Selective Induction of Apoptosis in Melanoma Cells by Tyrosinase Promoter-Controlled CD95 Ligand Overexpression Lothar F. Fecker, Christoph C. Geilen,

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Selective Induction of Apoptosis in Melanoma Cells by Tyrosinase Promoter-Controlled CD95 Ligand Overexpression Lothar F. Fecker, Christoph C. Geilen, Amir M. Hossini, Constanze Schwarz, Henry Fechner, David L. Bartlett, Constantin E. Orfanos, Jürgen Eberle Journal of Investigative Dermatology Volume 124, Issue 1, Pages 221-228 (January 2005) DOI: 10.1111/j.0022-202X.2004.23572.x Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 High tyrosinase promoter activity in melanoma cell lines characterized by high expression of endogenous tyrosinase mRNA. Relative luciferase activities (arbitrary units) were determined 48 h after transient transfection of melanoma cell lines SK-Mel-13, SK-Mel-19, MeWo, A-375, and M-5 as well as of non-melanoma cell lines MCF-7, PFSK-1, HeLa, and Hep-G2. Cells were transfected with luciferase reporter gene constructs under control of the SV40 promoter (white bars), the CMV promoter (gray) and the tyrosinase promoter (black). Relative luciferase activities were normalized by the median between the respective SV40 and CMV values. The median was set individually for each cell line to 100%. The complete experiment was performed three times for the tyrosinase promoter and for the SV40 promoter and was done twice for the CMV promoter, with each time triple values. Means and standard deviations calculated from the data of all experiments are presented here. Journal of Investigative Dermatology 2005 124, 221-228DOI: (10.1111/j.0022-202X.2004.23572.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Correlated expression of tyrosinase and microphthalmia-associated transcription factor (MITF) proteins. Expression of tyrosinase and of MITF was determined by Western analysis for melanoma cell lines SK-Mel-13, SK-Mel-19, MeWo, A-375, and M-5 as well as for non-melanoma cell lines MCF-7, PFSK-1, HeLa, and Hep-G2. Expression of the 70 kDa tyrosinase protein was restricted to the melanoma cell lines SK-Mel-13, SK-Mel-19, and MeWo. In these three melanoma cell lines, also MITF expression was detectable as two prominent protein bands of 55 and 60 kDa, which were also seen in a normal human melanocyte control culture (data not shown). Equal protein amounts (40 μg per lane) were loaded and consistent blotting was confirmed by ponceau staining and by control analysis of β-actin. Western blot experiments were performed twice using independent cell isolates giving highly similar results. Journal of Investigative Dermatology 2005 124, 221-228DOI: (10.1111/j.0022-202X.2004.23572.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Melanoma and non-melanoma cell lines represent CD95 cell surface expression. CD95 surface expression was determined by FACS analysis in non-melanoma and melanoma cell lines. Cells were labelled with a specific CD95 antibody (filled graphs) and with an isotypic control antibody (open graphs), respectively. A shift indicating relatively high CD95 surface expression is seen in non-melanoma cell lines PFSK-1, HeLa and Hep-G2 as well as in the melanoma cell lines SK-Mel-13, A-375, SK-Mel-19, and M-5. Relatively weak CD95 surface expression was seen in MeWo melanoma cells and in MCF-7 mammary carcinoma cells. Western blot experiments were performed twice using independent cell isolates giving highly similar results. Journal of Investigative Dermatology 2005 124, 221-228DOI: (10.1111/j.0022-202X.2004.23572.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Melanoma and non-melanoma cell lines are sensitive to CD95-dependent apoptosis. Relative DNA fragmentation rates of melanoma and non-melanoma cell lines treated for 16 h with a CD95 agonistic antibody (CH-11) are shown (gray bars). For each cell line basic apoptotic rates of untreated controls were individually set to 1 (white bars). Clear induction of CD95-mediated apoptosis was seen in all cell lines (p<0.01, Student's t test) with the exception of MeWo. The experiment was performed twice with both times triple values. Means and standard deviations of the values of both experiments are shown here. Journal of Investigative Dermatology 2005 124, 221-228DOI: (10.1111/j.0022-202X.2004.23572.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Selective induction of apoptosis in melanoma cells. Selective induction of apoptosis as determined by relative DNA fragmentation rates is shown in response to transient transfection of CD95 ligand (CD95L) expression constructs. (a) Relative DNA fragmentation rates of cell lines transiently transfected with a CMV promoter-CD95L construct (pIRES-CD95L; filled bars) as compared with a control plasmid (pIRES; open bars). Induction of apoptosis with the CMV promoter CD95L construct was statistically significant in all cell lines as compared with the control (p<0.02, Student's t test). (b) Relative DNA fragmentation rates of cell lines transiently transfected with a tyrosinase promoter CD95L construct (pTyr2-CD95L; filled bars) as compared with a control plasmid (pTyrex-2; open bars). DNA fragmentation rates of cell lines transfected with the respective control plasmid were set individually to 1. The experiments were performed three times for each construct with each time triple values. Means and standard deviations of the values of all three experiments are given here. Induction of apoptosis in SK-Mel-13, SK-Mel-19, MeWo, and in A-375 with the tyrosinase promoter CD95L construct was statistically significant (p<0.01, Student's t test). Journal of Investigative Dermatology 2005 124, 221-228DOI: (10.1111/j.0022-202X.2004.23572.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Determination of relative numbers of dead cells correlate with DNA fragmentation assays. Relative numbers of dead cells in melanoma cell lines SK-Mel-13 and in A-375 after transfection with the tyrosinase promoter CD95 ligand construct (Tyr CD95L) or with the control plasmid (Tyr C) were determined by typan blue staining (a) and by bisbenzimide nuclear staining (b). The experiments were performed in triplicate. Significant increase of dead and apoptotic cells, respectively, was found after transfection with the tyrosinase promoter CD95L construct as compared with the control (p<0.02, Student's t test). (c) Cell pictures after bisbenzimide staining are shown, several positive cells are indicated. Journal of Investigative Dermatology 2005 124, 221-228DOI: (10.1111/j.0022-202X.2004.23572.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 CD95L is expressed equally well in melanoma and non-melanoma cells after transient transfection. (a) Overexpression of CD95 ligand (CD95L) protein in SK-Mel-13, MCF-7 and PFSK-1 is shown by Western blot 48 h after transient transfection of pIRES-CD95L (CD95L). Vector-transfected cells are shown as negative controls (C). As positive control stably CD95L-transfected melanoma cells are shown (SKM13-CD95L) in which CD95L is inducible by doxycycline (+D), versus non-induced cells (-D). Equal amounts of proteins were loaded (40 μg per lane) and consistent blotting was confirmed by ponceau staining. For each cell line double experiments from independent cultures have been performed, giving highly similar results. (b) Surface expression of CD95L is shown 48 h after transient transfection of pIRES-CD95L (filled graphs) as determined by FACS analysis. Cells transiently transfected with a control plasmid (pIRES) were used as negative controls (open graphs). As a positive control, doxycycline-induced SKM13-CD95L cells (filled graph) are shown versus non-induced SKM13-CD95L cells (open graph). The overlay of the histograms of induced SKM13-CD95L cells versus the control revealed a shift to higher fluorescence in induced cells. This shift indicating CD95L surface expression is also seen in the overlays of transiently transfected melanoma and non-melanoma cells versus their respective controls. Journal of Investigative Dermatology 2005 124, 221-228DOI: (10.1111/j.0022-202X.2004.23572.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 Caspase activation and protein levels of Bcl-2 and Bax. Western blots for capase-8, caspase-3, caspase-7, Bcl-2, and Bax are shown for melanoma cell lines SK-Mel-13 and A-375. Proteins were extracted 48 h after transient transfection of cells with either pTyr2-CD95L (Tyr CD95 ligand (CD95L)), with pIRES-CD95L (CMV CD95L) or the respective control plasmids without the CD95L cDNA (Tyr C; CMV C). Identical protein amounts (25 μg per lane) were loaded in each lane, consistent blotting was confirmed by ponceau staining and by detection of β-actin. Molecular weights of caspase cleavage products (Casp), Bcl-2, Bax and β-actin are indicated on the right side. For each cell line double experiments from independent cultures have been performed, giving highly similar results. Journal of Investigative Dermatology 2005 124, 221-228DOI: (10.1111/j.0022-202X.2004.23572.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions