Basal-Cell Adhesion Molecule (B-CAM) is Induced in Epithelial Skin Tumors and Inflammatory Epidermis, and is Expressed at Cell–Cell and Cell–Substrate Contact Sites  Margarete Schön, Viktor Hogenkamp, B. Gregor Wienrich, Michael P. Schön  Journal of Investigative Dermatology  Volume 115, Issue 6, Pages 1047-1053 (December 2000) DOI: 10.1046/j.1523-1747.2000.00189.x Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 B-CAM is not expressed in normal epidermis, but is induced in epithelial tumors and normal-appearing skin overlying malignant tumors. Frozen sections of normal trunk skin (left panel, representative of 14 specimens), a solid BCC (middle panel, representative of 12 tumors), and a melanoma (right panel, representative of two cases) were stained with the B-CAM-specific MoAb VF18 as described in Materials and Methods. Scale bars: 20 μm. Journal of Investigative Dermatology 2000 115, 1047-1053DOI: (10.1046/j.1523-1747.2000.00189.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 B-CAM is upregulated in transformed keratinocytes in vitro and is induced during serial culture of normal human keratinocytes. (a) Lysates (10 μg of total protein per lane) of normal human keratinocytes (second passage, lane 1), SV40-transformed keratinocyte lines HaSV (lane 2), 425 (lane 3), and 130 (lane 4), and the spontaneously immortalized keratinocyte line HaCaT (lane 5) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, immunoblotted and stained with MoAb VF18 as outlined in Materials and Methods. Signals were visualized by the enhanced chemiluminescence method. The experiment shown is representative for three independent experiments. (b) Lysates were prepared from a primary culture (lane 1) and the first (lane 2) and second (lane 3) subculture of normal human keratinocytes obtained from the same donor, and equal amounts of protein were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, immunoblotted, and B-CAM was visualized using MoAb VF18. The experiment shown is representative for three independent experiments. Journal of Investigative Dermatology 2000 115, 1047-1053DOI: (10.1046/j.1523-1747.2000.00189.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 B-CAM is expressed de novo in the epidermis of inflammatory skin disorders. Five micrometer cryostat-sections of biopsies from an acute contact dermatitis (a, representative of four cases), a disseminated superficial actinic porokeratosis (b, representative of two cases) and Sweet's syndrome (c, representative of two cases) were stained with MoAb VF18 using the ABC immunoperoxidase method. In (d), a high power magnification of a horizontal section of a lesion from Sweet's syndrome demonstrates the basal (asterisk) and apicolateral (arrowheads) expression of B-CAM in the epidermis and in the dermal blood vessels. Scale bars: 20 μm. B-CAM expression in the three disorders depicted here is representative of other inflammatory disorders as outlined in the text. Journal of Investigative Dermatology 2000 115, 1047-1053DOI: (10.1046/j.1523-1747.2000.00189.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 B-CAM synthesis is decreased after inhibiting proliferation or inducing terminal differentiation in human keratinocytes, and is upregulated by UV irradiation. (a) SV40-transformed human keratinocytes (cell line 425) were incubated with normal medium (lanes 1, 4, and 7), 1.5 mM hydroxyurea (lanes 2, 5, and 8), or with 8 ng per ml mitomycin C (lanes 3, 6, and 9) and metabolically labeled with [35S]methionine. B-CAM was immunoprecipitated from cell lysates using MoAb VF18 (lanes 1–3) as outlined in Materials and Methods. Two unrelated cell-surface glycoproteins of 80 kDa (recognized by MoAb BT15, lanes 4–6) and 130 kDa (recognized by MoAb Q14, lanes 7–9) were also precipitated from the same cell lysates for comparison. (b) Normal human keratinocytes were serially cultured in serum-free medium with low concentration of divalent calcium cations (0.09 mM Ca2+ over three passages. Thereafter, parallel cultures were kept in 0.09 mM Ca2+ (lane 1) or 1 mM Ca2+ (lane 2) for 3 d as indicated. B-CAM expression was analyzed by western blot analysis (10 μg total protein per lane) using MoAb VF18. The experiment shown is representative for three independent experiments. (c) Subconfluent cultures of normal human keratinocytes were irradiated with 100 J per m2 UVB (squares) or 30 J per cm2 UVA (circles). B-CAM expression was assessed by fluorescein isothiocyanate analysis of untreated cultures, and 2, 4, 12, 24, or 48 h after irradiation. The values represent the relative B-CAM expression as calculated from the mean fluorescence intensities (untreated keratinocytes = 100%). The experiment shown is representative of three independent experiments showing similar results. Journal of Investigative Dermatology 2000 115, 1047-1053DOI: (10.1046/j.1523-1747.2000.00189.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Ultrastructural localization of B-CAM in tumor cells to cell–cell contact sites. Human squamous cell carcinoma cells (cell line SCL-2) were grown on a melamine resin foil, and stained under in vivo conditions with MoAb VF18, followed by gold-conjugated secondary antibody as described in Materials and Methods. The gold particles were visualized by TEM of whole cells. Scale bars: 1 μm. (a) Two SCL-2 cells in close vicinity to each other. Gold clusters (10 nm) are localized at the tips of cytoplasmic processes, which contact peripheral processes of the neighbor cell. (b) B-CAM expression at the contact between a peripheral cytoplasmic process and a SCL-2 cell body. TEM of vertical sections of immunogold (40 nm) labeled cell cultures show the spatial relationship of B-CAM to sites of cell–cell contact. (c) B-CAM reactivity at a cell contact site of SCL-2 cell bodies. TEM of vertical sections of immunogold (40 nm) labeled cell cultures. Journal of Investigative Dermatology 2000 115, 1047-1053DOI: (10.1046/j.1523-1747.2000.00189.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 B-CAM is expressed at peripheral cell processes of cultured squamous carcinoma cells. SCL-2 cells were cultured and labeled as outlined in Figure 5. Scale bars: 1 μm. (a) Whole mount TEM of SCL-2 cells labeled with MoAb VF18 and 40 nm gold. (b) Higher magnification of the peripheral cell process in (a). Journal of Investigative Dermatology 2000 115, 1047-1053DOI: (10.1046/j.1523-1747.2000.00189.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions