Formation Of DNA Photoproducts Research Articles

Data Processing for Predicting DNA Damaging Properties of Complex UV Sources.

A growing number of experimental evidence emphasizes that photobiological phenomena are not always the sum of the effect of individual wavelengths present in the emission spectrum of light sources. Unfortunately, tools are missing to identify such non-additive effects and predict effects of various exposure conditions. In the present work, we addressed these points for the formation of pyrimidine dimers in DNA upon co-exposure to UVC, UVB and UVA radiation. We first applied a combination index approach to determine whether mixtures of theses UV ranges exhibited additive, inhibitory or synergistic effects on the formation of cyclobutane pyrimidine dimers, (6-4) photoproducts and Dewar valence isomers. A predictive approach based on an experimental design strategy was then used to quantify the contribution of each wavelength range to the formation of DNA photoproducts. The obtained models allowed us to accurately predict the level of pyrimidine dimers in DNA irradiated under different conditions. The data were found to be more accurate than those obtained with the simple additive approach underlying the use of action spectra. Experimental design thus appears as an attractive concept that could be widely applied in photobiology even for cellular experiments.

DNA photoproducts released by repair in biological fluids as biomarkers of the genotoxicity of UV radiation.

UV-induced formation of photoproducts in DNA is a major initiating event of skin cancer. Consequently, many analytical tools have been developed for their quantification in DNA. In the present work, we extended our previous liquid chromatography-mass spectrometry method to the quantification of the short DNA fragments containing photoproducts that are released from cells by the repair machinery. We designed a robust protocol including a solid-phase extraction step (SPE), an enzymatic treatment aimed at releasing individual photoproducts, and a liquid chromatography method combining on-line SPE and ultra-high-performance liquid chromatography for optimal specificity and sensitivity. We also added relevant internal standards for a better accuracy. The method was validated for linearity, repeatability, and reproducibility. The limits of detection and quantification were found to be in the fmol range. The proof of concept of the use of excreted DNA repair products as biomarkers of the genotoxicity of UV was obtained first in in vitro studies using cultured HaCat cells and ex vivo on human skin explants. Further evidence was obtained from the detection of pyrimidine dimers in the urine of human volunteers collected after recreational exposure in summer. An assay was designed to quantify the DNA photoproducts released from cells within short fragments by the DNA repair machinery. These oligonucleotides were isolated by solid-phase extraction and enzymatically hydrolyzed. The photoproducts were then quantified by on-line SPE combined with UHPLC-MS/MS with isotopic dilution.

Skin Retention of Sorbates from an After Sun Formulation for a Broad Photoprotection

Overexposure to sunlight is widely accepted as the underlying cause of cutaneous melanoma. UV radiation induces the formation of DNA photoproducts that, if unrepaired, can induce carcinogenic mutations. Recent data indicate that sorbates can be useful to widen the protection against UV radiation by acting as a triplet-state quencher in the melanocyte. The aim of the present work was to prepare an after sun formulation containing ethylsorbate or sorbic acid in order to take advantage of the triplet-state quenching activity of these molecules and protect the skin from UV-induced damages. Ethylsorbate and sorbic acid were characterized in terms of solubility and partition coefficient, and their transdermal permeation and skin accumulation were studied in vitro from simple solutions and in the presence of cyclodextrins (alpha and hydroxypropylbeta) as a complexing agent. The goal was to reduce as much as possible sorbates permeation while sustaining their skin levels. The obtained results indicated that the addition of alphacyclodextrins determined a 6-folds (ethylsorbate ) or 4-folds (sorbic acid) reduction of the transdermal permeation. Sorbic acid and alphacyclodextrin (1:1 molar ratio) were then formulated in an after sun vehicle using 1.5% hyaluronic acid (sodium salt) as a thickener and hydrating agent. The addition of hyaluronic acid gave rise to a formulation with good cosmetic properties and good sorbate (0.2–0.3 µmol/cm2) skin levels (stratum corneum + viable epidermis) and thus a potential protection against post-exposure UV damage.

The use of suction blisters to measure sunscreen protection against UVR-induced DNA damage

The formation of DNA photoproducts caused by solar UVR exposure needs to be investigated in-vivo and in particular in order to assess sunscreens' level of protection against solar genotoxicity. The study's purposes were: i) to evaluate if the roof of suction blisters is an appropriate sampling method for measuring photoproducts, and ii) to measure in-vivo sunscreen protection against cyclobutane pyrimidine dimers. Skin areas on the interior forearms of eight healthy volunteers were exposed in-vivo to 2 MED of simulated solar radiation (SSR) and to 15 MED on a sunscreen protected area. After irradiation, six suction blisters were induced and the blister roofs were collected. Analysis of SSR-induced CPDs was performed by two independent methods: a chromatography coupled to mass spectroscopy (HPLC-MS/MS) approach and a 3D–imaging of CPD immunostaining by multiphoton microscopy on floating epidermal sheets. HPLC-MS/MS analyses showed that SSR-unexposed skin presented no CPD dimers, whereas 2 MED SSR-exposed skin showed a significant number of TT-CPD. The sunscreen covered skin exposed to 15 MED appeared highly protected from DNA damage, as the amount of CPD-dimers remained below the detection limit. The multiphoton-immunostaining analysis consistently showed that no CPD staining was observed on the non-SSR-exposed skin. A significant increase of CPD staining intensity and number of CPD-positive cells were observed on the 2 MED SSR-exposed skin. Sunscreen protected skin presented a very low staining intensity and the number of CPD-positive cells remained very close to non-SSR-exposed skin. This study showed that suction blister samples are very appropriate for measuring CPD dimers in-vivo, and that sunscreens provide high protection against UVR-induced DNA damage.

Insulin-like Growth Factor 1 Receptor Signaling Is Required for Optimal ATR-CHK1 Kinase Signaling in Ultraviolet B (UVB)-irradiated Human Keratinocytes

UVB wavelengths of light induce the formation of photoproducts in DNA that are potentially mutagenic if not properly removed by the nucleotide excision repair machinery. As an additional mechanism to minimize the risk of mutagenesis, UVB-irradiated cells also activate a checkpoint signaling cascade mediated by the ATM and Rad3-related (ATR) and checkpoint kinase 1 (CHK1) kinases to transiently suppress DNA synthesis and cell cycle progression. Given that keratinocytes in geriatric skin display reduced activation of the insulin-like growth factor 1 receptor (IGF-1R) and alterations in DNA repair rate, apoptosis, and senescence following UVB exposure, here we used cultured human keratinocytes in vitro and skin explants ex vivo to examine how IGF-1R activation status affects ATR-CHK1 kinase signaling and the inhibition of DNA replication following UVB irradiation. We find that disruption of IGF-1R signaling with small-molecule inhibitors or IGF-1 withdrawal partially abrogates both the phosphorylation and activation of CHK1 by ATR and the accompanying inhibition of chromosomal DNA synthesis in UVB-irradiated keratinocytes. A critical protein factor that mediates both ATR-CHK1 signaling and nucleotide excision repair is replication protein A, and we find that its accumulation on UVB-damaged chromatin is partially attenuated in cells with an inactive IGF-1R. These results indicate that mutagenesis and skin carcinogenesis in IGF-1-deficient geriatric skin may be caused by defects in multiple cellular responses to UVB-induced DNA damage, including through a failure to properly suppress DNA synthesis on UVB-damaged DNA templates.

DNA Photochemistry: Geometrically Unconstrained Pyrimidine (6–4) Pyrimidone Photoproducts Do Photoisomerize

Structural features are of major importance for the formation of mutagenic photoproducts in DNA. It was recently reported that lack of constraints between two adjacent nucleosidic units prevents the conversion of pyrimidine (6-4) pyrimidone photoproducts into their Dewar valence isomers. We here report that this is not the case for the thymidine photoproducts which, although unconstrained, are quantitatively converted into photolysis products identified as Dewar valence isomers by mass spectrometry and NMR and infrared spectroscopies.

The relative roles of DNA damage induced by UVA irradiation in human cells

UVA light (320-400 nm) represents approximately 95% of the total solar UV radiation that reaches the Earth's surface. UVA light induces oxidative stress and the formation of DNA photoproducts in skin cells. These photoproducts such as pyrimidine dimers (cyclobutane pyrimidine dimers, CPDs, and pyrimidine (6-4) pyrimidone photoproducts, 6-4PPs) are removed by nucleotide excision repair (NER). In this repair pathway, the XPA protein is recruited to the damage removal site; therefore, cells deficient in this protein are unable to repair the photoproducts. The aim of this study was to investigate the involvement of oxidative stress and the formation of DNA photoproducts in UVA-induced cell death. In fact, similar levels of oxidative stress and oxidised bases were detected in XP-A and NER-proficient cells exposed to UVA light. Interestingly, CPDs were detected in both cell lines; however, 6-4PPs were detected only in DNA repair-deficient cells. XP-A cells were also observed to be significantly more sensitive to UVA light compared to NER-proficient cells, with an increased induction of apoptosis, while necrosis was similarly observed in both cell lines. The induction of apoptosis and necrosis in XP-A cells using adenovirus-mediated transduction of specific photolyases was investigated and we confirm that both types of photoproducts are the primary lesions responsible for inducing cell death in XP-A cells and may trigger the skin-damaging effects of UVA light, particularly skin ageing and carcinogenesis.

DNA damage as a biological sensor for environmental sunlight

Solar ultraviolet (UV) radiation is widely known as an environmental genotoxic agent that affects ecosystems and the human population, generating concerns and motivating worldwide scientific efforts to better understand the role of sunlight in the induction of DNA damage, cell death, mutagenesis, and ultimately, carcinogenesis. In this review, general aspects of UV radiation at the Earth's surface are reported, considering measurements by physical and biological sensors that monitor solar UV radiation under different environmental conditions. The formation of DNA photoproducts and other types of DNA damage by different UV wavelengths are compared with the present information on their roles in inducing biological effects. Moreover, the use of DNA-based biological dosimeters is presented as a feasible molecular and cellular tool that is focused on the evaluation of DNA lesions induced by natural sunlight. Clearly, direct environmental measurements demonstrate the biological impact of sunlight in different locations worldwide and reveal how this affects the DNA damage profile at different latitudes. These tools are also valuable for the quantification of photoprotection provided by commercial sunscreens against the induction of DNA damage and cell death, employing DNA repair-deficient cells that are hypersensitive to sunlight. Collectively, the data demonstrate the applicability of DNA-based biosensors as alternative, complementary, and reliable methods for registering variations in the genotoxic impact of solar UV radiation and for determining the level of photoprotection sunscreens provided at the level of DNA damage and cell death.

Direct Effects of UV-B Radiation on the Freshwater Heterotrophic NanoflagellateParaphysomonassp

The formation of DNA photoproducts in organisms exposed to ambient levels of UV-B radiation can lead to death and/or reduced population growth in aquatic systems. Dependence on photoenzymatic repair to reverse DNA damage caused by UV-B radiation is demonstrated for Paraphysomonas sp., a member of a widely distributed genus of heterotrophic nanoflagellates. At 20 degrees C, Paraphysomonas sp. was exposed to a range of UV-B intensities encountered in natural systems. Populations of the flagellate survived and grew in a dose-dependent manner, but only when simultaneously exposed to photorepair radiation (PRR). In contrast, flagellates exposed to UV-B at 15 degrees C suffered 100% mortality except at the lowest UV-B level (with PRR) tested, which suggested a photorepair temperature optimum above 15 degrees C. After acute UV-B exposures, DNA damage (measured as the formation of pyrimidine dimers) was reduced only in organisms that underwent subsequent exposure to PRR. Populations kept in the dark after UV-B exposure maintained the initial levels of pyrimidine dimers. These results are the first to demonstrate the reliance of a heterotrophic flagellate on photoenzymatic DNA repair for survival from UV-B exposure.

Influence of cytosine methylation on ultraviolet-induced cyclobutane pyrimidine dimer formation in genomic DNA

The ultraviolet (UV) component of sunlight is the main cause of skin cancer. More than 50% of all non-melanoma skin cancers and >90% of squamous cell carcinomas in the US carry a sunlight-induced mutation in the p53 tumor suppressor gene. These mutations have a strong tendency to occur at methylated cytosines. Ligation-mediated PCR (LMPCR) was used to compare at nucleotide resolution DNA photoproduct formation at dipyrimidine sites either containing or lacking a methylated cytosine. For this purpose, we exploited the fact that the X chromosome is methylated in females only on the inactive X chromosome, and that the FMR1 (fragile-X mental retardation 1) gene is methylated only in fragile-X syndrome male patients. Purified genomic DNA was irradiated with UVC (254 nm), UVB (290–320 nm) or monochromatic UVB (302 and 313 nm) to determine the effect of different wavelengths on cyclobutane pyrimidine dimer (CPD) formation along the X-linked PGK1 (phosphoglycerate kinase 1) and FMR1 genes. We show that constitutive methylation of cytosine increases the frequency of UVB-induced CPD formation by 1.7-fold, confirming that methylation per se is influencing the probability of damage formation. This was true for both UVB sources used, either broadband or monochromatic, but not for UVC. Our data prove unequivocally that following UVB exposure methylated cytosines are significantly more susceptible to CPD formation compared with unmethylated cytosines.

How DNA lesions are turned into powerful killing structures: Insights from UV-induced apoptosis

Mammalian cells treated with ultraviolet (UV) light provide one of the best-known experimental systems for depicting the biological consequences of DNA damage. UV irradiation induces the formation of DNA photoproducts, mainly cyclobutane pyrimidine dimers (CPDs) and (6-4) pyrimidine-pyrimidone photoproducts [(6-4)PPs], that drastically impairs DNA metabolism, culminating in the induction of cell death by apoptosis. While CPDs are the most important apoptosis-inducing lesions in DNA repair proficient cells, recent data indicates that (6-4)PPs also signals for apoptosis in DNA repair deficient cells. The toxic effects of these unrepaired DNA lesions are commonly associated with transcription blockage, but there is increasing evidence supporting a role for replication blockage as an apoptosis-inducing signal. This is supported by the observations that DNA double-strand breaks (DSBs) arise at the sites of stalled replication forks, that these DSBs are potent inducers of apoptosis and that inhibition of S phase progression diminishes the apoptotic response. Reactive oxygen species, generated after exposure of mammalian cells to longer UV wavelengths, may also induce apoptotic responses. In this regard, emphasis is given to the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-OxoG), but indirect induced lesions such as lipoperoxide DNA adducts also deserve attention. ATR is the main established sensor molecule for UV-induced DNA damage. However, there is evidence that ATM as well as the MAPK pathway also play a role in the UV response by activating either the death receptor or the mitochondrial damage pathway. Adding more complexity to the subject, cells under stress suffer other types of processes that may result in cell death. Autophagy is one of these processes, with extensive cross-talks with apoptosis. No matter the mechanisms, cell death avoids cells to perpetuate mutations induced by genotoxic lesions. The understanding of such death responses may provide the means for the development of strategies for the prevention and treatment of cancer.

The Arylhydrocarbon Receptor Sees the Light

When cells are exposed to ultraviolet light (UVB) the UV response is initiated. This signaling response has two main pathways. The first occurs in the nucleus where UV irradiation causes the formation of DNA photoproducts. A second, DNA-independent, cytoplasmic signaling pathway activates members of the family of mitogen-activated protein kinases (MAPKs). Activation of MAPKs and the resulting inflammation of the skin are important in the development of skin cancers. Although the identity of the initial signaling molecule of this pathway is unknown, some clues pointed to the involvement of the arylhydrocarbon receptor (AhR). AhR is a cytosolic receptor that mediates the toxic effects of polycyclic aromatic hydrocarbons. Upon binding to such compounds, it dissociates from its chaperones, Hsp90 (heat shock protein 90 kD) and c-src, and translocates to the nucleus, where it activates the transcription of cytochrome P450 (also known as CYP ), resulting in the synthesis of CYP protein, an enzyme important in the breakdown of toxic substances. Exposure of human skin to UVB results in the synthesis of CYP, and in vitro UVB irradiation of human cells increases expression of CYP . Fritsche et al . exposed a human keratinocyte cell line (HaCaT), transfected with a plasmid encoding an AhR-green fluorescent protein (GFP) fusion protein, to UVB radiation and monitored (by fluorescent microscopy) translocation of AhR-GFP to the nucleus. Real-time polymerase chain reaction assays showed that UVB irradiation of HaCaT cells (which have endogenous AhR) resulted in increased transcription of CYP1A1 compared with that in cells that had been treated with either an AhR-specific inhibitor or an AhR-specific siRNA (AhR KO) before UVB irradiation. UVB irradiation causes internalization of epidermal growth factor receptors (EGFRs), and the authors used fluorescence microscopy and Western blotting to show that UVB irradiation of HaCaT cells, but not AhR KO cells, resulted in EGFR internalization and the phosphorylation of MAPKs. The internalization of growth factor receptors has been suggested as a crucial early step in the cytosolic UV response. AhR nuclear translocation and CYP1A1 expression were blocked in cells cultured in media depleted of tryptophan. Tryptophan is converted to the AhR ligand FICZ (6-formylindolo[3,2-b]carbazole) after exposure to UVB, but the authors are the first to show this process occurring in UVB-treated human cells. Both CYP1A1 expression and EGFR internalization were measurable in FICZ-treated HaCaT cells, but not in AhR KO cells. FICZ-mediated internalization of EGFR was blocked by inhibition of c-src activity. Finally, the authors showed that UVB irradiation of wild-type, but not AhR-deficient, mice resulted in the induction of CYP1A1 expression, establishing a role for AhR in mediating the UVB stress response in vivo. E. Fritsche, C. Schäfer, C. Calles, T. Bernsmann, T. Bernshausen, M. Wurm, U. Hübenthal, J. E. Cline, H. Hajimiragha, P. Schroeder, L.-O. Klotz, A. Rannug, P. Fürst, H. Hanenberg, J. Abel, J. Krutmann, Lightening up the UV response by identification of the arylhydrocarbon receptor as a cytoplasmic target for ultraviolet B radiation. Proc. Natl. Acad. Sci. U.S.A. 104 , 8851-8856 (2007). [Abstract] [Full Text]

Repair-Independent Chromatin Assembly onto Active Ribosomal Genes in Yeast after UV Irradiation

Chromatin rearrangements occur during repair of cyclobutane pyrimidine dimers (CPDs) by nucleotide excision repair (NER). Thereafter, the original structure must be restored to retain normal genomic functions. How NER proceeds through nonnucleosomal chromatin and how open chromatin is reestablished after repair are unknown. We analyzed NER in ribosomal genes (rDNA), which are present in multiple copies but only a fraction are actively transcribed and nonnucleosomal. We show that removal of CPDs is fast in the active rDNA and that chromatin reorganization occurs during NER. Furthermore, chromatin assembles on nonnucleosomal rDNA during the early events of NER but in the absence of DNA repair. The resumption of transcription after removal of CPDs correlates with the reappearance of nonnucleosomal chromatin. To date, only the passage of replication machinery was thought to package ribosomal genes in nucleosomes. In this report, we show that early events after formation of UV photoproducts in DNA also promote chromatin assembly.

Short-term and long-term cellular and molecular events following UV irradiation of skin: implications for molecular medicine.

Acute ultraviolet (UV) irradiation of normal human skin results in several clinical effects, including sunburn inflammation (erythema) and tanning, histological changes such as thickening of the epidermis, and local or systemic immunosuppression. Chronic UV irradiation leads to photoaging, sustained immunosuppression and photocarcinogenesis. Photocarcinogenesis involves the accumulation of genetic changes, as well as immune system modulation, and ultimately leads to the development of skin cancers. Recent advances in molecular and cellular biology have clarified the mechanisms of photocarcinogenesis, including the formation of DNA photoproducts, DNA repair, the mutation of proto-oncogenes and tumour suppressor genes, and UV-induced immunosuppression. Further investigation and a better understanding of photocarcinogenesis are critical to the development of effective prevention and intervention strategies for human skin cancer.

Molecular mechanisms of photocarcinogenesis.

Photocarcinogenesis represents the accumulation of genetic changes as well as immune system modulation, which ultimately lead to the development of skin cancers. The recent advances in molecular and cellular biology have clarified the mechanisms of photocarcinogenesis, including the formation of DNA photoproducts, DNA repair, mutation of proto-oncogenes and tumor suppressor genes, and UV-induced immunosuppression. The understanding and further investigation of photocarcinogenesis is critical to the development of effective prevention and intervention strategies for human skin cancer.

The DNA damage spectrum produced by simulated sunlight

The mutagenic effects of ultraviolet and solar irradiation are thought to be due to the formation of DNA photoproducts, most notably cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6–4) pyrimidone photoproducts ((6–4)PPs). Experimental systems for determining the levels and sequence dependence of photoproduct formation in DNA have often used high doses of short-wave (UVC) irradiation. We have re-assessed this issue by using DNA sequencing technologies and different doses of UVC as well as more physiologically relevant doses of solar irradiation emitted from a solar UV simulator. It has been questioned whether hot alkali treatment can detect (6–4)PPs at all sequence positions. With high UVC doses, the sequence distribution of (6–4)PPs was virtually identical when hot alkali or UV damage endonuclease (UVDE) were used for detection, which appears to validate both methods. The (6–4)PPs form at 5′-TpC and 5′CpC sequences but very low levels are seen at all other dipyrimidines including 5′-TpT. Contrary to expectation, we find that (6–4) photoproducts form at almost undetectable levels under conditions of irradiation for up to five hours with the solar UV simulator. The same treatment produces high levels of CPDs. In addition, DNA glycosylases, which recognize oxidized and ring-opened bases, did not produce significant cleavage of sunlight-irradiated DNA. From these data, we conclude that cyclobutane pyrimidine dimers are at least 20 to 40 times more frequent than any other DNA photoproduct when DNA or cells are irradiated with simulated sunlight.

15] Ultraviolet A- and singlet oxygen-induced mutation spectra

Publisher Summary Genetic stability is an important prerequisite for the maintenance of the integrity and functions of each cell. Several environmental agents, as well as endogenous sources, are known to play a significant role in the formation of DNA lesions. Most of these lesions are repaired, but a few may escape and can lead to mutagenesis during trans -lesion replication or to chromosomal aberrations, and, finally, to tumorigenesis or cell death. The causal relationship between solar ultraviolet (UV) radiation and carcinogenesis has been well established. UVB radiation (290-320 nm) causes the direct formation of DNA photoproducts and gives rise to mutations in proto-oncogenes and tumor suppressor genes that eventually lead to skin cancer. Genotoxicity of UVA radiation (320-400 nm), which represents more than 90% of the terrestrial UV solar energy, is most likely induced by indirect mechanisms. After UVA photon absorption by an unidentified endogenous photosensitizer, reactive oxygen species are generated that could react with DNA without further intermediates (type I photosensitized reaction) or via singlet oxygen (type II photosensitized reaction). The singlet oxygen appears to be an early intermediate of UVA cellular response, judging by its involvement in signal transduction and gene induction. It belongs to reactive oxygen species (ROS) that are ubiquitous oxidizing agents generated in all aerobic organisms, endogenously as byproducts of respiration and inflammatory response and also exogenously after exposure to a variety of agents. This chapter discusses data on DNA lesions, DNA repair, and mutation spectra, which characterize the genotoxic potential of both singlet oxygen and UVA radiation, essentially in mammalian species.

