Nucleotide Excision Repair Genes are Upregulated by Low-Dose Artificial Ultraviolet B: Evidence of a Photoprotective SOS Response? Tomoko Maeda, Prescillia.

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Presentation transcript:

Nucleotide Excision Repair Genes are Upregulated by Low-Dose Artificial Ultraviolet B: Evidence of a Photoprotective SOS Response? Tomoko Maeda, Prescillia P.S. Chua, Michelle T. Chong, Adrian B.T. Sim, Victor A. Tron Journal of Investigative Dermatology Volume 117, Issue 6, Pages 1490-1497 (December 2001) DOI: 10.1046/j.0022-202x.2001.01562.x Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 UV induced cell death in human keratinocytes. Human keratinocytes were irradiated with single dose of 100, 300, or 600 J per m2 UVB and stained with crystal violet after 72 h. Only adherent cells stain with crystal violet and absorb at 570 nm; 600 J per m2 was sufficient to kill most human keratinocytes in culture. The dose of UVB required to reduce survival to 37% (D37) of human keratinocytes was 410 J per m2. Journal of Investigative Dermatology 2001 117, 1490-1497DOI: (10.1046/j.0022-202x.2001.01562.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Flow cytometry analysis demonstrated apoptotic population at high dose. The numbers shown in the figure reflect the percentage of G0/G1, S, or G2/M. S-phase (cross-hatched area) assessment was conducted by Modifit LT program (p <0.01). Each flow gave %CV between 6.13 and 9.14. The shaded area represents sub-G0G1 population (= apoptotic cells), of which percentage was obtained by the CELLQuest program. At 24 h postdosing, there were a large number of apoptotic cells in cultures irradiated with 600 J per m2. Journal of Investigative Dermatology 2001 117, 1490-1497DOI: (10.1046/j.0022-202x.2001.01562.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Slot blot assay for UV-induced photoproducts in keratinocytes. (a) The repair of thymine dimers was accelerated at 100 J per m2 UVB. A slot blot assay was carried out to detect thymine dimers using monoclonal antibody. Thymine dimers were accumulated in a UVB dose-dependent manner when measured at 0 h (immediately following irradiation). DNA from cells irradiated with 600 or 300 J per m2 UVB had approximately 6-fold or 3-fold more thymine dimers than the DNA irradiated with 100 J per m2 UVB, respectively. In cells irradiated with 100 J per m2, repair was complete within 24 h. A slower rate of repair was observed in cells irradiated with 300 J per m2; thymine dimers were still unrepaired at 48 h, although the signal decreased with time. In cells irradiated with 600 J per m2 UVB, no repair of thymine dimers was evident even 48 h after irradiation. (b) (6–4) photoproducts were efficiently removed in each dose. A small number of (6–4) photoproducts were detected in cells irradiated with 100 J per m2, but approximately three times more (6–4) photoproducts were observed immediately following irradiation with 300 J per m2, and marked increase in the number of (6–4) photoproducts was detected in cells irradiated with 600 J per m2 UVB. In contrast to the repair of thymine dimers in irradiated cells (6–4) photoproducts were removed with a similar efficiency in cells irradiated with 100, 300, and 600 J per m2 UVB. Journal of Investigative Dermatology 2001 117, 1490-1497DOI: (10.1046/j.0022-202x.2001.01562.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Autoradiographs of NER-1 set and NER-2 set. The expression of NER genes was quantified at the mRNA level in cells dosed with 100, 300, or 600 J per m2 UVB. The genes that were analyzed were divided into two sets: (a) NER-1 includes XPG, DDB1, XPC, XPF, RPAp70, DDB2, hHR23B, XPA, RPAp32, RPAp14, L32, and GAPDH, and (b) NER-2 includes CSB, XPB, TFIIHp52, TFIIHp44, CSA, CDK7, CycH, TFIIHp34, ERCC1, XPD, L32, and GAPDH. Journal of Investigative Dermatology 2001 117, 1490-1497DOI: (10.1046/j.0022-202x.2001.01562.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 PCNA was upregulated at mRNA level after low- or intermediate-dose UVB, but not high dose. RNase protection assay was performed to examine PCNA level independently from NER-1 or NER-2 set, although the RNA sample was prepared at the same time. PCNA was upregulated 2.5-fold at 8 h with low dose (100 J per m2), and 3.5-fold at 48 h with intermediate dose (300 J per m2), but was downregulated with high dose (600 J per m2). After high-dose irradiation, PCNA mRNA level did not recover to constitutive level even after 48 h. Journal of Investigative Dermatology 2001 117, 1490-1497DOI: (10.1046/j.0022-202x.2001.01562.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 The expression of DR4 was markedly upregulated with 600 J per m2 UVB. The transcripts of DR4, one of the TNF receptors, showed marked upregulation at 600 J per m2 in undifferentiated keratinocytes (Ca2+). This supports that our observation in NER genes at 600 J per m2 are not nonspecific. Journal of Investigative Dermatology 2001 117, 1490-1497DOI: (10.1046/j.0022-202x.2001.01562.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Pre-exposure to low-dose UVB rescued cell death. The cells were pre-exposed to 100 J per m2 UVB or unirradiated, then exposed to 300 or 600 J per m2 on the next day. The cells pretreated with low-dose UV showed more survival than the cells without pretreatment. Bars at the data points indicate mean ± SD. Journal of Investigative Dermatology 2001 117, 1490-1497DOI: (10.1046/j.0022-202x.2001.01562.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 PCNA was induced only at low-dose UVB. PCNA protein was induced 8 h after low-dose UVB (100 J per m2) irradiation, then downregulated. This upregulation was confirmed by Coomassie stain of the gel and densitometry. Marked downregulation was observed after intermediate (300 J per m2) or high-dose (600 J per m2) UV. Journal of Investigative Dermatology 2001 117, 1490-1497DOI: (10.1046/j.0022-202x.2001.01562.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions