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Doxorubicin induces the DNA damage response in cultured human mesenchymal stem cells

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Abstract

Anthracyclines, including doxorubicin, are widely used in the treatment of leukemia. While the effects of doxorubicin on hematopoietic cells have been characterized, less is known about the response of human mesenchymal stem cells (hMSCs) in the bone marrow stroma to anthracyclines. We characterized the effect of doxorubicin on key DNA damage responses in hMSCs, and compared doxorubicin sensitivity and DNA damage response activation between isolated hMSCs and the chronic myelogenous leukemia cell line, K562. Phosphorylation of H2AX, Chk1, and RPA2 was more strongly activated in K562 cells than in hMSCs, at equivalent doses of doxorubicin. hMSCs were relatively resistant to doxorubicin such that, following exposure to 15 μM doxorubicin, the level of cleaved caspase-3 detected by western blotting was lower in hMSCs compared to K562 cells. Flow cytometric analysis of cell cycle progression demonstrated that exposure to doxorubicin induced G2/M phase arrest in hMSCs, while 48 h after exposure, 15.6 % of cells were apoptotic, as determined from the percentage of cells having sub-G1 DNA content. We also show that the doxorubicin sensitivity of hMSCs isolated from a healthy donor was comparable to that of hMSCs isolated from a chronic lymphocytic leukemia patient. Overall, our results demonstrate that high doses of doxorubicin induce the DNA damage response in hMSCs, and that cultured hMSCs are relatively resistant to doxorubicin.

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Abbreviations

hMSC:

Human mesenchymal stem cells

DDR:

DNA damage response

ATM:

Ataxia telangiectasia mutated

ATR:

ATM- and Rad3-related

DNA-PK:

DNA-dependent protein kinase

Chk1:

Checkpoint kinase 1

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Acknowledgments

This research was supported by grants from the Health Research Board (HRB grant number RP-2008-217) and from the Children’s Leukaemia Research Project. The Regenerative Medicine Institute, NUI Galway, is supported by funding from Science Foundation Ireland. AMP was supported in part by Galway County Council, and by the Thomas Crawford Hayes Fund, NUI Galway. We are grateful to Dr. Mark R.E. Coyne for providing patient-derived hMSCs, and to Dr. Eva Szegezdi for providing K562 cells.

Conflict of interest

The author(s) declare that they have no competing interests.

Author information

Author notes

  1. Séverine Cruet-Hennequart

    Present address: Microenvironment and Pathology Laboratory (MILPAT, EA 4652), Faculté de Médecine, Niveau 3, Avenue de la Côte de Nacre, 14032, Caen cedex, France

  2. Áine M. Prendergast

    Present address: Heidelberg Institute for Stem Cell Technologies and Experimental Medicine (HI-STEM) GmbH, DKFZ, Heidelberg, Germany

Authors and Affiliations

  1. DNA Damage Response Laboratory, Centre for Chromosome Biology and School of Natural Sciences, National University of Ireland Galway, Galway, Ireland

    Séverine Cruet-Hennequart, Áine M. Prendergast & Michael P. Carty

  2. Regenerative Medicine Institute, National University of Ireland Galway, Galway, Ireland

    Georgina Shaw & Frank P. Barry

Authors
  1. Séverine Cruet-Hennequart
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  2. Áine M. Prendergast
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  3. Georgina Shaw
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  4. Frank P. Barry
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  5. Michael P. Carty
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Corresponding author

Correspondence to Michael P. Carty.

Electronic supplementary material

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12185_2012_1196_MOESM1_ESM.pdf

Supplementary Figure 1: DNA damage responses and apoptosis induction in K562 cells treated with 1 μM doxorubicin. A. K562 cells were treated with 1 μM doxorubicin for the indicated times. DNA damage response proteins were analyzed by western blotting, as described in the legend to Fig. 1, and in Materials and Methods. B. Flow cytometry histograms from a representative experiment, showing the induction of sub-G1 cells, 48 h after mock-treatment or treatment of K562 cells with 1 μM doxorubicin. C. The percentage of sub-G1 K562 cells was determined following propidium iodide staining and flow cytometry, 48 h after treatment. Data represent the mean of two independent experiments; error bars represent one standard deviation (PDF 125 kb)

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Cruet-Hennequart, S., Prendergast, Á.M., Shaw, G. et al. Doxorubicin induces the DNA damage response in cultured human mesenchymal stem cells. Int J Hematol 96, 649–656 (2012). https://doi.org/10.1007/s12185-012-1196-5

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  • DOI: https://doi.org/10.1007/s12185-012-1196-5

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