doi:10.1016/j.jphotobiol.2004.09.010 
Copyright © 2004 Elsevier B.V. All rights reserved.
1,25-Dihydroxyvitamin D3 and analogues protect primary human keratinocytes against UVB-induced DNA damage
Petra De Haesa, b, Marjan Garmynb, c, Annemieke Verstuyfa, Pierre De Clercqd, Maurits Vandewalled, Hugo Degreefb, Katleen Vantieghema, Roger Bouillona, 
, 
and Siegfried Segaerta, b
aLaboratory for Experimental Medicine and Endocrinology, Onderwijs en Navorsing, Katholieke Universiteit Leuven, Gasthuisberg, Herestraat 49, Bus 902, 3000 Leuven, Belgium
bDepartment of Dermatology, Universitaire Ziekenhuizen Leuven, Kapucijnenvoer 33, 3000 Leuven, Belgium
cLaboratory of Dermatology, Katholieke Universiteit Leuven, Herestraat 49, Bus 818, 3000 Leuven, Belgium
dVakgroep voor Organische Chemie, Universiteit Gent, Krijgslaan 281 S4, 9000 Gent, Belgium
Received 18 August 2004;
revised 22 September 2004;
accepted 24 September 2004.
Available online 7 January 2005.
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Abstract
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.
Keywords: Ultraviolet-B; Keratinocytes; DNA damage; Cyclobutane pyrimidine dimers; 1,25-Dihydroxyvitamin D3; Vitamin D analogues
Fig. 1. 1,25(OH)2D3 protects human keratinocytes against UVB-induced CPD formation. Untreated keratinocytes (a), keratinocytes irradiated with UVB 16 mJ/cm2 (b) and keratinocytes pre-treated for 24 h with 1,25(OH)2D3 10−6 M prior to irradiation (c) were fixed with paraformaldehyde 30 min after irradiation and stained for CPDs as described in Section 2. Scale bar: 10 μM.
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Fig. 2. The protective effect of 1,25(OH)2D3 against UVB-induced CPD damage is dose- and time-dependent and parallels the anti-proliferative effect of 1,25(OH)2D3. (a) Keratinocytes were treated for 24 h with increasing doses 1,25(OH)2D3 (1,25D3) (a-1) or with 1,25D3 10−6 M for increasing periods of time (a-2) and subsequently irradiated with UVB 16 mJ/cm2. Immediately after irradiation cells were harvested for DNA and subjected to immuno dot blot analysis using an antibody directed against cyclobutane pyrimidine dimers (CPD). One representative blot of four independently performed experiments is shown. DNA dots were quantified by densitometric scanning and densities of duplicated samples of four independently performed experiments are expressed as percentage of control samples as mean ± SD. (b) Keratinocytes were treated for 24 h with increasing doses 1,25D3 (b-1) or with 1,25D3 10−6 M for increasing periods of time (b-2) and proliferation rates were determined with [3H]-thymidine incorporation as described in Section 2. Results are expressed as percentage of vehicle controls as mean ± SD of five replicates. * P < 0.05; *** P < 0.001 when comparing 1,25D3-treated cells with vehicle-treated cells.
Fig. 3. 1,25(OH)2D3 suppresses p53 accumulation in UVB-irradiated keratinocytes. Keratinocytes were irradiated with UVB 16 mJ/cm2 in the absence or presence of 1,25(OH)2D3 (1,25D3) 10−6 M. Lysates of the cells were harvested at the indicated time points after irradiation and subjected to western blot analysis using an antibody directed against p53. Representative blots of one of three independently performed experiments are shown. Blots from triplicate experiments were scanned and densitometric values, corrected for β-actin were plotted as mean ± SD. ** P < 0.01; *** P < 0.001 when comparing irradiated cells pre-treated with 1,25D3 or with vehicle.
Fig. 4. Cell-cycle arrest protects keratinocytes against UVB-induced CPD formation. (a) Keratinocytes were pre-treated for 24 h with vehicle (co), 1,25(OH)2D3 (1,25D3) 10−6 M, hydroxyurea (HU) 10−3 M, mimosine (mimo) 2 × 10−2 M or nocodazole (noco) 1.6 × 10−6 M before irradiation with UVB 16 mJ/cm2. Immediately after irradiation, cells were harvested for DNA and immuno dot blots were performed with an antibody directed against CPDs as described in Fig. 2. (b) Keratinocytes were treated for 24 h with vehicle (co), 1,25D3 10−6 M, HU 10−3 M, mimo 2 × 10−2 M or noco 1.6 × 10−6 M and proliferation rates were determined with [3H]-thymidine incorporation as described in Section 2. ** P < 0.01; *** P < 0.001 when comparing cells treated with anti-proliferative agents with vehicle-treated cells.
Fig. 5. Two 14-epi analogues of 1,25(OH)2D3 protect human keratinocytes against UVB-induced CPD formation. Keratinocytes were pre-treated for 24 h with increasing doses TX 522 (a) or TX 527 (b) before irradiation with UVB 16 mJ/cm2. Immediately after irradiation cells were harvested for DNA and an immuno dot blot was performed with an antibody directed against CPD as described in Fig. 2. * P < 0.05; ** P < 0.01; *** P < 0.001 when comparing TX 522- or TX 527-pre-treated irradiated cells with vehicle-pre-treated irradiated cells.
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