ATR inhibition selectively sensitizes G1 checkpoint-deficient cells to lethal premature chromatin condensation - PubMed (original) (raw)
ATR inhibition selectively sensitizes G1 checkpoint-deficient cells to lethal premature chromatin condensation
P Nghiem et al. Proc Natl Acad Sci U S A. 2001.
Abstract
Premature chromatin condensation (PCC) is a hallmark of mammalian cells that begin mitosis before completing DNA replication. This lethal event is prevented by a highly conserved checkpoint involving an unknown, caffeine-sensitive mediator. Here, we have examined the possible involvement of the caffeine-sensitive ATM and ATR protein kinases in this checkpoint. We show that caffeine's ability to inhibit ATR (but not ATM) causes PCC, that ATR (but not ATM) prevents PCC, and that ATR prevents PCC via Chk-1 regulation. Moreover, mimicking cancer cell phenotypes by disrupting normal G(1) checkpoints sensitizes cells to PCC by ATR inhibition plus low-dose DNA damage. Notably, loss of p53 function potently sensitizes cells to PCC caused by ATR inhibition by a small molecule. We present a molecular model for how ATR prevents PCC and suggest that ATR represents an attractive therapeutic target for selectively killing cancer cells by premature chromatin condensation.
Figures
Figure 1
Low-dose DNA damage or S phase-arresting agents induce PCC in cells in which ATR or Chk-1 has been inhibited. (A) Mitotic spreads showing three characteristic DNA-staining patterns were performed as described (see Materials and Methods). (B) Doxycycline-induced expression of a full-length kinase-inactive ATR point mutant (Asp-2475 → Ala, ATR-kd) caused asynchronous U2OS cells to undergo PCC by 24 h after treatment with UV (200 J/m2 of UV-B), ionizing radiation (IR, 10 Gy), hydroxyurea (HU, 1 mM), cisplatinum (cisplat., 600 nM), or aphidicolin (aphid., 2.5 μg/ml) (see Materials and Methods). (C) Cells that were not damaged or did not have ATR-kd induced did not undergo PCC. (D) Adenoviral expression of wild-type Chk-1 administered 24 h before UV irradiation completely rescued cells from PCC promoted by ATR-kd. Expression of a kinase-dead (kd) Chk-1 mutant under the same conditions augmented PCC and induced premature mitosis even if ATR-kd was not induced. In the indicated cases, doxycycline (1 mcg/ml) was added 2 days before cells were treated as specified. Nocodazole (100 ng/ml) was added at the time of damage (to block cells from exiting mitosis), and 24 h later (or at the times indicated in C) cells were harvested. The values represent the mean of two to five independent experiments or a representative experiment from within a dose-response curve. For each condition, over 2,000 cells were assessed, usually with >100 mitoses counted per condition. Agreement between experiments and three observers was excellent based on the criteria described (see Materials and Methods) to distinguish PCC and normal mitoses.
Figure 2
Involvement of ATR, but not ATM, in PCC. (A) Induction of ATR-kd expression by doxycycline promoted PCC in U2OS cells treated with caffeine (1 mM) and/or hydroxyurea (HU, 1 mM). (B) ATR-wt expression diminished PCC caused by the combination of caffeine and hydroxyurea. (C) 293T cells were transiently transfected (see Materials and Methods), and expression of CMV promoter-driven constructs of ATR or ATM were compared by Western blot to endogenous levels present in control (green fluorescent protein-transfected) cells. (D) Transient transfection in 293T cells of ATR-kd synergized with low-level UV (200 J/m2) to cause PCC, but ATM expression had no effect. (E) Neither ATM-wt nor ATM-kd transfection had any effect on PCC induced by caffeine and hydroxyurea, whereas transfection of the ATR-wt and ATR-kd constructs into 293T cells had effects similar to those shown in A and B in which their expression had been induced in the U2OS cell lines by doxycycline. (F) Analogously, neither ATM-wt nor ATM-kd transfection had any effect on PCC induced by ATR-kd and UV, whereas additional ATR-kd augmented and ATR-wt diminished PCC. Cells were transfected with 2 μg of DNA (1 μg of ATR-kd and 1 μg of the indicated construct) and were treated with UV (200 J/m2) and nocodazole (10 ng/ml) 48 h later, then harvested for mitotic spreads 24 h later (see Materials and Methods).
Figure 3
ATR is required after damage in mid-S phase. (A) U2OS cells were synchronized by using double thymidine block (see Materials and Methods) and harvested for propidium-iodide flow cytometry at the indicated times after release into normal medium. At the time of release (t = 0 h), 98% of cells were at the G1/S border, with progression into G2/M by 9 h later, as indicated. Arrowheads indicate 2C and 4C DNA content. (B) The effect of ATR-kd expression on the incidence of PCC in cells irradiated (UVB 200 J/m2) at different cell cycle phases. Cells irradiated in mid-S phase were most susceptible to undergoing PCC. Cells were harvested 24 h after UV. (C) When ATR-kd was induced, cells irradiated (UVB 200 J/m2) in mid-S phase underwent high levels of cell death, as evidenced by the increase in the fraction of cells with a sub-2C DNA content (shaded cell population, with percent of sub2C DNA content cells indicated).
Figure 4
Loss of G1 arrest promotes PCC induced by ATR inhibition and treatments that prolong DNA synthesis. (A) Promotion of G1 checkpoint function by overexpressing p21 or p27 completely rescued PCC induced by ATR inhibition and UV, compared with control adenoviral expression of green fluorescent protein (GFP). Interference with G1 checkpoint function via adenoviral expression of cyclin D1, cyclin E, CDK2, MDM2, or E6 markedly increased susceptibility to PCC following UV exposure and ATR-kd induction. (B) Similarly, a small molecule inhibitor of ATR, caffeine (here used at 300 μM), sensitized cells with G1 checkpoint deficiencies to PCC induction by UV. Values shown are means of three independent experiments, and standard errors of the mean were <5% in all cases. (C) A model of the role of ATR in preventing PCC. UV, IR, cisplatinum, hydroxyurea, and aphidicolin have in common the ability to increase the number of stalled replication forks and prolong DNA synthesis. The signal that DNA synthesis is not complete (and that chromatin condensation should not start) is sent by ATR after it is recruited to replication forks that have been primed by addition of an RNA primer via polymerase-α (13, 19). ATR-mediated phosphorylation of Chk-1 causes delay of mitotic entry and prevention of PCC. G1 checkpoints are lost in cancer cells by overexpression of cyclin D1, CDK2, cyclin E, MDM2, or E6 because of mutations in the p53 or Rb/p16 pathways. Because G1 checkpoint loss causes cells to enter S phase with more unrepaired damage, such cells would have a greater requirement for functional ATR to delay mitotic entry until all damage has been repaired and the DNA has been replicated. The chemical structure of caffeine, a small molecule inhibitor of ATR, is depicted. An animated interactive model for ATR function is available as Movie 1, which is published as supplemental data on the PNAS web site,
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