Induction Of Cyclobutane Pyrimidine Dimers Research Articles

Abstract 3813: DNA polymerase eta and a novel anticancer selenophene compound

Abstract Human DNA polymerase ≤ (pol α) plays a key role in replicating across the UV radiation induced cyclobutane pyrimidine dimers (CPD) in DNA and mutations in pol ≤ gene result in a syndrome called Xeroderma Pigmentosum Variant (XP-V). In the absence of pol β, the DNA replication forks collapse after prolonged stalling at CPD sites, which consequently lead to DNA breakages and cell death. Therefore, pol ≤ has been considered to serve as a cellular protection mechanism to enhance cell survival and reduce mutagenesis. In addition to UV-induced CPD lesion, pol ≤ has the ability to handle DNA lesions introduced by chemotherapeutic anticancer agents, such as platinum based compounds and nucleoside analogs. Such damage tolerance ability of pol ≤ has been shown to reduce cell sensitivity to these DNA targeting agents. D-501036 [2,5-bis(5-hydroxymethyl-2-selenienyl)-3-hydroxymethyl-N-methylpyrrole] is a selenophene compounds that demonstrated promising antitumor activity against various human cancer cells and in vivo xenograft mouse model. Mechanistic studies have shown that D-501036 induced DNA adduct(s) and caused genome instability, which result in cell cycle accumulation in the S-phase, DNA breakage, and subsequently lead to cell death. Interestingly, the IC50 for human fibroblast XP30RO cells derived from XP-V patients was >50 fold higher than the XP30RO cells that stably transfected with a pol ≤ expressing vector (XP30RO-EGFP-pol α). In addition, D-501036 caused an increased amount of DNA breakage in the XP30RO-EGFP-pol ≤ cells as compared to its parental XP30RO cells in both DNA fragmentation and comet assays. These results revealed a novel role of pol ≤ in the action of D-501036 that is very different from its protective role against UV irradiation or cisplatin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3813. doi:1538-7445.AM2012-3813

UVA1 Induces Cyclobutane Pyrimidine Dimers but Not 6-4 Photoproducts in Human Skin In Vivo

UVB readily induces cyclobutane pyrimidine dimers, mainly thymine dimers (TTs), and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) in DNA. These lesions result in "UVB signature mutations" found in skin cancers. We have investigated the induction of TTs and 6-4PPs in human skin in vivo by broadband UVA1, and have compared this with comparable erythemal doses of monochromatic UVB (300 nm). In vitro and ex vivo studies have shown the production of TTs, without 6-4PPs, by UVA1. We show that UVA1 induces TTs, without 6-4PPs, in the epidermis of healthy volunteers in vivo, whereas UVB induced both photoproducts. UVB induced more TTs than UVA1 for the same level of erythema. The level of UVA1-induced TTs increased with epidermal depth in contrast to a decrease that was seen with UVB. UVA1- and UVB-induced TTs were repaired in epidermal cells at a similar rate. The mechanism by which UVA1 induces TTs is unknown, but a lack of intra-individual correlation between our subjects' UVB and UVA1 minimal erythema doses implies that UVA1 and UVB erythema occur by different mechanisms. Our data suggest that UVA1 may be more carcinogenic than has previously been thought.

Resistance of the genome of Escherichia coli and Listeria monocytogenes to irradiation evaluated by the induction of cyclobutane pyrimidine dimers and 6-4 photoproducts using gamma and UV-C radiations

The effect of gamma and UV-C irradiation on the production of cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4 PPs) in DNA was investigated to compare the natural resistance of the genome of a Gram-positive bacterium and a Gram-negative bacterium against irradiation. Solution of pure DNA and bacterial strains Listeria monocytogenes and Escherichia coli were irradiated using gamma and UV-C rays. Extracted DNA from bacteria and pure DNA samples were then analysed by ELISA using anti-CPDs and anti-6-4 PPs monoclonal antibodies. The results show that gamma rays, as well as UV-C rays, induce the formation of CPDs and 6-4 PPs in DNA. During UV-C irradiation, the three samples showed a difference in their sensitivity against formation of CPDs (P≤0.05). Pure DNA was the most sensitive while the genome of L. monocytogenes was the most resistant. Also during UV-C irradiation, the genome of L. monocytogenes was the only one to show a significant resistance against formation of 6-4 PPs (P≤0.05). During gamma irradiation, for both types of lesion, pure DNA and the genome of E. coli did not show significant difference in their sensitivity (P>0.05) while the genome of L. monocytogenes showed a resistance against formation of CPDs and 6-4 PPs.

