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Single nucleotide polymorphisms modify the transporter activity of ABCG2

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Abstract

Single nucleotide polymorphism (SNP) analyses of the ABCG2 gene have revealed three nonsynonymous SNPs resulting in the amino acid changes at V12M, Q141K and D620N. To determine whether the SNPs have an effect on drug transport, human embryonic kidney cells (HEK-293) were stably transfected with full length ABCG2 coding wild-type or SNP variants of ABCG2. In 4-day cytotoxicity assays with mitoxantrone, topotecan, SN-38 or diflomotecan, cells transfected with wild-type R482 ABCG2 showed IC50 values up to 1.2-fold to 5-fold higher than cells expressing comparable levels of Q141K ABCG2, suggesting that the Q141K SNP affects drug transport. FTC-inhibitable mitoxantrone efflux normalized to ABCG2 surface expression as assayed by the anti-ABCG2 antibody 5D3 was significantly lower in cells transfected with Q141K ABCG2 than in those transfected with wild-type R482 ABCG2 (P=0.0048). Values for V12M and D620N ABCG2 were comparable to those for wild-type R482 ABCG2. The vanadate-sensitive ATPase activity of ABCG2 was assayed in Sf9 insect cells infected with wild-type or SNP variants of ABCG2. Basal ATPase activity in cells transfected with Q141K ABCG2 was 1.8-fold lower than in cells transfected with wild-type ABCG2, but was comparable among cells expressing wild-type, V12M or D620N ABCG2. Confocal studies of ABCG2 localization revealed higher intracellular staining in the Q141K transfectants than in cells transfected with wild-type or V12M ABCG2. Decreased transport of Hoechst 33342 was observed in Sf9 cells expressing V12M ABCG2; however, this was not true in HEK-293 cells expressing V12M ABCG2. These results suggest that the Q141K SNP affects the transport efficiency of ABCG2 and may result in altered pharmacokinetics or drug-resistance profiles in clinical oncology.

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Acknowledgements

We are very grateful to Gabriella Köblös for helping with the ATPase activity measurements and for Dr. Barry N. Elkind and Dr. László Homolya for help with the confocal studies. We also appreciate the technical assistance of Tim Nadjem.

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Authors and Affiliations

  1. Cancer Therapeutics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 9000 Rockville Pike, Bldg 10 Rm 12C103, Bethesda, MD, 20892, USA

    Kuniaki Morisaki, Robert W. Robey, Yasumasa Honjo, Orsolya Polgar, Kenneth Steadman & Susan E. Bates

  2. National Medical Center, Institute of Hematology and Immunology, Membrane Research Group, Hungarian Academy of Sciences, 1113, Budapest, Hungary

    Csilla Özvegy-Laczka & Balázs Sarkadi

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  1. Kuniaki Morisaki
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  2. Robert W. Robey
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  3. Csilla Özvegy-Laczka
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  4. Yasumasa Honjo
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  5. Orsolya Polgar
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  6. Kenneth Steadman
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  7. Balázs Sarkadi
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  8. Susan E. Bates
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Correspondence to Susan E. Bates.

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Csilla Özvegy-Laczka is a grantee of a postdoctoral fellowship (D 45957) of OTKA, Hungary, and the János Bolyai Scholarship of the Hungarian Academy of Sciences. Balázs Sarkadi is a recipient of a Howard Hughes International Scholarship. Susan E. Bates receives support from the Comissioned Corps of the USPHS

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Morisaki, K., Robey, R.W., Özvegy-Laczka, C. et al. Single nucleotide polymorphisms modify the transporter activity of ABCG2. Cancer Chemother Pharmacol 56, 161–172 (2005). https://doi.org/10.1007/s00280-004-0931-x

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