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FAP-1 in pancreatic cancer cells

functional and mechanistic studies on its inhibitory role in CD95-mediated apoptosis

¹ Research Unit Molecular Oncology, Clinic for General Surgery and Thoracic Surgery, Christian-Albrechts-University, 24105 Kiel, Germany
² Department of Medicine, Christian-Albrechts-University, 24105 Kiel, Germany
^* Author for correspondence (e-mail: hkalthoff{at}email.uni-kiel.de )

View larger version (34K): [in a new window]   Fig. 1. Resistance to CD95-induced apoptosis correlates with expression of FAP-1. (A) Relative sensitivity to CD95-mediated cell death in two pancreatic carcinoma cell lines. Cells were treated for 24 hours with various concentrations of the agonistic anti-CD95 antibody CH11 prior to measurement of DNA fragmentation using the JAM assay. Data are expressed as % viability, with untreated control cells set arbitrarily to 100, and values are means of six determinations. Standard deviations were below 10%. (B) RT-PCR analysis of FAP-1, c-FLIP and GAPDH mRNAs in Panc89 and Capan-1 cells. The inclusion of samples in which reverse transcriptase (RT) had been omitted (-) served to rule out false positive results by contaminating genomic sequences. Jurkat cells were used as a negative control for FAP-1 and c-FLIP. The parallel amplification of a GAPDH-specific sequence served to validate integrity of the input RNA. (C) Immunoblot analysis of FAP-1 in Panc89 and Capan-1 cells. Full-length FAP-1 from Panc89 cells migrates as a band of approximately 250 kDa. Specificity of the anti-FAP-1 antibody was confirmed by competition (Comp) with the respective blocking peptide.

View larger version (32K): [in a new window]   Fig. 2. Stable introduction of FAP-1 renders Capan-1 cells resistant to CD95-induced apoptosis. (A) Immunoblot analysis of FAP-1 and CD95 in Capan-1 transfectants. Data shown are derived from a single exposure of two different blots of which irrelevant lanes have been removed for clarity of presentation. 40 µg of protein was loaded for Capan-1, 8 µg for Panc89. An antibody to ß-actin was used as a loading control. (B) Relative sensitivity of FAP-1 transfectants to CD95-induced apoptosis (24 hour treatment with agonistic anti-CD95 antibody CH11, 100 ng/ml) measured with the JAM assay. Data for clone 2 are given as % viability (mean ± s.d.) relative to untreated cells set at 100%; equivalent data for clones 4, 12 and 40 are presented as the ratio of viability (%) of each FAP-1 transfectant over vector control. (C) Immunoblot analysis of PARP cleavage in Capan-1 (parental), vector-transfected control (vector) and Capan-1-FAP-1 clone 2 (clone 2) cells stimulated for 24 hours with CH11 (100 ng/ml). The appearance of the 85 kDa PARP cleavage product is indicative of caspase-3 activity. (D) Immunoblot analysis of caspase-8 cleavage after anti-CD95 (Ab) or TRAIL (Tr) stimulation. Capan-1 cells were left untreated (Co) or were treated for 24 hours with either CH11 antibody (Ab, 100 ng/ml) or TRAIL (Tr, 100 ng/ml). The degree of caspase-8 (casp 8) cleavage corresponds to a decrease in the procaspase-8-specific signal and should be estimated relative to the intensities of the ß-actin bands.

View larger version (16K): [in a new window]   Fig. 3. Inhibition of tyrosine phospatases renders pancreatic tumor cells sensitive to anti-CD95-induced cell death. Cells were pretreated for 0.5 hours with the indicated concentrations of sodium orthovanadate (SOV) prior to addition of CH11 antibody (100 ng/ml), TNF- (1000 U/ml) or 5-fluorouracil (50 µg/ml). After 24 hours cells were lysed and the extent of DNA fragmentation determined in the JAM assay. Data are expressed relative to untreated control cells set at 100% and are the mean of six or eight wells processed in parallel. Standard deviations were below 10% and were omitted from this figure for reasons of clarity. The depicted data are representative of three independent experiments.

