Archives of Toxicology

, Volume 92, Issue 6, pp 2119–2135 | Cite as

Loss of Wilms tumor 1 protein is a marker for apoptosis in response to replicative stress in leukemic cells

  • Miriam Pons
  • Claudia M. Reichardt
  • Dorle Hennig
  • Abinaya Nathan
  • Nicole Kiweler
  • Carol Stocking
  • Christian Wichmann
  • Markus Christmann
  • Falk Butter
  • Sigrid Reichardt
  • Günter Schneider
  • Thorsten Heinzel
  • Christoph Englert
  • Jörg Hartkamp
  • Oliver H. KrämerEmail author
  • Nisintha Mahendrarajah
Genotoxicity and Carcinogenicity


A remaining expression of the transcription factor Wilms tumor 1 (WT1) after cytotoxic chemotherapy indicates remaining leukemic clones in patients. We determined the regulation and relevance of WT1 in leukemic cells exposed to replicative stress and DNA damage. To induce these conditions, we used the clinically relevant chemotherapeutics hydroxyurea and doxorubicin. We additionally treated cells with the pro-apoptotic kinase inhibitor staurosporine. Our data show that these agents promote apoptosis to a variable extent in a panel of 12 leukemic cell lines and that caspases cleave WT1 during apoptosis. A chemical inhibition of caspases as well as an overexpression of mitochondrial, anti-apoptotic BCL2 family proteins significantly reduces the processing of WT1 and cell death in hydroxyurea-sensitive acute promyelocytic leukemia cells. Although the reduction of WT1 correlates with the pharmacological efficiency of chemotherapeutics in various leukemic cells, the elimination of WT1 by different strategies of RNA interference (RNAi) does not lead to changes in the cell cycle of chronic myeloid leukemia K562 cells. RNAi against WT1 does also not increase the extent of apoptosis and the accumulation of γH2AX in K562 cells exposed to hydroxyurea. Likewise, a targeted genetic depletion of WT1 in primary oviduct cells does not increase the levels of γH2AX. Our findings position WT1 as a downstream target of the apoptotic process that occurs in response to cytotoxic forms of replicative stress and DNA damage.


Apoptosis DNA damage Caspase Hydroxyurea Leukemia Replicative stress WT1 



We thank Christina Brachetti and Birgit Rasenberger (Department of Toxicology, UM Mainz, Germany), and Christiane Becker (UKE Aachen, Germany) for excellent technical support, and Richard Moriggl, Medical University Vienna, for BCL2/BCL-XL expression constructs. Gesine Bug (University Clinic Frankfurt/Main, Germany) and all members of the Department of Toxicology, UM Mainz were excellent discussion partners. Grant support: German Cancer Aid (#110909 and #110125 to OHK, and #109528 to CS/CE), Wilhelm Sander Foundation (#2010.078.2 to OHK), and Deutsche Forschungsgemeinschaft (#KR2291/5-1 and KR2291/7-1 to OHK).

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Supplementary material

204_2018_2202_MOESM1_ESM.tif (26.7 mb)
Fig. S1 Cell cycle profiles of leukemic cells exposed to replicative stress. aRepresentative flow cytometry profiles of PI-stained, fixed NB4 and K562 cells that were treated with 0.5 mM HU for 24 h. bHEL and MV4-11 cells were treated with 1 mM HU and BV-173 cells with 0.5 mM HU for 24 h. Immunoblots show WT1 expression; β-Actin, HSP90 and LDH, loading controls. Percentage of subG1 fractions in the cell cultures are stated below. cKCL-22 and KYO-01 cells were treated with 0.5 mM HU for 24 h. Immunoblots show expression of WT1; HSP90, loading control. dKCL-22 and KYO-01 cells treated as described were analyzed for cell cycle distributions via flow cytometry; n=3±SD; two-way ANOVA; Bonferroni’s multiple comparisons test; *p<0.5, ***p<0.001, ****p<0.0001 (TIF 27359 KB)
204_2018_2202_MOESM2_ESM.tif (26.1 mb)
Fig. S2 BAX and BAK expression after replicative stress induced by hydroxyurea. Cells were treated with 0.5 mM HU for 12 h. BAX and BAK levels were analyzed by immunoblot; α-Tubulin, loading control; Densitometric evaluation of BAK and BAX normalized to α-Tubulin; n=3±SD (BAK); n=6±SD (BAX/NB4); n=8±SD (BAX/K562) (TIF 26719 KB)
204_2018_2202_MOESM3_ESM.xlsx (66 kb)
Supplementary material 3 (XLSX 65 KB)
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Supplementary material 5 (XLSX 77 KB)
204_2018_2202_MOESM6_ESM.pdf (1.3 mb)
Supplementary material 6 (PDF 1357 KB)
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Supplementary material 7 (DOCX 18 KB)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Miriam Pons
    • 1
  • Claudia M. Reichardt
    • 2
  • Dorle Hennig
    • 3
    • 9
  • Abinaya Nathan
    • 2
  • Nicole Kiweler
    • 1
  • Carol Stocking
    • 4
  • Christian Wichmann
    • 5
  • Markus Christmann
    • 1
  • Falk Butter
    • 6
  • Sigrid Reichardt
    • 3
  • Günter Schneider
    • 7
  • Thorsten Heinzel
    • 3
  • Christoph Englert
    • 2
    • 3
  • Jörg Hartkamp
    • 8
  • Oliver H. Krämer
    • 1
    Email author
  • Nisintha Mahendrarajah
    • 1
  1. 1.Department of ToxicologyUniversity Medical CenterMainzGermany
  2. 2.Molecular GeneticsLeibniz Institute for Age Research, Fritz-Lipmann-InstituteJenaGermany
  3. 3.Department of Biochemistry, Institute of Biochemistry and Biophysics, Center for Molecular BiomedicineFriedrich-Schiller-University JenaJenaGermany
  4. 4.Heinrich Pette Institute, Leibniz Institute for Experimental VirologyHamburgGermany
  5. 5.Department of Transfusion Medicine, Cell Therapeutics and HemostaseologyLudwig-Maximilian University HospitalMunichGermany
  6. 6.Institute of Molecular Biology (IMB)MainzGermany
  7. 7.Klinik und Poliklinik für Innere Medizin IITechnical University of MunichMunichGermany
  8. 8.Institute of Biochemistry and Molecular Biology, Medical SchoolRWTH Aachen UniversityAachenGermany
  9. 9.Department of Molecular MedicineUniversity of Southern DenmarkOdenseDenmark

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