Hypomorphic PCNA mutation underlies a human DNA repair disorder

Numerous human disorders, including Cockayne syndrome, UV-sensitive syndrome, xeroderma pigmentosum, and trichothiodystrophy, result from the mutation of genes encoding molecules important for nucleotide excision repair. Here, we describe a syndrome in which the cardinal clinical features include short stature, hearing loss, premature aging, telangiectasia, neurodegeneration, and photosensitivity, resulting from a homozygous missense (p.Ser228Ile) sequence alteration of the proliferating cell nuclear antigen (PCNA). PCNA is a highly conserved sliding clamp protein essential for DNA replication and repair. Due to this fundamental role, mutations in PCNA that profoundly impair protein function would be incompatible with life. Interestingly, while the p.Ser228Ile alteration appeared to have no effect on protein levels or DNA replication, patient cells exhibited marked abnormalities in response to UV irradiation, displaying substantial reductions in both UV survival and RNA synthesis recovery. The p.Ser228Ile change also profoundly altered PCNA’s interaction with Flap endonuclease 1 and DNA Ligase 1, DNA metabolism enzymes. Together, our findings detail a mutation of PCNA in humans associated with a neurodegenerative phenotype, displaying clinical and molecular features common to other DNA repair disorders, which we showed to be attributable to a hypomorphic amino acid alteration.     

Abnormal ultraviolet mutagenic spectrum in plasmid DNA replicated in cultured fibroblasts from a patient with the skin cancer-prone disease, xeroderma pigmentosum.

A shuttle vector plasmid, pZ189, was utilized to assess the types of mutations that cells from a patient with xeroderma pigmentosum, complementation group D, introduce into ultraviolet (UV) damaged, replicating DNA. Patients with xeroderma pigmentosum have clinical and cellular UV hypersensitivity, increased frequency of sun-induced skin cancer, and deficient DNA repair. In comparison to UV-treated pZ189 replicated in DNA repair-proficient cells, there were fewer surviving plasmids, a higher frequency of plasmids with mutations, fewer plasmids with two or more mutations in the marker gene, and a new mutagenic hotspot. The major type of base substitution mutation was the G:C to A:T transition with both cell lines. These results, together with similar findings published earlier with cells from a xeroderma pigmentosum patient in complementation group A, suggest that isolated G:C to A:T somatic mutations may be particularly important in generation of human skin cancer by UV radiation.     

Clinical, genetic and DNA repair studies on a consecutive series of patients with xeroderma pigmentosum

We report clinical, genetic and biochemical findings in 13 families with the photosensitive genodermatosis, xeroderma pigmentosum. All patients had a defect in repair of DNA damage provoked by ultraviolet radiation. Eleven patients and their three affected sibs were defective in the excision repair of UVR induced DNA lesions while the other two were defective in post-replication repair. One in the former group was diagnosed prior to the development of permanent skin abnormalities and preventive measures succeeded for almost five years in maintaining a normal appearing skin. In addition, two cases were diagnosed prenatally and aborted therapeutically. Some patients' parents showed slightly reduced repair of UVR induced DNA damage. In xeroderma pigmentosum (XP), the defect in the excision of DNA lesions appears to be due to homozygosity for one of at least seven different mutations and, accordingly, XP patients can be assigned to seven so-called complementation groups, A to G. Of these, groups A, C and D are the most common. Somatic cell fusion allowed three of the families reported here to be assigned to group A, four to group C and four to group D. Fibroblasts of patients from these three groups were shown to differ not only in the degree and kinetics of their residual DNA repair but also in the kinetics with which their defect is complemented by fusion with normal or XP cells of other groups. This confirms that mutations of different genes play a role in XP and provides a basis for understanding how such genes interact to secure repair of DNA lesions in normal cells. We discuss the phenotype of XP from different complementation groups in relation to the severe neurological abnormalities which may develop and must be considered in genetic counselling. We also discuss the biochemical anomalies of XP and the cellular effects of physical and chemical agents which damage DNA. In the practical management of XP, the importance of early differential diagnosis and prompt initiation of treatment is emphasized. Lastly we review the relationship between DNA repair and skin cancer in XP.     

Clinical, Genetic and DNA Repair Studies on a Consecutive Series of Patients with Xeroderma Pigmentosum

We report clinical, genetic and biochemical findings in 13 familieswith the photosensitive genodermatosis, xeroderma pigmentosum. All patients had a defect in repair of DNA damage provoked byultraviolet radiation. Eleven patients and their three affectedsibs were defective in the excision repair of UVR induced DNAlesions while the other two were defective in post-replicationrepair. One in the former group was diagnosed prior to the developmentof permanent skin abnormalities and preventive measures succeededfor almost five years in maintaining a normal appearing skin.In addition, two cases were diagnosed prenatally and abortedtherapeutically. Some patients' parents showed slightly reducedrepair of UVR induced DNA damage. In xeroderma pigmentosum (XP), the defect in the excision ofDNA lesions appears to be due to homozygosity for one of atleast seven different mutations and, accordingly, XP patientscan be assigned to seven so-called complementation groups, Ato G. Of these, groups A, C and D are the most common. Somaticcell fusion allowed three of the families reported here to beassigned to group A, four to group C and four to group D. Fibroblastsof patients from these three groups were shown to differ notonly in the degree and kinetics of their residual DNA repairbut also in the kinetics with which their defect is complementedby fusion with normal or XP cells of other groups. This confirmsthat mutations of different genes play a role in XP and providesa basis for understanding how such genes interact to securerepair of DNA lesions in normal cells. We discuss the phenotypeof XP from different complementation groups in relation to thesevere neurological abnormalities which may develop and mustbe considered in genetic counselling. We also discuss the biochemicalanomalies of XP and the cellular effects of physical and chemicalagents which damage DNA. In the practical management of XP,the importance of early differential diagnosis and prompt initiationof treatment is emphasized. Lastly we review the relationshipbetween DNA repair and skin cancer in XP.     

Carcinogenesis through abnormalities of DNA repair genes

Mechanisms of carcinogenesis through abnormalities of DNA repair genes are overviewed. Inactivation of DNA mismatch repair(MMR) gene(s) observed in tumors of hereditary non-polyposis colorectal cancer induces frameshift mutator mutation in MMR genes themselves, growth inhibitory genes and apoptosis inhibitory genes providing favorable genetic background for a malignant clone to be expanded. Deficiency of nucleotide excision repair that is usually employed for the removal of pyrimidine dimer formed by ultraviolet-irradiation in xeroderma pigmentosum (XP) causes hypersensitivity of the skin to sunlight as well as increased risk of skin cancer. Strand specificity and absence of hot spots for p53 tumor suppressor gene mutations was reported in ultraviolet induced skin cancers of XP model mice.     

Complementation of the DNA repair deficiency in human xeroderma pigmentosum group a and C cells by recombinant adenovirus-mediated gene transfer

Nucleotide excision repair (NER) is one of the most versatile DNA repair mechanisms, ensuring the proper functioning and trustworthy transmission of genetic information in all living cells. The phenotypic consequences caused by NER defects in humans are autosomal recessive diseases such as xeroderma pigmentosum (XP). This syndrome is the most sun-sensitive disorder leading to a high frequency of skin cancer. The majority of patients with XP carry mutations in the XPA or XPC genes that encode proteins involved in recognition of DNA damage induced by UV light at the beginning of the NER process. Cells cultured from XPA and XPC patients are hypersensitive to UV light, as a result of malfunctioning DNA repair. So far there is no effective long-term treatment for these patients. Skin cancer prevention can only be achieved by strict avoidance of sunlight exposure or by the use of sunscreen agents. We have constructed recombinant adenoviruses carrying the XPA and XPC genes that were used to infect XP-A and XP-C immortalized and primary fibroblast cell lines. UV survival curves and unscheduled DNA synthesis confirmed complete phenotypic reversion in XP DNA repair deficient cells with no trace of cytotoxicity. Moreover, transgene expression is stable for at least 60 days after infection. This efficient adenovirus gene delivery approach may be an important tool to better understand XP deficiency and the causes of DNA damage induced skin cancer.     

DNA修复基因多态性与胃癌易感性的关系

目的分析中国西南地区人群DNA修复基因XRCCl和XPD基因多态性分布,探讨XRCCl和XPD基因多态性与胃癌发病风险的关系。方法采用1：1病例对照研究方法,采用基因测序法检测160例胃癌患者及160例正常人外周血GSTPl基因XPDAsp312Asn,XRCClArgl94Trp,和XRCClArg280His多态性,进行胃癌风险分析。结果胃癌组与正常对照组相比,XPD312、XRCCl280位点的多态性分布频率无明显差异,而XRCCl194Trp的分布频率在病例组和对照组中分别为17．2％和7．3％,具有统计学差异。XRCCl194Trp等位基因的个体其胃癌风险增高（OR=2．72,95％CI=1．04~7．24,P=0．027）。结论XRCClArgl94Trp基因多态性可增加人群患胃癌的风险,XPDAsp312Asn、XRCClArg280His基因多态性与胃癌易感性无关联。     

The preservation of a DNA repair disorder in continuous-line fibroblasts obtained from gout patients

DNA repair was explored in continuous cells withdrawn from gout patients. The data obtained were compared to those on primary cells (lymphocytes) from the same patients. Two continuous lines of fibroblasts obtained from the biopsy material of patients suffering from gout were examined for stability of reparation defects on long cell passage. The studies were made with 4 to 12 passages of patients' fibroblasts. The use of criteria reflecting certain stages of DNA repair (reparative synthesis of DNA, formation of induced DNA ruptures and their resynthesis during cell postincubation, reactivation and induced mutagenesis of measles vaccine virus in patients' cells) allowed confirmation of repair defect stability in gout patients' cells on their long passage. Based on the data on preservation of the repair defect on cell passage it is concluded that gout patients demonstrate the genetically determined impairment of the synthesis of DNA repair enzymes participating in the recovery of DNA impairments induced by UV radiation or UV mimetics.     

Contribution of DNA polymerase eta to immunoglobulin gene hypermutation in the mouse

The mutation pattern of immunoglobulin genes was studied in mice deficient for DNA polymerase eta, a translesional polymerase whose inactivation is responsible for the xeroderma pigmentosum variant (XP-V) syndrome in humans. Mutations show an 85% G/C biased pattern, similar to that reported for XP-V patients. Breeding these mice with animals harboring the stop codon mutation of the 129/Olain background in their DNA polymerase iota gene did not alter this pattern further. Although this G/C biased mutation profile resembles that of mice deficient in the MSH2 or MSH6 components of the mismatch repair complex, the residual A/T mutagenesis of pol eta-deficient mice differs markedly. This suggests that, in the absence of pol eta, the MSH2-MSH6 complex is able to recruit another DNA polymerase that is more accurate at copying A/T bases, possibly pol kappa, to assume its function in hypermutation.     

ERCC1 mutations impede DNA damage repair and cause liver and kidney dysfunction in patients

ERCC1-XPF is a multifunctional endonuclease involved in nucleotide excision repair (NER), interstrand cross-link (ICL) repair, and DNA double-strand break (DSB) repair. Only two patients with bi-allelic ERCC1 mutations have been reported, both of whom had features of Cockayne syndrome and died in infancy. Here, we describe two siblings with bi-allelic ERCC1 mutations in their teenage years. Genomic sequencing identified a deletion and a missense variant (R156W) within ERCC1 that disrupts a salt bridge below the XPA-binding pocket. Patient-derived fibroblasts and knock-in epithelial cells carrying the R156W substitution show dramatically reduced protein levels of ERCC1 and XPF. Moreover, mutant ERCC1 weakly interacts with NER and ICL repair proteins, resulting in diminished recruitment to DNA damage. Consequently, patient cells show strongly reduced NER activity and increased chromosome breakage induced by DNA cross-linkers, while DSB repair was relatively normal. We report a new case of ERCC1 deficiency that severely affects NER and considerably impacts ICL repair, which together result in a unique phenotype combining short stature, photosensitivity, and progressive liver and kidney dysfunction.     

Virus shedding by SV40-transformed human cells.

Cultures of human cells transformed by SV40 were found to release infectious virus, even after several passages in vitro. Virus shedding by these cultures did not depend on propagation of virus from cell to cell, as it was not affected by anti-SV40 antiserum that could effectively block virus propagation in acutely infected cells. Cells transformed by the 'early' temperature-sensitive mutant tsA30, and maintained at the restrictive temperature of 39 degrees, shed virus in reduced amount. Finally, xeroderma pigmentosum cells transformed by SV40 were also found to release virus, indicating that the enzymes of excision and repair of UV-induced damage to DNA probably were not involved in the molecular mechanism underlying virus shedding.     

