Overexpression of P-glycoprotein but not its mRNA in multidrug resistant cells selected with hydroxyrubicin

Previous studies have revealed that cultured cells treated with lipophilic natural products containing aromatic rings and basic amino group usually yielded multidrug resistant (MDR) variants. These MDR cells overexpress P-glycoprotein (P-gp), most often due to gene amplification or transcriptional activation of mdr/P-gp genes. Doxorubicin (Dox) is an anthracycline that belongs to this group of compounds. To explore the possible resistance mechanism(s) to anthracyclines that do not involve P-gp, we use a Dox analog, hydroxyrubicin (HyR) or WP159, which contains a C3' hydroxy group in replacement of the amino group in the sugar moiety of Dox thereby reducing basicity and eliminating positive charge in the parental compound to establish HyR-resistant cell lines. These resistant cells displayed the MDR phenotype and overexpressed P-gp as analyzed by Western blot analyses and immunohistochemical staining using two different anti-P-gp antibodies. Strikingly, the levels of P-gp mRNA in the majority of these MDR cells remained comparable to those in the drug-sensitive counterparts by slot blot hybridization. These results implicate that the basic center of the selecting agent is a critical determinant for generating diverse MDR variants, and that HyR may have a posttranscriptional effect on P-gp biosynthesis. This is the first report suggesting that cultured cells exposed to a particular selecting agent may give rise to particular subtype of MDR variants.