Molecular Aspects of Tanning

Tanning is commonly understood as increased melanization of the epidermis observed in skin following UV exposure. It is further understood to represent a host response that protects against future UV-induced damage. Although best studied in humans, tanning can also be observed in other mammals and even in more primitive animals such as sharks. The molecular basis of tanning was poorly understood well into the 1990s, although UV irradiation of murine melanoma cells had been shown to increase the number of cell surface receptors to a-melanocyte–stimulating hormone (a-MSH) and to increase the melanogenic response to a-MSH via increased protein levels and activity of tyrosinase, the rate-limiting enzyme in melanin synthesis (Bolognia et al., 1989; Chakraborty et al., 1991). However, the initiating molecular events were unknown. Identification of the responsible molecules ultimately evolved from studies of the UV action spectrum for critical events in human skin, as well as from a philosophical appreciation of tanning as a genome protective response. In the 1980s, investigators at Harvard’s Wellman Laboratories exposed normal skin of volunteers to a wide range of light doses using narrow band sources across the UV spectrum and observed them for several days to determine the lowest dose at each wavelength capable of producing a delayed tan (Parrish et al., 1982). In other experiments, they used the same approach to define the action spectrum for induction of cyclobutane pyrimidine dimers (CPDs), the most common form of DNA damage following UV exposure (Freeman et al., 1989). The spectra were virtually superimposable, peaking at B300 nm, suggesting a cause-and-effect relationship between the immediate DNA damage and subsequent increased production and dispersion of epidermal melanin. Eller et al. (1996) first documented that agents acting exclusively on DNA, such as DNA restriction enzymes, stimulate melanin production in cultured pigment cells, at least in part by increasing a-MSH binding and the melanogenic response to a-MSH. Although this did not exclude a role for UV-mediated membrane effects, it did directly implicate UV-mediated DNA damage in the tanning response. Similarly, accelerating DNA damage repair by providing UV-irradiated pigment cells with T4 endonuclease V, the bacterial enzyme that catalyzes the first step in CPD resolution, also enhanced the melanization response (Gilchrest et al., 1993). By the 1990s, DNA damage responses were recognized to be largely mediated by the transcription factor and tumor suppressor protein p53, also termed the ‘‘guardian of the genome’’ (Lane, 1992), and two groups independently asked whether p53 was involved in mediating the tanning response. Using p53 null osteosarcoma cells transfected with wild-type p53, Nylander et al. (2000) showed that p53 activation increased read-out from a transfected tyrosinase promoter linked to a reporter gene, implying that p53 could directly or indirectly stimulate tyrosinase transcription. This link between p53 and tanning was expanded and refined using both human melanoma cells and a mouse model. Compared with a p53-null parental melanoma line and to the same line transfected with an empty vector, melanoma cells transfected with wildtype p53 increased their tyrosinase mRNA levels progressively over 72 hours following p53 activation (Khlgatian et al., 1999; Khlgatian et al., 2002). Confirming an earlier report (Kichina et al., 1996), these studies also showed an inverse relationship between total p53 protein (inactive) and tyrosinase levels. The requirement for p53 activation to increase tyrosinase protein level and epidermal melanin content in vivo was confirmed by documenting the tanning ability of wild type versus p53 knockout mice (Khlgatian et al., 2002). The question remained whether p53 directly increased tyrosinase transcription, as no p53 consensus sequence had been identified in its promoter region. The key observation that the UV action spectrum for tanning is virtually identical to that for CPD production, had suggested that thymidine dinucleotide (abbreviated ‘‘TT’’), the obligate substrate for the majority of CPDs, might itself serve as a molecular signal for increased melanogenesis (tanning). Using various models, the responses to UV irradiation versus this DNA fragment were therefore compared. Indeed, TT increased mRNA and protein expression of tyrosinase as well as melanin content of cultured human and murine pigment cells and of intact guinea pig skin-containing melanocytes in the interfollicular epidermis, in a time frame similar to that observed after UV irradiation (Eller et al., 1994). As well, in guinea pig skin, the histological features were virtually identical following either treatment: increased total melanin with prominent

Diverse Roles of RNA Polymerase II-associated Factor 1 Complex in Different Subpathways of Nucleotide Excision Repair

Transcription-coupled repair (TCR) and global genomic repair (GGR) are two pathways of nucleotide excision repair (NER). In Saccharomyces cerevisiae, Rad26 is important but not absolutely required for TCR. Rpb4, a nonessential RNA polymerase II (Pol II) subunit that forms a subcomplex with Rpb7, and the Spt4-Spt5 complex, a transcription elongation factor, have been shown to suppress Rad26-independent TCR. The Pol II-associated factor 1 complex (Paf1C) has been shown to function in transcription elongation, 3'-processing of mRNAs, and posttranslational modification of histones. Here we show that Paf1C plays a marginal role in facilitating Rad26-dependent TCR but significantly suppresses Rad26-independent TCR. The suppression of Rad26-independent TCR is achieved by cooperating with Spt4-Spt5. We propose a model that, in the absence of Rad26, a lesion is "locked" in the active center of a Pol II elongation complex, which is stabilized by the coordinated interactions of Rpb4-Rpb7, Spt4-Spt5, and Paf1C with each other and with the core Pol II. We also found that Paf1C facilitates GGR, especially in internucleosomal linker regions. The facilitation of GGR is achieved through enabling monoubiquitination of histone H2B lysine 123 by Bre1, which in turn permits di- and trimethylation of histone H3 lysine 79 by Dot1. To our best knowledge, among the NER-modulating factors documented so far, Paf1C appears to have the most diverse functions in different NER pathways or subpathways.

Individual Photosensitivity of Human Skin and UVA-Induced Pyrimidine Dimers in DNA

Delineation of the DNA-damaging properties of UVA radiation is a major issue in understanding solar carcinogenesis. Emphasis was placed in this study on the formation of cyclobutane pyrimidine dimers (CPDs), which are now well established as the most frequent UVA-induced DNA lesions in human skin. The yield of CPDs was determined by a chromatographic assay following ex vivo UVA and UVB irradiation of biopsies taken from either phototype II or IV volunteers. A clear correlation was found between the frequency of UVB-induced CPDs and both the phototype and the minimum erythemal dose (MED). Similar results were obtained for the induction of CPDs upon exposure to UVA. Moreover, an excellent correlation was observed for each donor between the yield of DNA damage induced by either UVB or UVA. These observations show that the key parameters driving UVA-induced formation of CPDs are attenuation of radiation in the skin and the number of photons reaching skin cells rather than the cellular content in photosensitizers. In addition, the results show that both MED and phototype are good predictors of the vulnerability of DNA toward UVB and UVA in the skin. This result is of importance for the identification of individuals to be extensively protected.

Immunological detection of UV induced cyclobutane pyrimidine dimers and (6–4) photoproducts in DNA from reference bacteria and natural aquatic populations

UV light-caused DNA damage is a widespread field of study. As UV light has the biological effect of inactivating or killing bacteria, it is used for water disinfection. Due to this application, it is important to study the DNA damage efficiencies and regeneration capacities in bacteria after UV irradiation. Two monoclonal antibodies, anti-CPD and anti-(6–4) PP, were applied for an immunoassay of UV-induced DNA modifications. Cyclobutane pyrimidine dimer (CPD) and 6–4 photoproduct (6–4 PP) were detected in the reference bacteria Pseudomonas aeruginosa and Enterococcus faecium, and in natural water communities. The antibody-mediated detection signals increased with the UV doses from 100–400J/m2. Here, the CPD-specific signals were stronger than the (6–4) PP-specific signals. These immunological results were in accordance with parallel cultivation experiments. All UV-irradiated bacteria showed a reduction of their growth rate depending on UV application by several orders of magnitudes.The immunoassay was also applied to three types of natural aquatic habitats with different cell densities. Besides artificial UV irradiation, it was possible to visualize natural sunlight effects on these natural bacterial communities. Light-dependent and dark repair processes were distinguished using the established immunological assays. The antibody-mediated analyses presented are fast methods to detect UV-induced DNA lesions and repair capacities in selected bacterial species as well as in entire natural mixed populations.

Quantification of Cyclobutane Pyrimidine Dimers Induced by UVB Radiation in Conidia of the Fungi Aspergillus fumigatus, Aspergillus nidulans, Metarhizium acridum and Metarhizium robertsii

Conidia are responsible for reproduction, dispersal, environmental persistence and host infection of many fungal species. One of the main environmental factors that can kill and/or damage conidia is solar UV radiation. Cyclobutane pyrimidine dimers (CPD) are the major DNA photoproducts induced by UVB. We examined the conidial germination kinetics and the occurrence of CPD in DNA of conidia exposed to different doses of UVB radiation. Conidia of Aspergillus fumigatus, Aspergillus nidulans and Metarhizium acridum were exposed to UVB doses of 0.9, 1.8, 3.6 and 5.4 kJ m(-2). CPD were quantified using T4 endonuclease V and alkaline agarose gel electrophoresis. Most of the doses were sublethal for all three species. Exposures to UVB delayed conidial germination and the delays were directly related both to UVB doses and CPD frequencies. The frequencies of dimers also were linear and directly proportional to the UVB doses, but the CPD yields differed among species. We also evaluated the impact of conidial pigmentation on germination and CPD induction on Metarhizium robertsii. The frequency of dimers in an albino mutant was approximately 10 times higher than of its green wild-type parent strain after exposure to a sublethal dose (1.8 kJ m(-2)) of UVB radiation.

Amsacta moorei entomopoxvirus encodes a functional DNA photolyase (AMV025)

The major damage induced in DNA by ultraviolet light is the induction of cyclobutane pyrimidine dimers (CPDs). Amsacta moorei entomopoxvirus (AMEV) encodes a CPD photolyase (AMV025) with a putative role in converting these dimers back into monomers. In infected Lymantria dispar cells transcription of the AMV025 gene started 8 h post inoculation (p.i.) and continued through 38 h p.i. Transcription was inhibited by a DNA synthesis blocker. Transient expression in an Escherichia coli strain that lacks its endogenous photolyase, rescued growth of the UV-irradiated bacteria in a light-dependent manner, showing that AMV025 encodes a functional DNA photolyase.

Silenced yeast chromatin is maintained by Sir2 in preference to permitting histone acetylations for efficient NER

Very little is currently known about how nucleotide excision repair (NER) functions at the ends of chromosomes. To examine this, we introduced the URA3 gene into either transcriptionally active or repressed subtelomeric regions of the yeast genome. This enabled us to examine the repair of ultraviolet (UV)-induced cyclobutane pyrimidine dimers (CPDs) in identical sequences under both circumstances. We found that NER is significantly more efficient in the non-repressed subtelomere than the repressed one. At the non-repressed subtelomere, UV radiation stimulates both histones H3 and H4 acetylation in a similar fashion to that seen at other regions of the yeast genome. These modifications occur regardless of the presence of the Sir2 histone deacetylase. On the other hand, at the repressed subtelomere, where repair is much less efficient, UV radiation is unable to stimulate histone H4 or H3 acetylation in the presence of Sir2. In the absence of Sir2 both of these UV-induced modifications are detected, resulting in a significant increase in NER efficiency in the region. Our experiments reveal that there are instances in the yeast genome where the maintenance of the existing chromatin structures dominates over the action of chromatin modifications associated with efficient NER.

Site-specific analysis of UV-induced cyclobutane pyrimidine dimers in nucleotide excision repair-proficient and -deficient hamster cells: Lack of correlation with mutational spectra

Irradiation of cells with UVC light induces two types of mutagenic DNA photoproducts, i.e. cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts (6-4PP). To investigate the relationship between the frequency of UV-induced photolesions at specific sites and their ability to induce mutations, we quantified CPD formation at the nucleotide level along exons 3 and 8 of the hprt gene using ligation-mediated PCR, and determined the mutational spectrum of 132 UV-induced hprt mutants in the AA8 hamster cell line and of 165 mutants in its nucleotide excision repair-defective derivative UV5. In AA8 cells, transversions predominated with a strong strand bias towards thymine-containing photolesions in the non-transcribed strand. As hamster AA8 cells are proficient in global genome repair of 6-4PP but selectively repair CPD from the transcribed strand of active genes, most mutations probably resulted from erroneous bypass of CPD in the non-transcribed strand. However, the relative incidence of CPD and the positions where mutations most frequently arose do not correlate. In fact some major damage sites hardly gave rise to the formation of mutations. In the repair-defective UV5 cells, mutations were almost exclusively C > T transitions caused by photoproducts at PyC sites in the transcribed strand. Even though CPD were formed at high frequencies at some TT sites in UV5, these photoproducts did not contribute to mutation induction at all. We conclude that, even in the absence of repair, large variations in the level of induction of CPD at different sites throughout the two exons do not correspond to frequencies of mutation induction.

T4 endonuclease V: review and application to dermatology

Background: T4 endonuclease V was originally isolated from Escherichia coli infected with T4 bacteriophage. It has been shown to repair ultraviolet (UV)-induced cyclobutane pyrimidine dimers in DNA, which, when unrepaired, contribute to mutations that result in actinic keratoses and non-melanoma skin cancers (NMSC). This is a particular concern in patients with genetic defects in their DNA repair systems, especially those with xeroderma pigmentosum (XP). When packaged in liposomes and applied topically, T4 endonuclease V can traverse the stratum corneum and become incorporated within the cytoplasm and nucleus of epidermal keratinocytes and Langerhans cells. Objective: To review all major studies evaluating the efficacy of T4 endonuclease V in animals and humans, the toxicity and safety profile of the topical medication and its potential clinical uses. Methods: A literature search was performed through PubMed/Medline, using the keywords ‘T4N5’, ‘T4 endonuclease V’ and ‘dimericine’. Papers found in the bibliographies of those identified in the initial search and deemed relevant were also included. Conclusion: This enzyme increases the repair of UV-damaged DNA and produces other beneficial effects on UV-damaged cells. In clinical trials in XP patients, topical application of liposome-encapsulated T4 endonuclease V reduced the incidence of basal cell carcinomas by 30% and of actinic keratoses by > 68%. Adverse effects were minimal, and there was no evidence of allergic or irritant contact dermatitis. Although the photoprotective effect of T4N5 has been investigated only in XP patients, the possibility exists that it may benefit others likely to develop premalignant keratoses and NMSC, such as organ transplant recipients receiving immunosuppressive therapy and individuals who have had numerous psoralen plus UVA photochemotherapy treatments. It may be also be effective for normal individuals.

Early Events in UV Carcinogenesis—DNA Damage, Target Cells and Mutant p53 Foci†

Skin carcinomas are the most common cancers in fair-skinned populations of North West European descent. The risk is closely related to sun (UV) exposure and susceptibility to sunburn. Induction of squamous cell carcinomas (SCCs) in the skin of hairless mice by daily UVB exposure appears to emulate the genesis of these tumors in humans quite well. The carcinomas, and the UVB signature mutations that they carry in their p53 genes, can be linked most specifically to the induction of cyclobutane pyrimidine dimers (CPDs). The wavelength dependence of the induction of carcinomas parallels that of CPD induction over the UVB and UVA2 spectral regions. Microscopic clusters of cells overexpressing p53 with UVB signature mutations ("p53 patches") can be detected in the interfollicular epidermis long before the skin tumors arise. DNA repair--more precisely nucleotide excision repair--is a crucial line of defense against UV-induced p53 patches and skin carcinomas. Although chemoprevention of UV carcinogenesis, e.g. with difluoromethylornithine, may be successful by inhibiting the outgrowth of tumors, it may be better to counter the initial steps in tumor development. As the p53 patches appear to be potential precursors of SCCs, regression of p53 patches in unexposed skin should lower subsequent development of SCCs. However, "holoclonal" p53 patches might persist. Ablation of the interfollicular epidermis would be expected to abrogate development of SCC, and negation of this expectation [Faurschou A. et al., Exp. Dermatol. 2007;16:485-489] would indicate that SCCs stem from deep-seated cells in the hair follicles. Careful examination of archival material showed that although most small p53 patches arise interfollicularly, some may actually arise high up in a follicle, in the infundibulum.

Mechanisms of inhibitory effects of CoQ10 on UVB‐induced wrinkle formation in vitro and in vivo

Photodamaged skin exhibits wrinkles, pigmented spots, dryness and tumors. Solar UV radiation induces cyclobutane pyrimidine dimers (CPD) and further produces base oxidation by reactive oxygen species (ROS). ROS are thought to be a major factor to initiate the up-regulation of matrix metalloproteinases (MMPs) in keratinocytes and fibroblasts via activation of receptor proteins on the cell membrane of keratinocytes and fibroblasts, and to degrade fiber components in dermis, leading to wrinkle formation. Coenzyme Q10 (CoQ10) was reported to reduce ROS production and DNA damage triggered by UVA irradiation in human keratinocytes in vitro. Further, CoQ10 was shown to reduce UVA-induced MMPs in cultured human dermal fibroblasts. We speculated that UVB radiation-induced cytokine production in keratinocytes may be inhibited by CoQ10, resulting in the reduction of MMPs in fibroblasts leading to wrinkle reduction. Our in vitro studies showed that UVB-induced IL-6 production of normal human keratinocyte (NHKC) decreased in the presence of CoQ10. Furthermore, MMP-1 production of fibroblasts cultured with the medium containing CoQ10 collected from UVB-irradiated NHKC significantly decreased during 24 h culture. In the clinical trial study, we found that the use of 1% CoQ10 cream for five months reduced wrinkle score grade observed by a dermatologist. Taken together, our results indicate that CoQ10 may inhibit the production of IL-6 which stimulate fibroblasts in dermis by paracrine manner to up-regulate MMPs production, and contribute to protecting dermal fiber components from degradation, leading to rejuvenation of wrinkled skin.

An action spectrum (290–320 nm) for TNFα protein in human skinin vivosuggests that basal-layer epidermal DNA is the chromophore

Terrestrial solar UVB radiation ( approximately 295-320 nm) readily induces cyclobutane pyrimidine dimers (CPDs) in human skin DNA that result in characteristic mutations associated with nonmelanoma skin cancer. The proinflammatory cytokine TNFalpha is important in mouse skin chemical carcinogenesis and is thought to also play a role in UVR-induced skin cancer by its immunomodulatory properties. There is some in vitro evidence that CPDs initiate the production of TNFalpha, and we tested this hypothesis by comparing the wavelength dependence (action spectrum) for TNFalpha protein induction in human skin in vivo with our earlier in vivo action spectra for CPD induction in four different epidermal layers of human skin. Normal volunteers (n = 35) were irradiated with physiologically relevant doses of monochromatic UVB (290-320 nm), and TNFalpha concentration was assessed, by high-sensitivity ELISA, in exudates from skin suction blisters raised 8 h after irradiation. An action spectrum, constructed from the slopes of the dose-response curves at the different wavelengths, showed maximal efficacy at 300 nm. An excellent match was observed for TNFalpha and the CPD action spectrum for cells in the lower basal epidermis. These data strongly suggest that UVB-induced photodamage to DNA in the epidermal basal layer is a major trigger for TNFalpha production. The TNFalpha may originate directly from the keratinocytes in this layer or inflammatory cells that are rapidly recruited into the upper dermis (e.g., neutrophils) as a consequence of DNA photodamage to basal-layer keratinocytes.

Growth stimulation of UV-induced DNA damage retaining epidermal basal cells gives rise to clusters of p53 overexpressing cells

Ultraviolet (UV) radiation induces cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts ((6-4)PPs) in DNA, which may give rise to clusters of cells expressing mutant p53 (‘p53 patches’) and eventually to skin carcinomas. We have previously reported that some basal cells in murine skin accumulate CPDs upon chronic low-level UV exposure and that these CPD-retaining basal cells (CRBCs) encompass epidermal stem and progenitor cells. Through replication of their damaged DNA CRBCs may become mutagenic foci from which tumors might form. We therefore investigated whether CRBCs may give rise to p53 patches after forced proliferation by repeated applications of 12-O-tetradecanoylphorbol-13-acetate (TPA). CRBCs, induced in SKH-1 hairless mice by chronic low-level UV exposure (70J/m2 daily for 40 days), disappeared in the TPA-induced epidermal hyperplasia within 2 weeks and numerous clusters of epidermal cells with overexpressed p53 appeared after 4 weeks. Neither mutant p53 patches nor any foci of pErk1/2-overexpressing cells that could have caused reactive wild type p53 expression were found. In skin exposed to a single high UV dose (2.8kJ/m2) no CRBCs occurred, and no p53 clusters were observed after TPA treatment. These experiments suggest that CRBCs are a prerequisite for the formation of clusters of p53-overexpressing cells. The high frequency of these clusters (about 1 for every 3 CRBCs) precludes mutations in p53 as a likely cause. We surmise that forced proliferation of CRBCs gives rise to genomic instability that is propagated in daughter cells and evokes wild type p53 overexpression, signifying a potentially oncogenic process different from classic UV carcinogenesis involving mutant p53.

