Peuhu et al. BMC Cancer 2010, 10:521 http://www.biomedcentral.com/1471-2407/10/521 cells was also affected by cis-UCA. For this purpose, the cells were synchronized to G0/G1 by serum starvation, and the nuclei were analysed after the 2-h cis-UCA pulse followed by 24-h recovery for DNA content by PI labelling and flow cytometry. Interestingly, already 1% cis-UCA induced G2/M block, as the cells accumulated in this phase (Figure 3). Higher concentrations of cis- UCA slightly increased the fraction of cells in the G0/ G1 phase, potentially indicating a block at the cell cycle entry after synchronization. In addition, the higher con- centrations are also likely to induce a general cell cycle block due to short-circuits in cell cycle control. Western blotting studies showed that cis-UCA does not cause the accumulation of the cyclin-dependent kinase inhibi- tors p21(WAF1/Cip1) and p27(Kip1), which were highly expressed already in untreated 5637 cells (data not shown), indicating that the arrest is a consequence of general disturbances in cell control mechanisms rather than specific triggering of emergency break mechanisms, such as expression of p21 or p27. Page 5 of 9 cis-UCA reduces metabolic activity and promotes ERK and JNK signalling in 5637 bladder carcinoma cells The metabolic activity of the 5637 cells was followed for 4 h after the 2-h cis-UCA pulse using a MTS assay. A clear inhibition of the cumulative cellular reducing power was monitored by hourly measurements after 2% and 3% cis-UCA treatments (Figure 4A) without any visible effects on cell viability at these early time points. To study whether the observed metabolic inhibition was associated with activation of any major signalling path- ways, we measured the effects of cis-UCA on stress and mitogen-inducible protein kinases. For this purpose, we collected whole-cell lysates immediately after the 2-h pulse with 2% cis-UCA and analysed the phosphoryla- tion and overall expression levels of ERK1/2 and JNK1/2 by Western blotting. Interestingly, we detected a marked increase in the phosphorylated forms of ERK1/2 and JNK2 in response to cis-UCA treatment (Figure 4B), suggesting strong activation of these pathways. In con- trast, the phosphorylation of JNK1 was only increased to Figure 3 Cell cycle progression of 5637 bladder carcinoma cells is inhibited in response to cis-UCA. The cells were synchronized, treated with 0-3% cis-UCA or PIPES (corresponding to 3% cis-UCA equimolar concentration) for 2 h, and let to recover in the absence of treatments for 24 h. After incubation the collected cells were disrupted in sodium citrate buffer and the PI labelled nuclei were analysed by flow cytometry for DNA content. Cell cycle phases were gated and the percentage of cells in each phase quantified. Statistical analysis of the percentage of nuclei in G2/M cell cycle phase is presented (Mean ± SEM; n = 6; Student’s t-test n.s. non-significant, ** p < 0.01, *** p < 0.001).