Characterisation of Ercc1 deficiency in the liver and in conditional Ercc1-deficient primary hepatocytes in vitro

The ERCC1/XPF complex is responsible for incision at the 5' side of the lesion during nucleotide excision repair and is also involved in homologous recombination and interstrand cross-link repair. The aim of the current study was to set up a better model for examination of Ercc1 deficiency in the murine liver and to determine the DNA lesions responsible for the premature polyploidy observed. We used the Cre/lox system with an adenovirus carrying Cre recombinase to conditionally induce Ercc1 deficiency in murine hepatocytes in vitro. Increased levels of apoptosis were apparent in our Ercc1-deficient cultures, both spontaneously and after UV irradiation and oxidative DNA damage. Increased apoptosis was also observed in simple Ercc1-deficient livers and the time course of the development of polyploidy was characterised. Livers from simple Ercc1 knockout mice contained mitochondria with disrupted outer membranes. Lipid accumulation was observed in older Ercc1-deficient hepatocyte cultures and in young Ercc1-deficient and wild-type livers. Lipids disappeared from the wild-type livers with age, but persisted in Ercc1-deficient livers, suggesting that a reduced ability to repair oxidative DNA damage and a malfunction of oxidative pathways could be responsible for the Ercc1-deficient liver phenotype. Real-time RT-PCR was used to determine differences in expression of cell cycle regulation and survival genes between Ercc1-deficient and control livers. Higher mRNA levels of Igfbp2, a possible marker for polyploidy, and p21 were detected in Ercc1-deficient livers. The pro-apoptotic factor, Bax, showed increased levels of mRNA expression in young Ercc1-deficient livers. However, no elevation in the levels of reactive oxygen species, or of malondialdehyde DNA adducts, a product of oxidative DNA damage, were found in Ercc1-deficient liver and no elevated levels of genes involved in the oxidative damage response were seen.     

DNA repair gene Ercc1 is essential for normal spermatogenesis and oogenesis and for functional integrity of germ cell DNA in the mouse

Ercc1 is essential for nucleotide excision repair (NER) but, unlike other NER proteins, Ercc1 and Xpf are also involved in recombination repair pathways. Ercc1 knockout mice have profound cell cycle abnormalities in the liver and die before weaning. Subsequently Xpa and Xpc knockouts have proved to be good models for the human NER deficiency disease, xeroderma pigmentosum, leading to speculation that the recombination, rather than the NER deficit is the key to the Ercc1 knockout phenotype. To investigate the importance of the recombination repair functions of Ercc1 we studied spermatogenesis and oogenesis in Ercc1-deficient mice. Male and female Ercc1-deficient mice were both infertile. Ercc1 was expressed at a high level in the testis and the highest levels of Ercc1 protein occurred in germ cells following meiotic crossing over. However, in Ercc1 null males some germ cell loss occurred prior to meiotic entry and there was no evidence that Ercc1 was essential for meiotic crossing over. An increased level of DNA strand breaks and oxidative DNA damage was found in Ercc1-deficient testis and increased apoptosis was noted in male germ cells. We conclude that the repair functions of Ercc1 are required in both male and female germ cells at all stages of their maturation. The role of endogenous oxidative DNA damage and the reason for the sensitivity of the germ cells to Ercc1 deficiency are discussed.     

Inhibition of the ERCC1–XPF structure-specific endonuclease to overcome cancer chemoresistance

ERCC1–XPF is a structure-specific endonuclease that is required for the repair of DNA lesions, generated by the widely used platinum-containing cancer chemotherapeutics such as cisplatin, through the Nucleotide Excision Repair and Interstrand Crosslink Repair pathways. Based on mouse xenograft experiments, where ERCC1-deficient melanomas were cured by cisplatin therapy, we proposed that inhibition of ERCC1–XPF could enhance the effectiveness of platinum-based chemotherapy. Here we report the identification and properties of inhibitors against two key targets on ERCC1–XPF. By targeting the ERCC1–XPF interaction domain we proposed that inhibition would disrupt the ERCC1–XPF heterodimer resulting in destabilisation of both proteins. Using in silico screening, we identified an inhibitor that bound to ERCC1–XPF in a biophysical assay, reduced the level of ERCC1–XPF complexes in ovarian cancer cells, inhibited Nucleotide Excision Repair and sensitised melanoma cells to cisplatin. We also utilised high throughput and in silico screening to identify the first reported inhibitors of the other key target, the XPF endonuclease domain. We demonstrate that two of these compounds display specificity in vitro for ERCC1–XPF over two other endonucleases, bind to ERCC1–XPF, inhibit Nucleotide Excision Repair in two independent assays and specifically sensitise Nucleotide Excision Repair-proficient, but not Nucleotide Excision Repair-deficient human and mouse cells to cisplatin.     

