Melanin offers protection against induction of cyclobutane pyrimidine dimers and 6-4 photoproducts by UVB in cultured human melanocytes

The goal of this investigation was to correlate the melanin content in human pigmentary cells with the generation of UVB-induced photoproducts and to examine the relationship between the melanin content and the removal of the photoproducts. Cultured melanocytes from light-skinned individuals synthesized less melanin and produced more cyclobutane pyrimidine dimers and 6-4 photoproducts upon UVB exposure than did melanocytes from black skin. Tyrosine-stimulated melanogenesis provided protection against DNA damage in both cell types. In another set of pigmented cell lines a ratio between eumelanin and pheomelanin was determined. The assessment of association between DNA damage induction and the quantity and quality of melanin revealed that eumelanin concentration correlated better with DNA protection than pheomelanin. Skin type-I and skin type-VI melanocytes, congenital nevus (CN)-derived cells and skin type-II melanocytes from a multiple-melanoma patient were grown in media with low or high L-tyrosine concentration. The cells were irradiated with 200 J/m2 UVB, and the levels of the photoproducts were determined immediately and after 6 and 24 h. Once again the induction of the photoproducts was mitigated by increased melanogenesis, and it was inversely correlated with the skin type. No significant differences were found for the removal of photoproducts in the cultures of skin types I and VI and CN cells. No indications of a delay in the removal of photoproducts in the melanocytes from the multiple-melanoma patient were found either.     

Formation of cyclobutane pyrimidine dimers and 8-oxo-7,8-dihydro-2'-deoxyguanosine in mouse and organ-cultured human skin by irradiation with broadband or with narrowband UVB

Narrowband UVB (NB-UVB) is a newly developed UVB source that, in addition to the previously used broadband UVB (BB-UVB), has been effectively used in phototherapy of various skin diseases. Besides its therapeutic effectiveness, NB-UVB also has some adverse effects that should be evaluated. As with all phototherapies, the photocarcinogenic potential of NB-UVB is the major concern. To assess the carcinogenic potential we measured the DNA damage induced by the two UVB sources because exposure of cells to UVB directly or indirectly induces DNA damage such as cyclobutane pyrimidine dimers (CPD) or 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), respectively. These types of DNA damage cause mutations of oncogenes and tumor suppressor genes, which can lead to photocarcinogenesis. In the present study we measured the yield of CPD and the oxidative DNA damage marker, 8-oxodGuo, in organ-cultured human skin and in mouse skin after exposure to NB-UVB or BB-UVB at therapeutically equivalent doses. We show that a 10-fold higher dose of NB-UVB yields a similar amount of CPD compared with BB-UVB in two types of samples examined. In contrast to CPD, the formation of 8-oxodGuo after irradiation with NB-UVB at a 10-fold higher dose is 1.5-3 times higher than that caused by BB-UVB. These results suggest that although NB-UVB at equivalent erythema-edema doses is not more potent in inducing CPD formation than is BB-UVB, NB-UVB may generate a higher yield of oxidized DNA damage.     

Effects and Mechanism of Nicotinamide Against UVA‐ and/or UVB‐mediated DNA Damages in Normal Melanocytes

Melanoma incidences are increasing rapidly, and ultraviolet (UV) radiation from the sun is believed to be its major contributing factor. UV exposure causes DNA damage in skin which may initiate cutaneous skin cancers including melanoma. Melanoma arises from melanocytes, the melanin‐producing skin cells, following genetic dysregulations resulting into hyperproliferative phenotype and neoplastic transformation. Both UVA and UVB exposures to the skin are believed to trigger melanocytic hyperplasia and melanomagenesis. Melanocytes by themselves are deficient in repair of oxidative DNA damage and UV‐induced photoproducts. Nicotinamide, an active form of vitamin B3 and a critical component of the human body's defense system has been shown to prevent certain cancers including nonmelanoma skin cancers. However, the mechanism of nicotinamide's protective effects is not well understood. Here, we investigated potential protective effects and mechanism of nicotinamide against UVA‐ and/or UVB‐ induced damage in normal human epidermal melanocytes. Our data demonstrated an appreciable protective effect of nicotinamide against UVA‐ and/or UVB‐ induced DNA damage in melanocytes by decreasing both cyclobutane pyrimidine dimers and 8‐hydroxy‐2′‐deoxyguanosine levels. We found that the photoprotective response of nicotinamide was associated with the activation of nucleotide excision repair genes and NRF2 signaling. Further studies are needed to validate our findings in in vivo models.     

