The Total Amount of DNA Damage Determines Ultraviolet-radiation-induced Cytotoxicity After Uniformor Localized Irradiation of Human Cells  Kyoko Imoto, Nobuhiko Kobayashi, Sachiko Katsumi, Yoko Nishiwaki, Taka-aki Iwamoto, Aya Yamamoto, Yukio Yamashina, Toshihiko Shirai, Sachiko Miyagawa, Yoshiko Dohi, Shigeki Sugiura, Toshio Mori, PhD  Journal of Investigative Dermatology  Volume 119, Issue 5, Pages 1177-1182 (November 2002) DOI: 10.1046/j.1523-1747.2002.19514.x Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Micropore UV irradiation produces CPD in localized areas of the nucleus. The nucleus on the left has two CPD foci, whereas the nucleus on the right has three CPD foci. Journal of Investigative Dermatology 2002 119, 1177-1182DOI: (10.1046/j.1523-1747.2002.19514.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Determination of DNA photoproducts after localized or uniform UV irradiation using an ELISA. (A) MSU-2 cells. Localized UV irradiation with 100 or 200 J per m2 produced the same amounts of CPD as did uniform irradiation with 5.3 or 8.8 J per m2, respectively. Similarly, localized UV irradiation with 100 or 200 J per m2 produced the same amounts of 6–4PP as did uniform irradiation with 4.8 or 7.9 J per m2, respectively. (B) CS2OS cells. Localized irradiation with 100 or 200 J per m2 produced the equivalent amount of CPD as did uniform irradiation with 4.2 or 8.2 J per m2, respectively. Similarly, localized irradiation with 100 or 200 J per m2 formed the equivalent amount of 6–4PP as did uniform irradiation with 5.0 or 9.1 J per m2, respectively. Each point shows the mean ±SD of four independent experiments. L100 and L200 show localized UV irradiation with 100 or 200 J per m2, respectively. Journal of Investigative Dermatology 2002 119, 1177-1182DOI: (10.1046/j.1523-1747.2002.19514.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Localized UV irradiation with 100 or 200 J per m2 matched effects on cytotoxicity in MSU-2 cells induced by uniform irradiation with 5.2 or 8.3 J per m2, respectively. Similarly, localized irradiation with 100 or 200 J per m2 matched effects on cytotoxicity in CS2OS cells induced by uniform irradiation with 4.5 or 9.2 J per m2, respectively. Cytotoxicity was evaluated using a 4 d MTS assay. Each point shows the mean±SD of three independent experiments. Journal of Investigative Dermatology 2002 119, 1177-1182DOI: (10.1046/j.1523-1747.2002.19514.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Cytotoxicity is not significantly different between localized or uniform UV irradiation at doses inducing similar amounts of DNA damage both in MSU-2 and CS2OS cells. Comparisons of DNA damage formation and cytotoxicity between localized and uniform UV irradiation were made using data from Figure 2 and Figure 3. Journal of Investigative Dermatology 2002 119, 1177-1182DOI: (10.1046/j.1523-1747.2002.19514.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 No significant differences in repair kinetics for CPD or 6–4PP were observed following localized or uniform UV irradiation. Two hundred joules per m2 localized UV irradiation (•) or 10 J per m2 uniform UV irradiation (○) were performed in order to produce similar numbers of DNA damage. CPD (A) and 6–4PP (B) were then quantitated by ELISA immediately after irradiation and after UV incubation. Each point shows the mean ±SD of four independent experiments. Journal of Investigative Dermatology 2002 119, 1177-1182DOI: (10.1046/j.1523-1747.2002.19514.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Cell-cycle profiles do not differ significantly following localized or uniform UV irradiation at doses inducing similar amounts of DNA damage. Cell-cycle phase distribution was determined 96 h after localized or uniform UV irradiation using flow cytometry. Typical cell-cycle profiles are presented. Upper, uniform UV irradiation; lower, localized UV irradiation. Journal of Investigative Dermatology 2002 119, 1177-1182DOI: (10.1046/j.1523-1747.2002.19514.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions