The apical siting of BCRP on polarized cells is of particular relevance to the possible role or roles of BCRP in normal tissues. The protein is found in many tissues, including barrier sites, as outlined in Table II.
The highest BCRP expression is found in the placenta on the syncytiotrophoblast facing the maternal circulation. This suggests a role for the protein in the elimination of substrates from the fetus. This has been established for mouse Bcrpl; in both wild-type and P-gp knockout mice, inhibition of Bcrpl by GF120918 (a common inhibitor of human and mouse P-gp and BCRP) resulted in at least a twofold increase in the fetal uptake of orally administered topotecan, a BCRP substrate. BCRP is also expressed at more modest levels in the colon, small intestine, liver, ovary, and breast, where it may be concerned with elimination of material from these tissues. In a recent clinical study utilizing GF120918, it was shown that the oral bioavailability of topotecan more than doubled (from 40% to 97%) when the drug was coadministered with the inhibitor, thus underlining the functional significance of BCRP expression in the intestine.
This distribution of BCRP shows similarities to that of the multidrug transporter P-glycoprotein, which is also expressed in various epithelia, particularly in organs associated with drug absorption and disposition, such as hepato-cyte canalicular membrane and the intestinal mucosa. P-gp is thought to provide a first line of defense against the entry of many types of xenobiotics into the body. Knockout mice deficient in functional P-gp, although viable, fertile, and without obvious histological or developmental abnormalities, show significantly altered pharmacokinetics (and toxicity of several drugs) . The third subfamily of multidrug transporters, the MRPs, are widely distributed throughout the body in tissues including the choroid plexus, oral mucosa, small intestine, testis, and respiratory tract. Because there are several MRP homologs with overlapping substrate specificities, the importance of each for the elimination of particular substances is difficult to assess.
Both BCRP and P-gp are to be found on the endothelium lining the blood-brain barrier (see later discussion). The presence of multidrug transporters at such barrier sites creates "pharmacological sanctuaries" within the body, permitting certain organs and tissues to function in relative isolation from the rest of the body. Indeed, in P-gp knockout mice, the integrity of the blood-brain barrier is shown to be significantly compromised, with much higher brain penetration of P-gp substrates such as vinblastine and ivermectin being demonstrated. The relevance of BCRP at these sites is still under investigation (see later discussion).
The generation of the Bcrpl knockout mouse  has thrown new light on the putative physiological function of this transporter. Though these mice were anatomically normal and fertile, a defect was seen in their ability to handle a metabolite of chlorophyll, pheophorbide a, resulting in severe phototoxicity in mice exposed to light. They also exhibited a previously uncharacterized form of porphyria. Thus it became known that BCRP performs an essential function at the gut epithelium in effluxing toxic products of chlorophyll metabolism. BCRP knockout mice generated independently by Zhou et al.  were used to demonstrate that this transporter, rather than P-gp, is responsible for the dye efflux in the cells. This allows analysis of the "side-population," enriched in murine hematopoietic stem cells, which have high bone-marrow repopulating activity. The role BCRP plays at this location is still to be elucidated.
Expression of BCRP in Endothelia of Normal Tissues and of Tumors
BCRP differs from P-gp in being expressed on the endothelial lining of vascular beds in many tissues, not just at the blood-brain barrier. Interestingly, BCRP is evident in venules and capillaries (see Table II) but not in arterioles . Hence the transporter is distributed in the regions of the vasculature where the bulk of the exchange of materials between blood and tissues occurs. On endothelial cells of vasculature-supplying tumors (for example, testicular germ-cell tumors, endometrial, ovarian and colon carcinomas, and brain tumors), antibody staining for BCRP has been described as moderate to strong, stronger indeed than on the vascular endothelium in the surrounding normal regions . This raises the interesting possibility that BCRP expression is perhaps upregulated in the endothelium of blood vessels during neoplastic vasculogenesis.
Localization of BCRP in the Specialized Endothelium of the Blood-Brain Barrier
The presence of multidrug transporters is of particular importance in vascular endothelial cells at special barrier sites such as the blood-brain and blood-testis barriers. Here the vessels possess tight junctions that place severe restrictions on the free paracellular diffusion of many substances seen in peripheral endothelia.
Recent studies have explored the localization of BCRP in human brain material using fresh-frozen samples of both normal and tumor brain (meningiomas and gliomas) . Western blot results show a higher degree of expression of BCRP protein in the gliomas over the normal and menin-gioma samples. It could be seen by immunostaining that BCRP is primarily localized to blood vessels within the brain. In the case of two meningioma samples, notable heterogeneous staining for BCRP was seen in brain parenchymal cells in addition to endothelial cells. Diestra et al.  also reported a higher expression of BCRP in several unspecified brain tumors over normal brain parenchyma using immunohistochemical staining with a well-characterized anti-BCRP antibody.
By exploiting the powerful resolving capabilities of the confocal microscope, it has been possible to gain some understanding of the subcellular distribution of BCRP within brain microvessels. Utilizing the fact that the brain endothelial glucose transporter GLUT-1 is localized on both sides of brain endothelial cells (both luminal and abluminal membranes), dual-staining with antibodies for GLUT-1 and for BCRP revealed the main sites of BCRP expression in microvessels in both normal and tumor brain sections. The distribution of BCRP staining was seen to be inner to that of GLUT-1 in all microvessels viewed, which suggests that BCRP is localized toward the luminal membrane of human brain endothelial cells in the in vivo blood-brain barrier . It is probable therefore that BCRP, localized strategically at the luminal membrane of endothelial cells, has a protective function at the blood-brain barrier in limiting entry of substrates into the brain.
P-gp also has been localized to the luminal aspect of the brain capillary endothelium. It is already well documented that in this situation it performs what has been described as a "gatekeeper" role at the blood-brain barrier, pumping out a variety of xenobiotics that would otherwise gain access to the brain via the transcellular pathway due to their lipophilicity . The number of drugs known to be excluded from the brain by P-gp is large, ranging from nonsedating antihistamines, antiepileptics, and beta-blockers to anti-HIV reverse transcriptase inhibitors. What additional protection BCRP may bring to bear is as yet not well defined and would require the advent of knockout mice lacking several of the MDR transporters or use of combinations of specific inhibitors so that the influence of BCRP can be distinguished from that of P-gp in vivo.
The presence and importance of MRPs at the blood-brain barrier is even less clear. This is due both to the multiplicity of transporters in this family and to existing controversies in the literature. In contrast to BCRP, MRP1 is known to be functionally active in vivo at the epithelium of the choroid plexus, regulating the distribution of several xenobiotics into the CSF. But it has not been definitively localized in vivo at the blood-brain barrier. MRP1 does, however, become upregulated in cultured brain endothelial cells . This has allowed its functionality to be explored in vitro in brain endothelial cells cultured from several sources including human brain. In the case of MRP2, results of recent studies using in vivo microdialysis hint at a functional role for this protein at the rat blood-brain barrier, limiting the brain uptake of the anticonvul-sant phenytoin. However, these observations need further investigation.
A homolog of BCRP has been described in porcine brain endothelial cells by Eisenblatter et al. . This protein, named Brain Multidrug-Resistance Protein (BMDP), shows 86 percent amino acid identity with human BCRP and is predicted to have the typical architecture of a "reverse" halftransporter. BMDP was shown, using immunohistochem-istry, to be localized to the cell membrane of cultured porcine brain endothelial cells. A blood vessel location in vivo for the message was inferred from RNA isolation experiments in which the mRNA of BMDP appeared to be concentrated in the brain microvessels, with the levels of transcript higher in isolated capillaries than in homogenized brain tissue.
High levels of BMDP transcript were also detectable in cultured porcine brain endothelial cells. These appeared approximately 30 times higher than equivalent P-gp expression, suggesting that at least in the porcine endothelium, BMDP plays a more prominent role than P-gp. This was corroborated via functional studies using the radiolabeled substrate 3H-daunorubicin (a substrate common to both P-gp and BCRP) performed on cultured porcine brain endothelial cells grown as monolayers—GF120918 (which inhibits both P-gp and BCRP) abrogated almost completely the transport of daunorubicin from the basolateral to the apical side of the porcine brain endothelial cell monolayer. However, specific P-gp inhibitors gave only moderate inhibition. This strongly suggests that the contribution by BMDP to transport of substrates across the porcine blood-brain barrier may be greater than by P-gp. These studies are the first to report functional BCRP in cells derived from the blood-brain barrier.
Many questions still remain regarding the in vivo functionality of BCRP. Its ubiquitous expression in the endothe lium of veins and capillaries of every tissue so far examined suggest that it might efflux substrates that are potentially toxic to many tissues but are incapable of passing between endothelial cells. In particular, its expression at the blood-brain barrier may also be of paramount significance to limiting the brain penetration of substrates. The vast body of research available on P-gp and members of the MRP family has pointed to a number of specific-roles performed by these transporters at various sites in the body. There is still much to be learned about BCRP and the function it may perform in the microvessel endothelium.
The authors thank the Cancer Research Campaign for their contributions to the authors' own research work and the Cambridge Commonwealth Trust for assistance toward a studentship for HCC, who also holds an award from Universities UK.
1. Allen, J. D., and Schinkel, A. H. (2002). Multidrug resistance and pharmacological protection mediated by the Breast Cancer Resistance Protein (BCRP/ABCG2). Mol. Can. Ther. 1, 427-434. A very useful and comprehensive recent review on all aspects of BCRP.
2. Schinkel, A. H. (1999). P-Glycoprotein, a gatekeeper at the blood-brain barrier. Adv. Drug Deliv. Rev. 36, 179-194.
3. Jonker, J. W., Buitelaar, M., Wagenaar, E., van der Valk, M. A., Scheffer, G. L., Scheper, R. J., Plosch, T., Kuipers, F., Oude Elferink, R. P. J., Rosing, H., Beijnen, J. H., and Schinkel, A. H. (2002). The breast cancer resistance protein protects against a major chlorophyll-derived dietary phototoxin and protoporphyria. Proc. Natl. Acad. Sci. USA 99, 15649-15654.
4. Zhou, S., Morris, J. J., Barnes, Y., Lan, L., Schuetz, J. D., and Sorrentino, B. P. (2002). BCRP1 gene expression is required for normal numbers of side-population stem cells in mice, and confers relative protection to mitoxantrone in hematopoietic cells in vivo. Proc. Natl. Acad. Sci. USA 99, 12339-12344.
5. Maliepaard, M., Scheffer, G. L., Faneyte, I. F., van Gastelen M. A., Pijnenborg, A. C. L. M., Schinkel, A. H., van de Vijver, M. J., Scheper, R. J., and Schellens, J. H. M. (2001). Subcellular localization and distribution of the breast cancer resistance protein transporter in normal human tissues. Cancer Res. 61, 3458-3464.
6. Diestra, J. E., Scheffer, G. L., Catal, I., Maliepaard, M., Schellens, J. H. M., and Scheper, R. J. (2002). Frequent expression of the multidrug resistance associated protein BCRP/MXR/ABCP/ABCG2 in human tumors detected by the BXP-21 monoclonal antibody in paraffin-embedded material. J. Pathol. 198, 213-219.
7. Cooray, H. C., Blackmore, C. G., Maskell, L., and Barrand, M. A. (2002). Localization of Breast Cancer Resistance Protein in microvessel endothelium of human brain. Neuroreport 13, 2059-2063. Establishes a luminal localization for BCRP at the human blood—brain barrier.
8. Seetharaman, S., Barrand, M. A., Maskell, L., and Scheper, R. J. (1998). Multidrug resistance-related transport proteins in isolated human brain microvessels and in cells cultured from these isolates. J. Neurochem. 70, 1151-1159.
9. Eisenblatter, T., Huwel, S., and Galla, H. J. (2003). Characterisation of the brain multidrug resistance protein (BMDP/ABCG2/BCRP) expressed at the blood-brain barrier. Brain Res. 971, 221-231. The first paper to report a porcine homolog of BCRP highly expressed in cultured endothelial cells derived from the blood—brain barrier.
Litman, T., Brangi, M., Hudson, E., Fetsch, P., Abati, A., Ross, D. D., Miyake, K., Resau, J. H., and Bates, S. E. (2000). The multidrug-resistant phenotype associated with overexpression of the new ABC half-transporter, MXR (ABCG2). J. Cell. Sci. 113, 2011-2021.
Hiran C. Cooray is in the final year of his doctorate studying the expression and putative roles of BCRP in human brain material and in cultured endothelial cells.
Dr. Margery Barrand is a Senior Lecturer in the Department of Pharmacology in the University of Cambridge. Her group has strong research interests in multidrug transporters and, in particular, transport systems at the blood-brain barrier.
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