Ute Breitenbach, Jan P. Tuckermann, Christoffer Gebhardt, Karl H

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Keratinocyte-Specific Onset of Serine Protease BSSP Expression in Experimental Carcinogenesis Ute Breitenbach, Jan P. Tuckermann, Christoffer Gebhardt, Karl H. Richter, Peter Angel Journal of Investigative Dermatology Volume 117, Issue 3, Pages 634-640 (September 2001) DOI: 10.1046/j.0022-202x.2001.01437.x Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Identification of TPA-inducible genes in murine skin and SCC. A representative reverse northern analysis of triplicate filters is shown. Each lane is loaded with 100 ng of EcoRI digested plasmid DNAs of individual cDNA clones, derived from SSH between acetone-treated and 6 h TPA-treated dorsal skin of adult female C57BL/6 mice. Filters were hybridized with single-stranded 32P-labeled complex cDNA probes of equal specific activity obtained from acetone-control (co) or 6 h TPA-treated (TPA) dorsal skin (n = 3) as well as from SCC (SCC; n = 8). VL30, virus-like retrotransposon; MTI, metallothionein Ia; BSSP, brain and skin specific serine protease. Expressions of the housekeeping genes GAPDH and COX were used as internal controls for hybridization. Journal of Investigative Dermatology 2001 117, 634-640DOI: (10.1046/j.0022-202x.2001.01437.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 BSSP expression in TPA-treated mouse skin. After 0 (co), 1, 2, 4, 6, and 12 h TPA-treated mice were sacrificed and total RNA from skin was isolated as described in Materials and Methods. BSSP expression was analyzed by northern blot hybridization using the isolated SSH fragment as a probe. Each lane represents RNA pooled from three individuals. Rehybridization with a cDNA fragment of 18S RNA was performed, serving as a loading control. Journal of Investigative Dermatology 2001 117, 634-640DOI: (10.1046/j.0022-202x.2001.01437.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 BSSP expression in the skin of c-fos-deficient mice. c-fos-deficient mice (fos‒/‒) and their wild-type littermates (fos+/+) were treated with acetone (co), 10 nmol TPA (TPA), or 10 nmol TPA with 50 µg dexamethasone (T + D). After 6 h mice were sacrificed and total RNA from skin was isolated as described in Materials and Methods. BSSP expression was analyzed by northern blot hybridization using the isolated SSH fragment as a probe. Each lane represents RNA pooled from three individuals. Rehybridization with a cDNA fragment of 18S RNA was performed, serving as a loading control. Journal of Investigative Dermatology 2001 117, 634-640DOI: (10.1046/j.0022-202x.2001.01437.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Tissue-specific expression of BSSP in murine dorsal skin. Acetone (A, B), TPA (C, D), as well as TPA plus dexamethasone (E, F) were applied onto the skin of adult female C57BL/6 mice. After 6 h mice were sacrificed; skin biopsies were taken, paraformaldehyde-fixed and subsequently paraffin-embedded. The SSH-derived cDNA clone of BSSP was 35S-labeled and used as a riboprobe for in situ hybridization. BSSP expression can be detected in keratinocytes of the epidermis (ep) and hair follicle (hf), but not in fibroblasts of the dermis (de; arrows). Scale bar: 20 µm. Journal of Investigative Dermatology 2001 117, 634-640DOI: (10.1046/j.0022-202x.2001.01437.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Expression of BSSP in hyperplastic skin as well as benign and malignant skin tumors. Sections of hyperplastic adult skin (HS) and chemically induced papillomas (PA) and SCC (SCC) were hybridized with 35S-labeled riboprobes derived from the BSSP cDNA fragment and photographed under darkfield (a, d, g) and brightfield (b, e, h) conditions. Histochemical analysis of PCNA expression was performed using a PCNA antibody (c, f, i). ep, epidermis; bl, basal layer; st, stroma; cl, cornified layer; ci, carcinoma cell islet. Scale bar: 20 µm. Journal of Investigative Dermatology 2001 117, 634-640DOI: (10.1046/j.0022-202x.2001.01437.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions