Article Figures & Data
Figures
- 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.
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.
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.
- 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.
Tables
- Table 1.
Effect of AG14361 on topotecan-mediated growth inhibition in PARP-1+/+ and PARP-1−/− cells
GI50 (nmol/L) PF50 Topotecan Topotecan + 0.4 μmol/L AG14361 PARP-1+/+ 65.0 ± 7.0 19.5 ± 4.3* 3.4 ± 0.4 PARP-1−/− 20.8 ± 2.0*† 15.6 ± 0.6‡ 1.4 ± 0.1 Sensitivity ratio 3 ± 0.2 1.3 ± 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 alone Camptothecin + AG14361 30-min exposure 1 798 ± 351 934 ± 463 10 1,202 ± 620 1,311 ± 621 16-h exposure 1 206 ± 46 194 ± 29 10 457 ± 94 516 ± 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 alone Camptothecin + 0.4 μmol/L AG14361 3 0.021 ± 0.02 0.08 ± 0.05 2.4 ± 0.7 10 0.12 ± 0.05 0.15 ± 0.04 1.6 ± 0.5 15 0.17 ± 0.06 0.23 ± 0.02 1.6 ± 0.4 30 0.20 ± 0.04 0.26 ± 0.04* 1.3 ± 0.09 40 0.32 ± 0.06 0.37 ± 0.02 1.2 ± 0.17 100 0.32 ± 0.08 0.35 ± 0.04 1.2 ± 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 Camptothecin Camptothecin + 0.4 μmol/L AG14361 Camptothecin (30 nmol/L) 0 100 100 10 min 20 ± 5.4 50 ± 1.7* 2.5 ± 0.2 20 min 16 ± 2.8 22 ± 4.9 1.4 ± 0.4 Camptothecin (300 nmol/L) 0 100 100 1 h 15 ± 5.5 44 ± 5.1† 1.9 ± 0.1 16 h 10 ± 9.6 39 ± 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 line LC50 (nmol/L) Camptothecin Camptothecin + AG14361 AA8 25.0 ± 3.5 12.2 ± 2.4* EM9 5.6 ± 1.2 4.0 ± 1.1† V3 16.1 ± 4.0 10.2 ± 3.6‡ irs1SF 2.7 ± 0.7 1.7 ± 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.
Volume 11, Issue 23
