ATM-depletion sensitizes MCF-7 cells to iniparib Next, we asked whether ATM-depletion can sensitize MCF-7 cells to iniparib (BSI-201, SAR240550), a compound originally described as an irreversible inhibitor of PARP-1 [30], but recently shown to act as a nonselective PF-562271 modifier of cysteine-containing proteins [31, 32]. MCF7-ATMi and MCF7-ctr cells were treated with iniparib or its solvent,

DMSO, and analyzed for colony formation capacity, DNA content by FACS analysis, and BrdU assay. As shown in Figure 3A, ATM-depletion reduced the ability of MCF-7 cells to produce colonies after iniparib-treatment while no effect was observed in MCF7-ctr cells. At variance with olaparib-treatment, DNA content analysis did not reveal any significant difference between MCF7-ATMi LB-100 and MCF7-ctr cells in the appearance of hypodiploid, death cells, whereas only the MCF7-ATMi population experienced an accumulation of cells in the G2/M phase Selleckchem NU7026 of the cell cycle (Figure 3B). This effect on the cell cycle was confirmed by BrdU assays (Figure 3C). Together, these results suggest that ATM-depletion can also influence MCF-7 cell response to iniparib. Figure 3 MCF7-ATMi cells are more sensitive than MCF7-ctr cells to iniparib. (A) Quantitative

analyses of colony formation. The numbers of DMSO-resistant colonies in MCF7-ATMi and MCF7-ctr cells were

set to 100, while iniparib treated cel1s were presented as mean ± SD. (B) Flow cytometry analysis of cell-cycle distribution of MCF7-ATMi and MCF7-ctr cells treated with the indicated concentrations of iniparib for 48 hrs. (C) DNA synthesis was measured by BrdU incorporation assay 48 hrs after iniparib treatment. Data are represented as mean ± SD. Asterisks indicate statistical significant difference (*P < 0.1; **P < 0.05). ATM-depletion Roflumilast sensitizes ZR-75-1 breast cancer cells to olaparib but not to iniparib To further assess the impact of ATM-depletion in breast cancer cell response to olaparib and iniparib, we selected the ZR-75-1 line, whose cells, like the MCF-7 ones, are ER positive, HER2 negative, and wild-type for BRCA1/2 and TP53 genes [25]. Stable interference of ATM in ZR-75-1 cells was obtained as described for MCF-7 cells. Polyclonal populations, ZR-ATMi and ZR-ctr, were obtained by puromycin selection and ATM-depletion confirmed by Western blot analysis (Figure 4A). Next, dose–response viability assays were performed on ZR-ATMi and ZR-ctr cells upon incubation with olaparib, iniparib, or their solvent, DMSO. As shown in Figures 4B, ZR-ctr cells were strongly resistant to olaparib whereas their ATM-depleted counterpart became considerably sensitive and showed a partial accumulation in the G2/M phase of the cell cycle (Figure 4D).