Differential effects of poly(ADP-ribose) polymerase inhibition on DNA break repair in human cells are revealed with Epstein-Barr virus

Chromosome Stability Section, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 04/2012; 109(17):6590-5. DOI: 10.1073/pnas.1118078109
Source: PubMed


Poly(ADP-ribose) polymerase (PARP) inhibitors can generate synthetic lethality in cancer cells defective in homologous recombination. However, the mechanism(s) by which they affect DNA repair has not been established. Here we directly determined the effects of PARP inhibition and PARP1 depletion on the repair of ionizing radiation-induced single- and double-strand breaks (SSBs and DSBs) in human lymphoid cell lines. To do this, we developed an in vivo repair assay based on large endogenous Epstein-Barr virus (EBV) circular episomes. The EBV break assay provides the opportunity to assess quantitatively and simultaneously the induction and repair of SSBs and DSBs in human cells. Repair was efficient in G1 and G2 cells and was not dependent on functional p53. shRNA-mediated knockdown of PARP1 demonstrated that the PARP1 protein was not essential for SSB repair. Among 10 widely used PARP inhibitors, none affected DSB repair, although an inhibitor of DNA-dependent protein kinase was highly effective at reducing DSB repair. Only Olaparib and Iniparib, which are in clinical cancer therapy trials, as well as 4-AN inhibited SSB repair. However, a decrease in PARP1 expression reversed the ability of Iniparib to reduce SSB repair. Because Iniparib disrupts PARP1-DNA binding, the mechanism of inhibition does not appear to involve trapping PARP at SSBs.

Download full-text


Available from: Daniel Menendez,
  • Source
    • "Frequently, at high doses of irradiation, two such nicks are present in complementary DNA strands within one helical turn leading to DSBs (Milligan et al. 1995). There are about 10 SSBs for each DSB created by IR (Ma et al. 2012). IR breakage frequently leaves " dirty ends, " consisting of phosphoglycolates and terminal nucleotides, that cannot be ligated to " clean " ends consisting of a 5 0 phosphate and 3 0 -OH group, such as those created by endonucleases (Weinfeld and Soderlind 1991). "
    [Show abstract] [Hide abstract]
    ABSTRACT: DNA is subject to many endogenous and exogenous insults that impair DNA replication and proper chromosome segregation. DNA double-strand breaks (DSBs) are one of the most toxic of these lesions and must be repaired to preserve chromosomal integrity. Eukaryotes are equipped with several different, but related, repair mechanisms involving homologous recombination, including single-strand annealing, gene conversion, and break-induced replication. In this review, we highlight the chief sources of DSBs and crucial requirements for each of these repair processes, as well as the methods to identify and study intermediate steps in DSB repair by homologous recombination.
    Cold Spring Harbor perspectives in biology 08/2014; 6(9). DOI:10.1101/cshperspect.a016428 · 8.68 Impact Factor
  • Source
    • "We used two conditions to ensure that strand breaks were quantitated accurately: for PFGE, DNA was deproteinised at room temperature because extra strand breaks are created at higher temperatures [68], and hybridisation was carried out in dried gels because the transfer of large DNA fragments onto membranes [9], [10] is not quantitative [69]. In another study [70] published while this manuscript was in preparation, a significant amount of minichromosome DNA remained in the sample well of PFGE gels and was interpreted as nicked circles, but here little or no DNA remained in the wells and nicked circular DNA migrated slowly into the gel, possibly reflecting methodological differences. A Poisson distribution of strand breaks was assumed in [70], but is not consistent with our finding that only one double strand break is formed in minichromosome DNA in irradiated cells (Figure 1 and [43]); this assumption is not supported strongly by experimental evidence and does not take into account the variable conformations and microenvironments of chromatin in the nucleus. "
    [Show abstract] [Hide abstract]
    ABSTRACT: To obtain an overall picture of the repair of DNA single and double strand breaks in a defined region of chromatin in vivo, we studied their repair in a ∼170 kb circular minichromosome whose length and topology are analogous to those of the closed loops in genomic chromatin. The rate of repair of single strand breaks in cells irradiated with γ photons was quantitated by determining the sensitivity of the minichromosome DNA to nuclease S1, and that of double strand breaks by assaying the reformation of supercoiled DNA using pulsed field electrophoresis. Reformation of supercoiled DNA, which requires that all single strand breaks have been repaired, was not slowed detectably by the inhibitors of poly(ADP-ribose) polymerase-1 NU1025 or 1,5-IQD. Repair of double strand breaks was slowed by 20-30% when homologous recombination was supressed by KU55933, caffeine, or siRNA-mediated depletion of Rad51 but was completely arrested by the inhibitors of nonhomologous end-joining wortmannin or NU7441, responses interpreted as reflecting competition between these repair pathways similar to that seen in genomic DNA. The reformation of supercoiled DNA was unaffected when topoisomerases I or II, whose participation in repair of strand breaks has been controversial, were inhibited by the catalytic inhibitors ICRF-193 or F11782. Modeling of the kinetics of repair provided rate constants and showed that repair of single strand breaks in minichromosome DNA proceeded independently of repair of double strand breaks. The simplicity of quantitating strand breaks in this minichromosome provides a usefull system for testing the efficiency of new inhibitors of their repair, and since the sequence and structural features of its DNA and its transcription pattern have been studied extensively it offers a good model for examining other aspects of DNA breakage and repair.
    PLoS ONE 01/2013; 8(1):e52966. DOI:10.1371/journal.pone.0052966 · 3.23 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Alterations in checkpoint and DNA repair pathways may provide adaptive mechanisms contributing to acquired drug resistance. Here, we investigated the levels of proteins mediating DNA damage signaling and -repair in RPMI8226 multiple myeloma cells and its Melphalan-resistant derivative 8226-LR5. We observed markedly reduced steady-state levels of DNA glycosylases UNG2, NEIL1 and MPG in the resistant cells and cross-resistance to agents inducing their respective DNA base lesions. Conversely, repair of alkali-labile sites was apparently enhanced in the resistant cells, as substantiated by alkaline comet assay, autoribosylation of PARP-1, and increased sensitivity to PARP-1 inhibition by 4-AN or KU58684. Reduced base-excision and enhanced single-strand break repair would both contribute to the observed reduction in genomic alkali-labile sites, which could jeopardize productive processing of the more cytotoxic Melphalan-induced interstrand DNA crosslinks (ICLs). Furthermore, we found a marked upregulation of proteins in the non-homologous end-joining (NHEJ) pathway of double-strand break (DSB) repair, likely contributing to the observed increase in DSB repair kinetics in the resistant cells. Finally, we observed apparent upregulation of ATR-signaling and downregulation of ATM-signaling in the resistant cells. This was accompanied by markedly increased sensitivity towards Melphalan in the presence of ATR-, DNA-PK, or CHK1/2 inhibitors whereas no sensitizing effect was observed subsequent to ATM inhibition, suggesting that replication blocking lesions are primary triggers of the DNA damage response in the Melphalan resistant cells. In conclusion, Melphalan resistance is apparently contributed by modulation of the DNA damage response at multiple levels, including downregulation of specific repair pathways to avoid repair intermediates that could impair efficient processing of cytotoxic ICLs and ICL-induced DSBs. This study has revealed several novel candidate biomarkers for Melphalan sensitivity that will be included in targeted quantitation studies in larger patient cohorts to validate their value in prognosis as well as targets for replacement- or adjuvant therapies.
    PLoS ONE 02/2013; 8(2):e55493. DOI:10.1371/journal.pone.0055493 · 3.23 Impact Factor
Show more