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ABSTRACT

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-15 (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.

Abbreviations: CPD, cyclobutane pyrimidine dimmers; DMEM, Dulbecco modified Eagle medium; EDTA, ethylenediaminetetraacetic acid; EGF, epidermal growth factor; HPLC, high-performance liquid chromatography; NER, nucleotide excision repair; PBS, phosphate-buffered saline; UV-B, ultraviolet-B; UV-C, ultraviolet-C; XP, xeroderma pigmentosum.

?? 2004 American Society for Photobiology 0031-8655/04 $5.00 + 0.00

INTRODUCTION

Skin cells are directly exposed to UV irradiation, which induces DNA lesions, potentially leading to their transformation if they are not repaired. Ultraviolet-B (UV-B, 290-320 nm) is considered as the major wavelength range involved in skin carcinogenesis, although ultraviolet-A (320-400 nm) is also implicated. Individual DNA repair capacity strongly influences skin cancer susceptibility as illustrated in cancer prone DNA repair-deficient xeroderma pigmentosutn (XP) patients (1). In addition, polymorphisms in DNA repair genes have been shown to affect the DNA repair capacity, and some are over represented in certain cancer groups such as melanoma patients or patients with basal cell carcinoma (2-5).

The single-cell gel electrophoresis or comet assay allows the observation of DNA damage in isolated cells. The comet assay is a simple and sensitive microscopic method that can be performed on relatively small number of cells (20000-200000) (6,7), and different types of DNA damage can be observed according to the conditions chosen for lysis and electrophoresis. UV-B and ultraviolet-C (UV-C) irradiations generate lesions consisting mostly of cyclobutane pyrimidine dimmers (CPD) and pyrimidine (6-4) pyrimidone photoproducts (8). Both types of lesions are repaired through the nucleotide excision repair (NER) pathway, pyrimidine (6-4) pyrimidone photoproducts more rapidly than CPD (9). DNA single-strand breaks are transiently generated during the incision step of NER (10). Under alkaline conditions, causing DNA duplex denaturation, the stand breaks generated during the initial phase of DNA repair are observed by their effect on DNA migration. The number of DNA single-strand breaks are evaluated by the measurement of the tail moment values (product of tail length and tail DNA content). This parameter is most frequently used for UV studies (11,12) and is considered as a good and sensitive indicator of DNA breakage (13). Moreover, the tail moment value has been validated for the diagnosis of NER repair defects after UV exposure of deficient cells such as those from XP and trichotiodystrophy patients (14,15). Finally, the tail moment has recently been used to demonstrate the role of p53 in the base excision repair pathway (16).

In this work, we have analyzed the reliability and reproducibility of the comet assay after UV-B and UV-C irradiation of human skin cells, keratinocytes and dermal fibroblasts, from the same or from different normal donors. Together with interexperimental and interindividual variations, which have to be taken into account, we have observed some specificity in DNA response after UV exposure according to cell type.

MATERIALS AND METHODS

Cell culture. Human normal keratinocytes and fibroblasts were obtained from skin explants from patients undergoing mammary plastic surgery with their informed consent. All patients were Caucasian, and their skin phototype was between II and III according to the Fitzpatrick classification (17). In brief, to obtain primary keratinocytes, the skin was rinsed in phosphate-buffered saline (PBS; GIBCO-BRL, Cergy-Pontoise, France) and immersed in 0.25% trypsin (Boeringher Mannheim, Indianapolis, IN) at 4?¡ãC overnight. Tissue dissociation was stopped by addition of 10% fetal call serum (GIBCO-BRL). Harvested primary keratinocytes were cultured for the first passage with Dulbecco modified Eagle medium (DMEM)-Ham F12 medium (3:1, vol/vol) (GIBCO-BRL), supplemented with 10% fetal calf scrum, 10^sup -10^ M cholera toxin (Sigma-Aldrich, Saint-Quentin-Fallavier, France), 1.8 ?? 10^sup -4^ M adenine (Sigma-Aldrich), 5 mg/mL insulin (Sigma-Aldrich), 100 U/mL penicillin-streptomycin (GIBCO-BRL), 0.5 mg/mL hydrocortisone (Sigma-Aldrich) and 10 ng/mL epidermal growth factor (EGF, Sigma-Aldrich). Keratinocytes were seeded on irradiated (60 Gy) NIH3T3 feeder layers using a ^sup 137^Cs source emitting at 6 Gy/min (CIS Biointernational, IBL 437C, Gif-sur-Yvette, France). After the first passage, keratinocytes were cultured in keratinocyte-serum-free medium (GIBCO-BRL) supplemented with EGF 5 [eta]g/mL (GIBCO-BRL), bovine pituitary extract (GIBCO-BRL) 25 ??g/mL and penicillin-streptomycin 100 U/mL. To obtain normal human primary fibroblasts, skin explants were rinsed in PBS, minced and placed dermal side down in the petri dish. Attachment was allowed to occur at 37?¡ãC within a minimal volume of liquid (a drop of fetal calf serum) for 2 h, and then DMEM medium supplemented with 10% fetal calf serum and 100 U/mL penicillin-streptomycin was added. All skin cells were cultured for no more than two or three passages. NIH3T3 cells (obtained from H. Green, Boston, MA) were contact-inhibited NIH Swiss mouse embryo fibroblasts and were cultured in the same medium used for the primary human fibroblasts. All cells were cultured in 5% CO2 at 37?¡ãC.

UV sources. A germicidal lamp was used for UV-C (254 nm) irradiation (Philips, Suresnes, France), and fluence was established using a Vilbert Lnurmat (Marne La Vallee, France) dosimeter. The dose rate for the experiments was 0.3 J/m^sup 2^/s. The UV-B source was a Vilbert Lourmat TFX-35M, 6 ?? 15 W lamp with a [lambda]^sub max^ at 312 nm. This UV-B table emits less than 2% of the spectrum with wavelength between 270 and 290 nm (Fig. 1). Fluence rate at the site of cell irradiation was 10.25 J/m^sup 2^/s as determined by Parker chemical actinometric method. All UV irradiations were performed at room temperature.