Differential over‐expression of mdr1 genes in multidrug‐resistant rat glioblastoma cell lines selected with doxorubicin or vincristine

Abstract

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 0.5 cells. Northern and Western blot analysis of the mdrI 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 mdrI 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 mdrIa gene, whereas the C6 IV cells preferentially over-expressed the mdrIb 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 mdrIa gene product is more efficient than the mdrIb gene product in extruding anti-cancer drugs from the cells; and that the mdrIb gene product might preferentially extrude vincristine rather than doxorubicin.

Reinduction of Bevacizumab in Combination with Pegylated Liposomal Doxorubicin in a Patient with Recurrent Glioblastoma Multiforme Who Progressed on Bevacizumab/Irinotecan

“…Unfortunately, doxorubicin does not adequately penetrate the BBB [15] and is subject to resistance due to P-glycoprotein mediated efflux [16, 17]. However, pegylated liposomal doxorubicin selectively overcomes the BBB in the tumor areas, with accumulation more than 10 fold higher in the tumor than in normal brain tissue [18].…”

Puromycin selectively increases mdr1a expression in immortalized rat brain endothelial cell lines

“…Mdr1a over‐expression has already been described in the mouse J7.V3‐1 cell line during the course of stepwise selection for resistance to vinblastine (Hsu et al . 1989) as well as in a rat cell line derived from C6 glioma and selected for resistance to doxorubicin (Schott et al . 1993), although the mechanism of this specific mdr1a over‐expression could not be explained.…”

“…The mdr1 isoform expressed in rat brain capillaries is exclusively mdr1a (Schinkel et al 1994;Barrand et al 1995;Jette et al 1995;Regina et al 1998). Although the substrate specificity of mdr1a and mdr1b P-gp isoforms largely overlaps, functional differences with respect to drug resistance profiles and sensitivity to known P-gp substrates have been demonstrated by several laboratories Schott et al 1993;Tang-Wai et al 1995;Yamazaki et al 2001). An optimal in vitro rat BBB model would thus be expected to display a high P-gp activity associated with the selective expression of the mdr1a gene.…”

“…Although the substrate specificity of mdr1a and mdr1b P‐gp isoforms largely overlaps, functional differences with respect to drug resistance profiles and sensitivity to known P‐gp substrates have been demonstrated by several laboratories (Lothstein et al . 1989; Schott et al . 1993; Tang‐Wai et al .…”

In VivoModel Systems in P-Glycoprotein-Mediated Multidrug Resistance

“…42,94,95 Expression of either mdr1a or mdr1b seems to be controlled in the first place by the specific tissue cells in which drug resistance is induced and not by the selecting drug. 94,96 In some studies mdr1b was first expressed in the lowerresistant cell line, while in later stages of induction, at increased drug-resistance levels, mdr1a was expressed.…”