Figure 7
CD40-mediated modulation of ROS and apoptosis in normal, ‘para-malignant’ and transformed epithelial cells. (a) The basal levels of ROS in the panel of bladder cancer cell lines RT4 (well-differentiated, non-malignant), RT112 (moderately-differentiated, non-malignant) and EJ (undifferentiated, highly-malignant) were measured by H2DCFDA labelling. There was an apparent correlation between increased basal ROS expression and degree of anaplasty, with EJ>RT112>RT4 cells. Bars represent average relative fluorescence units (RFU) ±s.d. (n=3). (b) The RT4, RT112 and EJ cell lines were treated with the indicated concentrations of H2O2 and viability was assessed. The more anaplastic, malignant cells EJ exhibited the highest degree of susceptibility to oxidative stress in comparison to the highly differentiated, non-malignant RT4 cells that were the least susceptible. Bars represent mean % cell growth ±s.d. (n=5). (c) Ligation of CD40 by mCD40L in the cancer cell lines resulted in extensive apoptosis in the highly malignant EJ cells, whereas the non-anaplastic highly-differentiated RT4 cells showed relatively low susceptibility to CD40 ligation. RT112 cells that were engineered to express the CD40 receptor showed moderate susceptibility to CD40-mediated apoptosis. Bars represent mean % cell death ±s.d. (n=3). (d) The basal ROS in normal (NHU) cells, para-malignant NHU derivatives stably expressing hTERT (HU-hTERT) and the malignant cell line EJ were determined by H2DCFDA labelling and flow cytometry. Representative results of such analysis are shown in the overlay histogram (left), while the derived results for NHU, HU-hTERT, EJ and HCT116 from similar analyses are presented as average MFI (right). Basal ROS was higher in hTERT-expressing epithelial cells in comparison to their normal counterparts, but were lower than those observed in malignant (EJ and HCT116) cells. Bars represent mean MFI ±s.d. (n=3). (e) NHU, HU-hTERT, EJ and HCT116 cells were treated with the indicated concentrations of H2O2 and viability was assessed. The malignant cells EJ and HCT116 exhibited the highest degree of susceptibility to oxidative stress in comparison to normal NHU cells which were nearly completely refractory to the tested concentrations of H2O2. By contrast, the para-malignant HU-hTERT cells demonstrated significant susceptibility to oxidative stress, which was nevertheless lower than that of EJ and HCT116 cells. Bars represent mean % cell growth ±s.d. (n=5). (f) Ligation of CD40 by mCD40L caused substantial apoptosis in HU-hTERT cells, in comparison to their normal (NHU) counterparts that were refractory. Apoptosis in normal and para-malignant cells was compared to EJ and HCT116 cells. Bars represent mean fold change ±s.d. (n=5). (g) Immunoblotting experiments show that CD40 ligation induces Bak and to a lesser extent Bax expression (24 h) in para-malignant HU-hTERT cells in comparison to normal (NHU) cells. CK18 detection was used for comparison of controls (‘–’) versus mCD40L-treated cells (‘+’) and confirmed equal epithelial lysate loading. Results are representative of three independent experiments.