Short Interspersed DNA Element-mediated detection of UVB-induced DNA damage and repair in the mouse genome, in vitro, and in vivo in skin

We report a sensitive, SINE (Short Interspersed DNA Element)-mediated, PCR-based, DNA damage detection assay. Here, the SINE assay is used for detection of UVB-induced DNA damage and repair in cultured mouse cells and in vivo, in mouse skin. The unique feature of the SINE assay is its ability to support simultaneous amplification of multiple, random segments of genomic DNA. This can be accomplished due to the remarkable abundance, dispersion and conservation of SINEs in mammalian genomes. The most abundant SINEs in the mouse genome are the B1 elements, at a copy number of 50,000–80,000. Due to their strong sequence conservation, primers complementary to the B1 consensus sequence anneal to the majority of their targets in the genome. Consequently, long segments of genomic DNA located between pairs of B1 elements are efficiently amplified by PCR. Thus, in conjunction with the fact that many types of DNA adducts form blocks for thermostable polymerase, the B1 element anchored PCR makes a sensitive and versatile tool for assessing the overall integrity of the transcribed regions in mouse genome. We measured UVB-dose (0.1–3 kJ m−2) dependent formation of photoproducts in DNA from cultured cells, and after 20 h observed a substantial removal of damage at doses lower or equal to 0.6 kJ m−2. The sensitivity of detection of UVB-photoproducts formation and repair was compared to that of the conventional, single locus-targeting QPCR. Using the SINE assay we also have shown the distribution of UVB and UVC induced DNA adducts at a single nucleotide resolution within the B1 elements in mouse DNA. Lastly, we demonstrated that the sensitivity of the SINE assay is adequate for measurement of UVB-dose (1–6 kJ m−2) dependent formation and subsequent removal of photoproducts in vivo, in mouse skin.

DNA repair and survival in human lens epithelial cells with extended lifespan

Purpose. Ultraviolet-B radiation (290–320 nm) produces cataracts in animals and has been associated with human cataract formation in several epidemiological studies. UVB radiation decreases the long-term cell survival and changes the pattern of protein synthesis in cultured lens epithelial cells. However, the relationship between DNA photoproduct formation and long term cell survival in human lens epithelial cells is not known. In the present work, we used human lens epithelial cells with extended lifespan (HLE B-3 cells) to examine the kinetics of DNA repair and cell survival after UVB exposure. Methods. Cyclobutane pyrimidine dimers and pyrimidine-pyrimidone (6–4) photoproducts were analyzed by radioimmunoassay. Long-term survival of the cells was determined by measuring their ability form colonies when plated at low density. Results. HLE B-3 cells were repair competent after UVB (302 nm) exposure. Excision repair of the (6–4) photoproduct was more efficient than that of the cyclobutane dimer. Ninety five percent of the (6–4) photoproducts were repaired 24 h after 400 J/m2 UVB exposure, whereas 50% of the cyclobutane dimers were repaired during this time. When cells were split for the clonogenic assay immediately after irradiation, only 10% of the cells formed colonies following 7 days of culture in the serum-containing medium. When cells were split for the clonogenic assay after a 48 hour incubation in serum-containing medium, the colony-forming ability of the irradiated cells increased to 60% following culture in a serum-containing medium. Conclusions. These results indicate a close correlation between the repair of cyclobutane dimers and the increase in the long-term survival of the cells as measured by their colony-forming ability. The extended lifespan human lens epithelial cells HLE B-3 may be a useful model to investigate the mechanism and regulation of UVB-induced DNA repair in human lens cells.

Thymidine Dinucleotide Mimics the Effect of Solar Simulated Irradiation on p53 and p53-Regulated Proteins

The tumor suppressor protein p53 participates in DNA repair and cell cycle regulation in response to injuries like ultraviolet (UV) irradiation. We have previously reported that the thymidine dinucleotide (pTpT), a common target for DNA photoproduct formation by UV light, mimics many effects of UV irradiation in cultured skin-derived cells, at least in part through the activation of p53. In this report we compare the effects of solar-simulated irradiation and pTpT on p53 and p53-regulated proteins involved in cellular growth arrest and DNA repair in cultured human dermal fibroblasts. We find that, like UV irradiation, pTpT increases the levels of p53, p21, and proliferating-cell nuclear antigen. The magnitude and time course of the inductions are UV dose dependent and consistent with known regulatory interactions among these nuclear proteins. These data confirm and expand previous studies of UV effects on nuclear proteins involved in cell cycle regulation and DNA repair. Our observations suggest that such protective effects can also be induced by pTpT in the absence of initial DNA damage, rendering cells more capable of responding to subsequent DNA damage.