Correction of liver dysfunction in DNA repair-deficient mice with an ERCC1 transgene

The ERCC1 gene is essential for the repair of UV-induced DNA damage. Unlike most genes in the nucleotide excision repair (NER) pathway, ERCC1 is also involved in recombinational repair. Perhaps for this reason, ERCC1 knockout mice are not a model for the human NER deficiency disorder, xeroderma pigmentosum. Instead, ERCC1 null mice are severely runted and die before weaning from liver failure with accelerated hepatocyte polyploidy that is more reminiscent of a premature ageing disorder. To permit study of the role of ERCC1 in other tissues we have corrected the liver ERCC1 deficiency with a transgene under the control of a liver-specific promoter. The transgene alleviated runting and extended the lifespan. The elevated level of oxidative DNA damage and premature liver polyploidy were reversed and liver function was corrected. A widespread mitochondrial dysfunction was identified and an essential role for ERCC1 in the kidney was also revealed with transgene-containing ERCC1-deficient animals going on to die of renal failure. The nuclei of kidney proximal tubule cells became polyploid in a similar way to the premature liver polyploidy observed in younger ERCC1-deficient animals. We believe that this is a response to the accumulation of endogenous DNA damage in these particularly susceptible tissues which cannot be repaired in ERCC1-deficient animals.     

DDB2-Independent Role for p53 in the Recovery from Ultraviolet Light-Induced Replication Arrest

Ultraviolet light (UV light) induces helix distorting DNA lesions that pose a block to replicative DNA polymerases. Recovery from this replication arrest is reportedly impaired in nucleotide excision repair (NER)-deficient xeroderma pigmentosum (XP) fibroblasts and primary fibroblasts lacking functional p53. These independent observations suggested that the involvement of p53 in the recovery from UV-induced replication arrest was related to its role in regulating the global genomic subpathway of NER (GG-NER). Using primary human fibroblasts, we confirm that the recovery from UV-induced replication arrest is impaired in cells lacking functional p53 and in primary XP fibroblasts derived from complementation groups A or C (XP-A and XP-C) that are defective in GG-NER. Surprisingly, DNA synthesis recovered normally in GG-NER-deficient XP complementation group E (XP-E) cells that carry mutations in the p53 regulated DNA repair gene DDB2 and are specifically defective in the repair of cyclobutane pyrimidine dimers (CPD) but not pyrimidine (6-4) pyrimidone photoproducts. Disruption of p53 in these XP-E fibroblasts prevented the recovery from UV-induced replication arrest. Therefore, the roles of p53 and GG-NER in the recovery from UV-induced replication are separable and DDB2-independent. These results further indicate that primary human fibroblasts expressing functional p53 efficiently replicate DNA containing CPD whereas p53-deficient cells do not, consistent with a role for p53 in permitting translesion DNA synthesis of these DNA lesions.     

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.     

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.     

Growth retardation, early death, and DNA repair defects in mice deficient for the nucleotide excision repair enzyme XPF

Xeroderma pigmentosum (XP) is a human genetic disease which is caused by defects in nucleotide excision repair. Since this repair pathway is responsible for removing UV irradiation-induced damage to DNA, XP patients are hypersensitive to sunlight and are prone to develop skin cancer. Based on the underlying genetic defect, the disease can be divided into the seven complementation groups XPA through XPG. XPF, in association with ERCC1, constitutes a structure-specific endonuclease that makes an incision 5' to the photodamage. XPF-ERCC1 has also been implicated in both removal of interstrand DNA cross-links and homology-mediated recombination and in immunoglobulin class switch recombination (CSR). To study the function of XPF in vivo, we inactivated the XPF gene in mice. XPF-deficient mice showed a severe postnatal growth defect and died approximately 3 weeks after birth. Histological examination revealed that the liver of mutant animals contained abnormal cells with enlarged nuclei. Furthermore, embryonic fibroblasts defective in XPF are hypersensitive to UV irradiation and mitomycin C treatment. No defect in CSR was detected, suggesting that the nuclease is dispensable for this recombination process. These phenotypes are identical to those exhibited by the ERCC1-deficient mice, consistent with the functional association of the two proteins. The complex phenotype suggests that XPF-ERCC1 is involved in multiple DNA repair processes.     

p53 Prevents the Accumulation of Double-Strand DNA Breaks at Stalled-Replication Forks Induced by UV in Human Cells

To investigate the mechanism by which UV irradiation causes S-phase-dependent chromosome aberrations and thereby genomic instability, we have developed an assay to study the DNA structure of replication forks (RFs) in UV-irradiated mammalian cells, using pulse-field gel electrophoresis for the DNA analysis. We demonstrate that replication stalling at UV-induced pyrimidine dimers results in the formation of single-strand DNA (ssDNA) regions and incomplete RF structures. In normal and in excision-repair-defective xeroderma pimentosum (XP) cells, stalling at dimers is rapid and prolonged and recovery depends on dimer repair or bypass. By contrast, XP variant (XPV) cells, defective in replication of a UV-damaged template due to mutation of bypass-polymerase ?, fail to arrest at dimers, resulting in a much higher frequency of ssDNA regions in the stalled RFs. We show that the stability of UV-arrested RFs depends directly on functional p53, and indirectly on NER and pol ?. In p53-deficient cells, the stalled sites give rise to double-strand DNA breaks (DSBs), at a frequency inversely correlated with repair capacity of the cell. In normal cells only a fraction of the stalled sites give rise to DSBs, while in XPASV, XPDSV and also XPVSV all the sites do. XPVSV cells, although repair proficient, accumulate almost double the number of DSBs, suggesting that a high frequency of ssDNA regions in UV-arrested forks cause RF instability. These replication-associated DSBs do not accumulate in p53-proficient human cells. We propose that a major mechanism by which p53 maintains genome stability is the prevention of DSB accumulation at long-lived ssDNA regions in stalled-replication forks.

Supplemental material for this paper can be found at the following link:

http://www.landesbioscience.com/journals/cc/squiresCC3-12-sup.pdf     

First reported patient with human ERCC1 deficiency has cerebro-oculo-facio-skeletal syndrome with a mild defect in nucleotide excision repair and severe developmental failure

Nucleotide excision repair (NER) is a genome caretaker mechanism responsible for removing helix-distorting DNA lesions, most notably ultraviolet photodimers. Inherited defects in NER result in profound photosensitivity and the cancer-prone syndrome xeroderma pigmentosum (XP) or two progeroid syndromes: Cockayne and trichothiodystrophy syndromes. The heterodimer ERCC1-XPF is one of two endonucleases required for NER. Mutations in XPF are associated with mild XP and rarely with progeria. Mutations in ERCC1 have not been reported. Here, we describe the first case of human inherited ERCC1 deficiency. Patient cells showed moderate hypersensitivity to ultraviolet rays and mitomycin C, yet the clinical features were very severe and, unexpectedly, were compatible with a diagnosis of cerebro-oculo-facio-skeletal syndrome. This discovery represents a novel complementation group of patients with defective NER. Further, the clinical severity, coupled with a relatively mild repair defect, suggests novel functions for ERCC1.     

The knock-down of ERCC1 but not of XPF causes multinucleation

Excision repair cross complementing gene 1 (ERCC1) associated with xeroderma pigmentosum group F (XPF) is a heterodimeric endonuclease historically involved in the excision of bulky helix-distorting DNA lesions during nucleotide excision repair (NER) but also in the repair of DNA interstrand crosslinks. ERCC1 deficient mice show severe growth retardation associated with premature replicative senescence leading to liver failure and death at four weeks of age. In humans, ERCC1 is overexpressed in hepatocellular carcinoma and in the late G1 phase of hepatocyte cell cycle. To investigate whether ERCC1 could be involved in human hepatocyte cell growth and cell cycle progression, we knocked-down ERCC1 expression in the human hepatocellular carcinoma cell line Huh7 by RNA interference. ERCC1 knocked-down cells were delayed in their cell cycle and became multinucleated. This phenotype was rescued by ERCC1 overexpression. Multinucleation was not liver specific since it also occurred in HeLa and in human fibroblasts knocked-down for ERCC1. Multinucleated cells arose after drastic defects leading to flawed metaphase and cytokinesis. Interestingly, multinucleation did not appear after knocking-down other NER enzymes such as XPC and XPF, suggesting that NER deficiency was not responsible for multinucleation. Moreover, XPF mutant human fibroblasts formed multinucleated cells after ERCC1 knock-down but not after XPF knock-down. Therefore our results seem consistent with ERCC1 being involved in multinucleation but not XPF. This work reveals a new role for ERCC1 distinct from its known function in DNA repair, which may be independent of XPF. The role for ERCC1 in mitotic progression may be critical during development, particularly in humans.     

Synergistic interactions between XPC and p53 mutations in double-mutant mice: neural tube abnormalities and accelerated UV radiation-induced skin cancer

The significance of DNA repair to human health has been well documented by studies on xeroderma pigmentosum (XP) patients, who suffer a dramatically increased risk of cancer in sun-exposed areas of their skin [1] and [2]. This autosomal recessive disorder has been directly associated with a defect in nucleotide excision–repair (NER) [1] and [2]. Like human XP individuals, mice carrying homozygous mutations in XP genes manifest a predisposition to skin carcinogenesis following exposure to ultraviolet (UV) radiation [3], [4] and [5]. Recent studies have suggested that, in addition to roles in apoptosis [6] and cell-cycle checkpoint control [7] in response to DNA damage, p53 protein may modulate NER [8]. Mutations in the p53 gene have been observed in 50% of all human tumors [9] and have been implicated in both the early [10] and late [11] stages of skin cancer. To examine the consequences of a combined deficiency of the XPC and the p53 proteins in mice, we generated double-mutant animals. We document a spectrum of neural tube defects in XPC p53 mutant embryos. Additionally, we show that, following exposure to UV-B radiation, XPC p53 mutant mice have more severe solar keratosis and suffer accelerated skin cancer compared with XPC mutant mice that are wild-type with respect to p53.     

Deregulation of DNA methyltransferases and loss of parental methylation at the insulin-like growth factor II (Igf2)/H19 loci in p53 knockout mice prior to tumor development

To ascertain whether p53 deficiency in vivo leads to the deregulation of DNA methylation machinery prior to tumor development, we investigated the expression profile of DNA methyltransferases in the thymus and the liver of p53(+/+), p53(+/-), and p53(-/-) mice at 7 weeks of age before tumor development. The expression of DNA methyltransferases was examined in the thymus at 7 weeks of age, since the malignant T-cell lymphoma develops most frequently in p53(-/-) mice around 20 weeks of age. Both mRNA and protein levels of Dnmt1 and Dnmt3b were increased in the thymus and the liver of p53-deficient mice. The expression of Dnmt3a was also increased in the liver but not in the thymus of p53-deficient mice. Dnmt3L expression was reduced in the thymus of p53(+/-) and p53(-/-) mice. The total 5-methylcytosine (5-MeC) in the genomic DNA of p53(+/+), p53(+/-), and p53(-/-) mice was quantitated by dot-blot using antibody against 5-MeC. Global methylation was increased in the thymus and the liver of p53-deficient mice. To correlate the deregulated expression of DNA methyltransferases with the disturbance of the epigenetic integrity, we examined the DNA methylation of the imprinting control region (ICR) at the insulin-like growth factor II (Igf2)/H19 loci in the thymus and the liver of p53(+/+), p53(+/-), and p53(-/-) mice. The region containing two CCCTC binding factor (CTCF) binding sites in the 5'-ICR tended to be hypomethylated in the thymus of p53(-/-) mice, but not in the liver. The expression profile of Igf2 and H19 indicated that the thymus-specific changes of Igf2 and H19 expression were coherent to the hypomethylation of the ICR in the thymus. Our results suggest that p53 is required for the maintenance of DNA methylation patterns in vivo.     

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.     

Role of p53 tumor suppressor gene in the toxicity of TCDD, endrin, naphthalene, and chromium (VI) in liver and brain tissues of mice

It has been postulated that tumor suppressor genes are involved in the cascade of events leading to the toxicity of diverse xenobiotics. Therefore, we have assessed the comparative effects of 0.01, 0.10, and 0.50 median lethal doses (LD(50)) of 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD), endrin, naphthalene, and sodium dichromate (VI) [Cr(VI)] on lipid peroxidation, DNA fragmentation, and enhanced production of superoxide anion (cytochrome c reduction) in liver and brain tissues of p53-deficient and standard C57BL/6NTac mice to determine the role of p53 gene in the toxic manifestations produced by these diverse xenobiotics. In general, p53-deficient mice are more susceptible to all four xenobiotics than C57BL/6NTac mice, with dose-dependent effects being observed. Specifically, at a 0.50 LD(50) dose, naphthalene and Cr(VI) induced the greatest toxicity in the liver tissue of mice, and naphthalene and endrin exhibited the greatest effect in the brain tissue. At this dose, TCDD, endrin, naphthalene, and Cr(VI) induced 2.3- to 3.7-fold higher increases in hepatic lipid peroxidation and 1.8- to 3.0-fold higher increases in brain lipid peroxidation in p53-deficient mice than in C57BL/6NTac mice. At a 0. 10 LD(50) dose, TCDD, endrin, naphthalene, and Cr(VI) induced 1.3- to 1.8-fold higher increases in hepatic lipid peroxidation and 1.4- to 1.9-fold higher increases in brain lipid peroxidation in p53-deficient mice than in C57BL/6NTac mice. Similar results were observed with respect to DNA fragmentation and cytochrome c reduction (superoxide anion production). For example, at the 0.10 LD(50) dose, the four xenobiotics induced increases of 1.6- to 3. 0-fold and 1.5- to 2.1-fold in brain and liver DNA fragmentation, respectively, and increases of 1.5- to 2.3-fold and 1.4- to 2.5-fold in brain and liver cytochrome c reduction (superoxide anion production), respectively, in p53-deficient mice compared with control C57BL/6NTac mice. These results suggest that the p53 tumor suppressor gene may play a role in the toxicity of structurally diverse xenobiotics.     