View larger version (43K): [in a new window]   Fig. 4. Subcellular localization of FAP-1 in Panc89 cells as revealed by confocal laser scanning microscopy. (A-C) Distribution of ß-COP (A) and FAP-1 (B) in untreated Panc89 cells. Arrows indicate a cisternal structure positive for both proteins (A-C). (D-F) Distribution of ß-COP (D) and FAP-1 (E) in Panc89 cells treated with 1 µg/ml brefeldin A for 60 minutes. Cells were labelled by indirect immunofluorescence staining using a polyclonal antibody against FAP-1 and detected by a secondary antibody labelled with red fluorescence. ß-COP was visualised with a monoclonal antibody and detected with a green labelled secondary antibody. Colocalization of both labels results in yellow colouring (C,F). FAP-1 and ß-COP clearly colocalize in untreated cells at juxtanuclear cisternal sites (C). Arrowheads depict individual vesicular staining for FAP-1 (C,F). After brefeldin A treatment, the Golgi cisternae were disrupted and ß-COP (D) and FAP-1 (E) were dispersed throughout the cell and totally separated (F).

View larger version (77K): [in a new window]   Fig. 5. Effect of anti-CD95 stimulation on the intracellular distribution of CD95 and FAP-1 in Panc89 cells. Under control conditions (A,G,K) the majority of FAP-1 immunoreactivity (depicted in red) is localised in the Golgi area (A, arrowhead). Some additional peripheral vesicular staining is also seen. After stimulation of Panc89 cells with agonistic APO1-3 antibody (2 µg/ml) for 5 minutes (B,H) or 15 minutes (C,I) this staining pattern remains principally unaltered (B,C, arrowheads), however, immunoreactivity is strongly enhanced after 15 minutes. CD95 immunoreactivity (depicted in green) is found at the plasma membrane but is predominantly intracellular under control conditions (D,G,K). Within 5 minutes of APO1-3 incubation of Panc89 cells (E,H), immunoreactivity for CD95 is enhanced, and pronounced plasma membrane staining is found, especially at sites of cell-cell-contact (see arrows in E). After 15 minutes of CD95 stimulation, strong immunoreactivity for CD95 is spread all over the cell and is concentrated in the Golgi area (arrowhead in F), thus showing strong colocalization with FAP-1 (yellow colouring, arrowhead in I) while plasma membrane staining for CD95 is reduced. In a parallel experiment Panc89 cells were stimulated with 100 ng/ml TRAIL (control, K) for 5 minutes (L) and 15 minutes (M). No significant changes were observed in CD95 (green) and FAP-1 (red) staining patterns.

View larger version (36K): [in a new window]   Fig. 6. Effect of anti-CD95 stimulation on intracellular distribution of CD95 in Capan-1 cells. Cells were stimulated with agonistic APO1-3 antibody (2 µg/ml) for 5 minutes (B) or 15 minutes (C), as described in the legend to Fig. 5. Note the absence of enhanced intracellular immunoreactivity upon anti-CD95 treatment and the increase in plasma membrane staining at 15 minutes.

View larger version (37K): [in a new window]   Fig. 7. Effect of brefeldin A on anti-CD95-induced intracellular distribution of CD95 and FAP-1 (A-D) and on anti-CD95-induced apoptosis (E) in Panc89 cells. The immunostaining experiment was carried out as described in the legend to Fig. 5 except that the cells were challenged with APO1-3 for 1 hour in the absence (A,C) or presence (B,D) of brefeldin A. Note the absence of colocalization of FAP-1 and CD95 and the increase in plasma membrane staining upon combined anti-CD95/brefeldin A treatment (D). Apoptosis of cells treated for 4 hours with either brefeldin A (BFA) or anti-CD95 (CH11) alone or in combination was measured by binding of FITC-labeled annexin V (E). FITC-positive cells were detected by fluorescence flow cytometry. Shown are representative data (calculated as percentage of treated versus untreated cells) from one out of three independently performed experiments.

View larger version (87K): [in a new window]   Fig. 8. Expression of FAP-1 in pancreatic adenocarcinoma tissue revealed by immunohistochemistry using the C-20 antibody. Note the positive staining in the cytoplasm of pancreatic duct tumor cells of a stroma-rich tumor (a). Immunostaining is absent from an adjacent tissue section where the C-20 antibody had been preincubated with the respective blocking peptide (b), from the stromal compartment of tumor tissue and from pancreatic duct epithelium of a healthy individual (e). Two further tumor tissue specimens with positive staining are illustrated (c,d) of which one displayed a particularly strong immunoreactivity (c). Counterstained with hemalum. Magnification 135x (a,b,d,e); 270x (c).