by eye with a cutoff of 50 viable cells. The surviving fraction was calculated as / × plating efficiency , where PE was defined as /. Immunofluorescence for γ H2AX PC3 and DU145 cells were grown on sterile cover slips in six well plates with 3 ml medium. After 24 h, the cells were incubated with DMSO or 60 nM AZD1152. After 48 h of incubation, cells were irradiated with either 0 or 5 Gy. BMS-754807 IGF-1R inhibitor Either 30 min or 6 h after irradiation, the cover slips were washed with cold PBS, and cells were fixed with 4% formaldehyde for 10 min at room temperature. Cells were then washed twice in PBS and placed on cover slips in ice cold wells. Then 2 ml of ice cold Triton X 100 solution was added. After 15 min, the cover slips were washed three times in PBS. The cover slips were then blocked in 1% BSA/ PBS overnight at 4°C.
The next day, mouse anti human γ H2AX was added at a dilution of 1:100 in antibody buffer and incubated at 37°C for 30 min. Cells were washed three times in PBS and incubated with a rhodamine green labeled goat antimouse IgG secondary antibody AZD1152-HQPA 722544-51-6 at a dilution of 1:100 in antibody buffer at room 37°C for 30 min in the dark. The cover slips were then washed three times in PBS and placed on ice. The cells were then counterstained with 2 ml of 4, 6 diamidino 2 phenylindole for 5 min. The cover slips were washed three times in PBS and mounted using Vectashield on microscope slides. Three random regions of 50 cells each were examined under a microscope with 100× magnification. Nuclei containing �?0 foci were counted as positive for γ H2AX focus formation.
The percentages of positive cells were calculated and plotted. Statistical Analysis All assays performed in this study, including cell culture, immunoblotting, cell cycle quantification, clonogenic analysis and immunofluorescence, were performed in triplicate for each culture of cells randomized to one of the following treatment conditions: Niermann et al. Page 3 Radiat Res. Author manuscript, available in PMC 2012 April 1. NIH PA Author Manuscript NIH PA Author Manuscript NIH PA Author Manuscript control, drug alone, radiation alone and drug and radiation combined. This provided 80% power to detect a difference between two groups using a two group t test with a 0.05 significance level, assuming an average difference of 15% between any of the two groups and a standard deviation of 5%.
Standard deviations for the PC3 and DU145 cells were based on preliminary data obtained in our laboratory. The experimental observations were performed by analysts who were blinded to each of the various treatment conditions. The statistical software SPSS was used for all statistical analyses. All tests were two tailed. Values are expressed as means ± SD. RESULTS AZD1152 Results in Decreased Phosphorylation of Histone H3 in PC3 and DU145 Prostate Cancer Cells PC3 and DU145 cells were treated with varying concentrations of the Aurora kinase B inhibitor AZD1152 for a total of 48 h. Western blot analysis was used to quantify resulting AURKB. The functionality of AURKB was also assessed by quantifying p H3, the active, phosphorylated form of histone H3 required for normal chromosomal condensation. As is shown in Fig.
1A, the AURKB expression was stable at all doses for both PC3 and DU145 cells, however, AZD1152 concentrations of at least 60 nM of AZD1152 resulted in diminution of p H3, consistent with inhibition of AURKB H3 phosphorylating activity. Thus a concentration of 60 nM AZD1152 reached the threshold required to inhibit the activity of AURKB without affecting its expression. PC3 and DU145 cells were exposed to 60 nM AZD1152 for various times to determine the maximal time dependence of AURKB inhibition. Figure 1B shows that AURKB inhibition was stable at all exposure times tested. However, PC3 cells demonstrated significantly diminished p H3 levels with 48 h or more of exposure to AZD1152, and DU145 cells demonstrated significantly diminished levels of p H3 with 12 or more hours of exposure