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1.
Xeroderma pigmentosum variant (XPV) patients with mutations in the DNA polymerase eta (pol eta) gene are hypersensitive to sunlight and have greatly increased susceptibility to sunlight-induced skin cancer. Consistent with the ability of Pol eta to efficiently bypass UV light-induced cyclobutane pyrimidine dimers, XPV cells lacking Pol eta have diminished capacity to replicate UV-damaged DNA and are sensitive to UV light-induced killing and mutagenesis. To better understand these and other Pol eta functions, we generated Pol eta-deficient mice. Mice homozygous for a null mutation in pol eta are viable, fertile, and do not show any obvious spontaneous defects during the first year of life. However, fibroblasts derived from these mutant mice are sensitive to killing by exposure to UV light, and all Pol eta-deficient mice develop skin tumors after UV irradiation, in contrast to the wild-type littermate controls that did not develop such tumors. These results and biochemical studies of translesion synthesis by mouse Pol eta indicate that Pol eta-dependent bypass of cyclobutane pyrimidine dimers suppresses UV light-induced skin cancer in mice. Moreover, 37.5% of pol eta heterozygous mice also developed skin cancer during 5 months after a 5-month exposure to UV light, suggesting that humans who are heterozygous for mutations in pol eta may also have an increased risk of skin cancer.  相似文献   

2.
Cancer chemopreventive effects of polyphenols from green tea (GTP) in mouse models of photocarcinogenesis are established. The present study is extended from mouse model to human system in vivo to determine the effect of topical application of GTP to human individuals against UV light-induced DNA damage in the form of cyclobutane pyrimidine dimers (CPDs) in the skin. UVB-induced CPDs were detected by immunohistochemical technique using monoclonal antibodies to thymine dimers. With the gradual increase in UVB dose, both erythema response and CPD formation in the skin was increased. GTP treatment inhibited both UVB-induced erythema response as well as CPD formation. Topical treatment with GTP (approximately 1 mg/cm2 of skin area) 20 min before human buttock skin (sun-protected site) exposure to UVB inhibited CPD formation in epidermis by 81, 70, 60, and 60% at 0.5, 1.0, 2.0, and 4.0 minimal erythema dose of UV exposure, respectively. Treatment of human skin with varying doses of GTP (1-4 mg/2.5 cm2 of skin area) before a single dose of UVB exposure (4.0 minimal erythema dose) decreased dose dependently the formation of UVB-induced CPDs in both epidermis and dermis. The inhibition of UVB-induced CPDs by GTP treatment may be, at least in part, responsible for the inhibition of photocarcinogenesis. Our data suggest that GTP may be used as a novel chemopreventive candidate and possible strategy to reduce UV-induced skin cancer risk in the human population.  相似文献   

3.
Chronic exposure to sunlight causes skin cancer in humans, yet little is known about how habitual exposure to low doses of ultraviolet B radiation (UVB) affects DNA damage in the skin. We treated Skh-1 hairless mice with daily doses of suberythemal UVB for 40 days and analyzed the amount and distribution of DNA photodamage using RIAs and immunofluorescence micrography. We found that DNA damage accumulated in mouse skin as a result of chronic irradiation and that this damage persisted in the dermis and epidermis for several weeks after the chronic treatment was terminated. Although the persistent damage was evenly distributed throughout the dermis, it remained in the epidermis as a small number of heavily damaged cells at the dermal-epidermal boundary. Rates of DNA damage induction and repair were determined at different times over the course of chronic treatment in response to a higher challenge dose of UVB light. The amount of damage induced by the challenge dose increased in response to chronic exposure, and excision repair of cyclobutane pyrimidine dimers and pyrimidine(6-4)pyrimidone dimers was significantly reduced. The sensitization of mouse epidermal DNA to photoproduct induction, the reduction in excision repair, and the accumulation of nonrepairable DNA damage in the dermis and epidermis suggest that chronic low-dose exposure to sunlight may significantly enhance the predisposition of mammalian skin to sunlight-induced carcinogenesis.  相似文献   

4.
5.
Photolyase absorbs blue light and employs the energy to remove UV-induced DNA damage, cyclobutane pyrimidine dimers, or pyrimidine pyrimidone (6-4) lesions. These enzymes have been found in many living organisms ranging from bacteria to aplacental mammals, but their photoreactivation effect, such as survival increase of UV-irradiated cells by light-illumination, has not been identified in placental mammals, including humans. Therefore, we introduced a photolyase gene derived from the marsupial rat kangaroo, Potorous tridactylus, into HeLa cells and established the first human cell line capable of photorepairing UV-induced pyrimidine dimers. Several clones were found to increase cell survival after UV irradiation when illuminated by fluorescent light. The induction of apoptosis by UV irradiation was investigated in these photoreactivation-proficient cells. Several typical features of the programmed cell death, such as internucleosomal DNA degradation, presence of subdiploid cells, loss of membrane integrity, and chromosomal condensation, were found to be induced by UV in the HeLa cells, but they can be reduced by photorepair. This implicates that cyclobutane pyrimidine dimers cause UV-induced apoptosis in human cells.  相似文献   

6.
Wavelength dependence of oxidative DNA damage induced by UV and visible light   总被引:17,自引:2,他引:17  
Kielbassa  C; Roza  L; Epe  B 《Carcinogenesis》1997,18(4):811-816
DNA damage induced by UV radiation and visible light (290-500 nm) in AS52 Chinese hamster cells was analysed by an alkaline elution assay with specific repair endonucleases. Cells were exposed to extensively filtered monochrome or broad-band radiation. Between 290 and 315 nm, the ratio of base modifications sensitive to Fpg protein (i.e. 8- hydroxyguanine and formamidopyrimidines) and T4 endonuclease V (i.e. cyclobutane pyrimidine dimers) was constant (approximately 1:200), indicating that the direct excitation of DNA is responsible for both types of damage in this range of the spectrum. While the yield of pyrimidine dimers per unit dose continued to decrease exponentially beyond 315 nm, the yield of Fpg-sensitive modifications increased to a second maximum between 400 and 450 nm. The damage spectrum in this wavelength range consisted of only a few other modifications (strand breaks, abasic sites and pyrimidine modifications sensitive to endonuclease III) and is attributed to endogenous photosensitizers that give rise to oxidative DNA damage via singlet oxygen and/or type I reactions. The generation of Fpg-sensitive modifications by visible light was not linear with dose but followed a saturation curve. It is calculated that the exposure of the cells to low doses of solar radiation results in the formation of cyclobutane pyrimidine dimers and Fpg-sensitive modifications in a ratio of 10:1.   相似文献   

7.
Sunscreens are known to protect against sunlight-induced erythema and sunburn, but their efficiency at protecting against skin cancer is still a matter of debate. Specifically, the capacity of sunscreens to prevent or reduce tissue and DNA damage has not been thoroughly investigated. The present study was undertaken to assess the ability of a chemical broad-spectrum sunscreen to protect human skin against tissue and DNA damage after solar UV radiation. Engineered human skin was generated and either treated or not with a broad-spectrum SPF 30 sunscreen and exposed to increasing doses of simulated sunlight (SSL). Immediately after irradiation, histological, immunohistochemical, and molecular quantitative analyses were performed. The unprotected irradiated engineered human skin showed significant epidermal disorganization accompanied by a complete absence of laminin deposition. The sunscreen prevented SSL-induced epidermal damage at low doses and allowed laminin deposition at almost all SSL doses tested. The frequencies of cyclobutane pyrimidine dimers, pyrimidine (6-4) pyrimidone photoproducts, and photooxidative lesions measured by alkaline gel electrophoresis and radioimmunoassay were significantly reduced by the sunscreen. Thus, tissue and DNA damage may provide excellent quantitative end points for assessing the photoprotective efficacy of sunscreens.  相似文献   

8.
V T T?rm?nen  G P Pfeifer 《Oncogene》1992,7(9):1729-1736
Mutations in ras proto-oncogenes have been found in human skin cancers. Since ultraviolet light is implicated in the development of skin cancers, we have investigated the formation of UV-induced photoproducts along exons 1 and 2 of the three ras proto-oncogenes, H-ras, K-ras, and N-ras, in UV-irradiated human cells. The two major types of DNA photoproducts, cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone photoproducts [(6-4) photoproducts], were mapped at the DNA sequence level by ligation-mediated polymerase chain reaction (LMPCR). No significant differences were seen between irradiated purified DNA and irradiated cells, implying that local chromatin structure does not influence the distribution of photoproducts along exons 1 and 2 of the three ras genes. We find that the transcribed strand near codon 61 in H-ras, K-ras and N-ras shows a high frequency of potentially mutagenic cyclobutane dimers and (6-4) photoproducts. Codon 12 of H-ras, K-ras and N-ras displays only barely detectable photoproducts at a CpC dinucleotide. In human skin cancers, mutations were most frequently detected at codon 12 of H-ras and K-ras. These results imply that the initial frequency distribution of a mutagenic DNA adduct may not correlate with mutation spectra in human tumors.  相似文献   

9.
McVean  M; Liebler  DC 《Carcinogenesis》1997,18(8):1617-1622
Ultraviolet B (UVB, 290-320 nm) exposure results in a variety of cellular insults including induction of cyclobutane pyrimidine dimers in DNA. Accumulation of these lesions can lead to mutations in critical genes and contribute to the development of nonmelanoma skin cancer. Topically applied alpha-tocopherol (vitamin E) has previously been shown to prevent the induction of skin tumors in UVB irradiated female C3H/HeNTac mice. We hypothesized that alpha-tocopherol, which absorbs strongly in the UVB, may act as a sunscreen to prevent photodamage. To explore possible mechanisms of photoprotection, we topically applied alpha-tocopherol dispersed in a neutral cream vehicle to the dorsal epidermis of female C3H/HeNTac mice and exposed them to 2.5 J/m2/s of UVB for 60 min. Immediately after exposure, we analyzed thymine dimer levels in DNA by capillary gas chromatography with electron capture detection. Epidermal DNA from mice receiving this UVB dose contained 247 +/- 42 pmol thymine dimers/micromol thymine. Topical application of alpha-tocopherol inhibited dimer formation in a dose-dependent manner. A 1% alpha-tocopherol dispersion inhibited the formation of thymine dimers to 43% of levels in vehicle controls. Several vitamin E compounds, including alpha-tocopherol acetate, alpha-tocopherol methyl ether, gamma-tocopherol, and delta-tocopherol also inhibited thymine dimer formation, but were five- to ten-fold less potent than alpha- tocopherol. A variety of commercially available sunscreens were also less potent than alpha-tocopherol in their ability to reduce dimer formation. These results suggest that DNA photoprotection is an important mechanism by which topically applied alpha-tocopherol can inhibit UVB induced skin cancer. Alpha-Tocopherol acetate, the most common form of vitamin E in commercial skin care products, conferred less protection, perhaps due to its lower absorptivity in the UVB. Our results further underscore the importance of determining which forms of vitamin E can inhibit specific lesions involved in photocarcinogenesis.   相似文献   

10.
Demonstration of UV-dimers in human skin DNA in situ 3 weeks after exposure   总被引:2,自引:0,他引:2  
Data on DNA repair rates of specific types of DNA lesions are very limited in humans in situ. Rate of repair of UV-induced DNA damage was followed in the skin of 17 volunteers up to 3 weeks of UV exposure, using a (32)P-postlabelling technique for the determination of specific photoproducts. The subjects of skin phototypes I and IV were exposed to 40 mJ/cm(2) of solar simulating radiation on buttock skin, and biopsies were taken at 0 h, 48 h and 3 weeks of exposure for the analysis of two cyclobutane pyrimidine dimers, TT=C and TT=T, and two 6-4 photoproducts, TT-C and TT-T, as trinucleotides. Repair rates were heterogeneous for different photoproducts. T=T dimers were repaired slower than C=T dimers, and 2.3-9.0% of the initial T=T damage remained unrepaired after 3 weeks, and was detectable in 16/17 subjects. The identity of the identified photoproducts was confirmed by a photochemical reversion assay. Damage level correlated with skin types, type I being more sensitive than type IV in an age-matched comparison. This is the first time the persistence of defined human DNA damage is demonstrated up to 3 weeks. Long-lasting DNA damage increases the likelihood of mutations.  相似文献   

