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Human concentrative nucleoside transporter 3 is a determinant of fludarabine transportability and cytotoxicity in human renal proximal tubule cell cultures

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Abstract

Purpose

Interpatient variability in renal elimination of fludarabine (9-β-D-arabinosyl-2-fluoroadenine) by renal human nucleoside transporters (hNTs) may contribute to unpredictable toxicities including rare nephrotoxicities. This study assessed relationships between hNT levels and fludarabine uptake and cytotoxicity in cultures of human renal proximal tubule cells (hRPTCs) that produce multiple transporter types.

Methods

hRPTC cultures were established from ten different individuals and their hNT characteristics were assessed by measuring RNA expression by TaqMan™ reverse transcriptase polymerase chain reaction, protein abundance by quantitative immunoblotting of cell surface protein preparations, and uptake by radiolabeled nucleoside uptake assays. Fludarabine cytotoxicity against hRPTC cultures was quantified using methoxyphenyl tetrazolium inner salt (MTS) assays.

Results

RNA, protein and activities for human equilibrative NT 1 (hENT1) and 2 (hENT2) and human concentrative NT 3 (hCNT3) were identified in cultures of hRPTCs from ten different individuals. Significant differences in hCNT3 activities were exhibited among hRPTC cultures and correlated positively with cell surface levels of hCNT3 protein, but did not correlate with hCNT3 mRNA levels.

Conclusions

Observed differences in hCNT3-mediated uptake activities, hNT-mediated fludarabine uptake activities, and fludarabine cytotoxicities correlated positively with each other, suggesting that hCNT3 is a primary determinant of fludarabine uptake and cytotoxicity in hRPTC cultures. Variations in hCNT3 abundance in renal proximal tubules, and hence nucleoside reabsorption, may explain interpatient variability in fludarabine’s pharmacokinetics and toxicities.

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Abbreviations

IC50 :

50% Inhibitory concentration

EC50 :

50% Cell killing concentration

CNT:

Concentrative nucleoside transporter

ENT:

Equilibrative nucleoside transporter

hCNT1:

Human concentrative nucleoside transporter 1

hCNT2:

Human concentrative nucleoside transporter 2

hCNT3:

Human concentrative nucleoside transporter 3

hENT1:

Human equilibrative nucleoside transporter 1

hENT2:

Human equilibrative nucleoside transporter 2

hENT3:

Human equilibrative nucleoside transporter 3

hENT4:

Human equilibrative nucleoside transporter 4

hNT:

Human nucleoside transporter

hRPTC:

Human renal proximal tubule cell

MTS:

Methoxyphenyl tetrazolium inner salt

NBMPR:

Nitrobenzylmercaptopurine ribonucleoside

NT:

Nucleoside transporter

PCR:

Polymerase chain reaction

RT-PCR:

Reverse transcriptase polymerase chain reaction

SNP:

Single nucleotide polymorphism

SDS:

Sodium dodecyl sulfate

SLC28:

Solute Carrier 28

SLC29:

Solute Carrier 29

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Author information

Authors and Affiliations

  1. Membrane Protein Research Group, University of Alberta, Edmonton, AB, Canada

    Adam N. Elwi, Vijaya L. Damaraju, James D. Young & Carol E. Cass

  2. Department of Biochemistry, University of Alberta, Edmonton, AB, Canada

    Carol E. Cass

  3. Department of Oncology, University of Alberta, Edmonton, AB, Canada

    Adam N. Elwi, Vijaya L. Damaraju, Michelle L. Kuzma, Michael B. Sawyer & Carol E. Cass

  4. Department of Physiology, University of Alberta, Edmonton, AB, Canada

    James D. Young

  5. Department of Experimental Oncology, Cross Cancer Institute, Edmonton, AB, Canada

    Adam N. Elwi, Vijaya L. Damaraju, Michelle L. Kuzma & Carol E. Cass

  6. Department of Medical Oncology, Cross Cancer Institute, Edmonton, AB, Canada

    Michael B. Sawyer

  7. Astbury Centre for Structural Molecular Biology Institute of Membrane and Systems Biology, University of Leeds, Leeds, UK

    Stephen A. Baldwin

  8. Department of Oncology, Cross Cancer Institute, 11560 University Avenue NW, Edmonton, AB, T6G 1Z2, Canada

    Carol E. Cass

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  1. Adam N. Elwi
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  2. Vijaya L. Damaraju
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  3. Michelle L. Kuzma
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  4. Stephen A. Baldwin
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  5. James D. Young
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  7. Carol E. Cass
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Corresponding author

Correspondence to Carol E. Cass.

Additional information

This research was funded by the Alberta Cancer Board Research Initiative Program and National Cancer Institute of Canada. C.E.C. is Canada Research Chair in Oncology. J.D.Y. is Heritage Scientist of the Alberta Heritage Foundation for Medical Research. M.B S. received an American Society of Clinical Oncology Career Development Award. A.N.E. is supported by an Alberta Cancer Board Legacy Graduate Award and a Studentship from the Translational Research Training in Cancer Program jointly supported by the Canadian Institutes of Health Research, National Cancer Institute of Canada and Alberta Cancer Foundation.

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Elwi, A.N., Damaraju, V.L., Kuzma, M.L. et al. Human concentrative nucleoside transporter 3 is a determinant of fludarabine transportability and cytotoxicity in human renal proximal tubule cell cultures. Cancer Chemother Pharmacol 63, 289–301 (2009). https://doi.org/10.1007/s00280-008-0739-1

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