XP Complementation Groups Research Articles

Replication-related genes are upregulated in XP-A cells after UV-C irradiation

BackgroundXeroderma pigmentosum (XP) is hereditary disorder characterized by photosensitivity, predisposition to skin cancers of sun-exposed body sites and progressive neurologic symptoms in some cases. Cells from XP patients show higher sensitivity to ultraviolet radiation (UV) than normal cells. ObjectiveWe aimed to ascertain the genes differentially regulated in XP complementation group A (XP-A) cells after UV irradiation. MethodsXP-A cells were harvested at 4 or 12 h after a single exposure to low-dose UV-C radiation and subjected to transcriptome analysis by microarray. ResultsThe number of genes with significantly altered expression (≥2-fold difference) at 12 h was markedly higher in XP-A cells than that in normal cells, suggesting that the number of altered genes could be correlated to the amount of DNA damage. ConclusionWe recently reported that mitotic genes are induced in normal human fibroblasts after UV-C exposure, and similar results were observed in XP-A cells as normal cells. In addition, a majority of replication-related genes were significantly upregulated in XP-A cells, whereas no such expression pattern was observed in the normal control cells. Collectively, these results indicate that the XPA protein can transcriptionally inhibit the series of replication-related genes, and could possibly regulate replication and/or re-replication after UV irradiation.

Quantitative analysis of brain atrophy in patients with xeroderma pigmentosum group A carrying the founder mutation in Japan

IntroductionXeroderma pigmentosum (XP) is an inherited congenital disease presenting with dermatological and neurological manifestations. In Japan, XP complementation group A (XP-A) is most frequently observed in eight clinical subtypes, and the homozygous founder mutation, IVS3-1G>C in XPA, suffer from severe manifestations including progressive brain atrophy since childhood. In this study, we used magnetic resonance imaging (MRI) and applied volumetric analysis to elucidate the start and the progression of the brain atrophy in these patients. Material and methodsTwelve Japanese patients with XP-A carrying the founder mutation and seven controls were included. MRI was performed for each patient once or more. Three-dimensional T1 weighted images were segmented to gray matter, white matter, and cerebrospinal fluid, and each volume was calculated. ResultsConventional MRI demonstrated progressive whole brain atrophy in patients with XP-A. Moreover, volumetric analysis showed that reductions of total gray matter volumes (GMV) and total brain volumes (TBV) started at the age of five. The slope of reduction was similar in all cases. The GMV and TBV values in controls were higher than those in XP-A cases after the age of five. ConclusionsThis is the first quantitative report presenting with the progression of brain atrophy in patients with XP-A. It is revealed that the brain atrophy started from early childhood in Japanese patients with XP-A carrying the homozygous founder mutation.

The Role of Altered Nucleotide Excision Repair and UVB-Induced DNA Damage in Melanomagenesis

UVB radiation is the most mutagenic component of the UV spectrum that reaches the earth’s surface and causes the development of DNA damage in the form of cyclobutane pyrimidine dimers and 6-4 photoproducts. UV radiation usually results in cellular death, but if left unchecked, it can affect DNA integrity, cell and tissue homeostasis and cause mutations in oncogenes and tumour-suppressor genes. These mutations, if unrepaired, can lead to abnormal cell growth, increasing the risk of cancer development. Epidemiological data strongly associates UV exposure as a major factor in melanoma development, but the exact biological mechanisms involved in this process are yet to be fully elucidated. The nucleotide excision repair (NER) pathway is responsible for the repair of UV-induced lesions. Patients with the genetic disorder Xeroderma Pigmentosum have a mutation in one of eight NER genes associated with the XP complementation groups XP-A to XP-G and XP variant (XP-V). XP is characterized by diminished repair capacity, as well as a 1000-fold increase in the incidence of skin cancers, including melanoma. This has suggested a significant role for NER in melanoma development as a result of UVB exposure. This review discusses the current research surrounding UVB radiation and NER capacity and how further investigation of NER could elucidate the role of NER in avoiding UV-induced cellular death resulting in melanomagenesis.

