Skip to main content

Advertisement

Log in

Expression of the ATP-binding Cassette Membrane Transporter, ABCG2, in Human and Rodent Brain Microvessel Endothelial and Glial Cell Culture Systems

  • Research Paper
  • Published:
Pharmaceutical Research Aims and scope Submit manuscript

Abstract

Purpose

The function of ABCG2 (BCRP), a member of the ATP-binding cassette (ABC) superfamily of membrane-associated drug transporters, at the blood-brain barrier remains highly controversial. This project investigates the functional expression of endogenous ABCG2 in cultures of human and rodent brain cellular compartments.

Materials and Methods

RT-PCR, western blot and fluorescent immunocytochemical analyses were performed on ABCG2-overexpressing human breast cancer (MCF-MX100) cells, human and rat brain microvessel endothelial (HBEC and RBE4, respectively), and rat glial cells.

Results

RT-PCR analysis detected ABCG2 mRNA in all the cell culture systems. Western blot analysis with anti-ABCG2 monoclonal BXP-21 antibody detected a robust band at ∼72 kDa in the ABCG2-overexpressing MCF-MX100 cell line, whereas low expression was found in human and rat brain cell systems. Immunofluorescence microscopy detected predominant plasma membrane localization of ABCG2 in MCF-MX100 cells but weak signal in all brain cellular compartments. In the presence of ABCG2 inhibitors, the accumulation of 3H-mitoxantrone and pheophorbide A, two established ABCG2 substrates, was significantly increased in MCF-MX100 cells but not in the human and rodent brain cell culture systems.

Conclusions

Our data show low endogenous ABCG2 protein expression, localization and activity in cultures of human and rat brain microvessel endothelial and glial cells.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10
Fig. 11
Fig. 12
Fig. 13
Fig. 14
Fig. 15
Fig. 16
Fig. 17
Fig. 18

Similar content being viewed by others

Abbreviations

ABC:

ATP-binding cassette

BCRP:

breast cancer resistance protein

HBEC:

human brain endothelial cells

MRP:

multidrug resistance-associated protein

P-gp:

P-glycoprotein

References

  1. L. A. Doyle, W. Yang, L. V. Abruzzo, T. Krogmann, Y. Gao, A. K. Rishi, and D. D. Ross. A multidrug resistance transporter from human MCF-7 breast cancer cells. Proc. Natl. Acad. Sci. U. S. A. 95:15665–15670 (1998).

    Article  PubMed  CAS  Google Scholar 

  2. J. D. Allen, R. F. Brinkhuis, J. Wijnholds, and A. H. Schinkel. The mouse Bcrp1/Mxr/Abcp gene: amplification and overexpression in cell lines selected for resistance to topotecan, mitoxantrone, or doxorubicin. Cancer Res. 59:4237–4241 (1999).

    PubMed  CAS  Google Scholar 

  3. T. Eisenblatter, S. Huwel, and H. J. Galla. Characterisation of the brain multidrug resistance protein (BMDP/ABCG2/BCRP) expressed at the blood-brain barrier. Brain Res. 971:221–231 (2003).

    Article  PubMed  CAS  Google Scholar 

  4. S. Hori, S. Ohtsuki, M. Tachikawa, N. Kimura, T. Kondo, M. Watanabe, E. Nakashima, and T. Terasaki. Functional expression of rat ABCG2 on the luminal side of brain capillaries and its enhancement by astrocyte-derived soluble factor(s). J. Neurochem. 90:526–536 (2004).

    Article  PubMed  CAS  Google Scholar 

  5. K. E. Berge, H. Tian, G. A. Graf, L. Yu, N. V. Grishin, J. Schultz, P. Kwiterovich, B. Shan, R. Barnes, and H. H. Hobbs. Accumulation of dietary cholesterol in sitosterolemia caused by mutations in adjacent ABC transporters. Science 290:1771–1775 (2000).

    Article  PubMed  CAS  Google Scholar 

  6. J. Xu, Y. Liu, Y. Yang, S. Bates, and J. T. Zhang. Characterization of oligomeric human half-ABC transporter ATP binding cassette G2. J. Biol. Chem. 279:19781–19789 (2004).

