Figure 1.
Decreased clonogenic survival of PCa cells exposed to radiation and rucaparib.
A, Radiation and rucaparib dose response in PC3, C4-2, DU145, VCaP and LNCaP cells was established by clonogenic cell survival assays. Left panels indicate the response to radiation, those on the right to rucaparib. B, Synergistic effect of the combination of radiation and rucaparib on clonogenic survival in LNCaP, PC3, C4-2, and VCaP cells, where CI <1 represents synergy and CI >1 an antagonistic interaction between the two treatments. Error bars represent SD of mean (n = 3).
Figure 2.
Cellular senescence is activated in PTEN-deficient cells.
Percentage of β- galactosidase positive cells was determined following radiation (A) and rucaparib (B),treatment in PTEN-deficient LNCaP, C4-2, and PC3 cells. The percentage of β-galactosidase-positive cells was determined by counting ≥five different fields (∼70 cells/sample). C, Synergy analyses for senescence in LNCaP, PC3, and C4-2 cells for the combination treatment. See Figs. S2 and 3 for β-galactosidase staining and additional synergy analyses. D, Percentage of β-galactosidase positive C4-2 cells was determined after 12 days of treatment with radiation ± rucaparib. E, parental and TMPRSS2-ERG fusion gene-expressing PC3 cells were quantified for SA-β-galactosidase staining after 6 days following 4 Gy radiation, 2.5 µM rucaparib, and the DNA-PKcs inhibitor NU7441 (500 nM), alone or in combination. Error bars represent SD of mean (n = 3).
Figure 3.
Combination of rucaparib with radiation increases χH2AX and 53BP1 foci leading to persistent DNA damage.
χH2AX (A) and 53BP1 (B) foci were determined by immunofluoresecence microscopy at 24 h following combined treatment with radiation and rucaparib in PC3 and LNCaP cells (left panel), with time-dependent kinetics shown (right panel). C, Rad51 foci were visualized and quantified in PC3 cells similarly after 24 h of treatment. Error bars represent SD of mean (n = 3).
Figure 4.
LDR is effective in sensitizing PCa Cells.
A, Clonogenic survival assays were performed for PC3 (left panel) and C4-2 cells (right panel) at different dose-rates ±1.25 µM rucaparib. B, C4-2 cells were exposed to different dose rates of radiation from an Ir-192 source ±2.5 µM rucaparib. A total radiation dose of 5 Gy (left panel) and 10 Gy (right panel) was delivered; therefore the time of exposure differed for the various dose-rates used. Error bars represent SD of mean (n = 3).
Figure 5.
LDR induces enhanced DNA damage in the presence of rucaparib.
A, Confocal immunostaining for χH2AX foci, enumarated in PC3 and C4-2 cells following radiation at the indicated dose rates. B, Quantitation of dose-dependent formation of γH2AX foci. Error bars represent SD of mean (n = 3).
Figure 6.
LDR, when combined with rucaparib, augments DNA damage-induced 53BP1 IRIF.
A, Confocal immunostaining of 53BP1 foci in PC3 and C4-2 cells following radiation at the indicated dose rates. B, Graphical representation of dose-dependent 53BP1 IRIF generation. Error bars represent SD of mean (n = 3).
Figure 7.
The TMPRSS2-ERG fusion gene provides enhanced sensitivity to PARP inhibition, comparable to that of DNA-PKcs inhibition in parental cells.
A, Clonogenic survival assay in PC3 cells following 4 Gy radiation ±2.5 µM rucaparib and the DNA-PKCs inhibitor NU7441 (500 nM; left panel). B, Clonogenic survival assays in PC3 cells and derivatives expressing TMPRSS2-ERG fusion III at different dose-rates ±2.5 µM rucaparib. C, Immunostaining for χH2AX and 53BP1 in PC3 cells expressing the fusion gene following radiation ± rucaparib treatment for 24 h. Error bars represent SD of mean (n = 3).