Expression and Purification of the Human Xeroderma Pigmentosum F (XPF/ERCC1) Complex in Bacterial Cells for Protein Binding Assays and Crystallographic Studies

Mutations in dna repair enzymes of the XP complementation group, XPA‐XPG, leads to a number of genetic disorders such as Xeroderma Pigmentosum, Cockayne's Syndrome, and Trichothiodystrophy. The structure specific dna repair endonuclease responsible for the 5′ incision during dna repair, XPF, is involved in homologous recombination that assists in removing interstrand cross‐link. XP‐F is an autosomal recessive disease characterized by hypersensitivity of the skin to sunlight followed by high incidence of skin cancer and frequent neurologic abnormalities. I expressed and purified human recombinant XPF and ERCC1 proteins in E.Coli Rosetta and BL21 cells, these results will be scaled up for use in interaction and crystallographic studies with proteins of the XP family.

Suppression of UV-induced apoptosis by the human DNA repair protein XPG

The severe xeroderma pigmentosum/Cockayne syndrome (XP/CS) syndrome is caused by mutations in the XPB, XPD and XPG genes that encode the helicase subunits of TFIIH and the 3' endonuclease of nucleotide excision repair (NER). Because XPB and XPD have been implicated in p53-mediated apoptosis, we examined the possible involvement of XPG in this process. After ultraviolet light (UV) irradiation, primary fibroblasts of XP complementation group G (XP-G) individuals with CS enter apoptosis more readily than other NER-deficient cells, but this is unlinked to unrepaired damage. These XP-G/CS cells accumulate p53 post-UV but they fail to accumulate the 90/92 kDa isoforms of Mdm2 and their cellular distribution of Mdm2 is impaired. Apoptosis levels revert to wild type, Mdm2 90/92 kDa isoforms accumulate, and Mdm2 regains its normal post-UV nuclear location in transduced XP-G/CS cells expressing wild-type XPG, but not an XPG catalytic site mutant. These results suggest that XPG suppresses UV-induced apoptosis and that this suppression, most simply, requires its endonuclease function.

A re-examination of the intragenome distribution of repaired sites in proliferating xeroderma pigmentosum complementation group C fibroblasts

We find that rapidly proliferating fibroblasts from xeroderma pigmentosum complementation group C (XP-C) patients, cells that have a small residual DNA excision repair capacity, repair DNA in localized regions of the genome in a clustered pattern rather than at single sites in dispersed locations. This finding is similar to that observed earlier for nodividing cells but is in contrast to published results that indicate that the residual repair in proliferating XP-C cells is dispersed throughout the genome in a non-clustered pattern. While we detect the same amount of repair in both proliferating and nondividing cells, we also observe no shift from the clustered pattern of repair to a more dispersive pattern when nondividing cells are stimulated to proliferate by fresh serum addition. We have no obvious explanation for these discrepancies with the published results. We have noted previously that proliferating XP-C cells are very UV sensitive relative to normal cells while nondividing cells that exhibit the same amount of repair activity are relatively UV resistant. There is not satisfactory explanation for this change in relative response to the lethal effects of UV, a change not observed for cell strains from other XP complementation groups. However, we argue that clustered repair in specific genomic regions promotes survival in nondividing XP-C cells but does not promote survival in proliferating cells.

Transformation and immortalization of diploid xeroderma pigmentosum fibroblasts

Diploid xeroderma pigmentosum (XP) skin fibroblast strains from various XP-complementation groups (B, C, G, and H) were transformed with an origin-defective SV40 early region or with the pSV3gpt plasmid. In the latter case, transfected cells were selected for their ability to express the dominant xgpt gene. Immortalized cell lines were obtained from XP-complementation groups C (8CA, 3MA, and 20MA; XP3MA and XP20MA were formerly considered to belong to complementation group I), G (2BI and 3BR), and H (2CS). No immortalized cells could be isolated from complementation group B (11BE). The immortalization frequency of wild-type diploid fibroblasts and diploid cultures from XP patients was not significantly increased by cotransfection with the SV40 early region plus several selected viral and cellular oncogenes. In fact, co-transfection with some of the oncogenes caused a marked decrease of the transformation frequency. The observed immortalization occurred at a frequency of approximately 5 × 10 −8.

Heat effects on UV-killing of cultured normal and XP complementation group A (XP-A) fibroblasts

Heat effects on UV-killing of cultured normal and XP complementation group A (XP-A) fibroblasts

Relationship between pyrimidine dimers, 6-4 photoproducts, repair synthesis and cell survival: Studies using cells from patients with trichothiodystrophy

Trichothiodystrophy is a genetic disease which in the majority of cases studied is associated with a deficiency in the ability to repair UV damage in cellular DNA. Three categories of UV response have been identified. In type 1 the response is completely normal, whereas type 2 cells are deficient in excision-repair, with properties indistinguishable from those of XP complementation group D. Type 3 cells have normal survival following UV-irradiation and normal rates of removal of cyclobutane pyrimidine dimer sites. Nevertheless repair synthesis is reduced by 50% in these cell strains and this is associated with a marked reduction in the repair of 6-4 photoproducts from cellular DNA. The present results show that 50% or more of repair synthesis at early times after irradiation of normal primary human fibroblasts is attributable to repair of 6-4 products. They also suggest that repair of cyclobutane dimers is crucial for cell survival.

Xeroderma pigmentosum patients from Germany: repair capacity of 45 XP fibroblast strains of the Mannheim XP Collection as measured by colony-forming ability and unscheduled DNA synthesis following treatment with methyl methanesulfonate and N-methyl-N-nitrosourea.

A total of 45 XP fibroblast strains from the Mannheim XP Collection (representatives of XP complementation groups A, C, D, E, F or G, I, and XP variants) were investigated for colony-forming ability (term: D0) after treatment with up to ten doses of the methylating carcinogen MeSO2OMe. As controls 16 fibroblast strains from normal donors were used. Except for 4 XP strains (1 from group C and 3 from group D) which, however, were borderline cases, none of the remaining 41 XP strains was found to be more sensitive than normal controls. This held true within the limits of an experimental accuracy (experimental variability of D0 values) of +/- 7%. When weighted means were calculated for XP complementation groups and compared with that of normal donors at a significance level of 5%, no significant difference was detected. In contrast, after exposure of 6 XP group D strains to MeNOUr, a weighted mean D0 value was obtained which was significantly decreased by 27%. Unscheduled DNA synthesis (term: G0 which serves as a measure of excision repair) after exposure to MeNOUr was quantitatively the same (experimental variability: +/- 8%) both in the group of normal strains and in most of the XP complementation groups. Exceptions were group E and group F (or G) which had higher, and group I which had lower repair. Analogous G0 values measured after exposure to MeSO2OMe (experimental variability: +/- 13%), however, differed from that of the control strains: they were lower in XP complementation groups A, D, E, F (or G), and I. However, groups A, E, F (or G), and I including only 3 individual strains or less may be considered to be possibly ill-represented. Yet, group D including 11 XP strains did show reduction of the mean G0 value by 35%. From this it is concluded that there are repair defects in XP group D strains with regard to MeSO2OMe-induced adducts. These defects seem to be small.

Microinjection of Escherichia coli UvrA, B, C and D proteins into fibroblasts of xeroderma pigmentosum complementation groups A and C does not result in restoration of UV-induced unscheduled DNA synthesis

The UV-induced unscheduled DNA synthesis (UDS) in cultured human fibroblasts of repair-deficient xeroderma pigmentosum complementation groups A and C was assayed after injection of identical activities of either Uvr excinuclease (UvrA, B, C and D) from Escherichia coli or endonuclease V from phage T4. Under conditions where the T4 enzyme was able to induce repair synthesis in both XP complementation groups in agreement with earlier observations (de Jonge et al., 1985), no effect of the UvrABCD excinuclease could be observed either when the enzymatic complex was injected into the cytoplasm, or when it was delivered directly into the nucleus. In additon, no effect of the E. coli excinuclease was found on the repair ability of normal repair-proficient human fibroblasts. We conclude that the UvrABCD excinuclease may not work on DNA lesions in human chromatin.

