. These data indicate that the inhibition of AURKB by AZD1152 is both dose and timedependent. AZD1152 Induces G2/M Cell Cycle Arrest and Polyploidy Because AURKB normally facilitates progression beyond the G2/M cell cycle transition point, SRT1720 1001645-58-4 we used flow cytometry to measure the effects of AURKB inhibition on the distribution of cell cycle phases in PC3 and DU145 cells exposed to AZD1152 for 48 h. Figure 2A shows the resulting percentages of each of the cell cycle phases in PC3 cells. At low concentrations of AZD1152, there was a relatively high level of G0/G1 phase cells and a relatively low level of G2/M phase cells , indicative of fully functional AURKB. However, as the concentrations were increased from 3 nM to 30 nM, G2/M phase cells reached levels above 50% and G0/G1 phase cells represented less than 5% of cells.
Additionally, the Barasertib 722544-51-6 fraction of polyploid cells increased at concentrations of 30 nM. At AZD1152 concentrations above 30 nM, to the maximum tested concentration of 1000 nM, these cell cycle effects were sustained. Cells in the S phase and sub G0 phase each represented less than 10% of the total population at all dose levels. With AZD induced AURKB inhibition, DU145 cells similarly demonstrated a dose dependent decreased percentage of G0/G1 phase cells and increased percentage of polyploid cells , the transition in cell cycle composition over a concentration range from 10 nM to 100 nM AZD1152. The percentage of G2/M phase cells increased to a maximal level of 35% at a concentration of 60 nM, with higher concentrations resulting in a somewhat lower G2/M fraction, but still higher than baseline, at concentrations of 100 nM or greater.
These cell Niermann et al. Page 4 Radiat Res. Author manuscript, available in PMC 2012 April 1. NIH PA Author Manuscript NIH PA Author Manuscript NIH PA Author Manuscript cycle analyses indicated that AZD1152 induced AURKB inhibition is maximized at concentrations of 60 nM for both PC3 and DU145 prostate cancer cell populations exposed to AZD1152 for 48 h. Next, the cell cycle effects of AZD1152 treatment were tested in both PC3 and DU145 cells using a fixed concentration of 60 nM AZD1152 but varying the duration of treatment. As shown in Fig. 2B, top panel, PC3 cells demonstrated a time dependent decrease in the fraction of G0/G1 cells , an increased fraction of G2/M cells , and an increased fraction of polyploid cells .
Maximal treatment effects were seen with a treatment time of 24 to 48 h. S phase and sub G0 phase cells each comprised less than 15% of the total fraction at all treatment times. The cell cycle effects of durations of varying treatment AZD1152 on DU145 cells is shown in Fig. 2B, bottom panel. As observed in PC3 cells, increasing treatment time resulted in a gradually decreased fraction of G0/G1 phase cells . Like PC3 cells, DU145 cells showed peak levels of G2/M phase cells at 24 h and a maximal fraction of polyploid cells at 48 to 72 h. Optimal inhibition of AURKB was seen with 60 nM for 48 h for both PC3 and DU145 cells. Neoadjuvant AZD1152 Followed by Radiation Results in Increased and Sustained DNA Damage Employing the optimal regimen of 60 nM AZD1152 for 48 h, PC3 and DU145 cells were exposed to radiation and the resulting DNA damage was quantified.
Figure 3 shows that PC3 cells not receiving radiation AZD1152 alone demonstrated minimal evidence of DNA double strand breaks, as indicated by low levels of γ H2AX foci . However, 68% of the PC3 cell population that received 5 Gy radiation alone exhibited evidence of DNA damage. Those PC3 cells that received the combination of AZD1152 and 5 Gy radiation had DNA damage in the entire population of cells, demonstrating a level of DNA damage that was significantly higher than cells exposed to radiation without AZD1152 . Furthermore, the significantly increased amounts of γ H2AX foci in PC3 cells were sustained 6 h after radiation treatment . Again, unirradiated cells, either with or without AZD1152, demonstrated minima