Silvia Selleri, Holger Seltmann, Silvia Gariboldi, Yuri F

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Doxorubicin-Induced Alopecia Is Associated with Sebaceous Gland Degeneration Silvia Selleri, Holger Seltmann, Silvia Gariboldi, Yuri F. Shirai, Andrea Balsari, Christos C. Zouboulis, Cristiano Rumio Journal of Investigative Dermatology Volume 126, Issue 4, Pages 711-720 (April 2006) DOI: 10.1038/sj.jid.5700175 Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Semithin sections of sebaceous glands from untreated rats. Control samples were collected at (a) T5, (b) T7, and (c) T10. The gland maintains the same morphological features at every time point. Bar=10μm. Journal of Investigative Dermatology 2006 126, 711-720DOI: (10.1038/sj.jid.5700175) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Sebaceous gland regression accompanying hair loss induced by DXR treatment. Semithin sections (left) and alkaline phosphatase reactions (right) are shown. (a, b) Control, (c, d) Time point T5. The sebaceous gland is markedly reduced in size but still present. (e, f) Time point T7. The sebaceous gland appears very small, and the semithin section shows the presence of clustered, condensed cells. (g, h) Time point T10. Animals show alopecia and the rare hair follicles appear very small, shrunken, and damaged. Sebaceous glands are undetectable. Bar=10μm. Journal of Investigative Dermatology 2006 126, 711-720DOI: (10.1038/sj.jid.5700175) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Electron microscopy of sebaceous glands at different time points. (a) Control, (b, c, d) 5, 7, 10 days, respectively, after the start of treatment. In (d) the condensed material obstructing the pilary canal is sebum. Bar=1μm. Journal of Investigative Dermatology 2006 126, 711-720DOI: (10.1038/sj.jid.5700175) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Red Oil staining of rat sebaceous glands showing a progressive regression. (a) Control animal. (b, c, d) 5, 7, 10 days, respectively, after the start of DXR treatment. Bar=10μm. Journal of Investigative Dermatology 2006 126, 711-720DOI: (10.1038/sj.jid.5700175) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Measurement of sebaceous gland area in control and DXR-treated animals. Value means and standard deviation are shown for T5, T7, and T10 days after the start of treatment. Journal of Investigative Dermatology 2006 126, 711-720DOI: (10.1038/sj.jid.5700175) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Detection of apoptosis in rat sebaceous glands. The percentage of apoptotic cells was evaluated by the TUNEL assay in both untreated and DXR-treated samples at T5 and T7. The total number of cells, stained with 4,6-diamidino-2-phenylindole was compared to the cells positive for TUNEL staining. Journal of Investigative Dermatology 2006 126, 711-720DOI: (10.1038/sj.jid.5700175) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 The sebaceous glands of C57BL/6 mice 7 days after DXR treatment. (a) Control animal. (b) DXR-treated animal. Bar=10μm. (c) Measurement of the sebaceous gland area of both control and DXR-treated animals. Value means and standard deviation are shown. Journal of Investigative Dermatology 2006 126, 711-720DOI: (10.1038/sj.jid.5700175) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 Measurement of sebaceous gland area in C57BL/6 mice (a) and in rats (b) during anagen and catagen stage of the hair follicle. Value means and standard deviations are shown. Journal of Investigative Dermatology 2006 126, 711-720DOI: (10.1038/sj.jid.5700175) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 9 SZ95 sebocyte cultures treated with different DXR concentrations (upper panel). In the lower panel, the respective volumes of the cytoplasm and nucleus as well as the cytoplasm/nuclear ratio are presented. Already at 10−7M DXR an increased cytoplasm volume can be observed. This further increases in a dose-dependent manner. The nuclear volume is not affected by DXR at the concentrations tested. ***P<0.001. Journal of Investigative Dermatology 2006 126, 711-720DOI: (10.1038/sj.jid.5700175) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 10 In vitro experiments on SZ95 sebocytes depicting (a) cell numbers, (b) caspase-3/7 activity as marker of apoptosis, and (c) lactate dehydrogenase (LDH) release as marker of dead cells. A DXR dose-dependent reduction of cell number is associated with marked apoptosis and cell death, where apoptosis preceded cell death. Asterisks indicate statistical significance, ***P<0.001. Journal of Investigative Dermatology 2006 126, 711-720DOI: (10.1038/sj.jid.5700175) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 11 Proliferation of SZ95 sebocyte cultures treated with different DXR concentrations. DXR concentrations greater than 10−6M were 100% toxic after only 2 days of treatment. In contrast, a DXR dose of 10−7M was subtoxic and dependent on the duration of treatment. *P<0.05, **P<0.01, ***P<0.001. Journal of Investigative Dermatology 2006 126, 711-720DOI: (10.1038/sj.jid.5700175) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 12 Detection of apoptosis on SZ95 sebocytes cell line as assessed by TUNEL assay. (a) Untreated cells; (b) treated cells with 10−7M, (c) 10−6M, and (d) 10−5M DXR. (e) The graph and the table indicate the mean values and standard deviations of the percentage of TUNEL-positive cells that have undergone apoptosis. Journal of Investigative Dermatology 2006 126, 711-720DOI: (10.1038/sj.jid.5700175) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 13 Cell cycle analysis of SZ95 sebocytes either untreated or treated with DXR at a concentration of 10−6M, with respect to untreated controls, in the G2–M and S phases of the cell cycle was observed. This suggested that cells were entering apoptosis. Journal of Investigative Dermatology 2006 126, 711-720DOI: (10.1038/sj.jid.5700175) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions