Ultraviolet Irradiation Induces Apoptosis in Human Immature, But Not in Skin Mast Cells Sven Guhl, Silke Tapkenhinrichs, Alina Smorodchenko, Andreas Grützkau,

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Ultraviolet Irradiation Induces Apoptosis in Human Immature, But Not in Skin Mast Cells Sven Guhl, Silke Tapkenhinrichs, Alina Smorodchenko, Andreas Grützkau, Beate M. Henz, Torsten Zuberbier Journal of Investigative Dermatology Volume 121, Issue 4, Pages 837-844 (October 2003) DOI: 10.1046/j.1523-1747.2003.12480.x Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 UV irradiation induces apoptosis of HMC-1 cells in a time- and dose-dependent manner. HMC-1 cells were exposed to different doses of UVB (A), UVA1 (B), and PUVA1 (C), incubated for 0 to 48 h, and apoptosis assessed by flow cytometric analysis of cell membrane integrity. Data represent the mean±SEM of four to seven experiments. Journal of Investigative Dermatology 2003 121, 837-844DOI: (10.1046/j.1523-1747.2003.12480.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Increasing number of dead HMC-1 cells as a postapoptotic event after UV irradiation. HMC-1 cells were exposed to different doses of UVB (A), UVA1 (B), and PUVA1 (C), incubated for 0 to 48 h, and cell death assessed by FACS analysis. Data represent the mean±SEM of four to seven experiments. Journal of Investigative Dermatology 2003 121, 837-844DOI: (10.1046/j.1523-1747.2003.12480.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Ultrastructure of HMC-1 cells undergoing apoptosis after treatment with UV irradiation. (A) Untreated control HMC-1 cells exhibit a normal morphology with an intact nucleus and regularly distributed chromatin. (B) After irradiation with 75 mJ per cm2 UVB and subsequent incubation for 12 h, HMC-1 cells show morphologic features typical of apoptosis such as the formation of apoptotic bodies containing chromatin fragments (arrow 1) and excessive membrane blebbing (arrow 2) (original magnification × 6000). Journal of Investigative Dermatology 2003 121, 837-844DOI: (10.1046/j.1523-1747.2003.12480.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 UV irradiation activates caspase-3 in HMC-1 cells in a time- and dose-dependent manner. HMC-1 cells were treated with different doses of UVB (A), UVA1 (B), and PUVA1 (C) irradiation, incubated for 0 to 48 h, and activity of caspase-3 assessed by fluorimetric measurement of DEVD-AMC cleavage. Data represent the mean of three representative independent experiments UVB and single experiments for UVA1 and PUVA1. Journal of Investigative Dermatology 2003 121, 837-844DOI: (10.1046/j.1523-1747.2003.12480.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 UV irradiation elevates p53 protein levels in nuclear extracts of HMC-1 cells. After treatment of HMC-1 cells with UV irradiation and subsequent incubation for 0 to 24 h, nuclear protein was extracted and immunoblotting performed with anti-p53 antibody. (A) Lanes 1–5, unirradiated sham controls after an incubation of 0, 4, 8, 12, and 24 h; lanes 6 to 10, cells irradiated with 75 mJ per cm2 UVB after an incubation of 0, 4, 8, 12, and 24 h; (B) lanes 1 to 5, unirradiated sham controls after an incubation of 4, 8, 12, and 24 h; lanes 6 to 10, cells irradiated with 20 J per cm2 UVA1 after an incubation of 0, 4, 8, 12, and 24 h; (C) lanes 1 to 5, unirradiated sham controls pretreated with 8-MOP after an incubation of 0, 4, 8, 12, and 24 h; lanes 6 to 10, cells irradiated with 5 J per cm2 PUVA1 after an incubation of 0, 4, 8, 12, and 24 h. Data are representative of three independent experiments with similar results. Journal of Investigative Dermatology 2003 121, 837-844DOI: (10.1046/j.1523-1747.2003.12480.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 UV irradiation leads to an enhanced cleavage of PARP in nuclear extracts of HMC-1 cells. After treatment of HMC-1 cells with UV irradiation and subsequent incubation for 0 to 24 h, nuclear protein was extracted and immunoblotting performed with anti-PARP antibody. (A) Lanes 1 to 5, unirradiated sham controls after an incubation of 0, 4, 8, 12, and 24 h; lanes 6 to 10, cells irradiated with 75 mJ per cm2 UVB after an incubation of 0, 4, 8, 12, and 24 h; (B) lanes 1 to 5, unirradiated sham controls after an incubation of 4, 8, 12, and 24 h; lanes 6 to 10, cells irradiated with 20 J per cm2 UVA1 after an incubation of 0, 4, 8, 12, and 24 h; (C) lanes 1 to 5, unirradiated sham controls pretreated with 8-MOP after an incubation of 0, 4, 8, 12, and 24 h; lanes 6 to 10, cells irradiated with 5 J per cm2 PUVA1 after an incubation of 0, 4, 8, 12, and 24 h. Data from two experiments with similar results. Journal of Investigative Dermatology 2003 121, 837-844DOI: (10.1046/j.1523-1747.2003.12480.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Caspase-3 activation in different cell types in response to UVB, staurosporine, and cycloheximide. Mature dermal mast cells (Mc) poorly respond to any apoptotic stimuli applied. Only staurosporine is sufficient to activate caspase-3 in a moderate fashion (n=9, p≤0.05). In contrast in mature but proliferating dermal mast cells (cultured with SCF±IL-4 for 2 wk) especially UVB was able to induce of caspase-3 activation (single experiment). The proliferating cell lines HMC-1 and KU 812 are highly sensitive to UV light and staurosporine (n=3, p≤0.05), but less to cycloheximide treatment. Similar to mast cells, freshly isolated keratinocytes (n=6, p≤0.05) moderately react on UVB, but on the other side they are sensitive to cycloheximide. HaCaT cells were more sensitive to UVB as compared with primary keratinocytes, but poorly respond to staurosporine. Journal of Investigative Dermatology 2003 121, 837-844DOI: (10.1046/j.1523-1747.2003.12480.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions