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Cancer Therapy: Preclinical

The Novel Poly(ADP-Ribose) Polymerase Inhibitor, AG14361, Sensitizes Cells to Topoisomerase I Poisons by Increasing the Persistence of DNA Strand Breaks

Lisa M. Smith, Elaine Willmore, Caroline A. Austin and Nicola J. Curtin
Lisa M. Smith
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Elaine Willmore
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Caroline A. Austin
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Nicola J. Curtin
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DOI: 10.1158/1078-0432.CCR-05-1224 Published December 2005
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  • Fig. 1.
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    Fig. 1.

    Effect of AG14361 on topo I poison-induced growth inhibition and cytotoxicity in PARP-1+/+, PARP-1−/−, and human leukemia cell lines. A, camptothecin-induced growth inhibition in exponentially growing K562 cells in the presence (□) or absence (▪) of 0.4 μmol/L AG14361. Cells were exposed to drugs for 16 hours followed by 5-day growth in drug-free medium or medium containing 0.4 μmol/L AG14361. Cell growth was measured by cell counting and expressed as a percentage of the relevant DMSO or 0.4 μmol/L AG14361 alone control. Points, mean of three independent experiments; bars, SE. B, cytotoxicity in exponentially growing K562 cells exposed to camptothecin in the presence (□) or absence (▪) of AG14361 for 16 hours. Cytotoxicity was measured by colony formation in 0.125% agarose and expressed as a percentage of the relevant DMSO or 0.4 μmol/L AG14361 alone control. Points, mean of four independent experiments; bars, SE.

  • Fig. 2.
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    Fig. 2.

    Effect of AG14361 on camptothecin-stabilized cleavable complex induction and removal. A, K562 cells were treated with 10 μmol/L camptothecin alone (white column) or camptothecin + 0.4 μmol/L AG14361 (black column) for 30 minutes. Gray column, untreated control. Levels of cleavable complexes were measured using the trapped in agarose DNA immunostaining assay. Columns, mean FITC-associated fluorescence values from seven independent experiments; bars, SE. B, K562 cells were treated with 10 μmol/L camptothecin in the presence (black column) or absence (white column) of 0.4 μmol/L AG14361 for 30 minutes and then allowed to reverse for 10 minutes in drug-free medium or medium containing 0.4 μmol/L AG14361. Hatched column, a control sample treated with camptothecin and not allowed to repair; gray column, DMSO control. Cleavable complexes were measured by SDS precipitation. Columns, mean 14C-associated radioactivity (DPM) from three independent experiments; bars, SE.

  • Fig. 3.
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    Fig. 3.

    Repair of camptothecin-induced DNA single-strand breaks in K562 cells in the presence or absence of AG14361. K562 cells were exposed to 30 nmol/L camptothecin for 30 minutes followed by repair in drug-free medium (white columns) or medium containing 0.4 μmol/L AG14361 (black columns) for 0, 10, or 20 minutes. DNA strand breaks were measured by alkaline elution. Relative elution was calculated by comparison with DMSO or 0.4 μmol/L AG14361 alone control as appropriate. Columns, mean of three independent experiments; bars, SE.

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    Fig. 4.

    Proposed role of PARP-1 in the repair of topo I poison-mediated DNA damage. Topo I poisons, exemplified by camptothecin (CPT), lead to the formation of a cleavable complex in which topo I is covalently attached to the 3′ phosphate. PARP-1 binds the resultant free 5′ OH DNA end, stimulating its automodification activity and recruiting XRCC1. XRCC1 in turn recruits tyrosyl DNA phosphodiesterase-1 (TDP-1), which removes the topo I from the DNA leaving a 3′ phosphate terminus. Before removal, topo I may be partially degraded by Cullin 3 or SUMO-dependent mechanisms, leaving a peptide fragment that is removed by tyrosyl DNA phosphodiesterase-1. XRCC1 also recruits polynucleotide kinase (PNK), which converts the DNA ends to 3′ OH and 5′ phosphate. Finally, XRCCI acts as a scaffold for DNA polymerase β (Pol β) and DNA ligase III (Lig III) to fill and ligate the gap, completing the repair of the camptothecin-induced DNA damage.

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  • Table 1.

    Effect of AG14361 on topotecan-mediated growth inhibition in PARP-1+/+ and PARP-1−/− cells

    GI50 (nmol/L)
    PF50
    TopotecanTopotecan + 0.4 μmol/L AG14361
    PARP-1+/+65.0 ± 7.019.5 ± 4.3*3.4 ± 0.4
    PARP-1−/−20.8 ± 2.0*†15.6 ± 0.6‡1.4 ± 0.1
    Sensitivity ratio3 ± 0.21.3 ± 0.5
    • NOTE: Growth of cells treated with topotecan in the presence or absence of AG14361 for 5 days continuously determined by sulforhodamine B assay and expressed as a percentage of the relevant DMSO or 0.4 μmol/L AG14361-treated control. Mean ± SE GI50 of three independent experiments. PF50 is the potentiation factor, the ratio of GI50 topotecan alone to GI50 topotecan plus AG14361 calculated from GI50 of individual experiments.

    • ↵* P < 0.001, Student's paired t test compared with PARP-1+/+ treated with camptothecin alone.

    • ↵† Not significantly different from PARP-1+/+ treated with topotecan plus AG14361.

    • ↵‡ P < 0.05, Student's paired t test compared with PARP-1-/- treated with camptothecin alone.

  • Table 2.

    Effect of AG14361 on formation of camptothecin-induced protein-DNA complexes

    Camptothecin (μmol/L)Protein-DNA complexes (% control)
    Camptothecin aloneCamptothecin + AG14361
    30-min exposure
        1798 ± 351934 ± 463
        101,202 ± 6201,311 ± 621
    16-h exposure
        1206 ± 46194 ± 29
        10457 ± 94516 ± 62
    • NOTE: K562 cells were exposed to 1 or 10 μmol/L camptothecin in the presence or absence of 0.4 μmol/L AG14361 for 30 minutes or 16 hours. Levels of cleavable complexes were measured by SDS precipitation and expressed as a percent of the DMSO or 0.4 μmol/L AG14361-treated control as appropriate. Mean ± SE of three independent experiments.

  • Table 3.

    Effect of AG14361 on camptothecin-induced DNA strand breaks after 16-hour exposure

    Camptothecin (nmol/L)DNA single-strand breaks (relative elution)
    Fold increase in strand breaks
    Camptothecin aloneCamptothecin + 0.4 μmol/L AG14361
    30.021 ± 0.020.08 ± 0.052.4 ± 0.7
    100.12 ± 0.050.15 ± 0.041.6 ± 0.5
    150.17 ± 0.060.23 ± 0.021.6 ± 0.4
    300.20 ± 0.040.26 ± 0.04*1.3 ± 0.09
    400.32 ± 0.060.37 ± 0.021.2 ± 0.17
    1000.32 ± 0.080.35 ± 0.041.2 ± 0.14
    • NOTE: K562 cells were exposed to camptothecin in the presence or absence of 0.4 μmol/L AG14361 for 16 hours before determination of DNA single-strand breaks by alkaline elution. Relative elution was calculated by comparison with DMSO-treated or 0.4 μmol/L AG14361 alone control as appropriate. Mean ± SE for three independent experiments. Fold increase was calculated from mean ± SE relative elution values for individual experiments.

    • ↵* P < 0.005, paired t test compared with camptothecin alone.

  • Table 4.

    Effect of AG14361 on repair of camptothecin-induced DNA strand breaks in K562 cells

    Time after camptothecin removal% Unrepaired DNA
    Fold increase in strand breaks
    CamptothecinCamptothecin + 0.4 μmol/L AG14361
    Camptothecin (30 nmol/L)
        0100100
        10 min20 ± 5.450 ± 1.7*2.5 ± 0.2
        20 min16 ± 2.822 ± 4.91.4 ± 0.4
    Camptothecin (300 nmol/L)
        0100100
        1 h15 ± 5.544 ± 5.1†1.9 ± 0.1
        16 h10 ± 9.639 ± 6.2‡2.5 ± 0.85
    • NOTE: K562 cells were exposed to 30 or 300 nmol/L camptothecin for 30 minutes followed by repair in drug-free (control) medium or medium containing 0.4 μmol/L AG14361 for the times indicated. DNA strand breaks were measured by alkaline elution. Relative elution was calculated by comparison with DMSO or 0.4 μmol/L AG14361 alone control as appropriate. Mean ± SE of three independent experiments.

    • ↵* P < 0.005, Student's paired t test compared with camptothecin alone.

    • ↵† P = 0.07, Student's paired t test compared with camptothecin alone.

    • ↵‡ P = 0.02, Student's paired t test compared with camptothecin alone.

  • Table 5.

    Effect of AG41631 on survival of AA8, EM9, V3, and irs1SF cell lines following a 16-hour exposure to camptothecin

    Cell lineLC50 (nmol/L)
    CamptothecinCamptothecin + AG14361
    AA825.0 ± 3.512.2 ± 2.4*
    EM95.6 ± 1.24.0 ± 1.1†
    V316.1 ± 4.010.2 ± 3.6‡
    irs1SF2.7 ± 0.71.7 ± 0.4§
    1.4 ± 0.4∥
    • NOTE: Cells were treated with camptothecin ± 0.4 μmol/L AG14361 and survival was determined using a clonogenic assay. Mean ± SE LC50 of at least four independent experiments. Mean ± SE PF50 values calculated using LC50 of individual experiments.

    • ↵* P < 0.01, compared with camptothecin alone, Student's paired t test.

    • ↵† Not significantly different from camptothecin alone.

    • ↵‡ P < 0.05, compared with camptothecin alone, Student's paired t test.

    • ↵§ AG14361 (15 nmol/L) was used.

    • ↵∥ AG14361 (70 nmol/L) was used.

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Clinical Cancer Research: 11 (23)
December 2005
Volume 11, Issue 23
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The Novel Poly(ADP-Ribose) Polymerase Inhibitor, AG14361, Sensitizes Cells to Topoisomerase I Poisons by Increasing the Persistence of DNA Strand Breaks
Lisa M. Smith, Elaine Willmore, Caroline A. Austin and Nicola J. Curtin
Clin Cancer Res December 1 2005 (11) (23) 8449-8457; DOI: 10.1158/1078-0432.CCR-05-1224

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The Novel Poly(ADP-Ribose) Polymerase Inhibitor, AG14361, Sensitizes Cells to Topoisomerase I Poisons by Increasing the Persistence of DNA Strand Breaks
Lisa M. Smith, Elaine Willmore, Caroline A. Austin and Nicola J. Curtin
Clin Cancer Res December 1 2005 (11) (23) 8449-8457; DOI: 10.1158/1078-0432.CCR-05-1224
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