Abstract
Xenobiotic-induced interstrand DNA–DNA cross-links (ICL) interfere with transcription and replication and can be converted to toxic DNA double strand breaks. In this work, we investigated cellular responses to 1,4-bis-(guan-7-yl)-2,3-butanediol (bis-N7G-BD) cross-links induced by 1,2,3,4-diepoxybutane (DEB). High pressure liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI+-MS/MS) assays were used to quantify the formation and repair of bis-N7G-BD cross-links in wild-type Chinese hamster lung fibroblasts (V79) and the corresponding isogenic clones V-H1 and V-H4, deficient in the XPD and FANCA genes, respectively. Both V-H1 and V-H4 cells exhibited enhanced sensitivity to DEB-induced cell death and elevated bis-N7G-BD cross-links. However, relatively modest increases of bis-N7G-BD adduct levels in V-H4 clones did not correlate with their hypersensitivity to DEB. Further, bis-N7G-BD levels were not elevated in DEB-treated human clones with defects in the XPA or FANCD2 genes. Comet assays and γ-H2AX focus analyses conducted with hamster cells revealed that ICL removal was associated with chromosomal double strand break formation, and that these breaks persisted in V-H4 cells as compared to control cells. Our findings suggest that ICL repair in cells with defects in the Fanconi anemia repair pathway is associated with aberrant re-joining of repair-induced double strand breaks, potentially resulting in lethal chromosome rearrangements.
Highlights
The anticancer activity of many clinically useful drugs including platinum compounds, mitomycin C, psoralens, and antitumor nitrogen mustards is attributed to their ability to form chromosomal interstrand DNA–DNA cross-links (ICLs) [1]
Our results reflect significantly increased levels of bis-N7G-BD adducts in the nucleotide excision repair (NER)-deficient V-H1 cell lines compared to isogenic control at all three time points during drug treatment (T1, T2, the 3 h exposure (T3) in Figure 4, p < 0.05) while Fanconi anemia (FA)-deficient VH-4 cells showed a significant increase in ICLs only at the time of drug removal (T3 in Figure 4, p < 0.05)
Bis-N7G-BD Adduct Formation in DEB-Treated Human Cells The presence of elevated levels of ICLs in DEB-sensitive V-H4 hamster cells as compared to the isogenic, DEB-resistant V79 clone is consistent with the hypothesis that defects in adduct removal may underlie cellular sensitivity of the former cells to DEB
Summary
The anticancer activity of many clinically useful drugs including platinum compounds, mitomycin C, psoralens, and antitumor nitrogen mustards is attributed to their ability to form chromosomal interstrand DNA–DNA cross-links (ICLs) [1]. EcOiatnutncerurdeimnawsbitiieintarhgsl ranging between 0 and 2.6 adducts per 107 nucleotides (Figure 3) While this approach does not allow us to distinguish interstrand from intrastrand bis-N7G-BD crosslinks, our previous studies have shown that SS-, RR-DEB used in this work almost exclusively forms interstrand DNA–DNA cross-links [4,36]. Bis-N7G-BD Adduct Formation in DEB-Treated Human Cells The presence of elevated levels of ICLs in DEB-sensitive V-H4 hamster cells as compared to the isogenic, DEB-resistant V79 clone is consistent with the hypothesis that defects in adduct removal may underlie cellular sensitivity of the former cells to DEB. We performed an additional series of experiments to examine ICL formation and removal in wild-type and FA or XP-deficient human cell lines These results revealed that the kinetics of ICL formation and removal in FANCD2- or XPA-deficient clones did not significantly differ from that observed in isogenic wild-type control lines (Figure S2).
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