A further definition of characteristics of DNA-excision repair in xeroderma pigmentosum complementation group A strains

The location in the genome of excision repair following exposure to UV (254 nm) of two XP complementation group A strains, XP12BE and XP8LO, that differ considerably in their excision-repair rates, have been determined. Capacity for repair in XP8LO has also been determined. Sites repaired in DNA in a 24-h post-UV period were located relative to the remaining pyrimidine dimers using the M. luteus UV-endonuclease to nick partially repaired DNA and sedimentation in alkaline sucrose to size the resulting DNA. Repair in group A occurs randomly throughout the genome in a manner similar to that observed for normal cells but in contrast to domain-limited repair in group C strains. This observation defines a further similarity of the excision repair detected in group A compared to normal cells that is in addition to the previously reported related characteristics of the respective excision rate curves. A reduced repair capacity in XP8LO relative to normal cells was detected. This strain, which repairs DNA at an initial rate identical to that of normal strains when irradiated with doses of 5 J/m 2 or less, repairs DNA at a slower than normal but constant rate at higher doses. This leads to the suggestion that XP8LO is defective in the number of repair enzyme complexes compared to normal cells.

Interspecies complementation analysis of xeroderma pigmentosum and UV-sensitive chinese hamster cells

Complementation analysis was performed 24 h after fusion of UV-sensitive CHO cells (CHO 12 RO) with XP cells of complementation groups A, B, C, D, F and G. The parental cells are characterized by low levels of unscheduled DNA synthesis (UDS). In all combinations, the UDS levels observed in heterokaryons were higher than those in parental mutant cells, clearly indicating cooperation of human and Chinese hamster repair functions. In heterokaryons of CHO 12 RO with XPA and XP-C cells, the UDS values reached about the normal human level, whereas in heterokaryons with XP-B, XP-D and XP-F, UDS was restored at a level approaching that in wild-type CHO cells. The results obtained after fusion of CHO cells with two representative cell strains from the XP-G group, XP 2 BI and XP 3 BR, were inconsistent. Fusion with XP 3 BR cells yielded UDS levels ranging from wild-type Chinese hamster to normal human, whereas fusion with XP 2 BI cells resulted in a slight increase in UDS which even after 48 h remained below the level found in wild-type CHO cells. The occurrence of complementation in these interspecies heterokaryons indicates that the genetic defect in the CHO 12 RO cells is different from the defects in the XP complementation groups tested.

Factors modifying 3-aminobenzamide cytotoxicity in normal and repair-deficient human fibroblasts.

3-Aminobenzamide (3-AB), an inhibitor of poly(ADP-ribosylation), is lethal to human fibroblasts with damaged DNA. Its cytotoxicity was determined relative to a number of factors including the types of lesions, the kinetics of repair, and the availability of alternative repair systems. A variety of alkylating agents, UV or gamma irradiation, or antimetabolites were used to create DNA lesions. 3-AB enhanced lethality with monofunctional alkylating agents only. Within this class of compounds, methylmethanesulfonate (MMS) treatments made cells more sensitive to 3-AB than did treatment with methylnitrosourea (MNU) or methylnitronitrosoguanidine (MNNG). 3-AB interfered with a dynamic repair process lasting several days, since human fibroblasts remained sensitive to 3-AB for 36-48 hours following MMS treatment. During this same interval, 3-AB caused these cells to arrest in G2 phase. Alkaline elution analysis also revealed that this slow repair was delayed further by 3-AB. Human mutant cells defective in DNA repair differed in their responses to 3-AB. Among mutants sensitive to monofunctional alkylating agents, ataxia telangiectasia cells were slightly more sensitive to 3-AB than control cells, while Huntington's disease cells had a near-normal response. Among UV-sensitive strains, xeroderma pigmentosum variant (XPV) cells were more sensitive to 3-AB after MMS than were XP complementation group A (A) cells, which responded normally. Greater lethality with 3-AB could be dependent on inability of the mutant cells to repair damage by other processes.

