There is a crucial need for development of prognostic and predictive biomarkers in human bladder carcinogenesis in order to personalize preventive and therapeutic strategies and improve outcomes. increase in phosphorylation of H1 linker histones from normal human bladder epithelial cells to low-grade superficial to high-grade invasive bladder cancer cells. This finding was further validated by immunohistochemical staining of the normal epithelium and transitional cell cancer from human bladders. Cell cycle analysis of histone H1 phosphorylation by western blotting showed an increase of phosphorylation from G0/G1 phase to M phase again supporting this as a proliferative marker. Changes in histone H1 phosphorylation status may further clarify epigenetic changes during bladder carcinogenesis and provide diagnostic and prognostic biomarkers or targets for future therapeutic interventions. reported that the p-T146 antibody stained HeLa cells undergoing mitosis 36. Therefore the cell cycle dependence of T146 phosphorylation in bladder cancer was examined by western blotting synchronized UMUC3 cells against the p-T146 antibody (Figure 6). Cells were in G0/G1 early S late S early G2/M and late G2/M phases at 0 2 4 7 and 9 hours after release respectively. Cells were blocked in mitosis with nocodazole treatment. Western blot analysis revealed that H1 phosphorylation increased with time as more of the cellular population progressed to M phase. Maximum phosphorylation was observed with the sample blocked during M as expected for a CDK-dependent site 28. The staining in S phase is likely due to a small proportion of the cells already cycling to M. The cell cycle dependence of p-T146 can be seen by immunohistochemical staining of the formalin fixed paraffin embedded cell block of unsynchronized UMUC3 cells (Supplementary Figure 17). Figure 6 Cell cycle dependence of p-T146 in the invasive human bladder cancer cell line UMUC3. Cells were synchronized by double thymidine block and then released. For each time point two plates of cells were grown. One plate was used for PHA-665752 cell cycle analysis … H1 p-T146 is a potential biomarker of human bladder cancer progression As the high grade invasive bladder cell lines demonstrate increased phosphorylation compared to non-invasive low-grade bladder cancer and transformed normal bladder lines immunohistochemical analysis for p-T146 and Ki67 a well characterized biomarker of proliferation 37 was conducted on human non-cancerous normal appearing PHA-665752 bladder urothelium non-invasive low-grade non-invasive high-grade and invasive high-grade bladder cancer tissues (n ≥ 8 for each tissue type) (Figure 7). The percentage of positively stained nuclei was quantified in representative images for each case. ANOVA analysis demonstrated significant differences in percentage of p-T146 staining between grades (p<0.001). Pairwise comparisons indicate PHA-665752 that invasive high-grade (21.5% +/? 2.9%) and non-invasive high grade (16.8% +/? 2.3%) were significantly greater than non-cancer (1.2% +/? 0.7%) (p≤0.001) and that invasive high-grade was significantly greater than noninvasive low-grade cancer (8.4% +/? 2.9%) (p=0.002). Although there was a trend in higher nuclear p-T146 staining for non-invasive high-grade as compared to non-invasive low-grade this did not reach statistical significance (p=0.073). The difference in percentage of positive nuclear staining with grade is strongly correlated with traditional markers of proliferation including Ki67 (p<0.001). Invasive high-grade (36.8% +/?6.6%) and non-invasive high-grade (48.2% +/? 9.3%) was greater than non-cancer (7.9% +/? 5.0%) (p<0.05) FN1 and the non-invasive high-grade was greater than non-invasive low-grade (17.3% +/? 5.0%) (p=0.01). Figure 7 Tissues ranged from non-cancerous normal appearing bladder urothelium to non-invasive low-grade non-invasive high-grade and invasive high-grade human bladder cancers were used. N ≥ 8 for each tissue type. (experiments. The striking differences in H1 phosphorylation of variants H1.5 H1.2 and H1.4 between superficial (non-invasive) and invasive cell lines may be useful in bladder cancer screening and/or predictive biomarkers of recurrence invasiveness progression and response to treatment. Of course all these potential implications of these findings require future confirmatory large-scale studies. During the cell cycle of invasive UMUC3 bladder cancer PHA-665752 cells H1 phosphorylation gradually increases from G1 to M transition with the most significant increase occurring during G2/M stage and the maximum phosphorylation being observed during M 28. Initial H1.