l evidence of DNA damage at 6 h. The response of DU145 cells to single agent and combination therapy with radiation and AZD1152 was similar to the response exhibited INO-1001 PARP inhibitor by PC3 cells: 69.3% of DU145 cells treated with radiation alone demonstrated γ H2AX foci 30 min after irradiation compared to 100% of DU145 cells treated with a combination of radiation and ACS1152 . These levels of DNA damage were sustained at 6 h after the completion of radiation treatment . Again, unirradiated cells, either with or without AZD1152, demonstrated minimal evidence of DNA damage. Inhibition of Aurora Kinase B with AZD1152 Results in Radiosensitization of PC3 and DU145 Prostate Cancer Cells To investigate whether AZD1152 radiosensitizes PC3 and DU145 cells, clonogenic assays were performed on cells treated with the optimal treatment for AZD1152 and varying doses of radiation .
PC3 cells receiving AZD1152 in combination with radiation had increased sensitivity to the lethal effect of radiation at all doses tested, with a drug enhancement ratio of 1.53 . DU145 cells demonstrated significant radiosensitivity PLX-4720 918505-84-7 with increasing dose, with a DER of 1.71 at a surviving fraction of 0.4 . The DER was calculated at a surviving fraction of 0.4 Niermann et al. Page 5 Radiat Res. Author manuscript, available in PMC 2012 April 1. NIH PA Author Manuscript NIH PA Author Manuscript NIH PA Author Manuscript because the fraction of control treated DU145 cells never reached the level of 0.1 after 1 to 6 Gy radiation. DISCUSSION One of the goals of this study was to elucidate the mechanism by which AZD1152, an AURKB inhibitor, affects cell cycling in human derived PC3 and DU145 prostate cancer cells.
AURKB is an interesting therapeutic target because of its ability to facilitate and control cell cycle progression. AURKB phosphorylates histone H3, inducing chromosome condensation and facilitating cytokinesis . Several studies have shown that AZD1152 is capable of inhibiting phosphorylation of histone H3 . While our findings regarding the AZD1152 mediated effects on histone H3 were consistent with the published results for other cell lines, the data presented here did reveal some differences in the response of the PC3 and DU145 cells to AZD1152. We found that p H3 levels are both dose and timedependent with a trend toward decreased levels of p H3 by 60 nM for 48 h in both cell lines.
Consistent with previous reports detailing the effects of AURKB inhibition, including cell cycle arrest , our results showed that AZD1152 maximizes the proportion of cells in G2/M phase and polyploidy in PC3 and DU145 cells. The maximum in G2/M phase and polyploid cells occurred at 48 h, also in agreement with previously published data . Previous studies have shown that the expression of p53 appears to predict the effects of AZD1152. Among HCT116 colon cancer cells, those that have a double p53 knockout demonstrate increased polyploidy compared to wild type cells . Although we found that AZD1152 resulted in increased levels of both polyploid and G2/Mphase cells in PC3 cells, which are p53�?�? the G2/M phase showed overall predominance. For the DU145 cells, which are characteristically p53+/+, our results showed a prevalence of polyploid cells.
This is not entirely unanticipated, however, because DU145 cells express heterozygous 233Leu and 274Phe p53 mutations, neither of which behaves as a dominant negative mutation. Some studies have suggested that mutations expressed simultaneously are able to completely inactivate p53 growth suppressive function . Thus it is plausible that p53 dysfunctionality is responsible for the accumulation of polyploid cells in the presence of an AURKB inhibitor. Previous reports of the effects of AZD1152 on cells of acute myeloid leukemia cell lines showed increased fractions of both G2/M phase and polyploid cells and a simultaneous increase in S phase cells . In contrast, our data for PC3 and DU145 prostate cancer cells showed decreases in S phase cells in resp