Defective thymine dimer excision from xeroderma pigmentosum chromatin and its characteristic catalysis by cell-free extracts.

Specific excision of thymine dimers from isolated normal human and xeroderma pigmentosum (XP complementation groups A, C, D and G) chromatin was investigated under cell-free conditions. Crude extracts derived from unirradiated XP groups A, C and G cells were unable to excise dimers from their own nuclear sonicates, native chromatin and whole-cell sonicates prepared after exposure to 100 J/m2 of u.v. radiation at 254 nm, while normal-cell extracts were able to do so from all substrates including purified DNA. However, the extracts of XP groups A, C and G cells became capable of excising thymine dimers from chromatin preparations depleted of loosely bound nonhistone proteins with 0.35 M NaCl and from purified DNA. Extracts of XP group D cells catalyzed normal levels of excision from nuclear sonicates, native chromatin and 0.35 M NaCl-treated chromatin. These results suggest that none of the XP groups examined is deficient in a dimer-specific u.v. endonuclease. XP groups A, C and G cells are apparently defective in 'XP factors' present in the non-histone protein fraction, which are required for the excision of thymine dimers from chromatin. The XP group D factor appears to be different from the others. Extracts from XP groups A, C and G cells were able to complement each other with respect to dimer excision from chromatin. Novobiocin (200 micrograms/ml) completely inhibited dimer excision effected by extracts of normal cells or by complementing extracts of XP cells.

Three complementation groups in Cockayne syndrome.

After 16 Jm-2 of UV-irradiation non-dividing normal cells recover normal rates of RNA synthesis within 24 h, whereas in cells from donors with Cockayne syndrome (CS) the rate of RNA synthesis gradually declines. Cultures of a mixed population from 2 CS donors were fused with polyethylene glycol; subsequently they were UV-irradiated and RNA synthesis was measured autoradiographically in mono-, bi-, and multinuclear cells. Genetic complementation was indicated by high levels of RNA synthesis in bi- and multinuclear cells when compared with mononuclear cells. Using this assay, 11 CS strains have been assigned to three complementation groups: 2 into group A, 8 into group B and 1 into group C. The strain in group C is derived from an individual who also had xeroderma pigmentosum (XP), and was the sole known representative of XP-complementation group B.

DNA strand breaking and rejoining in response to ultraviolet light in normal human and xeroderma pigmentosum cells.

We describe a reproducible technique for measuring DNA strand breaking and rejoining in cells after treatment with U.V.-light. Results obtained with normal human cells, xeroderma pigmentosum cells (XP, complementation group A) and XP variant cells suggest that all three of these cell-types can carry out single-strand incision with equal rapidity. However, the breaks so induced appeared to be only slowly rejoined in the XP variant cells and rejoined not at all in XP complementation group A cells. Furthermore, parental strand rejoining was inhibited by caffeine in XP variant cells but not in normal cells.

Repair by human cells of adenovirus-2 damaged by psoralen plus near ultraviolet light treatment

Adenovirus-2 is damaged by treatment with psoralen plus near-ultraviolet (UV) light as shown by reduced ability to infect human fibroblasts. The apparent sensitivity of the virus to this treatment depended upon the strain of cells used. The virus was 3–4 times more sensitive to the treatment when infecting xeroderma pigmentosum (XP complementation groups A or D) fibroblasts than when infecting normal fibroblasts. DNA extracted from virus preparations that had undergone such treatment was analyzed for treatment-induced crosslinks by gel electrophoresis and sedimentation in alkaline sucrose gradients. The fraction of adenovirus DNA molecules remaining non-crosslinked after treatment was found to correlate with the survival of the virus in normal fibroblasts. This result showed that the psoralen plus near-UV treatment gave rise to non-cross-link lesions (presumably psoralen-DNA mono-adducts), that were repairable by normal but not by XP fibroblasts, and suggested the possibility that normal fibroblasts cannot repair this type of crosslink in the DNA of an infecting adenovirion.