To verify whether ATM-depletion has a functional impact on MCF-7 cells, we assessed the sensitivity learn more of ATM-depleted and control cells to IR and doxorubicin treatment, that are known to induce different outcomes in ATM proficient and defective

cells. In particular, radiosensitivity is a defining feature of ATM-defective cells [26] whereas, in a wild-type p53 context, doxorubicin-resistance was shown to characterize ATM-deficient cells in vitro [27] and in breast cancer patients [28]. As shown in Figure 1B and 1C, MCF7-ATMi cells were more sensitive to IR and more resistant to doxorubicin than MCF7-ctr cells. The contribution of ATM in the latter result was confirmed in MCF-7 parental cells by KU 55933-induced ATM inactivation (Figure 1D). These results Quisinostat concentration were further confirmed by evaluating the cell cycle profiles (Figure 1E). After 24 hrs from irradiation, both MCF7-ctr and MCF7-ATMi cells show the expected enrichment into the G2/M phase. After 48 hrs from irradiation, MCF7-ctr cells repair the damage and re-enter into the cell cycle; in contrast,

MCF7-ATMi cells, which are known to have defects in sensing and repairing DNA double strand breaks [26], show a delay in re-entering into the cell cycle. In contrast, as expected from the data reported by Jiang and co-workers [27], the ATMi cells were more resistant to doxorubicin and a lower proportion of cells underwent cell death. Figure 1 MCF-7 transduction with shATM-carrying vectors elicits a phenotype compatible with ATM defective cells. (A) MCF-7 cells were transfected with shATM-carrying vector (MCF7-ATMi) and its siR5 negative control (MCF7-ctr). ATM

protein levels in MCF-7-ATMi and MCF-7-ctr cells were analyzed by Western blot. α-tubulin was used as an internal control. B-D Cell viability of MCF7-ATMi and MCF7-ctr cells upon treatment with IR (B) and doxorubicin (C). (D) MCF7-ctr cells were pre-treated with ATM inhibitor KU 55933 or its solvent before AG-881 supplier addition of doxorubicin as in (C). Data are represented as mean ± standard deviation (SD). (E) Flow cytometry analysis of cell-cycle distribution of MCF7-ATMi and MCF7-ctr cells upon treatment with IR and doxorubicin at indicated times. Asterisks indicate statistical significant difference (*P < 0.1; **P < 0.05). Altogether, IKBKE these results show that MCF-7 transduction with shATM-carrying vectors interferes with ATM expression and elicits some aspects of a phenotype compatible with ATM-deficient cells. ATM-depletion sensitizes MCF-7 cells to olaparib To evaluate whether ATM-depletion modifies MCF-7 response to PARP inhibitors, we first used olaparib (AZD2281, Ku-0059436), an orally bioavailable compound whose effectiveness in BRCA1/2 mutated breast and ovarian cancers was studied in phase II clinical trials and, for ovarian cancers is under further evaluation in phase III clinical studies [12].