Attenuation of DNA damage in the dermis and epidermis of the albino hairless mouse by chronic exposure to ultraviolet-A and -B radiation

Mammalian skin is vulnerable to the photocarcinogenic and photoaging effects of solar UV radiation and defends itself using a variety of photoprotective responses including epidermal thickening, tanning and the induction of repair and antiradical systems. We treated Skh-1 albino hairless mice for 60 days with ultraviolet-A (UVA) or ultraviolet-B (UVB) radiation and measured the frequency of cyclobutane pyrimidine dimers and pyrimidine(6-4)pyrimidone photoproducts induced by a single acute sunburn dose of UVB at different stages of the chronic treatment. We found that both UVA and UVB exposure produced a photoprotective response in the dermis and epidermis and that the degree of photoproduct attenuation was dependent on dose, wavelength and the type of damage induced. Although epidermal thickening was important, our data suggest that UV protective compounds other than melanin may be involved in mitigating the damaging effects of sunlight in the skin.     

Molecular Regulation of UV‐Induced DNA Repair

Ultraviolet (UV) radiation from sunlight is a major etiologic factor for skin cancer, the most prevalent cancer in the United States, as well as premature skin aging. In particular, UVB radiation causes formation of specific DNA damage photoproducts between pyrimidine bases. These DNA damage photoproducts are repaired by a process called nucleotide excision repair, also known as UV‐induced DNA repair. When left unrepaired, UVB‐induced DNA damage leads to accumulation of mutations, predisposing people to carcinogenesis as well as to premature aging. Genetic loss of nucleotide excision repair leads to severe disorders, namely, xeroderma pigmentosum (XP), trichothiodystrophy (TTD) and Cockayne syndrome (CS), which are associated with predisposition to skin carcinogenesis at a young age as well as developmental and neurological conditions. Regulation of nucleotide excision repair is an attractive avenue to preventing or reversing these detrimental consequences of impaired nucleotide excision repair. Here, we review recent studies on molecular mechanisms regulating nucleotide excision repair by extracellular cues and intracellular signaling pathways, with a special focus on the molecular regulation of individual repair factors.     

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.     

Physiological Doses of Ultraviolet Irradiation Induce DNA Strand Breaks in Cultured Human Melanocytes, as Detected by Means of an Immunochemical Assay

Abstract— An immunochemical assay, i.e. sandwich enzyme-linked immunosorbent assay, has been modified to detect UV-induced damage in cellular DNA of monolayer-grown human melanocytes. The method is based on the binding of a monoclonal antibody to single-stranded DNA. The melanocytes derived from human foreskin of skin type II individuals were suspended and exposed to UVA, UVB, solar-simulated light or γ-rays. Following physiological doses of UVA, UVB or solar-simulated light, a dose-related DNA unwinding comprising a considerable number of single-strand breaks (ssb) was observed. No correlation was found between different seeded cell densities or different culturing periods and the UVA sensitivity of the cells. After UVA irradiation, 0.07 ssb/10¹⁰ Da/kJ/m² were detected and after UVB irradiation 1.9 ssb/10¹⁰ Da/kJ/m² were seen. One minimal erythema dose of solar-simulated light induced 2.25 ssb/10¹⁰ Da. Our results from melanocytes expressed in ssb/Da DNA are comparable and have the same sensitivity toward UVA as well as toward UVB as nonpigmented skin cells. As low doses of UVA have already been shown to induce detectable numbers of ssb, this assay is of great interest for further investigations about the photoprotecting and/or photosensitizing effects of melanins in human melanocytes derived from different skin types.     

Cell Cycle Kinetics Following UVA Irradiation in Comparison to UVB and UVC Irradiation

Abstract— There is limited information about the carcinogenic effect of longwave ultraviolet radiation (UVA: 315-400 nm). In particular very little is known about the relevant genotoxic damage caused by physiological doses of UVA radiation. A general response of cells to DNA damage is a delay or arrest of the cell cycle. Conversely, such cellular responses after UVA irradiation would indicate significant genotoxic damage. The aim of this study is to compare cell cycle kinetics of human fibroblasts after UVC (190-280 nm radiation), UVB (280-315 nm radiation) and UVA irradiation. Changes in the cell cycle kinetics were assessed by bivariate flow cytometric analysis of DNA synthesis and of DNA content. After UVC, UVB or UVA irradiation of human fibroblasts a suppression was seen of bromodeoxyuridine (BrdU) incorporation at all stages of S phase. The magnitude of this suppression appeared dose dependent. Maximum suppression was reached at 5-7 h after UVB exposure and directly after UVA exposure, and normal levels were reached 25 h after UVB and 7 h after UVA exposure. The lowered BrdU uptake corresponded with a lengthening of the S phase. No dramatic changes in percentages of cells in G₁, S and G₂/M were seen after the various UV irradiations. Apparently, UVA irradiation, like UVB and UVC irradiation, can temporarily inhibit DNA synthesis, which is indicative of genotoxic damage.     

Interexperimental and interindividual variations of DNA repair capacities after UV-B and UV-C irradiations of human keratinocytes and fibroblasts

DNA repair plays a central role in the cellular response to UV. In this work we have studied the response of skin cells (i.e. fibroblasts and keratinocytes) from the same or from different individuals after both ultraviolet-B (UV-B) and ultraviolet-C (UV-C) irradiations using the comet assay to characterize the specific cellular response to UV-induced DNA damage. Cells were irradiated with increasing doses of UV-B or UV-C. To study the UV dose dependency of initial steps of DNA repair, namely recognition and incision at DNA damage level, the comet assay was performed, under alkaline conditions, 60 min after UV irradiation to allow detection of DNA strand breaks. Comparative analysis of tail moment values after UV exposure of cells from the same or from different individuals showed interexperimental and interindividual variations, implying that repeated assays are necessary to characterize the individual DNA repair capacity. With increasing doses of UV in keratinocytes, a plateau was rapidly reached after irradiation, whereas in fibroblasts a linear dose-effect relationship was observed. These interindividual variations associated with cellular specificity in DNA response may be of significance in skin cell and individual susceptibility toward UV-induced carcinogenesis.     

An Experimental Population Study of Nucleotide Excision Repair as a Risk Factor for UVB‐induced Melanoma

Nucleotide excision repair (NER) is the primary defense against the DNA damage implicit in skin cancer formation and is negatively affected by chronic exposure to UVB radiation. However, in situ and in vitro studies consistently yield equivocal results when addressing individual DNA repair capacity and melanoma susceptibility. The primary objective of this study was to determine if individual global NER capacity is a risk factor for melanoma formation in a prominent UVB‐inducible melanoma model, hybrid Xiphophorus fishes. After neonatal UVB irradiation, adult tumor‐bearing and tumor‐free fish were given a challenge UVB dose and (6–4) photoproduct repair was quantified in individual fish at 24 h using radioimmunoassay. Despite considerable inter‐individual variation in repair capacity, ranging from 13% to 91%, we found no difference in mean NER capacity between fish with and without melanomas, thus detaching global NER from melanomagenesis. Furthermore, despite epidemiological data indicating that sex and age are important risk factors underlying melanoma susceptibility, we found no difference in mean NER rates among the sexes or as a function of age. We conclude with a discussion of the apparent paradox of how inter‐individual variation in NER is not a risk factor given the clear evidence that DNA damage underlies melanoma susceptibility.     

PYRIMIDINE DIMER FORMATION IN HUMAN SKIN

Cyclobutyl pyrimidine dimers are major photoproducts formed upon irradiation of DNA with ultraviolet light. We have developed a method for detecting as few as one pyrimidine dimer per million bases in about 50 ng of non-radioactive DNA, and have used this method to quantitate dimer yields in human skin DNA exposed in situ to UV. We found that UVA radiation (320–400 nm) produces detectable levels of dimers in the DNA of human skin. We also measured UVB-induced dimer yields in skin of individuals of differing sun sensitivity and found higher yields in individuals with higher UVB minimal erythema doses and greater sun sensitivity. These approaches should provide important information on damage induced in human skin upon exposure to natural or artificial sources of ultraviolet radiation.     

THE EPIDERMAL CELL KINETIC RESPONSE TO ULTRAVIOLET B IRRADIATION COMBINES REGENERATIVE PROLIFERATION AND CARCINOGEN ASSOCIATED CELL CYCLE DELAY

The cell cycle traverse of epidermal basal cells 24 h after in vivo exposure of ultraviolet B (UVB) irradiation was studied by immunochemical staining of incorporated bromodeoxyuridine (BrdU) and bivariate BrdU/DNA flow cytometric analysis. The results were compared with the cell kinetic patterns following topical application of the skin carcinogen methylnitrosourea (MNU) as well as the skin irritant cantharidin. Hairless mice were injected intraperitoneally with BrdU 24 h after treatment of their back skin with either a minimal erythema dose of UVB, or a single application of MNU or cantharidin dissolved in acetone. The cell cycle traverse of the BrdU-labelled cohorts of epidermal basal cells were then followed for the subsequent 12 h. At 6 h after BrdU-injection, when all labelled cells in the control group as well as in the cantharidin group had left the S phase, the bivariate distributions of the UVB-exposed and the MNU group showed that BrdU-positive cells were still present in S phase. Hence, UVB irradiation, similar to the carcinogen MNU, prolonged the S phase duration in some of the basal cells. At 12 h after pulse labelling, however, BrdU-positive cells from UVB-exposed mice were re-entering S phase from G1 phase, indicating that UVB irradiation induced a shortening of the cell cycle time as well, similar to the response observed after cantharidin. The present data can not tell whether these cells also were delayed in S phase. Thus, the cell cycle traverse in hairless mouse epidermis 24 h after in vivo exposure to UVB seemed to be a combination of the cell kinetic effects following chemical skin carcinogens and skin irritants. UVB irradiation induced both a delay in transit time through S phase, probably due to DNA damage and subsequent repair, as well as a reduction in the total cell cycle time consistent with rapid regenerative proliferation.     

UV Radiation Increases Carcinogenic Risks for Oral Tissues Compared to Skin

People can expose their oral cavities to UV (290–400 nm) by simply opening their mouths while outdoors. They can also have their oral cavities exposed to UV indoors to different UV‐emitting devices used for diagnoses, treatments and procedures like teeth whitening. Because the World Health Organization declared UV radiation as a complete human carcinogen in 2009, we asked if oral tissues are at a similar or higher carcinogenic risk compared to skin tissue. To understand the UVB (290–320 nm)‐related carcinogenic risks to these tissues, we measured initial DNA damage in the form of cyclobutane pyrimidine dimers (CPD), the repair rate of CPD (24 h) and the number of apoptotic dead cells over time resulting from increasing doses of erythemally weighted UV radiation. We used commercially available 3D‐engineered models of human skin (EpiDerm?), gingival (EpiGingival?) and oral (EpiOral?) tissues and developed an analytical approach for our tri‐labeling fluorescent procedure to identify total DNA, CPD and apoptotic cells so we can simultaneously quantify DNA repair rates and dead cells. Both DNA repair and apoptotic cell numbers are significantly lower in oral cells compared with skin cells. The combined results suggest UVB‐exposed oral tissues are at a significantly higher carcinogenic risk than skin tissues.     

1,25-Dihydroxyvitamin D3 and analogues protect primary human keratinocytes against UVB-induced DNA damage

Exposure to UVB irradiation is a major risk factor for the development of skin cancer. Therefore, it is important to identify agents that can offer protection against UVB-caused damage. Photocarcinogenesis is caused largely by mutations at sites of incorrectly repaired DNA photoproducts, of which the most common are the cyclobutane pyrimidine dimers (CPDs). In this study, we demonstrated that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] protects primary human keratinocytes against the induction of CPDs by UVB. This protection required pharmacologic doses 1,25(OH)2D3 and an incubation period of at least 8 h before irradiation. Furthermore, we provided arguments indicating that the anti-proliferative capacity of 1,25(OH)2D3 underlies its protective effect against UVB-induced DNA damage. Finally, we showed that 19-nor-14-epi-23-yne-1,25(OH)2D3 (TX 522) and 19-nor-14,20-bisepi-23-yne-1,25(OH)2D3 (TX 527), two low-calcemic analogues of 1,25(OH)2D3, were even 100 times more potent than the parent molecule in inhibiting UVB-caused DNA damage. These molecules are therefore promising candidates for the chemoprevention of UVB-induced skin cancer.     