    Article  PubMed  CAS  Google Scholar 

  7. F. Staud and P. Pavek. Breast cancer resistance protein (BCRP/ABCG2). Int. J. Biochem. Cell Biol. 37:720–725 (2005).

    Article  PubMed  CAS  Google Scholar 

  8. M. Maliepaard, G. L. Scheffer, I. F. Faneyte, M. A. van Gastelen, A. C. L. M. Pijnenborg, A. H. Schinkel, M. J. Van de Vijver, R. J. Scheper, and J. H. M. Schellens. Subcellular localization and distribution of the breast cancer resistance protein transporter in normal human tissues. Cancer Res. 61:3458–3464 (2001).

    PubMed  CAS  Google Scholar 

  9. H. C. Cooray, C. G. Blackmore, L. Maskell, and M. A. Barrand. Localisation of breast cancer resistance protein in microvessel endothelium of human brain. NeuroReport 13:2059–2063 (2002).

    Article  PubMed  CAS  Google Scholar 

  10. W. Zhang, J. Mojsilovic-Petrovic, M. F. Andrade, H. Zhang, M. Ball, and D. B. Stanimirovic. The expression and functional characterization of ABCG2 in brain endothelial cells and vessels. FASEB J. 17:2085–2087 (2003).

    PubMed  Google Scholar 

  11. Y. J. Lee, H. Kusuhara, J. W. Jonker, A. H. Schinkel, and Y. Sugiyama. Investigation of efflux transport of dehydroepiandrosterone sulfate and mitoxantrone at the mouse blood-brain barrier: A minor role of breast cancer resistance protein. J. Pharmacol. Exp. Ther. 312:44–52 (2005).

    Article  PubMed  CAS  Google Scholar 

  12. A. E. van Herwaarden, J. W. Jonker, E. Wagenaar, R. F. Brinkhuis, J. H. Schellens, J. H. Beijnen, and A. H. Schinkel. The breast cancer resistance protein (Bcrp1/Abcg2) restricts exposure to the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine. Cancer Res. 63:6447–6452 (2003).

    PubMed  Google Scholar 

  13. S. Cisternino, C. Mercier, F. Bourasset, F. Roux, and J. M. Scherrmann. Expression, up-regulation, and transport activity of the multidrug-resistance protein Abcg2 at the mouse blood-brain barrier. Cancer Res. 64:3296–3301 (2004).

    Article  PubMed  CAS  Google Scholar 

  14. P. Breedveld, D. Pluim, G. Cipriani, P. Wielinga, O. van Tellingen, A. H. Schinkel, and J. H. M. Schellens. The effect of Bcrp1 (Abcg2) on the in vivo pharmacokinetics and brain penetration of imatinib mesylate (gleevec): Implications for the use of breast cancer resistance protein and P-glycoprotein inhibitors to enable the brain penetration of imatinib in patients. Cancer Res. 65:2577–2582 (2005).

    Article  PubMed  CAS  Google Scholar 

  15. I. Becker, K. F. Becker, R. Meyermann, and V. Hollt. The multidrug-resistance gene MDR1 is expressed in human glial tumors. Acta Neuropathol. (Berl). 82:516–519 (1991).

    Article  CAS  Google Scholar 

  16. H. A. Volk and W. Loscher. Multidrug resistance in epilepsy: Rats with drug-resistant seizures exhibit enhanced brain expression of P-glycoprotein compared with rats with drug-responsive seizures. Brain 128:1358–1368 (2005).

    Article  PubMed  Google Scholar 

  17. A. Prat, K. Biernacki, B. Becher, and J. P. Antel. B7 expression and antigen presentation by human brain endothelial cells: Requirement for proinflammatory cytokines. J. Neuropathol. Exp. Neurol. 59:129–136 (2000).

    PubMed  CAS  Google Scholar 

  18. F. Roux, O. Durieu-Trautmann, N. Chaverot, M. Claire, P. Mailly, J. M. Bourre, A. D. Strosberg, and P.-O. Couraud. Regulation of gamma-glutamyl transpeptidase and alkaline phosphatase activities in immortalized rat brain microvessel endothelial cells. J. Cell. Physiol. 159:101–113 (1994).

