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Research ArticleCHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY

Topotecan Triggers Apoptosis in p53-Deficient Cells by Forcing Degradation of XIAP and Survivin Thereby Activating Caspase-3-Mediated Bid Cleavage

Maja T. Tomicic, Markus Christmann and Bernd Kaina
Journal of Pharmacology and Experimental Therapeutics January 2010, 332 (1) 316-325; DOI: https://doi.org/10.1124/jpet.109.159962
Maja T. Tomicic
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Markus Christmann
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Bernd Kaina
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Abstract

The topoisomerase I inhibitor topotecan (TPT) is used in the therapy of different tumors including high-grade gliomas. We previously showed that TPT-induced apoptosis depends on p53 with p53 wild-type (wt) cells being more resistant because of p53-controlled degradation of topoisomerase I. Here, we show that p53-deficient (p53−/−) fibroblasts undergo excessive mitochondrial apoptosis featuring H2AX phosphorylation, Bcl-xL decline, cytochrome c release, caspase-9/-3/-2 activation, and cleavage of Bid. In wt and apaf-1−/− cells, caspase-2 did not become activated and Bid was not cleaved. In addition, p53−/− cells cotreated with TPT and caspase-3 inhibitor showed neither caspase-2 activation nor Bid cleavage, implying that caspase-2 is processed downstream of the apoptosome by caspase-3. Although processing of caspase-9/-3 was similar in wt and p53−/− cells, only p53−/− cells displayed active caspase-3. This was due to the proteasomal degradation of X-chromosome-linked inhibitor of apoptosis (XIAP) and survivin that inhibits caspase-3 activity. Accordingly, TPT-induced apoptosis in wt cells was increased after XIAP/survivin knockdown. Silencing of Bid led to reduction of TPT-triggered apoptosis. Data obtained with mouse fibroblasts could be extended to human glioma cells. In U87MG (p53wt) cells cotreated with TPT and pifithrin-α, or transfected with p53-siRNA, caspase-2 and Bid were significantly cleaved and XIAP/survivin was degraded. Furthermore, the knockdown of XIAP and survivin led to increased TPT-triggered apoptosis. Overall, the data show that p53-deficient/depleted cells are hypersensitive to TPT because they down-regulate XIAP and survivin, and thus amplify the intrinsic apoptotic pathway via caspase-3-mediated Bid cleavage. Therefore, in gliomas harboring wild-type p53, TPT-based therapy might be improved by targeted down-regulation of XIAP and survivin.

Footnotes

  • This work was supported by the German Research Foundation [Grants DFG KA724, DFG CH665/2-1].

  • Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

    doi:10.1124/jpet.109.159962

  • ↵Embedded Image The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.

  • ABBREVIATIONS:

    TPT
    topotecan
    IAP
    inhibitor of apoptosis protein
    DMEM
    Dulbecco's minimal essential medium
    DSB
    double-strand break
    FBS
    fetal bovine serum
    mAb
    monoclonal antibody
    MEF
    mouse embryonic fibroblast
    pAb
    polyclonal antibody
    PIDD
    p53-induced protein with a death domain
    PI
    propidium iodide
    Pth
    pifithrin-α
    siRNA
    small interfering RNA
    topoI
    topoisomerase I
    XIAP
    X-chromosome linked inhibitor of apoptosis
    zVAD-fmk
    benzyloxy-carbonyl-Val-Ala-Asp-fluoromethylketone
    zDEVD-fmk
    benzyloxy-carbonyl-Asp-Glu-Val-Asp-fluoromethylketone
    zLEHD-fmk
    benzyloxy-carbonyl-Leu-Glu-His-Asp-fluoromethylketone.

    • Received August 5, 2009.
    • Accepted October 6, 2009.
  • © 2010 by The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics: 332 (1)
Journal of Pharmacology and Experimental Therapeutics
Vol. 332, Issue 1
1 Jan 2010
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Research ArticleCHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY

Topotecan Triggers Apoptosis in p53-Deficient Cells by Forcing Degradation of XIAP and Survivin Thereby Activating Caspase-3-Mediated Bid Cleavage

Maja T. Tomicic, Markus Christmann and Bernd Kaina
Journal of Pharmacology and Experimental Therapeutics January 1, 2010, 332 (1) 316-325; DOI: https://doi.org/10.1124/jpet.109.159962

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Research ArticleCHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY

Topotecan Triggers Apoptosis in p53-Deficient Cells by Forcing Degradation of XIAP and Survivin Thereby Activating Caspase-3-Mediated Bid Cleavage

Maja T. Tomicic, Markus Christmann and Bernd Kaina
Journal of Pharmacology and Experimental Therapeutics January 1, 2010, 332 (1) 316-325; DOI: https://doi.org/10.1124/jpet.109.159962
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