Protection of normal human reconstructed epidermis from UV by catalase overexpression

Abstract

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.

Catalase overexpression reduces UVB-induced apoptosis in a human xeroderma pigmentosum reconstructed epidermis

“…High efficiency of lentiviral vector-mediated gene transfer in normal and XPC keratinocytes As previously shown, lentiviral vectors provide high efficiency and sustained transgene expression in normal human keratinocytes. [22][23][24] In this study, the same type of lentivectors was used for the transduction of XPC keratinocytes to overexpress either the marker gene EGFP (TPEW) or the 'therapeutic' gene catalase (TPCATW) (Figure 2a). The percentage of EGFPpositive cells assessed by cytofluometry 5 days post-transduction reached 85-95% both in normal and XPC cells (Figure 2b).…”

“…22,24 Briefly, the UVB lamp emitted a continuous spectrum between 280 and 380 nm, with a major peak at 312 nm. After rinsing with PBS, RE were irradiated at a dose of 180 or 220 mJ cm À2 UVB.…”

“…The following plasmids were used: a packaging plasmid (pCMVD8.91), an envelope plasmid (pMD.G) from the vesicular stomatitis virus glycoprotein envelope (VSV-G) and transfer vectors (TPW, TPEW or TPCATW) as described previously. 22,24 After 48 h, the viral supernatant was harvested, filtered through 0.22 mm filter, centrifuged at 35 000 g for 4 h and frozen in aliquots. Determination of the titer of each viral supernatant was performed by assessing EGFP expression by flow cytometry and p24 using an enzyme-linked immunosorbent assay.…”

“…We recently demonstrated that overexpression of catalase had a protective effect against UVB-induced apoptosis in cultured normal human keratinocytes as well as reconstructed epidermis (RE). 22,24 To test the hypothesis that reinforcement of internal photoprotectors can affect the phenotypic expression of severe photosensitive disorders, we extended our previous studies to epidermis reconstructed with XPC keratinocytes (XP-RE). We found that catalase overexpression can reduce UVB-induced apoptosis in XP-RE, and that reduced apoptosis is accompanied by a decrease in p53 accumulation.…”

“…[16][17][18][19][20] Several studies have shown that keratinocyte exposure to UVB irradiation generates ROS in excessive quantities that quickly overwhelm various antioxidant defense systems including non-enzymatic (a-tocopherol and vitamin C) and enzymatic antioxidants (catalase and superoxide dismutase). [21][22][23][24] UVB-induced ROS production leads to cytotoxicity through proteins, lipids and DNA oxidation both in vitro and in vivo. 21,[25][26][27] Unlike lipids and proteins, damaged DNA molecules cannot be replaced and need to be repaired.…”