Cancer Gene Therapy (2007) 14, 174–186 r 2007 Nature Publishing Group All rights reserved 0929-1903/07 $30.00 www.nature.com/cgt ORIGINAL ARTICLE Protection of normal human reconstructed epidermis from UV by catalase overexpression HR Rezvani1, M Cario-Andre´1, C Pain1, C Ged1, H deVerneuil1 and A Taı¨ eb1,2 1Inserm E 0217, University Victor Segalen Bordeaux 2, Bordeaux, France and 2CHU Bordeaux, Department of Dermatology, Hoˆpital St Andre´, Bordeaux, France Reactive oxygen species (ROS) generated by ultraviolet (UV) irradiation are counterbalanced by endogenous antioxidant systems. To test the hypothesis of a novel photoprotective approach, we irradiated epidermis reconstructed with normal human keratinocytes overexpressing sustainably lentivirus-mediated catalase (CAT), copper/zinc superoxide dismutase (CuZnSOD) or manganese superoxide dismutase (MnSOD) enzymes. We found that following UVB irradiation there was a marked decrease in sunburn cell formation, caspase-3 activation and p53 accumulation in human reconstructed epidermis overexpressing CAT. Moreover, UVA-induced hypertrophy and DNA oxidation (8-oxodeoxyguanosine) were decreased by CAT overexpression. These effects were not achieved by overexpression of CuZnSOD or MnSOD. In conclusion, vector-mediated CAT overexpression could be a promising photoprotective tool against deleterious effects of UV irradiation such skin cancer especially in monogenic/polygenic photosensitive disorders characterized by ROS accumulation. Cancer Gene Therapy (2007) 14, 174–186. doi:10.1038/sj.cgt.7701000; published online 20 October 2006 Keywords: gene transfer; superoxide dismutase; photoprotection; lentiviral vector Introduction damage under the control of the tumor suppressor protein p53.6,7 However, several studies on different cells have The solar ultraviolet (UV) radiation reaching the earth’s shown that UVBalso acts as an inducer of death 8,9 10,11 surface is the most important risk factor for the receptors and ROS, both of which participate in 12 development of skin cancer. Non-melanoma skin cancer UVB-induced apoptosis. consists of basal cell carcinoma (BCC), the most prevalent Thus, UVA and UVBcan induce ROS such as the À form of skin cancer, and squamous cell carcinoma superoxide anion radical (O2K) and hydrogen peroxide 1,2 (SCC). The rapid rising incidence of both BCC and (H2O2) which could be further converted to the highly SCC over the past decades, suggests that atmospheric reactive hydroxyl radical (KOH) via the Fenton 2 þ - 3 þ K À ozone depletion may further increase these rate. Thus, (Fe þ H2O2 Fe þ OH þ OH) and Haber–Weiss À - K À 13 there is a vital requirement to develop a strategy for (O2K þ H2O2 O2 þ OH þ OH) reactions. In order to prevention and therapy of skin cancer. neutralize these ROS, living cells have acquired various Solar UV radiation includes UVC (200–280 nm), UVB defense systems including non-enzymatic (a-tocopherol, À (280–320 nm) and UVA (320–400 nm) light. Whereas vitamin C) and enzymatic antioxidants. In particular, O2K UVC is absorbed by the atmospheric layer, UVA and is converted to less reactive H2O2 and O2 by superoxide UVBboth function as initiator and promoter in photo- dismutase (SOD), an enzyme present in three forms in carcinogenesis.2 UVA, which is the major component of humans, namely a cytosolic copper/zinc superoxide solar UV radiation, is poorly absorbed by DNA and dismutase (CuZnSOD), a mitochondrial manganese affects various cell components such as activator protein- superoxide dismutase (MnSOD) and an extracellular 3 4 1 (AP-1) and heme oxygenase, mainly through reactive one (EC-SOD). H2O2 is converted to H2O and O2 either oxygen species (ROS) production.4,5 UVBis initially by catalase (CAT) located in peroxisomes or glutathione 13 absorbed by DNA and causes direct DNA damage. It was peroxidase located in cytoplasm and mitochondria. originally thought that sunburn cell (SBC) formation was Heme oxygenase decreases iron availability and thereby 14 only the consequence of UVB-induced DNA photo- restricts KOH formation. However, an imbalance between ROS-producing levels and the antioxidant defense is in favor of the former upon UV irradiation Correspondence: Professor A Taı¨ eb, Inserm E 0217, Universite´ and leads to cytotoxicity by proteins, lipids and DNA Victor Segalen Bordeaux 2, 146 rue Le´ o Saignat, 33076 Bordeaux 15–18 cedex, France. oxidation both in vitro and in vivo. It has been shown E-mail: [email protected] that UVA and UVBirradiation of murine skin results in Received 1 May 2006; revised 13 June 2006; accepted 19 August impairment of enzymatic and non-enzymatic antioxi- 5 2006; published online 20 October 2006 dants. In addition, several studies have demonstrated Protective effect of catalase against UV HR Rezvani et al 175 that an increase in antioxidant defense systems can green fluorescent protein (EGFP), CAT, CuZnSOD and reduce the deleterious effects of UV-induced ROS. MnSOD was driven by the human phosphoglycerate The protective effects of non-enzymatic antioxidant or kinase (PGK) promoter. Lentiviral particles were pro- radical scavenger addition on UVB-,18–20 UVA-20 and duced by transient transfection of 293T cells as described UVA þ B-21 induced damage have been reported. We previously.26 To determine the titer of each viral vector, have recently demonstrated that overexpression of CAT serial dilutions were used for transduction of 293T cells. reduces UVB-induced apoptosis in human keratino- Following transduction, EGFP expression was measured cytes.22 Furthermore, a severe disturbance in antioxidant directly by cytofluorimetric analysis. Typical titers after balance with diminished antioxidant enzymes was found concentration for lentiviral vectors were between 2 Â 108 in non-melanoma tumors.23 Taken together, these data and 2 Â 109/ml. Enzyme-linked immunosorbent assays of suggest that increase in antioxidant defense may protect p24 were used to determine the concentration of viral p24 keratinocytes from UV-induced oxidative stress. Mean- protein in the different viral supernatants. By comparing while, controversial results from various isolated cells the p24 of different supernatants and specific titers of point out that in vivo data are necessary to understand the EGFP, the titration of different viral supernatants was complex of oxidative stress and antioxidant defense in determined. response to UV irradiation. Thus, we decided to investigate the effects of UVA and UVBirradiation on Transduction of keratinocytes reconstructed epidermis (RE) made with human kerati- A total of 5 Â 105 cells were plated in T25 flasks and nocytes transduced by CuZnSOD, MnSOD or CAT, incubated for 24 h in complete medium. Before infection, because this system is more similar to human skin than medium was removed and cells were infected with viral keratinocyte monolayers. supernatants for 24 h at 371C in the presence of 8 mg/ml protamine sulfate. After 5 days, the cells were trypsinized and used to prepare RE. Before reconstruction, the percentage of EGFP-positive cells was analyzed by Materials and methods cytofluorimetry. Source of keratinocytes and epidermal reconstruction Keratinocytes were isolated from normal human skin in Irradiation procedure 22 patients undergoing plastic surgery as already described. UVBand UVA irradiation was delivered with a Biotronic Briefly, fresh skin fragments were immediately cut into device (Vilber Lourmat, Marne la Valle´ e, France) 5 Â 5 mm pieces and treated with trypsin-ethylenediamine- equipped with a dosimeter as already described.21,22 tetraacetic acid (EDTA) for 3 h at 371C or overnight at Briefly, the UVB lamp emitted a continuous spectrum 41C in order to separate the epidermis from the dermis. between 280 and 380 nm with a major peak at 312 nm. 5 Keratinocytes were seeded at a concentration of 10 cells The UVA lamp delivered UV in the range of 312–400 nm 2 per cm in MCDB153 medium, which included hydro- with a major peak at 365 nm. After rinsing in phosphate- cortisone (0.5 mg/ml), epidermal growth factor (10 ng/ml), buffered saline (PBS), RE were irradiated at a dose of 180 insulin (5 mg/ml) and bovine pituitary extract. This or 220 mJ cmÀ2 UVBand at a dose of 10 J cm À2 UVA. medium was changed three times a week. When the These doses were chosen after preliminary tests in order to culture reached 70–80% confluence, the cells were induce significant levels of apoptosis (20 and 40%) in the detached with trypsin-EDTA and then resuspended in basal layer of normal RE. Following irradiation, samples MCDB153 medium or were used to reconstruct epidermis. were rinsed in PBS and incubated in fresh medium. Preparation of RE Histological and immunohistochemical studies Normal skin samples were cut into 1 Â 1 cm pieces and Reconstructs were fixed in 4% formaldehyde, embedded then kept at 371C for 15 days. Then, dermis was separated in paraffin, cut in 5 mm sections and stained with from epidermis and conserved at À801C. Normal human hematoxylin–eosin to assess the general morphology of keratinocytes were seeded at 4 Â 105 cells per cm2 on the the epidermis and the amount of SBCs. We counted 100 dead dermis. Twenty-four hours later, the dermis was cells each time on 10 different areas for quantification of immersed in culture medium for 72 h to allow cell SBC. The sections of epidermis reconstructed with proliferation. Then, it was placed at the air/liquid keratinocytes transduced by EGFP (RE/EGFP) were interface for 7 days to allow differentiation.24 counterstained with hematoxylin and analyzed under fluorescence microscopy to quantify transduction. The Construction and production of lentiviral vectors monoclonal antibody against CAT (Rockland, Tebu-Bio, The pRRL.SIN.PPT.PGK.MCS.WPRE and the Le Perray en Yvelines, France) was also used for this pRRL.SIN.PPT.PGK.EGFP.WPRE vectors, hereafter purpose. DNA oxidation was detected 1 h after UVA termed TPW and TPEW, were a generous gift from D irradiation with a monoclonal anti-8-oxodeoxyguanosine Trono (Universite´ de Gene` ve, Switzerland).25 The vectors (anti-8-oxodG) antibody (RD systems, Lille, France) (TPCATW, TPCuZnSODW and TPMnSODW, see according to the manufacturer’s recommendations.