Functional and physical interactions between ERCC1 and MSH2 complexes for resistance to cis-diamminedichloroplatinum(II) in mammalian cells

Bulky DNA lesions are mainly repaired by nucleotide excision repair (NER), in which the interaction of ERCC1 with XPA protein recruits the ERCC1-XPF complex, which acts as a structure-specific endonuclease in the repair process. However, additional functions besides NER have been suggested for the ERCC1-XPF complex, because ERCC1- or XPF-deficient rodent cells are significantly more sensitive to DNA interstrand cross-linking (ICL) agents such as cis-diamminedichloroplatinum(II) (CDDP) than any other NER-deficient cells and because ERCC1-deficient mice suffer a more severe phenotype than XPA-deficient mice. By using RNA interference we show here that suppression of ERCC1 expression increases the sensitivity of xeroderma pigmentosum group A (XPA)-deficient human cells to CDDP but not to UV. This increased sensitivity to CDDP is observed in mouse cells defective in Xpa as well but not in cells defective both in Xpa and the mismatch repair gene Msh2. These data suggest that ERCC1 and MSH2 are involved co-operatively in CDDP resistance in mammalian cells. As a possible molecular basis, we show further a physical interaction between endogenous ERCC1 and MSH2 complexes in HeLa cell extracts. Using tagged ERCC1 in COS7 cells, the minimum region in ERCC1 necessary for the immuno-precipitation of MSH2 is turned out to be the carboxyl-terminal domain between the 184th and 260th amino acid, which is partly overlapping with the XPF-binding domain of ERCC1. This interaction may be important in additional functions of ERCC1-XPF including the repair of CDDP-induced DNA damage.     

Deficiency in the DNA repair protein ERCC1 triggers a link between senescence and apoptosis in human fibroblasts and mouse skin

ERCC1 (excision repair cross complementing‐group 1) is a mammalian endonuclease that incises the damaged strand of DNA during nucleotide excision repair and interstrand cross‐link repair. Ercc1^?/Δ mice, carrying one null and one hypomorphic Ercc1 allele, have been widely used to study aging due to accelerated aging phenotypes in numerous organs and their shortened lifespan. Ercc1^?/Δ mice display combined features of human progeroid and cancer‐prone syndromes. Although several studies report cellular senescence and apoptosis associated with the premature aging of Ercc1^?/Δ mice, the link between these two processes and their physiological relevance in the phenotypes of Ercc1^?/Δ mice are incompletely understood. Here, we show that ERCC1 depletion, both in cultured human fibroblasts and the skin of Ercc1^?/Δ mice, initially induces cellular senescence and, importantly, increased expression of several SASP (senescence‐associated secretory phenotype) factors. Cellular senescence induced by ERCC1 deficiency was dependent on activity of the p53 tumor‐suppressor protein. In turn, TNFα secreted by senescent cells induced apoptosis, not only in neighboring ERCC1‐deficient nonsenescent cells, but also cell autonomously in the senescent cells themselves. In addition, expression of the stem cell markers p63 and Lgr6 was significantly decreased in Ercc1^?/Δ mouse skin, where the apoptotic cells are localized, compared to age‐matched wild‐type skin, possibly due to the apoptosis of stem cells. These data suggest that ERCC1‐depleted cells become susceptible to apoptosis via TNFα secreted from neighboring senescent cells. We speculate that parts of the premature aging phenotypes and shortened health‐ or lifespan may be due to stem cell depletion through apoptosis promoted by senescent cells.     