Targeting DNA damage and repair: embracing the pharmacological era for successful cancer therapy

DNA is under constant assault from genotoxic agents which creates different kinds of DNA damage. The precise replication of the genome and the continuous surveillance of its integrity are critical for survival and the avoidance of carcinogenesis. Cells have evolved an arsenal of repair pathways and cell cycle checkpoints to detect and repair DNA damage. When repair fails, typically cell cycle progression is halted and apoptosis is initiated. Here, we review the different sources and types of DNA damage including DNA replication stress and oxidative stress, the repair pathways that cells utilize to repair damaged DNA, and discuss their biological significance, especially with reference to cancer induction and cancer therapy. We also describe the main methodologies currently used for the detection of DNA damage with their strengths and limitations. We conclude with an outline as to how this information can be used to identify novel pharmacological targets for DNA repair pathways or enhancers of DNA damage to develop improved treatment strategies that will benefit cancer patients.     

DNA polymerase eta is involved in hypermutation occurring during immunoglobulin class switch recombination

Base substitutions, deletions, and duplications are observed at the immunoglobulin locus in DNA sequences involved in class switch recombination (CSR). These mutations are dependent upon activation-induced cytidine deaminase (AID) and present all the characteristics of the ones observed during V gene somatic hypermutation, implying that they could be generated by the same mutational complex. It has been proposed, based on the V gene mutation pattern of patients with the cancer-prone xeroderma pigmentosum variant (XP-V) syndrome who are deficient in DNA polymerase eta (pol eta), that this enzyme could be responsible for a large part of the mutations occurring on A/T bases. Here we show, by analyzing switched memory B cells from two XP-V patients, that pol eta is also an A/T mutator during CSR, in both the switch region of tandem repeats as well as upstream of it, thus suggesting that the same error-prone translesional polymerases are involved, together with AID, in both processes.     

Genetic correction of DNA repair-deficient/cancer-prone xeroderma pigmentosum group C keratinocytes

Xeroderma pigmentosum (XP) is a rare photosensitive and cancer-prone syndrome transmitted as an autosomal recessive trait. Most cancers developed by XP patients are basal and squamous cell carcinoma strikingly restricted to sun-exposed parts of the skin. Cells from patients with classic XP are deficient in nucleotide excision repair, a versatile biochemical mechanism for removal of ultraviolet-induced DNA lesions. Among the seven classic XP complementation groups known to date (XP-A to XP-G), XP-C is the most common one in Europe and North Africa and XP-C patients remain free of neurologic problems often seen in other XP complementation groups. This has prompted us to undertake genetic correction of XP-C fibroblasts and particularly keratinocytes, which are the most relevant cells in relation to skin cancer and have proven recently to be capable of reconstructing XP-C skin in vitro. In this study, we demonstrate that DNA repair capacity, cell survival properties, and transition from proliferative to abortive keratinocyte colonies toward UVB irradiation can be fully recovered in keratinocytes from patients with XPC transduced with a retroviral vector stably driving the expression of the wild-type XPC protein. In addition, we show that in the absence of UV, XP-C keratinocytes exhibit intrinsic cell cycle abnormalities, and beta(1)-integrin overexpression, defects that are also both fully reversed after genetic correction. Together with full correction of the defects in XP-C corrected keratinocytes, in vitro reconstruction of skin from these cells open a rational perspective to XP tissue therapy.     

Induction and repair of DNA double-strand breaks using constant-field gel electrophoresis and apoptosis as predictive markers for sensitivity of cancer cells to cisplatin

This study was designed to evaluate some parameters that may play a role in the prediction of cancer cells sensitivity to cisplatin (CIS). Sensitivity, induction and repair of DNA double-strand breaks (DSB), cell cycle regulation and induction of apoptosis were measured in four cancer cell lines with different sensitivities to CIS. Using a sulphorhodamine-B assay, the cervical carcinoma cells (HeLa) were found to be the most sensitive to CIS followed by breast carcinoma cells (MCF-7) and liver carcinoma cells (HepG2). Colon carcinoma HCT116 cells were the most resistant. As measured by constant-field gel electrophoresis (CFGE), DSB induction, but not residual DSB exhibited a significant correlation with the sensitivity of cells to CIS. Flow cytometric DNA ploidy analysis revealed that 67% of HeLa cells and 10% of MCF-7 cells shift to sub-G1 phase after incubation with CIS. Additionally, CIS induced the arrest of MCF-7 cells in S-phase and the arrest of HepG2 and HCT116 cells in both S phase and G2/M phase. Determination of the Fas-L level and Caspase-9 activity indicated that CIS-induced apoptosis results from the mitochondrial (intrinsic) pathway. These results, if confirmed using clinical samples, indicate that the induction of DNA DSB as measured by CFGE and the induction of apoptosis should be considered, along with other predictive markers, in future clinical trials to develop predictive assays for platinum -based therapy.     