11.
XPD exon 10 and 23 polymorphisms and DNA repair in human skin in situ   总被引:16,自引:0,他引:16  
Forty-four Finnish volunteers who were previously studied with regard to the repair rate of UV-specific cyclobutane pyrimidine dimers in the skin were genotyped for XPD polymorphisms at codons 312 (exon 10 G-->A, Asp-->Asn) and 751 (exon 23 A-->C, Lys-->Gln). The repair rate was measured at 24 h for two different cyclobutane dimers. The data did not show consistent XPD genotype-specific differences in DNA repair rates among all subjects. The combined exon 10 AA and exon 23 CC genotype was associated with an approximately 50% depression of repair rate but this was of borderline statistical significance. However, the exon 23 C allele was associated with depressed repair among subjects aged 50 years or older and the result was consistent with both dimers.  相似文献   

12.
In the present study, we for the first time investigated the formation of ultraviolet (UV) photoproducts, cyclobutane pyrimidine dimers (CPDs), pyrimidine-pyrimidone (6–4) photoproducts (64PPs) and Dewar isomers, in vivo in shaved and depilated C3H/HeN mouse skin exposed to natural sunlight (NSL) at noon for 5 min to 1 h in mid-summer, using a highly sensitive immunohistochemical method. This method permits the quantitative analysis of UV-photoproducts in formalin-fixed, paraffin-embedded sections with specific antibodies against CPDs, 64PPs and Dewar isomers. We demonstrated that the induction of CPDs in vivo in mouse skin by NSL was exposure time-dependent, but the accumulation of 64PPs or Dewar isomers was comparatively low in the skin sections from mice exposed to NSL in vivo. The results indicate that CPDs are the main photoproducts in vivo induced by sunlight and that their formation and repair may be important in connection with carcinogenesis in sun-exposed areas of human skin.  相似文献   

13.
Cleaver  James E. 《Carcinogenesis》1989,10(9):1691-1696
Human flbroblasts were embedded in microscopic-sized agarosebeads and lysed in situ to produce chromosome-sized DNA trappedinside the beads. Conventional alkaline agarose gel electrophoresisof trapped DNA from cells damaged by X-rays before embeddinggave mol. wts similar to those obtained with alkaline sucrosegradients. When cells were irradiated with UV light before embeddingin agarose and incubated with UV endonuclease to cleave cyclobutanepyrimidine dimer sites, UV damage was detectable by gel electrophoresisover a range of 2.6–52 J/m2 as a linear function of dose,and repair was detectable within 6 h of irradiation. Two independentlyderived UV-resistant xero-derma pigmentosum (XP) revertantsdid not remove cyclobutane dimers up to 48 h after irradiation.Incubation of normal and XP revertant cells with cytosine arabinosideafter UV irradiation resulted in similar numbers of single-strandbreaks; these breaks represent repair of non-dimer pbotoproducts.Evidently, excision of cyclobutane pyrimidine dimers from thewhole genome is not necessary for survival of human cells afterUV irradiation; rather, repair of non-dimer photoproducts suchas [6–4] photoproducts in the genome as a whole or cyclobutanedimers in a small number of genes may be more biologically important.  相似文献   

14.
Ultraviolet (UV) radiation induces cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts (6-4PPs) in DNA, which through gene mutations (e.g. in P53) may lead to skin carcinogenesis. Upon chronic low-level UV exposure, certain basal cells in mouse epidermis were reported to accumulate CPDs. These observations raised questions on whether these cells were fully DNA-repair deficient, and whether they were stem or progenitor cells, as suggested by their long residence time. We found that CPD-retaining basal cells (CRBCs) in SKH-1 hairless mice were repair proficient as accumulation of (6-4)PP, which is a hallmark for complete nucleotide excision repair-deficiency in rodents, was not observed. Accumulation of 6-4PP as well as CPD did, however, occur in basal cells in the epidermis of DNA repair-deficient Xpc-/- mice. Chronic UV exposure of DDB2 transgenic mice and DDB2 knockout mice revealed that the occurrence of CRBCs was inversely correlated with DDB2-expression, indicating that a boost in DNA repair lowered CPD accumulation. Stem cells are quiescent cells and can be identified as 5-bromo-2'-deoxyuridine-label retaining cells (BrdU-LRCs). Induction of BrdU-LRCs followed by chronic UV irradiation showed that all BrdU-label retaining stem cells were also CPD-retaining cells. As most CRBCs were not BrdU-labeled we surmized that these cells must include BrdU-negative stem cells and early progenitor cells. In confirmation of the latter, we found that CRBCs occurred among MTS24+ hair follicle progenitor cells. These findings provide the first evidence that epidermal stem and progenitor cells are prone to the accumulation of UV-induced DNA-damage and can be a prominent target in skin carcinogenesis.  相似文献   

15.
Human 8-oxoguanine DNA glycosylase-1 (hOGG1) is the key DNA repair enzyme responsible for initiating repair of UV radiation-induced 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG). Previously we have shown that basal cells in human epidermis are particularly sensitive to UVA-mediated DNA damage probably due to low expression of hOGG1. Here we investigate some aspects of the regulatory role of Cockayne syndrome B (CSB) on hOGG1 expression and function. Cockayne syndrome B and hOGG1 genes were knocked down by miRNA technology in the HaCaT human keratinocyte cell line. Loss of the CSB gene decreased hOGG1 mRNA, and loss of hOGG1 increased CSB, indicating that they influence each other's expression. Protein levels were assessed in cells grown into engineered human skin using immunohistochemistry. This confirmed that CSB knockdown with miRNA reduced hOGG1 protein levels, but hOGG1 knockdown did not influence expression of CSB protein. Using comet assay we found that both hOGG1 and CSB knockdown reduced repair of both UVA- and UVB-induced 8-oxo-dG, consistent with CSB downregulation of hOGG1 mRNA and protein. In contrast, CSB but not hOGG1 knockdown reduced repair of UVB- and UVA-induced cyclobutane pyrimidine dimer photolesions. In engineered human skin, repair of UVA-induced 8-oxo-dG was inhibited by both hOGG1 and CSB knockdown, confirming the functional role of both proteins in cells with 3-D cellular contacts. These findings directly indicate that hOGG1 and CSB influence each other's expression. CSB is required for maintaining hOGG1 enzyme levels and function. Cockayne syndrome B could therefore be required for 8-oxo-dG repair due to its regulatory effect on hOGG1 expression. Cockayne syndrome B but not hOGG1 is also required for efficient repair of cyclobutane pyrimidine dimers. Cockayne syndrome B regulation of DNA repair could contribute to the effect of UVA in causing mutations that lead to skin cancer in humans.  相似文献   

16.
Mirzayans R  Pollock S  Scott A  Gao CQ  Murray D 《Oncogene》2003,22(36):5562-5571
We investigated the effects of metabolic labeling with [(3)H]thymidine, [(3)H]uridine, and [(14)C]thymidine on human cells in terms of cell growth, p53 signaling, and nucleotide excision repair. Labeling with [(3)H] nucleosides resulted in growth inhibition by both p53-dependent and -independent mechanisms. Tritium labeling also led to nuclear accumulation of p53 and induction of the p53-regulated gene p21(WAF1) and its encoded protein (p21). ATM-deficient cells, however, did not increase their p53 and p21 protein levels in response to radiolabeling. Thus, labeling of human cells with tritiated nucleosides activates the radiation-responsive, ATM-dependent, DNA-damage surveillance network. Labeling of normal cells with [(3)H]thymidine significantly accelerated the repair of ultraviolet (UV) light-induced cyclobutane pyrimidine dimers, as monitored by a sensitive immunofluorescence assay. Unlike [(3)H] labeling, [(14)C] labeling did not produce any impact on proliferation, p53 signaling, or DNA repair. In the light of these findings, the validity of results obtained with nucleic acid synthesis and DNA repair assays that involve [(3)H] and [(14)C] labeling is discussed. Our immunofluorescence approach detected pyrimidine dimers after exposure to UV fluences as low as 1 J/m(2) (the lowest fluence examined). This approach may prove particularly useful for monitoring DNA damage and its repair following exposure to extremely low levels of genotoxic agents.  相似文献   

