Figure 1.
Panel A: representative flow cytometry of LNCaP cells stained with anti-PSMA or with J591 mAb. Controls are represented by cells goat anti-mouse-FITC IgG only. Panel B: flow cytometry of LNCaP cells incubated with biotinylated J591 mAb (B-J591) with or without pretreatment (45 min on ice) with anti-PSMA at the indicated concentrations or with non-biotinylated J591 mAb as control. Antibody binding was revealed by FITC-Avidin (F-Avidin). The percentage of positive cells and the Mean Fluorescence Intensity observed under the different conditions are representative of two independent experiments performed with independent cultures. Panel C: Western blot analysis of LNCaP cell lysates immuneprecipitated with the indicated mAbs, and then probed with the anti-PSMA mAb followed by a goat anti-mouse antiserum labelled with horseradish peroxidase. The apparent molecular weight of PSMA band is indicated by the arrow.
Figure 2.
Kinetics of ERK1/21/2, p38, RAC 1 and RAS phosphorylation induced by PSMA cross-linking.
LNCaP cells were left untreated or subjected to PSMA cross-linking for the indicated times at 37°C. An anti-VCAM-1 mAb was used as the isotype matched control. Panels A and B: p38 and ERK1/2 activation was assessed in crude lysates. Equal amounts (20 µg) of total proteins were boiled in sample buffer and separated by SDS-PAGE. Following immunoblotting with an anti-phospho-p38 or anti-phospho-ERK1/2 mAb, immunoreactive bands were visualized by using horseradish peroxidase-conjugated secondary antibody and the ECL system. Panels C and D: RAC1 and RAS activation was evaluated by a specific assay as described in the Material and Methods section. Bound active GTP-RAC and GTP-RAS molecules were analyzed by Western blotting using an anti-RAC1 or anti-Ras mAb and visualized as above. Total amount of ERK1/2, p38, RAC1 and RAS in crude lysates are shown as loading control at the bottom of each gel. Results are representative of one of three independent experiments.
Table 1.
Kinetics of ERK1/2, p38, RAC1 and RAS activation in LNCaP cells following cross-linking of anti-PSMA, anti-VCAM-1 or 7E11c mAba).
Figure 3.
NF-κB activation induced by PSMA crosslinking.
LNCaP cells were subjected to stimulation by cross-linking of PSMA or of VCAM-1 for 45 min at room temperature and then cultured for the indicated times at 37°C. The cells were then harvested, washed, permeabilized and stained with a PE-labelled anti-phosphorylated NF-κB p65 antibody. Cells were then analyzed by flow cytometry. The MFI was recorded. An isotype-matched IgG1 antibody was used to mock-treat LNCaP cells; the Figure illustrates a representative experiment out of three performed independently. MFI values of controls were: untreated cells 9.22±0.9, cells treated with an anti-VCAM-1 mAb 5.74+0.09, cells treated with a mouse IgG1 mAb 5.02±0.3. No substantial differences in the control samples were observed at the various time points of the experiments. MFI of Anti-PSMA cross-linked cells were: 22.03±2.2 and 15.6±1.3 at 15 min and 30 min, respectively. p values of samples treated with anti-PSMA mAb were <0.01 at 15 min and 30 min, respectively, with respect to the untreated cells. The first panel from top (mouse IgG1) is the plot corresponding to time 0.
Figure 4.
Inducing activity of PSMA cross-linking on IL-6 and CCL5 expression and role of PSMA-activated p38 and ERK1/2 .
IL-6 (panel A) and CCL5 (panel B): production by LNCaP cells left untreated (open bars), stimulated by cross-linking via anti PSMA mAb (filled bars) or anti-VCAM-1 mAb (dashed bars) in the presence or in the absence of single or combined inhibitors (SB and PD) at the indicated doses (SB50 = 50 µg/ml, SB25 = 25 µg/ml; PD50 = 50 µg/ml, PD25 = 25 µg/ml; SB/PD25 = combinations of SB+PD at 25 µg/ml each). Control and treated cell cultures were washed, cultured for further 24 h before collecting cells and cell-free supernatants. IL-6 and CCL5 accumulation in each supernatant was measured by ELISA and expressed as pg/mg of cell lysates/24 h. Inhibitors were added to the cells 6 h prior to stimulation and maintained throughout the duration of the experiment. Values represent the mean±SEM of results of four experiments performed in duplicates. Stars on top of the bars indicate statistical significance as follows: p<0.001, ***; p<0.01, **; p<0.05, *.
Figure 5.
Effects of pro-proliferative stimuli on LNCaP cells.
A) LNCaP proliferation induced by treatment with CCL5 and/or IL-6. 3H-TdR uptake of cells after 24 h of culture with CCL5 (grey bars), 72 h of culture with IL-6 (hatched bars) or 24 h of culture with combinations of CCL5 and IL-6 (crossed bars) at the indicated concentrations. B) 3H-TdR uptake of cells after 24 h culture with 5 ng/ml of CCL5 in the presence or in the absence of anti-CCR5 or 7E11c antibodies at the indicated concentrations; C) Western blot analysis of cell extracts (20 µg/lane) of LNCaP cells treated with CCL5 (5 ng/ml) and probed with anti-STAT 5 or with anti-cyclin D1 antibodies. Equal protein loading in all the lanes is confirmed by not specific bands shown in the middle gel. Arrows indicate phosphorylated STAT5 proteins. Western blotting results are representative of two independent experiments. D) 3H-TdR uptake of cells 72 h after binding or cross-linking of 7E11c or anti-PSMA antibodies. Values represent the mean±SEM of three independent experiments performed in triplicates. Stars on the bars indicate statistical significance as in Fig. 4.