Abstract
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.
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Acknowledgements
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).
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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)
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)
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Pons, M., Reichardt, C.M., Hennig, D. et al. Loss of Wilms tumor 1 protein is a marker for apoptosis in response to replicative stress in leukemic cells. Arch Toxicol 92, 2119–2135 (2018). https://doi.org/10.1007/s00204-018-2202-3
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DOI: https://doi.org/10.1007/s00204-018-2202-3