17.
Xeroderma pigmentosum genes: functions inside and outside DNA repair   总被引:1,自引:0,他引:1  
Sugasawa K 《Carcinogenesis》2008,29(3):455-465
Xeroderma pigmentosum (XP) is an autosomal recessive disease, which is characterized by susceptibility to ultraviolet light (UV)-induced skin cancer. Among eight genes so far identified as responsible for XP, XPA through XPG are involved in nucleotide excision repair of DNA damage induced by UV as well as various chemical carcinogens. Since this repair system removes a major UV photoproduct, the cyclobutane pyrimidine dimer, quite slowly from the global genome, this lesion must be accurately bypassed during replication by DNA polymerase eta, encoded by the XPV gene. Recent studies have revealed that each of these XP genes possesses additional functions, some of which are concerned with other DNA repair pathways and/or cellular DNA damage responses. Such differential functions not only explain clinical heterogeneity among different genetic complementation groups but also have implications for the promotion of carcinogenic processes in XP patients.  相似文献   

18.
Pyrimidine dimer formation and repair in human skin   总被引:7,自引:0,他引:7  
Cyclobutyl pyrimidine dimers have been detected in the DNA of human skin following in vivo irradiation with suberythemal doses of ultraviolet (UV) radiation from FS-20 sun lamp fluorescent tubes. Dimers were assayed by treatment of extracted DNA with Micrococcus luteus UV-specific endonuclease, alkaline agarose electrophoresis, and ethidium bromide staining. This technique, in contrast to conventional dimer assays, can be used with nonradioactive DNA and is optimal at low UV light doses. M. luteus endonuclease-sensitive sites were determined after exposure of untanned skin in two volunteers to UV light (0.97, 1.94, or 3.88 X 10(3) J/sq m; lambda, 290 to 360 nm). At 20 min postirradiation (dose, 1.94 X 10(3) J/sq m), fewer M. luteus endonuclease-sensitive sites were found in the DNA than immediately after the irradiation. Even fewer endonuclease-sensitive sites were found at 20 min when the UV-irradiated skin was subsequently irradiated with visible light than when the area was kept in the dark. These data suggest that some dimer disappearance by excision repair occurs within 20 min of UV irradiation and that photoreactivation of dimers can make a contribution to the total repair process.  相似文献   

19.
Xeroderma pigmentosum (XP) is a rare recessive disorder that is characterized by extreme sensitivity to UV light. UV light exposure results in the formation of DNA damage such as cyclobutane dimers and (6-4) photoproducts. Nucleotide excision repair (NER) orchestrates the removal of cyclobutane dimers and (6-4) photoproducts as well as some forms of bulky chemical DNA adducts. The disease XP is comprised of 7 complementation groups (XP-A to XP-G), which represent functional deficiencies in seven different genes, all of which are believed to be involved in NER. The main clinical feature of XP is various forms of skin cancers; however, neurological degeneration is present in XPA, XPB, XPD and XPG complementation groups. The relationship between NER and other types of DNA repair processes is now becoming evident but the exact relationships between the different complementation groups remains to be precisely determined.Using gene expression analysis we have identified similarities and differences after UV light exposure between the complementation groups XP-A, XP-C, XP-D, XP-E, XP-F, XP-G and an unaffected control. The results reveal that there is a graded change in gene expression patterns between the mildest, most similar to the control response (XP-E) and the severest form (XP-A) of the disease, with the exception of XP-D. Distinct differences between the complementation groups with neurological symptoms (XP-A, XP-D and XP-G) and without (XP-C, XP-E and XP-F) were also identified. Therefore, this analysis has revealed distinct gene expression profiles for the XP complementation groups and the first step towards understanding the neurological symptoms of XP.  相似文献   

20.
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