Depletion of ATR selectively sensitizes ATM-deficient human mammary epithelial cells to ionizing radiation and DNA-damaging agents

Abstract

DNA damage response (DDR) to double strand breaks is coordinated by 3 phosphatidylinositol 3-kinase-related kinase (PIKK) family members: the ataxia-telangiectasia mutated kinase (ATM), the ATM and Rad3-related (ATR) kinase and the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs). ATM and ATR are central players in activating cell cycle checkpoints and function as an active barrier against genome instability and tumorigenesis in replicating cells. Loss of ATM function is frequently reported in various types of tumors, thus placing more reliance on ATR for checkpoint arrest and cell survival following DNA damage. To investigate the role of ATR in the G2/M checkpoint regulation in response to ionizing radiation (IR), particularly when ATM is deficient, cell lines deficient of ATM, ATR, or both were generated using a doxycycline-inducible lentiviral system. Our data suggests that while depletion of ATR or ATM alone in wild-type human mammary epithelial cell cultures (HME-CCs) has little effect on radiosensitivity or IR-induced G2/M checkpoint arrest, depletion of ATR in ATM-deficient cells causes synthetic lethality following IR, which correlates with severe G2/M checkpoint attenuation. ATR depletion also inhibits IR-induced autophagy, regardless of the ATM status, and enhances IR-induced apoptosis particularly when ATM is deficient. Collectively, our results clearly demonstrate that ATR function is required for the IR-induced G2/M checkpoint activation and subsequent survival of cells with ATM deficiency. The synthetic lethal interaction between ATM and ATR in response to IR supports ATR as a therapeutic target for improved anti-cancer regimens, especially in tumors with a dysfunctional ATM pathway.

Keywords: ATM and Rad3-related (ATR); ATM, the ataxia-telangiectasia mutated kinase; ATP, adenosine triphosphate; ATR, the ATM and Rad3-related; CHK1, the checkpoint kinase 1; CHK2, the checkpoint kinase 2; DAPI, 4′,6-diamidino-2-phenylindole; DDR, DNA damage response; DNA damage response; DNA-PKcs, the catalytic subunit of the DNA-dependent protein kinase; DSBs, double strand breaks; G2/M checkpoint; HME-CCs, human mammary epithelial cell cultures; IR, ionizing radiation; RMI, relative mitotic index; SSBs, single strand breaks; WT, Wild-type; ionizing radiation; synthetic lethality.

Figure 1.

Validation of ATR depletion in HME-CCs and characterization of cell proliferation. (A) Quantification of ATR expression in HME-CC cell lines with representative protein gel blots. Whole cell extracts were used and results are presented in mean Relative ATR Protein Levels ±SE (n = 3). *indicates significant difference compared to LacZ-NS (P < 0.05). (B) Cell proliferation and doubling times of wild-type HME-CC (LacZ-NS) and HME-CCs deficient of ATR (LacZ-ATR), ATM (ATM1-NS) or both (ATM1-ATR). The doubling time for each cell line is displayed as mean Doubling time±SE (n = 3). *compared to LacZ-NS (P < 0.05); **compared to ATM1-NS (P < 0.05).

Figure 2.

Simultaneous depletion of ATR and ATM in HME-CC cells (ATM1-ATR) results in (A) Synthetic lethality to γ-radiation (Viability was evaluated using CellTiter-Blue viability assay kit at 48 hr post treatment) and (B) Abrogation of IR-induced G₂/M checkpoint (2 hr post treatment). Cell viability was estimated by comparing the fluorescent signal in irradiated samples to that in the time-matched control of the same cell line. Relative mitotic index was calculated by dividing the percentage of mitotic cells in the treated sample by the percentage of mitotic cells in its respective untreated sample. The data for LacZ-NS following 3 Gy of IR treatment is not showing in the graph because the measurement is 0, which means complete G₂/M checkpoint arrest. Results are presented in mean ± SE (n = 3). *means significantly different from the wild-type (LacZ-NS) (P < 0.05); **means change in cell viability due to ATM/ATR double depletion is significantly greater than the sum of that due to ATM or ATR depletion alone (P < 0.05).

Figure 3.

Regulation of checkpoint signaling at 2 hr following 2Gy of IR in wild-type HME-CCs and HME-CCs deficient in ATM, ATR or both. Whole cell extract was used for protein gel blot. "*" corresponds to the actual p53 band.

Figure 4.

Regulation of checkpoint signaling at 2 hr following 2 Gy of IR in wild-type HME-CCs and HME-CCs deficient in ATM, ATR or both. Nuclear extract was used for western blot. "*" corresponds to the actual p53 band.

Figure 5.

IR-induced autophagy and apoptosis in HME-CCs. (A) IR-induced autophagy was inhibited in ATR-deficient HME-CCs 24 hr following IR treatment. (B) IR-induced apoptosis was enhanced in ATM and ATR double deficient cells (ATM1-ATR) 48 hr post treatment. Autophagy was evaluated at 24 hr post IR using Cyto-ID autophagy detection kit. Apoptosis Caspase3/7 activity was measured at 48 hr psot treatment using ApoTox-Glo Triplex Assay (Promega) and normalized to the number of viable cells. All of the presented values are relative to that of the untreated LacZ-NS and show in mean ± SE (n = 3). *means significantly different from wild-type (LacZ-NS) (P < 0.05); **means change in apoptosis due to ATM/ATR double depletion is significantly greater than the sum of that due to ATM or ATR depletion alone (P < 0.05).

Figure 6.

Depletion of ATR in ATM-deficient HME-CC cells sensitizes cells to DNA-damaging drugs (A) Bleomycin and (B) Etoposide. Cells were induced for ATR shRNA expression by doxycycline for 2 d prior to drug treatment and cell viability was evaluated by CellTiter-Blue assay at 48 h post drug treatment. All presented values were calculated by dividing the average fluorescent signal in treated cells with that in its own time-matched untreated counterpart. *means cell viability is significantly different from wild-type (LacZ-NS) (P < 0.05); **means change in cell viability due to ATM/ATR double depletion is significantly greater than the sum of that due to ATM or ATR depletion alone (P < 0.05).