Mice with DNA repair gene Ercc1 deficiency in a neural crest lineage are a model for late-onset Hirschsprung disease

The Ercc1 gene is essential for nucleotide excision repair and is also important in recombination repair and the repair of interstrand crosslinks. We have previously used a floxed Ercc1 allele with a keratinocyte-specific Cre recombinase transgene to inactivate Ercc1 in the epidermal layer of the skin and so generate a mouse model for UV-induced non-melanoma skin cancer. Now, in an attempt to generate a model for UV-induced melanoma, we have used the floxed Ercc1 allele in combination with a Cre transgene under the control of the tyrosinase gene promoter to produce mice with Ercc1-deficient melanocytes that are hypersensitive to UV irradiation. These animals developed normally, but died when 4–6 months old with severe colonic obstruction. Melanocytes are derived from the neural crest and the tyrosinase promoter is also expressed in additional neural crest-derived lineages, including the progenitors of the parasympathetic nervous system that innervates the gastrointestinal tract and controls gut peristalsis. A functional enteric nervous system developed in floxed Ercc1 mice with the tyrosinase Cre transgene, but was found to have degenerated in the colons of affected mice. We suggest that accumulating unrepaired endogenous DNA damage in the Ercc1-deficient colonic parasympathetic ganglia leads to the degeneration of this network and results in a colonic obstructive disorder that resembles late-onset Hirschsprung disease in man.     

The formation of UV-induced chromosome aberrations involves ERCC1 and XPF but not other nucleotide excision repair genes

ERCC1-XPF, through its role in nucleotide excision repair (NER), is essential for the repair of DNA damage caused by UV light. ERCC1-XPF is also involved in recombinational repair processes distinct from NER. In rodent cells chromosome aberrations are a common consequence of UV irradiation. We have previously shown that ERCC1-deficient cells have a lower ratio of chromatid exchanges to breaks than wild type cells. We have now confirmed this result and have shown that XPF-deficient cells also have a lower ratio than wild type. However, cells deficient in the other NER genes, XPD, XPB and XPG, all have the same ratio of exchanges to breaks as wild type. This implies that ERCC1-XPF, but not other NER proteins, is involved in the formation of UV-induced chromosome aberrations, presumably through the role of ERCC1-XPF in recombinational repair pathways rather than NER. We suggest that ERCC1-XPF may be involved in the bypass/repair of DNA damage in replicating DNA by an exchange mechanism involving single strand annealing between non-homologous chromosomes. This mechanism would rely on the ability of ERCC1-XPF to trim non-homologous 3' tails.     

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

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.     

An XPG DNA repair defect causing mutagen hypersensitivity in mouse leukemia L1210 cells.

One of the most widely used antitumor drugs is cis-diamminedichloroplatinum(II) (cisplatin), and mechanisms of cisplatin resistance have been investigated in numerous model systems. Many studies have used mouse leukemia L1210/0 as a reference wild-type cell line, and cisplatin-resistant subclones have been derived from it. Increased DNA excision repair capacity is thought to play a key role in the acquired cisplatin resistance, and this has influenced development of drugs for clinical trials. We report here that the L1210/0 line is in fact severely deficient in nucleotide excision repair of damaged DNA in vivo and in vitro. L1210/0 cell extracts could be complemented by extracts from repair-defective human xeroderma pigmentosum (XP) or rodent excision repair cross-complementing (ERCC) mutant cells, except for XPG/ERCC5 mutants. Purified XPG protein could restore repair proficiency to L1210/0 extracts. Expression of mouse XPG mRNA was similar in all L1210 lines studied, suggesting a point mutation or small alteration of XPG in L1210/0 cells. The DNA repair capacity of a cisplatin-resistant subline, L1210/DDP10, is similar to that of type culture collection L1210 cells and to those of other normal mammalian cell lines. Nucleotide excision repair of DNA is thus clearly important in the intrinsic cellular defense against cisplatin. However, in contrast to what is generally believed, enhancement of DNA repair above the normal level in these rodent cells does not appear to be a mechanism of acquired resistance to the drug.     

CITED2 silencing sensitizes cancer cells to cisplatin by inhibiting p53 trans-activation and chromatin relaxation on the ERCC1 DNA repair gene

In this study, we show that silencing of CITED2 using small-hairpin RNA (shCITED2) induced DNA damage and reduction of ERCC1 gene expression in HEK293, HeLa and H1299 cells, even in the absence of cisplatin. In contrast, ectopic expression of ERCC1 significantly reduced intrinsic and induced DNA damage levels, and rescued the effects of CITED2 silencing on cell viability. The effects of CITED2 silencing on DNA repair and cell death were associated with p53 activity. Furthermore, CITED2 silencing caused severe elimination of the p300 protein and markers of relaxed chromatin (acetylated H3 and H4, i.e. H3K9Ac and H3K14Ac) in HEK293 cells. Chromatin immunoprecipitation assays further revealed that DNA damage induced binding of p53 along with H3K9Ac or H3K14Ac at the ERCC1 promoter, an effect which was almost entirely abrogated by silencing of CITED2 or p300. Moreover, lentivirus-based CITED2 silencing sensitized HeLa cell line-derived tumor xenografts to cisplatin in immune-deficient mice. These results demonstrate that CITED2/p300 can be recruited by p53 at the promoter of the repair gene ERCC1 in response to cisplatin-induced DNA damage. The CITED2/p300/p53/ERCC1 pathway is thus involved in the cell response to cisplatin and represents a potential target for cancer therapy.     

Reduced hematopoietic reserves in DNA interstrand crosslink repair-deficient Ercc1-/- mice

The ERCC1-XPF heterodimer is a structure-specific endonuclease involved in both nucleotide excision repair and interstrand crosslink repair. Mice carrying a genetic defect in Ercc1 display symptoms suggestive of a progressive, segmental progeria, indicating that disruption of one or both of these DNA damage repair pathways accelerates aging. In the hematopoietic system, there are defined age-associated changes for which the cause is unknown. To determine if DNA repair is critical to prolonged hematopoietic function, hematopoiesis in Ercc1-/- mice was compared to that in young and old wild-type mice. Ercc1-/- mice (3-week-old) exhibited multilineage cytopenia and fatty replacement of bone marrow, similar to old wild-type mice. In addition, the proliferative reserves of hematopoietic progenitors and stress erythropoiesis were significantly reduced in Ercc1-/- mice compared to age-matched controls. These features were not seen in nucleotide excision repair-deficient Xpa-/- mice, but are characteristic of Fanconi anemia, a human cancer syndrome caused by defects in interstrand crosslink repair. These data support the hypothesis that spontaneous interstrand crosslink damage contributes to the functional decline of the hematopoietic system associated with aging.     

USP45 deubiquitylase controls ERCC1–XPF endonuclease-mediated DNA damage responses

Reversible protein ubiquitylation plays important roles in various processes including DNA repair. Here, we identify the deubiquitylase USP45 as a critical DNA repair regulator. USP45 associates with ERCC1, a subunit of the DNA repair endonuclease XPF–ERCC1, via a short acidic motif outside of the USP45 catalytic domain. Wild-type USP45, but not a USP45 mutant defective in ERCC1 binding, efficiently deubiquitylates ERCC1 in vitro, and the levels of ubiquitylated ERCC1 are markedly enhanced in USP45 knockout cells. Cells lacking USP45 are hypersensitive specifically to UV irradiation and DNA interstrand cross-links, similar to cells lacking ERCC1. Furthermore, the repair of UV-induced DNA damage is markedly reduced in USP45-deficient cells. ERCC1 translocation to DNA damage-induced subnuclear foci is markedly impaired in USP45 knockout cells, possibly accounting for defective DNA repair. Finally, USP45 localises to sites of DNA damage in a manner dependent on its deubiquitylase activity, but independent of its ability to bind ERCC1–XPF. Together, these results establish USP45 as a new regulator of XPF–ERCC1 crucial for efficient DNA repair.     

Drosophila ERCC1 is required for a subset of MEI-9-dependent meiotic crossovers

Drosophila MEI-9 is the catalytic subunit of a DNA structure-specific endonuclease required for nucleotide excision repair (NER). The enzymatic activity of this endonuclease during NER requires the presence of a second, noncatalytic subunit called ERCC1. In addition to its role in NER, MEI-9 is required for the generation of most meiotic crossovers. To better understand the role of MEI-9 in crossover formation, we report here the characterization of the Drosophila Ercc1 gene. We created an Ercc1 mutant through homologous gene targeting. We find that Ercc1 mutants are identical to mei-9 mutants in sensitivity to DNA-damaging agents, but have a less severe reduction in the number of meiotic crossovers. MEI-9 protein levels are reduced in Ercc1 mutants; however, overexpression of MEI-9 is not sufficient to restore meiotic crossing over in Ercc1 mutants. We conclude that MEI-9 can generate some meiotic crossovers in an ERCC1-independent manner.