Peuhu et al. BMC Cancer 2010, 10:521 http://www.biomedcentral.com/1471-2407/10/521 Page 4 of 9 Figure 2 cis-UCA induces both apoptotic and necrotic cell death in 5637 bladder carcinoma cells. The cells were treated with indicated concentrations of cis-UCA in buffer solutions adjusted to pH 7.4 or pH 6.5 for 2 h and let to recover in the absence of cis-UCA for 20 h. The cell culture supernatants and cell lysates were analysed for formation of cytoplasmic histone-associated DNA fragments with a photometric enzyme- immunoassay (A.). Alternatively, the cells treated with cis-UCA or PIPES (corresponding to 2% cis-UCA equimolar concentration) at pH 6.5 were collected and labelled with TMRM for mitochondrial membrane potential (B.), analysed for caspase-3 activation (C.) or labelled with PI for cell permeabilization as a measure of non-apoptotic cell death (D.). The samples were analysed by flow cytometry (Mean ± SEM; n = 3; Student’s t-test * p < 0.05, ** p < 0.01, *** p < 0.001). The pictures are representative of two independent experiments. cell-released DNA-histone complexes, measured in the extracellular medium, increased with 2% and 4% cis- UCA (data not shown). To eliminate the possibility that these effects were caused by increased extracellular osmolality due to high concentrations of cis-UCA, cells were treated with an equimolar concentration of PIPES, which is otherwise nontoxic to cells. In these experi- ments, PIPES affected neither cell viability nor cellular markers of apoptosis and necrosis (Figure 2). To further analyse the biochemical events that take place after 2% cis-UCA treatment, the nuclei were labelled with DAPI and imaged by confocal microscopy (Additional file 1: Figure S1). The results showed that a large fraction of the cis-UCA-exposed 5637 cells dis- played nuclei with characteristic apoptotic features, including nuclear condensation and fragmentation. To investigate caspase signalling upstream of caspase-3, the activation-induced cleavage of caspase-8 was monitored by Western blotting. Interestingly, we were unable to detect caspase-8 cleavage, indicating that caspase-8 is not activated in response to cis-UCA (data not shown). This suggests that the extrinsic apoptotic pathway is probably not involved in cis-UCA-induced cell death. A modest cleavage of poly(ADP-ribose) polymerase-1 (PARP1) after treatment with 2% cis-UCA was detected (data not shown), suggesting that in our experimental setup, the cleavage of PARP1 is more likely to indicate general cellular stress than progression of classical apop- totic cell death. 5637 bladder carcinoma cells enter cell cycle arrest in response to cis-UCA As cis-UCA was able to trigger cell death, we sought to determine whether cell cycle progression of the 5637