Transcription coupled nucleotide excision repair in Escherichia coli can be affected by changing the arginine at position 529 of the β subunit of RNA polymerase

The proposed mechanism for transcription coupled nucleotide excision repair (TCR) invokes RNA polymerase (RNAP) blocked at a DNA lesion as a signal to initiate repair. In Escherichia coli, TCR requires the interaction of RNAP with a transcription-repair coupling factor encoded by the mfd gene. The interaction between RNAP and Mfd depends upon amino acids 117, 118, and 119 of the β subunit of RNAP; changing any one of these to alanine diminishes the interaction [1]. Using direct assays for TCR, and the lac operon of E. coli containing UV induced cyclobutane pyrimidine dimers (CPDs) as substrate, we have found that a change from arginine to cysteine at amino acid 529 of the β subunit of the RNAP inactivates TCR, but does not prevent the interaction of RNAP with Mfd. Our results suggest that this interaction may be necessary but not sufficient to facilitate TCR.

Development and Application of a Novel Immunoassay for Measuring Oxidative DNA Damage in the Environment¶

We developed a facile, cost-effective competitive binding assay for the analysis of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) in DNA, using a polyclonal rabbit antiserum raised against an 8-oxodGuo hapten coupled to bovine serum albumin and radiolabeled synthetic ligand containing multiple 8-oxodGuo residues. This radioimmunoassay (RIA) displays a high affinity for 8-oxodGuo in DNA, with a detection limit of ∼1 adduct in 105 bases of DNA. 8-oxodGuo standards for RIA were quantified by high-performance liquid chromatography and electrochemical detection in DNA diluted in methylene blue and exposed to visible light. As an initial application we quantified 8-oxodGuo in dosimeters deployed at increasing depths in the Southern Ocean during the austral spring of the 1998 field season or at the surface at Palmer Station, Antarctica, throughout the 1999 field season. Cyclobutane pyrimidine dimers (CPD) were quantified using an established RIA. We found that the frequency of both photoproducts decreased with depth. However, CPD induction was attenuated at a faster rate than 8-oxodGuo, correlating with the differential attenuation of solar ultraviolet wavelengths in the water column. CPD induction was closely related with ultraviolet-B radiation (UVB) attenuation, whereas the lower attenuation of 8-oxodGuo suggests that oxidative damage is more closely related to ultraviolet-A radiation (UVA) irradiance. The ratio of 8-oxodGuo : CPD was also found to covary with changes in stratospheric ozone concentrations at Palmer Station. These data demonstrate the usefulness of these assays for environmental photobiology and the potential for their use in studying the relative impacts of UVB versus UVA, including ozone depletion events.

Melanin Offers Protection Against Induction of Cyclobutane Pyrimidine Dimers and 6-4 Photoproducts by UVB in Cultured Human Melanocytes¶

Melanin Offers Protection Against Induction of Cyclobutane Pyrimidine Dimers and 6-4 Photoproducts by UVB in Cultured Human Melanocytes¶

Ultraviolet-B-Induced Cyclobutane-pyrimidine Dimer Formation and Repair in Arctic Marine Macrophytes¶

The significance of ultraviolet-B radiation (UVBR: 280–315 nm)–induced DNA damage as a stress factor for Arctic marine macrophytes was examined in the Kongsfjord (Spitsbergen, 78°55.5′N, 11°56.0′E) in summer. UVBR penetration in the water column was monitored as accumulation of cyclobutane-pyrimidine dimers (CPD) in bare DNA. This showed that UVBR transparency of the fjord was variable, with 1% depths ranging between 4 and 8 m. In addition, induction and repair kinetics of CPD were studied in several subtidal macrophytes obtained from the Kongsfjord (5–15 m). Surface exposure experiments demonstrated CPD accumulation in Palmaria palmata, Devaleraea ramentacea, Phycodrys rubens, Coccotylus truncatus and Odonthalia dentata. In artificial light, field collected material of P. palmata, D. ramentacea, P. rubens and Laminaria saccharina showed efficient CPD repair, with only 10% of the artificially induced CPD remaining after 5 h. No significant differences in repair rate were observed among these species. CPD repair was slower or absent in O. dentata, C. truncatus and Monostroma arcticum, indicating that fast repair mechanisms such as photolyase were not continuously expressed in these species. CPD repair rates were not directly related to the vertical distribution of algae in the water column and to the reported UV sensitivity of the examined species. Dosimeter incubations showed that maximal exposure to DNA damaging wavelengths was low for all examined species. Furthermore, most species collected below the 1% depth for DNA damage displayed efficient CPD repair, suggesting that UVBR-induced CPD currently impose a minor threat for mature stages of these species growing in the Kongsfjord, Spitsbergen.