Effect of cyclosporin A on DNA repair and cancer incidence in kidney transplant recipients

Cancer incidence is enhanced in transplant recipients. Decreased DNA repair ability is associated with increased cancer incidence. Transplanted patients with cancer were found to have reduced DNA repair. We hypothesized that immunosuppressive therapy may impair DNA repair and thus contribute to the increased cancer incidence in transplanted patients. The objectives of this study were (1) to investigate the effect of two immunosuppressive treatment protocols on DNA repair in kidney transplant recipients; (2) to evaluate the cancer incidence in these patients; and (3) to study the in vitro effect of cyclosporin A (CsA), azathioprine, and prednisolone-separately and in various combinations-on DNA repair. Three groups were studied: (1) a control group; (2) patients treated with azathioprine and prednisone (double-therapy group); and (3) patients treated with CsA, azathioprine, and prednisone (triple-therapy group). The two patient groups did not differ in age, gender, time on dialysis before transplantation, or kidney function or in the number of acute rejections. However, the interval from transplantation to the DNA repair study was shorter in the triple-therapy group (P <.01). DNA repair was induced in peripheral blood mononuclear cells (PBMCs) by ultraviolet irradiation and expressed as tritiated thymidine uptake by these cells. DNA repair in the triple-therapy group was 679 +/- 64 cpm/10(6) cells, significantly less than that in the control group (1049 +/- 69 cpm/10(6) cells, P <.02). In the double-therapy group, DNA repair was similar to that in the control group. The follow-up period was shorter in the triple-therapy group (116 +/- 19 months vs 174 +/- 29 months, P <.01). Five tumors developed in the triple-therapy group, but only one developed in the double-therapy group (P =.05). The in vitro study showed a dose-dependent reduction in PBMC DNA repair by CsA. Azathioprine and prednisolone reduced DNA repair slightly, but CsA reduced DNA repair significantly more than either one or a combination of them. In summary, triple therapy was associated with impaired PBMC DNA repair and increased cancer incidence. CsA was responsible in large part for the reduction in DNA repair ability found in the in vitro and in vivo studies. This may have partly contributed to the enhanced cancer incidence in the kidney transplant recipients.     

Oncogenic transformation by cellular DNA isolated from human cytomegalovirus-infected cells.

Delayed replication of human cytomegalovirus (CMV) was initiated in human embryonic fibroblasts using partially ultraviolet light-inactivated virus stock. Cellular [high molecular weight (HMW)] DNA extracted from CMV-infected cell cultures demonstrated a substantial increase in transforming activity after introduction into hamster embryo fibroblasts relative to HMW DNA extracted from mock-infected cells. The transforming activity of HMW DNA varied between 0.01 and 0.25 foci/micrograms DNA. HMW DNA isolated from CMV-infected cells after initiation of viral DNA synthesis demonstrated a significant decrease in the induction of morphologically transformed foci. The transforming activity of HMW DNA was unaffected by HindIII or XbaI endonuclease digestion, but it was sensitive to sonication and EcoRI endonuclease and DNase treatment. Six cell lines were established from the foci of morphologically altered cells. Cells of these lines demonstrated loss of contact inhibition, replication in semisolid agarose or in medium containing low serum, a high saturation density, and tumorigenicity when implanted into hamsters. Histopathological examination of the tumors identified the tissues as fibrosarcomas. In situ hybridization of cells isolated from foci of morphologically altered cells or Southern blot analysis of DNA isolated from either the cell lines or tumors did not demonstrate the presence of sequences with homology to viral DNA using the transforming region or the entire viral DNA as a probe. The lack of viral DNA sequences as well as the similar phenotypic characteristics of cell lines and tumors suggest that alteration(s) induced by CMV may occur in specific region(s) of cellular DNA.     

Quantitation of DNA repair capacities of human tumor cells by estimation of transfer of DNA adducts to repair proteins

The repair of O6-methylguanine produced in DNA by alkylating agents is accomplished by a unique lesion reversal mechanism which recognizes the methyl group and transfers it to itself in a suicide reaction. Much of what we know about the importance of O6-methylguanine-DNA methyltransferase repair in human cells comes from the study of Mer- tumor cell strains which are deficient in transferase activity. The human transferase has a preference for repair of methyl groups, but will also act on other substrates. Assays for transferase activity detect either the loss of O6-methylguanine from DNA or the appearance of methylated protein. A new assay detects the recovery of a restriction site in a synthetic polymer following demethylation. Inhibition of transferase activity can be produced in cells by several methods.