine (C6 IV line). Each line displays a preferential 400-fold resistance towards the drug used for selection, the C6 IV line being especially weakly resistant to doxorubicin (13-fold). Verapamil completely restored doxorubicin sensitivity in the C6 IV line as well as vincristine resistance in the C6 0.5 line, but could not completely reverse doxorubicin resistance in the C6 0.5 line or vincristine resistance in the C6 IV line. This suggests that specific mechanisms of resistance against each drug were added to a common P-glycoprotein-mediated multidrug-resistance mechanism. Doxorubicin efflux was total within 2 hr in the C6 IV line, whereas it remained 8 to 10% of drug in the C6 0.5 line 4 hr after drug removal, despite a more rapid efflux of the drug in the first 30 min. This 2-compartment behavior could be related to a special sub-cellular distribution of doxorubicin in C6 O.S cells. Northern and Western blot analysis of the mdr1 gene and of the P-glycoprotein expressed by the 2 resistant cell lines made it possible to quantify their degree of over-expression; when compared with the C6 wild strain, the C6 0.5 line over-expressed both the mdr1 gene and the P-glycoprotein to a slightly higher level than the C6 IV line. Northern and Western blot analysis also suggested that C6 0.5 cell preferentially over-expressed the mdr1a gene, whereas the C6 IV cells preferentially over-expressed the mdr1b gene. This differential over-expression was confirmed after polymerase-chain-reaction amplification of the cDNA sequences transcribed from total RNA extracted from the 2 lines. It can be concluded therefore that the mdr1a gene product is more efficient than the mdr1b gene product in extruding anti-cancer drugs from the cells; and that the mdr1b gene product might preferentially extrude vincristine rather than doxorubicin. (C) 1993 Wiley-Liss, Inc.