    Article  PubMed  CAS  Google Scholar 

  19. R. Bendayan, G. Lee, and M. Bendayan. Functional expression and localization of P-glycoprotein at the blood-brain barrier. Microsc. Res. Tech. 57:365–380 (2002).

    Article  PubMed  CAS  Google Scholar 

  20. P. T. Ronaldson, M. Bendayan, D. Gingras, M. Piquette-Miller, and R. Bendayan. Cellular localization and functional expression of P-glycoprotein in rat astrocyte cultures. J. Neurochem. 89:788–800 (2004).

    Article  PubMed  CAS  Google Scholar 

  21. G. Lee, L. Schlichter, M. Bendayan, and R. Bendayan. Functional expression of P-glycoprotein in rat brain microglia. J. Pharmacol. Exp. Ther. 299:204–212 (2001).

    PubMed  CAS  Google Scholar 

  22. R. W. Robey, Y. Honjo, A. van de Laar, K. Miyake, J. T. Regis, T. Litman, and S. E. Bates. A functional assay for detection of the mitoxantrone resistance protein, MXR (ABCG2). Biochim. Biophys. Acta 1512:171–182 (2001).

    Article  PubMed  CAS  Google Scholar 

  23. R. W. Robey, K. Steadman, O. Polgar, and S. E. Bates. ABCG2-mediated transport of photosensitizers: Potential impact on photodynamic therapy. Cancer Biol. Ther. 4:187–194 (2005).

    Article  PubMed  CAS  Google Scholar 

  24. H. Minderman, A. Suvannasankha, K. L. O’Loughlin, G. L. Scheffer, R. J. Scheper, R. W. Robey, and M. R. Maer. Flow cytometric analysis of breast cancer resistance protein expression and function. Cytometry 48:59–65 (2002).

    Article  PubMed  CAS  Google Scholar 

  25. T. Litman, M. Brangi, E. Hudson, P. Fetsch, A. Abati, D. D. Ross, K. Miyake, J. H. Resau, and S. E. Bates. The multidrug-resistant phenotype associated with overexpression of the new ABC half-transporter, MXR (ABCG2). J. Cell Sci. 113:2011–2021 (2000).

    PubMed  CAS  Google Scholar 

  26. A. Gupta, Y. Zhang, J. D. Unadkat, and Q. Mao. HIV protease inhibitors are inhibitors but not substrates of the human breast cancer resistance protein (BCRP/ABCG2). J. Pharmacol. Exp. Ther. 310:334–341 (2004).

    Article  PubMed  CAS  Google Scholar 

  27. Y. Honjo, C. A. Hrycyna, Q. W. Yan, W. Y. Medina-Perez, R. W. Robey, A. van de Laar, T. Litman, M. Dean, and S. E. Bates. Acquired mutations in the MXR/BCRP/ABCP gene alter substrate specificity in MXR/BCRP/ABCP-overexpressing cells. Cancer Res. 61:6635–6639 (2001).

    PubMed  CAS  Google Scholar 

  28. A. Regina, A. Koman, M. Piciotti, B. El Hafny, M. S. Center, R. Bergmann, P.-O. Couraud, and F. Roux. Mrp1 multidrug resistance-associated protein and P-glycoprotein expression in rat brain microvessel endothelial cells. J. Neurochem. 71:705–715 (1998).

    Article  PubMed  CAS  Google Scholar 

  29. S. Dallas, L. Schlichter, and R. Bendayan. Multidrug resistance protein (MRP) 4- and MRP 5-mediated efflux of 9-(2-phosphonylmethoxyethyl)adenine by microglia. J. Pharmacol. Exp. Ther. 309:1221–1229 (2004).

    Article  PubMed  CAS  Google Scholar 

  30. A. van Loevezijn, J. D. Allen, A. H. Schinkel, and G. J. Koomen. Inhibition of BCRP-mediated drug efflux by fumitremorgin-type indolyl diketopiperazines. Bioorg. Med. Chem. Lett. 11:29–32 (2001).

    Article  PubMed  Google Scholar 

  31. R. W. Robey, K. Steadman, O. Polgar, K. Morisaki, M. Blayney, P. Mistry, and S. E. Bates. Pheophorbide a is a specific probe for ABCG2 function and inhibition. Cancer Res. 64:1242–1246 (2004).

