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Dynamic ABCG2 expression in human embryonic stem cells provides the basis for stress response

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Abstract

ABCG2 is a plasma membrane multidrug transporter with an established role in the cancer drug-resistance phenotype. This protein is expressed in a variety of tissues, including several types of stem cell. Although ABCG2 is not essential for life, knock-out mice were found to be hypersensitive to xenobiotics and had reduced levels of the side population of hematopoietic stem cells. Previously we have shown that ABCG2 is present in human embryonic stem cell (hESC) lines, with a heterogeneous expression pattern. In this study we examined this heterogeneity, and investigated whether it is related to stress responses in hESCs. We did not find any difference between expression of pluripotency markers in ABCG2-positive and negative hESCs; however, ABCG2-expressing cells had a higher growth rate after cell separation. We found that some harmful conditions (physical stress, drugs, and UV light exposure) are tolerated much better in the presence of ABCG2 protein. This property can be explained by the transporter function which eliminates potential toxic metabolites accumulated during stress conditions. In contrast, mild oxidative stress in hESCs caused rapid internalization of ABCG2, indicating that some environmental factors may induce removal of this transporter from the plasma membrane. On the basis of these results we suggest that a dynamic balance of ABCG2 expression at the population level has the advantage of enabling prompt response to changes in the cellular environment. Such actively maintained heterogeneity might be of evolutionary benefit in protecting special cell types, including pluripotent stem cells.

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Acknowledgments

The authors are grateful to Beáta Haraszti for excellent technical assistance and acknowledge useful suggestions of Katalin Kiss about immunocytochemical staining. The authors appreciate the gift of HUES9 cell line from Dr Douglas Melton, HHMI. This work was supported by grants from OTKA (NK83533), ETT (213-09, 211-09), ES2HEART (OM00203/2007), STEMKILL (OM00108/2008), KMOP-1.1.2-07/1-2008-0003, and TÁMOP-4.2.2-08/1-2008-0015. Tamás I. Orbán is a recipient of the János Bolyai Scholarship of the Hungarian Academy of Sciences.

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Authors and Affiliations

  1. Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis, University and National Blood Center, Diószegi u. 64., 1113, Budapest, Hungary

    Zsuzsa Erdei, Balázs Sarkadi, Kornélia Szebényi, Veronika Makó, Adrienn Péntek, Tamás I. Orbán & Ágota Apáti

  2. Institute of Molecular Pharmacology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary

    Balázs Sarkadi, Tamás I. Orbán & Ágota Apáti

  3. Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary

    Anna Brózik & György Várady

Authors
  1. Zsuzsa Erdei
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  2. Balázs Sarkadi
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  3. Anna Brózik
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  4. Kornélia Szebényi
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  5. György Várady
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  6. Veronika Makó
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  7. Adrienn Péntek
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  8. Tamás I. Orbán
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  9. Ágota Apáti
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Correspondence to Ágota Apáti.

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Special Issue: Structure, function, folding and assembly of membrane proteins—insight from biophysics.

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249_2012_838_MOESM1_ESM.tif

Suppl. Fig. 1 Effect of the ABCG2 inhibitor (Ko143) on cell proliferation. HUES9-eGFP cells without sorting were grown on MEF feeder cells in the presence or the absence of the ABCG2 inhibitor (Ko143, 5 μM). Cell proliferation was monitored based on eGFP fluorescence up to 7 days, data were obtained from three independent experiments (TIFF 1471 kb)

249_2012_838_MOESM2_ESM.tif

Suppl. Fig. 2 Effect of H 2 O 2 treatment on cell proliferation. HUES9-eGFP cells were cultured on Matrigel® coated surfaces and treated with different concentrations (0-1000 μM) of H2O2. Cell proliferation was monitored based on eGFP fluorescence at 24 h (TIFF 1308 kb)

249_2012_838_MOESM3_ESM.jpg

Suppl. Fig. 3 Effect of H 2 O 2 treatment on ABCG2 mRNA level. HUES9-eGFP cells were cultured on Matrigel® coated surfaces and treated with 150 μM H2O2 Samples were collected at 3, 6 and 24 h for analysis of ABCG2 mRNA, data were obtained from two independent experiments (JPEG 1961 kb)

249_2012_838_MOESM4_ESM.tif

Suppl. Fig. 4 Effect of H 2 O 2 on ABCG2 protein expression. HUES9 cells were cultured on Matrigel® coated chambers. Cells were treated with 150 μM H2O2 for 1, 2, 3 and 6 h respectively, fixed, permeabilized (for details see the “Materials and methods” section) and stained with the 5D3 antibody to visualize ABCG2 (green). Nuclei were counterstained with Hoechst33342 (blue). Stained samples were examined by an Olympus FV500-IX confocal laser scanning microscope. All measurements were carried out as four independent experiments, performed as triplicates (TIFF 18134 kb)

249_2012_838_MOESM5_ESM.tif

Suppl. Fig. 5 Localization of the Na/K ATPase after oxidative stress (H 2 O 2 treatment) in HUES9 cells. HUES9 cells were cultured on Matrigel® coated chambers. Cells were treated with 150 μM H2O2 for 3 h, fixed, permeabilized (for details see the “Materials and methods” section) and stained with the antibody against human Na/K ATPase (red) to visualize the localization of the protein. Stained samples were examined by an Olympus FV500-IX confocal laser scanning microscope (TIFF 1044 kb)

249_2012_838_MOESM6_ESM.tif

Suppl. Fig. 6 Localization of ABCG2 after oxidative stress (H 2 O 2 treatment) in HUES9 cells. HUES9 cells were cultured on Matrigel® coated chambers. Cells were treated with 150 μM H2O2 for 3 h, fixed, permeabilized (for details see the “Materials and methods” section) and stained with the 5D3 antibody to visualize ABCG2 (green). Representative markers of different intracellular compartments (red) were applied to define ABCG2-containing intracellular compartments, including polyclonal antibodies against LAMP-1 and Rab-5 proteins (labeling lysosomes and endosomes, respectively) as well as the WGA lectin recognizing intracellular membranes including the Golgi network. Nuclei were counterstained with Hoechst33342 (blue). Stained samples were examined by an Olympus FV500-IX confocal laser scanning microscope. Z-stack analyses were made along the violet (right panel) and the yellow (bottom panel) labeled line (TIFF 9069 kb)

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Erdei, Z., Sarkadi, B., Brózik, A. et al. Dynamic ABCG2 expression in human embryonic stem cells provides the basis for stress response. Eur Biophys J 42, 169–179 (2013). https://doi.org/10.1007/s00249-012-0838-0

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