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. 2005 Jun 8;33(10):3283-91.
doi: 10.1093/nar/gki639. Print 2005.

The XPF-ERCC1 endonuclease and homologous recombination contribute to the repair of minor groove DNA interstrand crosslinks in mammalian cells produced by the pyrrolo[2,1-c][1,4]benzodiazepine dimer SJG-136

Peter H Clingen  1 , Inusha U De Silva, Peter J McHugh, Farid J Ghadessy, Michael J Tilby, David E Thurston, John A Hartley
Affiliations

The XPF-ERCC1 endonuclease and homologous recombination contribute to the repair of minor groove DNA interstrand crosslinks in mammalian cells produced by the pyrrolo[2,1-c][1,4]benzodiazepine dimer SJG-136

Peter H Clingen et al. Nucleic Acids Res. .

Abstract

SJG-136, a pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimer, is a highly efficient interstrand crosslinking agent that reacts with guanine bases in a 5'-GATC-3' sequence in the DNA minor groove. SJG-136 crosslinks form rapidly and persist compared to those produced by conventional crosslinking agents such as nitrogen mustard, melphalan or cisplatin which bind in the DNA major groove. A panel of Chinese hamster ovary (CHO) cells with defined defects in specific DNA repair pathways were exposed to the bi-functional agents SJG-136 and melphalan, and to their mono-functional analogues mmy-SJG and mono-functional melphalan. SJG-136 was >100 times more cytotoxic than melphalan, and the bi-functional agents were much more cytotoxic than their respective mono-functional analogues. Cellular sensitivity of both SJG-136 and melphalan was dependent on the XPF-ERCC1 heterodimer, and homologous recombination repair factors XRCC2 and XRCC3. The relative level of sensitivity of these repair mutant cell lines to SJG-136 was, however, significantly less than with major groove crosslinking agents. In contrast to melphalan, there was no clear correlation between sensitivity to SJG-136 and crosslink unhooking capacity measured using a modified comet assay. Furthermore, repair of SJG-136 crosslinks did not involve the formation of DNA double-strand breaks. SJG-136 cytotoxicity is likely to result from the poor recognition of DNA damage by repair proteins resulting in the slow repair of both mono-adducts and more importantly crosslinks in the minor groove.

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Figures

Figure 1
Figure 1
Structures of melphalan, mono-melphalan, SJG-136 and mmy-SJG.
Figure 2
Figure 2
Melphalan and SJG-136 sensitivity in parental AA8, V79 and CHO-K1 cells, NER mutants XPB (UV23), XPD (UV42), XPF (UV47), CSB (UV61), ERCC1 (UV96) and XPG (UV135), recombination mutants XRCC2 (irs1), XRCC3 (irs1SF) and the NHEJ mutant XRCC5 (xrs5). Growth inhibition was determined using the SRB assay and the fraction of control A540 calculated as described. All results are the mean of at least three independent experiments and error bars show the standard error of the mean.
Figure 3
Figure 3
Mono-melphalan and mmy-SJG sensitivity of parental AA8, V79 and CHOK1 cells, NER mutants XPB (UV23), XPD (UV42), XPF (UV47), CSB (UV61), ERCC1 (UV96) and XPG (UV135), recombination mutants XRCC2 (irs1), XRCC3 (irs1SF) and the NHEJ mutant XRCC5 (xrs5). Growth inhibition was determined using the SRB assay and the fraction of control A540 calculated as described. All results are the mean of at least three independent experiments and error bars show the standard error of the mean.
Figure 4
Figure 4
Clonogenic Survival assays for parental AA8, NER mutants XPB (UV23), ERCC1 (UV96) and the recombination mutant XRCC3 (irs1SF) cells after treatment with melphalan or SJG-136. The surviving fraction was calculated as described. All results are the mean of two independent experiments and error bars show the standard error of the mean.
Figure 5
Figure 5
Decrease in comet tail moment (TM) with increasing concentration of (A) melphalan and (B) SJG-136 in AA8 cells. AA8 cells were treated with each compound for 1 h and analysed immediately. For each drug concentration, the TM of 50 cells was measured and the mean values calculated. The percentage decrease in TM in comparison with untreated samples was calculated. Results are the mean of at least three independent experiments and error bars show the standard error of the mean.
Figure 6
Figure 6
Kinetics of unhooking of (A) melphalan and (B) SJG-136 induced ICLs in parent (AA8) and NER mutant cell lines; XPB (UV23), XPG (UV135), XPF (UV47) and ERCC1 (9UV96). Cells were treated with 30 μM melphalan or 0.3 μM SJG-136 for 1 h, incubated in fresh medium and the level of ICLs measured at specific time points using the modified comet assay. Results are the mean of three independent experiments and error bars show the standard error of the mean.
Figure 7
Figure 7
Induction of DNA DSBs following treatment with melphalan or SJG-136. Exponentially growing AA8 cells were treated with increasing doses of either compound for 1 h, and DSBs analysed immediately by PFGE.
Figure 8
Figure 8
Repair kinetics of melphalan-induced DNA DSBs in parent AA8 cells, the NER mutant ERCC1 (UV96) and the recombination mutant XRCC3 (irs1SF). Cells were treated with 30 μM melphalan for 1 h and the level of DSBs assessed immediately, and 4, 8 and 24 h after drug treatment. Semi-quantitative analysis of the percentage of DNA released from the plugs from a typical experiment are shown. Results show the mean of two independent experiments and error bars show the standard error of the mean.
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