Figure 5: ATRi effect on cell cycle progression, chromosomal instability, DNA damage and apoptosis.
From: ATR inhibitors as a synthetic lethal therapy for tumours deficient in ARID1A
(a). Histogram of the cellular DAPI-stained DNA content, determined by FACS, in HCT116 ARID1A+/+ and ARID1A−/− cells at the indicated time points following release from synchronization in G1/early S phase by double thymidine block. Cells were released from thymidine block into media containing DMSO (blue) or VX-970 (0.5 μM, red). (b). Bar chart illustrating increased level of anaphase bridges in HCT116 ARID1A−/− and ARID1A+/+ cells exposed to VX-970 (0.5 μM, 8 h). Cells were stained with DAPI. A minimum of 50 anaphases were scored in three biological replicate experiments. *P=0.023, Student’s t-test. (c). Bar chart illustrating frequency of anaphase bridges in TOV21G cells exposed to VX-970 (0.5 μM) or DMSO for 8 h before fixation. Experiment performed as for (b). P=0.006 Student’s t-test. (d) Images of mitotic spreads from HCT116 ARID1A+/+ and ARID1A−/− cells following exposure to either DMSO or VX-970 (1 μM). Scale bar, 20 μm. (e) Bar chart illustrating extent of chromosomal abnormalities in HCT116 ARID1A+/+ and ARID1A−/− cells exposed to VX-970 (1 μM). *P<0.05, Student’s t-test. (f) Western blot illustrating γH2AX in HCT116 ARID1A+/+ and ARID1A−/− cells exposed to VX-970 (0.5 μM) for the indicated time before cell lyses. (g) Bar chart illustrating apoptotic fraction in cells exposed to increasing concentrations of VX-970 for 24 h. Experiment as per Fig. 3i. (h) Western blot illustrating PARP cleavage in HCT116 ARID1A+/+ and ARID1A−/− cells exposed to VX-970. Experiment performed as per Fig. 3h. (i) A model for the proposed mechanism driving the sensitivity of ARID1A-deficient cells to ATRi. Loss of ARID1A function results in: (i) a defect in the ability of cells to recruit TOP2A to chromatin; and (ii) cell cycle progression defects in both S and G2/M phases of the cell cycle. These factors combined or in isolation might render tumour cells sensitive to small molecule ATRi as these agents impair the ability of cells to mount adequate DDRs, while at the same time accelerating mitotic entry. FACS, fluorescence-activated cell sorting.
