Defects in interstrand cross-link uncoupling do not account for the extreme sensitivity of ERCC1 and XPF cells to cisplatin

Abstract

The anticancer drug cisplatin reacts with DNA leading to the formation of interstrand and intrastrand cross-links that are the critical cytotoxic lesions. In contrast to cells bearing mutations in other components of the nucleotide excision repair apparatus (XPB, XPD, XPG and CSB), cells defective for the ERCC1-XPF structure-specific nuclease are highly sensitive to cisplatin. To determine if the extreme sensitivity of XPF and ERCC1 cells to cisplatin results from specific defects in the repair of either intrastrand or interstrand cross-links we measured the elimination of both lesions in a range of nucleotide excision repair Chinese hamster mutant cell lines, including XPF- and ERCC1-defective cells. Compared to the parental, repair-proficient cell line all the mutants tested were defective in the elimination of both classes of adduct despite their very different levels of increased sensitivity. Consequently, there is no clear relationship between initial incisions at interstrand cross-links or removal of intrastrand adducts and cellular sensitivity. These results demonstrate that the high cisplatin sensitivity of ERCC1 and XPF cells likely results from a defect other than in excision repair. In contrast to other conventional DNA cross-linking agents, we found that the repair of cisplatin adducts does not involve the formation of DNA double-strand breaks. Surprisingly, XRCC2 and XRCC3 cells are defective in the uncoupling step of cisplatin interstrand cross-link repair, suggesting that homologous recombination might be initiated prior to excision of this type of cross-link.

γ-H2AX formation in response to interstrand crosslinks requires XPF in human cells

“…These results suggest that the entire NER pathway is not necessary to generate DSB in cells, and that the XPF protein specifically participates in an NER-independent function that is required and probably rate-limiting for DSB formation in human cells. This conclusion is generally in agreement with prior work, which indicated that the NER functions of XPF are distinct from its role in ICL processing [23,47,48]. Our work in human cells corroborates and complements prior work, all in Chinese hamster ovary cells, demonstrating that ERCC1, which normally is in a heterodimeric complex with XPF, is required for the initial incisions and γ-H2AX formation [32], and that XPF and ERCC1 are required for incisions leading to DSB in an in vitro assay [14].…”

“…Some precedent for differential processing of chemically distinct ICL exists. Cisplatin forms a small number of ICL and fails to induce DSB in rodent cells [48]. NER processing of psoralen ICL that depends on XPA and other NER proteins to uncouple one strand of the ICL [21,22], followed by translesion synthesis with a bypass polymerase may be one scenario that could account for reduced DSB formation in GM637 cells relative to XP-A cells [11,20,50,51].…”

Enhanced repair of DNA interstrand crosslinking in ovarian cancer cells from patients following treatment with platinum-based chemotherapy

“…This is the first step in the complex molecular mechanism of repair of DNA ICLs (McHugh et al, 2001) and the comet assay cannot determine if the repair process has gone to completion and correctly restored the integrity of both strands of the DNA. Mammalian cells defective in the unhooking step of cisplatin interstrand crosslink repair, as measured using the comet assay, include cells bearing mutations in nucleotide excision repair (e.g., XPB, XPD, XPG, ERCC1 and XPF) and homologous recombination (e.g., XRCC2 and XRCC3) (De Silva et al, 2002). Cells defective in ERCC1 are highly sensitive to cisplatin and several groups have investigated the influence of ERCC1 on resistance to platinum chemotherapy (Ferry et al, 2000;Reed, 2005) and suggest that ERCC1 is a good marker for cellular or clinical resistance to these drugs.…”

“…The method has also been used to measure cisplatin-induced ICLs in vitro (De Silva et al, 2002). In this study, we have used the method to compare the formation and repair (unhooking) of ICL's following ex vivo exposure to cisplatin in tumour cells and normal cells isolated from ovarian cancer patients who were either newly diagnosed, or had been previously treated with platinum-based chemotherapy.…”

Minor structural modifications to alchemix influence mechanism of action and pharmacological activity

“…The UV5 cell line is defective in the XPD protein, which is involved in NER and crosslink repair [37], but not homologous recombination [38]. Since only the UV4 and not the UV5 cell line was sensitive to ICT2901 and ICT2903, it implicates that homologous recombination is involved in the response to these agents.…”