    Article  PubMed  CAS  Google Scholar 

  32. J. W. Jonker, M. Buitelaar, E. Wagenaar, M. A. Van Der Valk, G. L. Scheffer, R. J. Scheper, T. Plosch, F. Kuiper, R. P. J Oude Elferink, H. Rosing, J. H. Beijnen, and A. H. Schinkel. The breast cancer resistance protein protects against a major chlorophyll-derived dietary phototoxin and protoporphyria. Proc. Natl. Acad. Sci. USA 99:15649–15654 (2002).

    Article  PubMed  CAS  Google Scholar 

  33. E. Aronica, J. A. Gorter, S. Redeker, E. A. van Vliet, M. Ramkema, G. L. Scheffer, R. J. Scheper, P. Van Der Valk, S. Leenstra, J. C. Baayen, W. G. M. Spliet, and D. Troost. Localization of breast cancer resistance protein (BCRP) in microvessel endothelium of human control and epileptic brain. Epilepsia 46:849–857 (2005).

    Article  PubMed  CAS  Google Scholar 

  34. E. Rocchi, A. Khodjakov, E. L. Volk, C. H. Yang, T. Litman, S. E. Bates, and E. Schneider. The product of the ABC half-transporter gene ABCG2 (BCRP/MXR/ABCP) is expressed in the plasma membrane. Biochem. Biophys. Res. Commun. 271:42–46 (2000).

    Article  PubMed  CAS  Google Scholar 

  35. X. Decleves, A. Regina, J. L. Laplanche, F. Roux, B. Boval, J. M. Launay, and J. M. Scherrmann. Functional expression of P-glycoprotein and multidrug resistance-associated protein (Mrp1) in primary cultures of rat astrocytes. J. Neurosci. Res. 60:594–601 (2000).

    Article  PubMed  CAS  Google Scholar 

  36. S. Dallas, X. Zhu, S. Baruchel, L. Schlichter, and R. Bendayan. Functional expression of the multidrug resistance protein 1 in microglia. J. Pharmacol. Exp. Ther. 307:282–290 (2003).

    Article  PubMed  CAS  Google Scholar 

  37. C. Ozvegy, A. Varadi, and B. Sarkadi. Characterization of drug transport, ATP hydrolysis, and nucleotide trapping by the human ABCG2 multidrug transporter. modulation of substrate specificity by a point mutation. J. Biol. Chem. 277:47980–47990 (2002).

    Article  PubMed  CAS  Google Scholar 

  38. B. Bauer, A. M. Hartz, G. Fricker, and D. S. Miller. Pregnane X receptor up-regulation of P-glycoprotein expression and transport function at the blood–brain barrier. Mol. Pharmacol. 66:413–419 (2004).

    PubMed  CAS  Google Scholar 

  39. R. Bendayan, P. T. Ronaldson, D. Gingras, and M. Bendayan. In Situ localization of P-glycoprotein (ABCB1) in human and rat brain. J. Histochem. and Cytochem. 54:1159–1167 (2006).

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work is supported by grants to Reina Bendayan from the Canadian Institutes of Health Research (MOP-56976) and the Ontario HIV Treatment Network (OHTN). Gloria Lee is a recipient of an OHTN Studentship Award. Alexander Prat holds the Donald Paty Career Award from the Multiple Sclerosis Society of Canada as well as “Chercheur Boursier Junior” from the Fonds de la Recherche en Sante du Quebec. Karolina Wosik is a recipient of a fellowship from the Multiple Sclerosis Society of Canada. We would like to thank Dr. J. Henderson for the help and advice with the undertaking of fluorescence microscopy studies and Drs S. Bates and F. Roux for providing the MCF-7 and RBE4 cell culture systems, respectively.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Reina Bendayan.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lee, G., Babakhanian, K., Ramaswamy, M. et al. Expression of the ATP-binding Cassette Membrane Transporter, ABCG2, in Human and Rodent Brain Microvessel Endothelial and Glial Cell Culture Systems. Pharm Res 24, 1262–1274 (2007). https://doi.org/10.1007/s11095-007-9244-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11095-007-9244-1

Key words

Navigation