Medical UV Exposures and HIV Activation

This paper presents the first attempt to evaluate the potential of clinical UV exposures to induce the human immunodeficiency (HIV) promoter and, thus, to upregulate HIV growth in those skin cells that are directly affected by the exposure. Using the data for HIV promoter activation in vitro, we computed UVB and psoralen plus UVA (PUVA) doses that produce 50% of the maximal promoter activation (AD₅₀). Then, using (a) literature data for UV transmittance in the human skin, (b) a composite action spectrum for HIV promoter and pyrimidine dimer induction by UVB and (c) an action spectrum for DNA synthesis inhibition by PUVA, we estimated the distribution of medical UVB and PUVA doses in the skin. This allowed us to estimate how deep into the skin the HIV-activating doses might penetrate in an initial and an advanced stage of UVB or PUVA therapy. Such analysis was done for normal type II skin and for single exposures. The results allow us to predict where in the skin the HIV promoter may be induced by selected small and large therapeutic UVB or PUVA doses. To accommodate changes in skin topography due to disease and UV therapy, our considerations would require further refinements. For UVB we found that, when the incident dose on the surface of the skin is 500 J/m² (290–320nm) (initial stage of the therapy), the dose producing 50% of the maximal HIV promoter activation (AD^UVB₅₀) is limited to the stratum corneum. However, with an incident dose of 5000 J/m² (an advanced stage of the therapy), AD^UVB₅₀ may be delivered as far as the living cells of the epidermis and even to some parts of the upper dermis. For PUVA we found that, when the incident UVA doses are 25 or 100 kJ/m² (320–400nm) (an initial and an advanced stage of therapy, respectively), and the 8-methoxypsoralen concentration in the blood is 0.1 μg/mL (the desired level), the combined doses to the mid epidermis (and some areas of the upper dermis) are well below the 50% HIV promoter-activating PUVA dose (AD^PUVA₅₀). Only under the worst scenario conditions, i. e. an exceptionally high drug concentration in the patient's tissues and localization of HIV in the nearest proximity to the skin surface, would the combined PUVA dose expected during photochemotherapy exceed AD^PUVA₅₀. These results suggest that the probability of HIV activation in the epidermis by direct mechanisms is higher for UVB than for PUVA treatment. However, complexities of the UV-inducible HIV activation and immunomodulatory phenomena are such that our results by themselves should not be taken as an indication that UVB therapy carries a higher risk than PUVA therapy when administered to HIV-infected patients.     

Green tea phenol extracts reduce UVB-induced DNA damage in human cells via interleukin-12

Green tea chemoprevention has been a focus of recent research, as a polyphenolic fraction from green tea (GTP) has been suggested to prevent UV radiation-induced skin cancer. Recently, it was demonstrated that GTP reduced the risk for skin cancer in a murine photocarcinogenesis model. This was accompanied by a reduction in UV-induced DNA damage. These effects appeared to be mediated via interleukin (IL)-12, which was previously shown to induce DNA repair. Therefore, we studied whether GTP induction of IL-12 and DNA repair could also be observed in human cells. KB cells and normal human keratinocytes were exposed to GTP 5 h before and after UVB. UVB-induced apoptosis was reduced in UVB-exposed cells treated with GTP. GTP induced the secretion of IL-12 in keratinocytes. The reduction in UV-induced cell death by GTP was almost completely reversed upon addition of an anti-IL-12-antibody, indicating that the reduction of UV-induced cell death by GTP is mediated via IL-12. The ability of IL-12 to reduce DNA damage and sunburn cells was confirmed in "human living skin equivalent" models. Hence the previously reported UV-protective effects of GTP appear to be mediated in human cells via IL-12, most likely through induction of DNA repair.     