SW480, a p53 double-mutant cell line retains proficiency for some p53 functions

During certain types of cellular stress, the p53 tumor suppressor protein binds to DNA and transactivates a variety of genes that regulate critical responses including apoptosis, cell cycle checkpoints, differentiation, and angiogenesis. In addition, functional p53 is known to be required for efficient nucleotide excision repair (NER) of bulky DNA adducts generated through exposure to environmental mutagens such as UV light. Nonetheless, we previously showed that the model p53-mutated human adenocarcinoma strain SW480 is proficient in the removal of UV-induced cyclobutane pyrimidine dimers (CPD) via NER. We undertook the present study to begin probing the molecular basis for this unexpected repair phenotype. Cytogenetic analysis indicated that SW480 is stable at the chromosomal level, i.e. manifests a karyotypic profile very similar to that revealed for this line as far back as 14 years ago. After fluorescence in situ hybridization (FISH), using a probe complementary to the p53 gene, we found that 98% of the SW480 interphase nuclei contains three copies of the gene, later revealed to be localized on intact short arms of three chromosomes 17. DNA sequence analysis further showed that all three p53 copies in SW480 carry two point mutations (R273H and P309S), and levels of the corresponding mutated p53 protein are about 20-fold higher than in the closely related p53 wild-type strain LoVo. Using an electrophoretic mobility shift assay (EMSA), we demonstrated that R273H/P309S p53 is able to bind with wild-type affinity to its consensus DNA sequence in vitro. Analysis of p21(Cip1/WAF1) expression and in vivo footprinting by ligation-mediated PCR (LMPCR) showed that, in wild-type LoVo cells, an exposure to cellular stress (e.g. UV or ionizing radiation) is necessary for p53 activation of the p21(Cip1/WAF1) promoter. In contrast, the R273H/P309S-mutated p53 protein in SW480 constitutively activates p21(Cip1/WAF1) in the absence of stress through an unknown mechanism. A similar phenomenon whereby mutated p53 in SW480 is able to induce NER-related proteins might explain the normal DNA repair phenotype previously observed in this strain. For now we conclude that, in general, results obtained using SW480 as a p53-deficient cell line should be interpreted very cautiously.     

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.     

UV-induced skin carcinogenesis in xeroderma pigmentosum group A (XPA) gene-knockout mice with nucleotide excision repair-deficiency

Nucleotide excision repair (NER) removes a wide variety of lesions from the genome and is deficient in the genetic disorder, xeroderma pigmentosum (XP). In this paper, an in vitro analysis of the XP group A gene product (XPA protein) is reported. Results of an analysis on the pathogenesis of ultraviolet (UV)-B-induced skin cancer in the XPA gene-knockout mouse are also described: (1) contrary to wild type mice, significant bias of p53 mutations to the transcribed strand and no evident p53 mutational hot spots were detected in the skin tumors of XPA-knockout mice. (2) Skin cancer cell lines from UVB-irradiated XPA-knockout mice had a decreased mismatch repair activity and an abnormal cell cycle checkpoint, suggesting that the downregulation of mismatch repair helps cells escape killing by UVB and that mismatch repair-deficient clones are selected for during the tumorigenic transformation of XPA (-/-) cells. (3) The XPA-knockout mice showed a higher frequency of UVB-induced mutation in the rpsL transgene at a low dose of UVB-irradiation than the wild type mice. CC-->TT tandem transition, a hallmark of UV-induced mutation, was detected at higher frequency in the rpsL transgene in the XPA-knockout mice than the wild type mice. This rpsL/XPA mouse system will be useful for further analysing the role of NER in the mutagenesis induced by various carcinogens. (4) The UVB-induced immunosuppression was greatly enhanced in the XPA-knockout mice. It is possible that an enhanced impairment of the immune system by UVB irradiation is involved in the high incidence of skin cancer in XP.     

Role of p21WAF1 in the cellular response to UV

UV or g irradiation mediated DNA damage activates p53 and induces cell cycle arrest. Induction of cyclin dependent kinase inhibitor p21WAF1 by p53 after DNA damage plays an important role in cell cycle arrest after gamma irradiation. The p53 mediated cell cycle arrest has been postulated to allow cells to repair the DNA damage. Repair of UV damaged DNA occurs primarily by the nucleotide excision pathway (NER). It is known that p21WAF1 binds PCNA and inhibits PCNA function in DNA replication. PCNA is also required for repair by NER but there have been conflicting reports on whether p21WAF1 can inhibit PCNA function in NER. It has therefore been difficult to integrate the UV induced cell cycle arrest by p21 in the context of repair of UV damaged DNA. A recent study reported that p21WAF1 protein is degraded after low but not high doses of UV irradiation, that cell cycle arrest after UV is p21 independent, and that at low dose UV irradiation p21WAF1 degradation is essential for optimal DNA repair. These findings shed new light on the role of p21 in the cellular response to UV and clarify some outstanding issues concerning p21WAF1 function.