Mrp4 Confers Resistance to Topotecan and Protects the Brain from Chemotherapy

Abstract

The role of the multidrug resistance protein MRP4/ABCC4 in vivo remains undefined. To explore this role, we generated Mrp4-deficient mice. Unexpectedly, these mice showed enhanced accumulation of the anticancer agent topotecan in brain tissue and cerebrospinal fluid (CSF). Further studies demonstrated that topotecan was an Mrp4 substrate and that cells overexpressing Mrp4 were resistant to its cytotoxic effects. We then used new antibodies to discover that Mrp4 is unique among the anionic ATP-dependent transporters in its dual localization at the basolateral membrane of the choroid plexus epithelium and in the apical membrane The endothelial cells of the brain's capillaries are tightly joined to form a hydrophobic permeability barrier (32) termed the blood-brain barrier. Pgp expression in these cells limits the movement of hydrophobic cationic drugs from the blood into the brain (36,42,43). However, in vitro, these capillary endothelial cells also transport organic anions unidirectionally toward the capillary lumen in an energy-dependent fashion (5, 25, 41). Therefore, the capillary endothelial cells appear to express an unidentified anionic ABC transporter. Currently, it is unknown whether an anionic ABC transporter is expressed at functional levels in vivo in the endothelium of brain capillaries.The ABC transporter Mrp4, originally described as a nucleotide transporter (37), is known to transport a diverse array of compounds (2,7,34) and is capable of transporting organic anions as well as antiviral and antiretroviral compounds that do not easily penetrate the central nervous system (CNS) (2, 3, 9, 27, 37). Mrp4 expression was previously demonstrated on the basolateral membrane of the prostate gland and the apical membrane of the kidney (21, 44). Studies in cultured epithelial cells have demonstrated basolateral localization of Mrp4 (22). Transporters typically route to one surface in polarized cells. For instance, the Mrp (ABCC) subfamily members localize to either the basolateral or apical membrane, but not to both. MRP1 is restricted to the basolateral membrane of the choroid plexus and intestine, whereas MRP2 is found on the apical membrane in the intestine and liver (26,29). Mrp4 might be unique among the Mrp transporters in having cell-or tissuedependent polarized expression, but the biological importance of this unique ability to localize either apically or basolaterally remains unknown.We have developed Mrp4 knockout mice, and here we report their first use to show that Mrp4 is expressed in the lumen of brain capillaries and in the basolateral membrane in the choroid plexus epithelium. In vivo, Mrp4 restricts topotecan movement from the blood into the CSF and from the capillaries into the brain tissue by virtue of its unique ability to traffic to either the apical or basolateral membrane. We further show that Mrp4 overexpression confers resistance to the camptothecin topotecan. These studies have specific therapeutic implications for targeting the CNS that might harbor tumors but have more general impl...

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