Ultraviolet B irradiation modulates the immune system of fish (Rutilus rutilus, Cyprinidae). Part III: Lymphocytes

The effects of short-term exposure to ultraviolet B (UVB) radiation on lymphocyte-related parameters were studied under controlled laboratory conditions using roach (Rutilus rutilus), a cyprinid teleost, as the model fish. In vitro lymphoproliferative responses stimulated with a T-cell-specific mitogen, concanavalin A (ConA), or a B-cell-specific activator, lipopolysaccharide (LPS), were decreased in exposed fish. Also nonstimulated proliferation was lower than in unexposed fish. ConA-activated responses returned to normal levels within 7 days after exposure, but LPS-activated responses were reduced throughout the 14 day follow-up. The capability of UVB-exposed fish to produce an antibody response was studied by intraperitoneal immunization with bovine gamma-globulin (BGG). The concentration of anti-BGG antibodies in plasma as well as the number of anti-BGG-specific antibody-secreting cells in the spleen or blood were not decreased in fish exposed either to a single dose of UVB prior to immunization, or to single dose of UVB prior to immunization followed by three additional doses after immunization. Immunoglobulin M (IgM) production, when assayed as plasma IgM level or as the number of IgM-secreting cells in the spleen or blood, was not suppressed after exposure to UVB irradiation. These results indicate that a single dose of UVB or short-term exposure to UVB irradiation has no negative effects on IgM production or reactivity against antigen administered via the intraperitoneal route. However, the suppression of in vitro lymphoproliferative responses suggest that exposure to UVB has the potential to interfere with lymphocyte-related functions in fish.     

No adaptation to UV-induced immunosuppression and DNA damage following exposure of mice to chronic UV-exposure

It is well known that ultraviolet (UV) radiation induces erythema, immunosuppression and carcinogenesis. We hypothesized that chronic exposure to solar UV radiation induces adaptation that eventually prevents the suppression of acquired immunity. We studied adaptation for UV-induced immunosuppression after chronic exposure of mice to a suberythemal dose of solar simulated radiation (SSR) with Cleo Natural lamps, and subsequent exposure to an immunosuppressive dose of solar or UVB radiation (TL12). After UV dosing, the mice were sensitized and challenged with either diphenylcyclopropenone (DPCP) or picryl chloride (PCl). To assess the adaptation induced by solar simulated radiation, we measured the proliferative response and cytokine production of skin-draining lymph node cells after immunization to DPCP, the contact hypersensitivity (CHS) response to PCl, and thymine-thymine (T-T) cyclobutane dimers in the skin of mice. After induction of immunosuppression by SSR or by TL12 lamps, the proliferative response of draining lymph node cells after challenge with DPCP, or the CHS after challenge with PCl, showed significant suppression of the immune response. Chronic irradiation from SSR preceding the immunosuppressive dose of UV failed to restore the suppressed immune response. Reduced lipopolysaccharide-triggered cytokine production (of IL-12p40, IFN-gamma, IL-6 and TNF-alpha) by draining lymph node cells of mice sensitized and challenged with DPCP indicated that no adaptation is induced. In addition, the mice were not protected from T-T dimer DNA damage after chronic solar irradiation. Our studies reveal no evidence that chronic exposure to low doses of SSR induces adaptation to UV-induced suppression of acquired immunity.     

Nucleotide excision repair proteins rapidly accumulate but fail to persist in human XP-E (DDB2 mutant) cells

The xeroderma pigmentosum (XP-E) DNA damage binding protein (DDB2) is involved in early recognition of global genome DNA damage during DNA nucleotide excision repair (NER). We found that skin fibroblasts from four newly reported XP-E patients with numerous skin cancers and DDB2 mutations had slow repair of 6-4 photoproducts (6-4PP) and markedly reduced repair of cyclobutane pyrimidine dimers (CPD). NER proteins (XPC, XPB, XPG, XPA and XPF) colocalized to CPD and 6-4PP positive regions immediately (<0.1 h) after localized UV irradiation in cells from the XP-E patients and normal controls. While these proteins persist in normal cells, surprisingly, within 0.5 h these repair proteins were no longer detectable at the sites of DNA damage in XP-E cells. Our results indicate that DDB2 is not required for the rapid recruitment of NER proteins to sites of UV photoproducts or for partial repair of 6-4PP but is essential for normal persistence of these proteins for CPD photoproduct removal.