Coimbatore S. Sreevidya, Atsushi Fukunaga, Noor M

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Agents that Reverse UV-Induced Immune Suppression and Photocarcinogenesis Affect DNA Repair Coimbatore S. Sreevidya, Atsushi Fukunaga, Noor M. Khaskhely, Taro Masaki, Ryusuke Ono, Chikako Nishigori, Stephen E. Ullrich Journal of Investigative Dermatology Volume 130, Issue 5, Pages 1428-1437 (May 2010) DOI: 10.1038/jid.2009.329 Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Reversal of immune suppression and acceleration of DNA repair by platelet-activating factor (PAF) and serotonin (5-HT) receptor antagonists. Effect of agents that repair DNA damage, neutralize cis-urocanic acid (cis-UCA) activity, or block PAF and 5-HT from binding to their receptors on immune suppression. Mice were exposed to 15kJm−2 of ultraviolet B (UVB) radiation and then treated with (a) T4N5-containing liposomes, (b) monoclonal anti-cis-UCA, (c) PAF receptor antagonist, and (d) 5-HT receptor antagonist. The effect the various treatments had on UV-induced immune suppression was determined using a delayed-type hypersensitivity (DTH) assay. The data are expressed as mean footpad swelling±SEM. The asterisk denotes a significant difference (P<0.05) from the positive control. Effect of PAF and 5-HT receptor antagonists on the numbers of cyclobutane pyrimidine dimmer (CPD) and pyrimidine (6–4) pyrimidinone photoproducts (6–4 photoproducts). Mice were exposed to 2kJm−2 of UVB radiation and then immediately injected with 1μM of PCA-4248 or 1μM of 1-(1-naphthyl)piperazine (1-NPZ). At 1 and 24hours post irradiation, skin samples were removed, and (e) CPD or (f) 6–4 photoproducts (PP) were visualized by immunohistochemistry. Bar=50μm. High-magnification views ( × 400) are shown in the inserts. At various times post irradiation, skin samples were taken, DNA was isolated, and (g) CDP and (h) 6–4 photoproducts were measured by ELISA. *Indicates a significant difference (P<0.05) from the UV-only control (Student's t-test, n=5). Journal of Investigative Dermatology 2010 130, 1428-1437DOI: (10.1038/jid.2009.329) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Unscheduled DNA synthesis is accelerated by treating UV-irradiated mice with PAF and serotonin (5-HT) receptor antagonists. (a) Pam 212 cells were treated with a PAF (PCA-4248; 50μM) or a 5-HT (1-(1-naphthyl)piperazine (1-NPZ); 25μM) receptor antagonist, washed, and then exposed to 200Jm−2 of ultraviolet B (UVB) radiation. The cells were incubated with BrdU (3hours, 37°C) and then incubated with anti-BrdU antibody. The left panels show anti-BrdU staining (red). The center panels show 4′-6-diamidino-2-phenylindole (DAPI) staining of the nuclei. The right panel shows a merge of the BrdU and DAPI staining. Arrowheads illustrate nuclei in the S phase; arrows indicate punctate nuclear staining characteristic of cells undergoing unscheduled DNA synthesis (UDS). The percentage of cells undergoing UDS in each group is indicated. Bar=50μm. (b) Quantification of UDS. The number of individual grains per nuclei was counted. *Indicates a significant difference (P<0.05) from the UV-only control (Student's t-test, 50 cells per sample). Journal of Investigative Dermatology 2010 130, 1428-1437DOI: (10.1038/jid.2009.329) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Failure to reverse cyclobutane pyrimidine dimmer (CPD) formation in PAF and serotonin (5-HT) receptor antagonist-treated XPA-/- mice. Wild-type (WT) or XPA-/- mice were exposed to 2kJm−2 of ultraviolet B (UVB) radiation and immediately injected with 1μM of PCA-4248 or 1μM of 1-(1-naphthyl)piperazine (1-NPZ). (a) CPDs were detected by immunohistochemistry. Bar=50μm. High-magnification views ( × 400) are shown in the inserts. (b) Apoptosis was detected by TUNEL. Bar=200μm. Journal of Investigative Dermatology 2010 130, 1428-1437DOI: (10.1038/jid.2009.329) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Serotonin (5-HT) and cis-urocanic acid (cis-UCA) induced reactive oxygen species (ROS) formation. Keratinocytes were exposed to ultraviolet B (UVB) radiation (200Jm−2), or cultured with 10μM of carbamyl-PAF (cPAF), 50μM of 5-HT, or 50μM of cis-UCA. Some of the cells were pretreated with PCA-4248 (50μM) or 1-(1-naphthyl)piperazine (1-NPZ) (25μM). (a) ROS induction was measured by flow cytometry or (b) fluorescence microscopy. Bar=50μm. Journal of Investigative Dermatology 2010 130, 1428-1437DOI: (10.1038/jid.2009.329) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Reduced 8-oxo-deoxyguanosine (8-oxo-dG) lesions following treatment with platelet-activating factor (PAF) and serotonin (5-HT) receptor antagonists. Mice were exposed to 2kJm−2 of ultraviolet B (UVB) radiation, or injected with 500nM of carbamyl-PAF (cPAF) or 5μM of cis-urocanic acid (cis-UCA). Some mice were injected with 1μM of PCA-4248 or 1μM of 1-(1-naphthyl)piperazine (1-NPZ) immediately following irradiation. Skin samples were also collected after dermal injection of 500nM of cPAF or 5μM of cis-UCA. All samples were collected 24hours post treatment. (a) 8-oxo-dG was detected by immunohistochemistry. (b) 8-oxo-dG was measured by ELISA. Data are expressed as ng of 8-oxo-dG per 100mg total DNA. P-values were determined by Student's t-test (n=5). (c) Apoptosis (TUNEL) in response to dermal cPAF and cis-UCA injection. Bar=200μm. (d) PARP cleavage is blocked by PAF and 5-HT receptor antagonists. Keratinocytes were pretreated with PCA 4248 (50μM) or 1-NPZ (25μM), washed, and then exposed to 200Jm−2 of UVB radiation. Total cellular protein was isolated 1 and 24hours post irradiation. Cleaved PARP was measured by western blotting. Equal gel loading was confirmed by staining with a glyceraldehyde 3-phosphate dehydrogenase (GAPDH)-specific antibody. Journal of Investigative Dermatology 2010 130, 1428-1437DOI: (10.1038/jid.2009.329) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions