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First published online 14 November 2007
doi: 10.1242/jcs.009761

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Interferon- and tumor necrosis factor- sensitize primarily resistant human endometrial stromal cells to Fas-mediated apoptosis

Herbert Fluhr^1,2,*, Stefanie Krenzer^2,*, Gerburg M. Stein³, Björn Stork³, Margarita Deperschmidt², Diethelm Wallwiener², Sebastian Wesselborg³, Marek Zygmunt^1, and Peter Licht^2,4

¹ Department of Obstetrics and Gynecology, University of Greifswald, Wollweberstr. 1, 17475 Greifswald, Germany
² Department of Obstetrics and Gynecology, University of Tübingen, Calwerstr. 7, 72076 Tübingen, Germany
³ Department of Internal Medicine I, University of Tübingen, Otfried-Müller-Str. 10, 72076 Tübingen, Germany
⁴ Fertility Center Nuremberg, Agnesgasse 2, 90403 Nuremberg, Germany

View larger version (18K): [in this window] [in a new window]   Fig. 1. Human ESCs are resistant to Fas-mediated apoptosis (in contrast to Jurkat cells) independently of their state of differentiation. (A) Human ESCs were incubated with increasing concentrations (0, 50, 100 and 200 ng/ml) of an activating anti-Fas antibody for 24 (white bars) and 48 (black bars) hours. Treatment of ESCs with 200 µg/ml mitomycin C (Mito) was used as a positive control. **P<0.01 for both 24 and 48 hours mitomycin (Mito) treated versus the respective untreated control. (B) Jurkat cells were also incubated with increasing concentrations of an activating anti-Fas antibody for 24 hours. **P<0.01 for treated cells versus untreated control. (C) Undifferentiated (white bars) and decidualized (black bars) human ESCs were incubated with increasing concentrations (0, 50, 100 and 200 ng/ml) of an activating anti-Fas antibody for 24 hours. The rate of apoptosis was measured by flow-cytometric detection of hypodiploid nuclei. Bars show the mean ± s.e.m.

View larger version (21K): [in this window] [in a new window]   Fig. 2. IFN- and TNF- sensitize human ESCs to Fas-mediated apoptosis. Decidualized (black bars) as well as undifferentiated (white bars) ESCs were treated with 50 ng/ml IFN-, 25 ng/ml TNF- and 1 U/ml hCG alone and in the indicated combinations for 24 hours before stimulating the cells with 100 ng/ml of an activating anti-Fas antibody for another 24 hours. The rate of apoptosis was determined by flow-cytometric detection of hypodiploid nuclei (*P<0.05 for both 6 and 7 versus 0-5). Bars show the mean ± s.e.m.

View larger version (27K): [in this window] [in a new window]   Fig. 3. Human ESCs express Fas on the cell surface at a lower level than do Jurkat cells. Fas expression does not change during decidualization in vitro. (A) Human ESCs and Jurkat cells were stained with a monoclonal anti-Fas antibody (filled histograms) and compared to cells stained with an IgG1 isotype control (white histograms) using flow cytometry. (B) The expression level of Fas as measured by MCFI. The signal derived from undifferentiated ESCs was set to 100 (**P<0.01: Jurkat versus undifferentiated and decidualized ESCs). (C) Fas mRNA expression was measured by semiquantitative real-time RT-PCR to compare undifferentiated and decidualized ESCs (n.s., non significant). Bars show the mean ± s.e.m. (D) Fas protein level was determined by flow cytometry to compare undifferentiated and decidualized ESCs. Filled histograms, ESCs stained with a monoclonal anti-Fas antibody; white histograms, ESCs stained with an IgG1 isotype control.

View larger version (17K): [in this window] [in a new window]   Fig. 4. IFN- and TNF- cause an upregulation of Fas expression in human ESCs. Human ESCs were treated with 50 ng/ml IFN-, 25 ng/ml TNF- and 1 U/ml hCG alone and in various combinations for 24 hours (mRNA) and 48 hours (protein) and were subsequently analyzed for the expression of Fas. (A) Fas mRNA level was determined by semiquantitative real-time RT-PCR (*P<0.05: 6 versus 0). (B) The amount of membrane-bound Fas was determined by flow cytometry (**P<0.01: each 6 and 7 versus 0-5). The expression level as measured by MCFI is shown. The signal derived from the untreated control was set to 100. Bars show the mean ± s.e.m.

View larger version (20K): [in this window] [in a new window]   Fig. 5. Caspase 8 is expressed in human ESCs but is not regulated by IFN-, TNF- or hCG. (A) ESCs were stained intracellularly to determine total caspase 8 levels and were analyzed by flow cytometry (filled histogram, caspase 8 staining; white histogram, isotype control). (B) ESCs were treated with 50 ng/ml IFN-, 25 ng/ml TNF- and 1 U/ml hCG alone and in various combinations for 24 hours and were subsequently analyzed for the expression of caspase 8 by flow cytometry. The expression level as measured by MCFI is shown. The signal derived from the untreated control was set to 100. Bars show the mean ± s.e.m.

View larger version (15K): [in this window] [in a new window]   Fig. 6. Caspase 8, caspase 9 and caspase 3 are activated in human ESCs upon apoptotic Fas signaling. Human ESCs treated with 50 ng/ml IFN- and 25 ng/ml TNF- for 24 hours (black dots) before stimulating the cells with 100 ng/ml of an activating anti-Fas antibody for the indicated time periods were compared to ESCs without pre-treatment with cytokines (white dots). The enzymatic activity of caspase 8 (A), caspase 9 (B) and caspase 3 (C) was measured using specific luminescent activity assays [**P<0.01: ESCs with cytokine pre-treatment versus cells without pre-treatment; values of control (0 hours) were set 1].

View larger version (21K): [in this window] [in a new window]   Fig. 7. FLIP mRNA expression is upregulated in ESCs by IFN- and TNF-. Undifferentiated ESCs were treated with IFN-, TNF- and hCG alone or in various combinations for 24 hours and the expression of FLIP mRNA was measured by semiquantitative real-time RT-PCR (**P<0.01: 6 and 7 each versus 0-5). Bars show the mean ± s.e.m.