Article Figures & Data
Figures
- Figure 1.
Uncoupling of the G2 DNA damage checkpoint by UCN-01. A, UCN-01 can induce precocious cell cycle progression in damaged cells. HeLa cells were irradiated and incubated for 16 hours. The cells were then treated with caffeine, UCN-01, and RO3306 as indicated. After 12 hours, the cells were fixed and analyzed by flow cytometry. B, cells were treated as shown in A. Cell-free extracts were prepared and the expression of the indicated proteins was detected by immunoblotting. Equal loading of lysates was confirmed by immunoblotting for actin. C, unscheduled mitotic entry after CHK1 depletion. HeLa cells were transfected with control or CHK1 siRNA. After 6 hours, the cells were irradiated, grown for 28 hours, and analyzed by flow cytometry. D, cells were treated as shown in C. Lysates were prepared analyzed by immunoblotting.
- Figure 2.
Various cell fates are induced by UCN-01. A, mitotic catastrophe and survival after UCN-01 challenge. HeLa cells expressing histone H2B-GFP and mRFP1-APC/C biosensor were untreated (n = 62), irradiated and grown for 16 hours (n = 66), UCN-01–treated (n = 48), or irradiated (16 hours) followed by UCN-01 treatment (n = 62). The cells were then subjected to time-lapse microscopy to track individual cells for 12 hours. Gray, interphase; black, mitosis (from DNA condensation to anaphase) or mitotic catastrophe; truncated bars, cell death. The time of cell death after mitosis is defined by the death of one of the daughters. The percentages of cells that completed mitosis or underwent mitotic catastrophe (MC) are shown [a portion of cells that were still trapped in an extremely long mitosis at the end of the 12-hour imaging was also categorized as MC—longer imaging revealed that they subsequently also underwent apoptosis (data not shown)]. B, Representative still images from time-lapse microscopy of IR- and UCN-01–treated cells. Cells were treated and imaged as shown in A. An example of a cell that could complete mitosis is shown. The full video can be found in Supplementary data (Supplementary Video S2). C, mitosis is extended in UCN-01–treated cells. Cells were treated and imaged as shown in A. The duration of mitosis in control (n = 50), cells treated with IR (n = 7, only a very small number of cells could enter mitosis), or IR followed by UCN-01 (n = 35) are shown (mean ± 90% CI). D, anaphase is delayed in UCN-01–induced mitosis. Control (n = 50) or IR- and UCN-01–treated cells (n = 35) were analyzed by time-lapse microscopy as shown in A. The time of prometaphase–metaphase and metaphase–anaphase was quantified (mean ± 90% CI).
- Figure 3.
Both the onset and extent of mitotic catastrophe are dependent on the level of DNA damage. A, high dose of IR prohibits UCN-01–induced G1 entry. HeLa cells were irradiated (15 or 40 Gy), grown for 16 hours, and treated with UCN-01 as indicated. At 12 and 24 hours after UCN-01 addition, DNA contents were analyzed by flow cytometry. B, dose-dependent activation of CHK1. HeLa cells were irradiated (15 or 40 Gy), grown for 16 hours, and treated with UCN-01 as indicated. Nocodazole was included to trap checkpoint-bypassed cells in mitosis. At the indicated time points after UCN-01 addition, the cells were harvested and analyzed by immunoblotting. C, Delayed mitotic entry and massive mitotic catastrophe after IR (40 Gy) and UCN-01 treatment. Cells were treated with IR (40 Gy) followed by UCN-01 as shown in A. Time-lapse microscopy was used to track individual cells for 12 hours. Key and definition are the same as given in Fig. 2A (n = 50). D, massive mitotic catastrophe in cells treated with IR (40 Gy) and UCN-01. Cells were treated with IR (40 Gy), UCN-01, and analyzed as shown in A (n = 50). Cells that completed mitosis without dying (mitosis), die during mitosis (mitotic catastrophe), or did not enter mitosis throughout the imaging period (interphase) were quantified (mean ± SD of 3 experiments).
- Figure 4.
Expression of p31comet attenuates UCN-01–mediated mitotic catastrophe. A, mitosis induced by checkpoint bypass is abolished by p31comet. FLAG-p31comet/HeLa cells were grown in the absence or presence of doxycycline for 30 hours to turn on or off the expression of FLAG-p31comet, respectively. They were treated with IR (16 hours) followed by UCN-01 (12 hours) as indicated. Lysates were prepared and analyzed by immunoblotting. B, p31comet suppresses UCN-01–mediated cell death. Cells were treated as shown in A. Cell viability was determined with trypan blue exclusion analysis (mean ± SD of 3 experiments). C, expression of MAD2 shRNA or p31comet suppresses mitotic catastrophe. HeLa cells expressing histone H2B-GFP were transfected with control vector (n = 50), plasmids expressing FLAG-p31comet (n = 52), or MAD2 shRNA (n = 74) together with a YFP marker. The cells were irradiated, grown for 16 hours, before treated with UCN-01. Time-lapse microscopy was used to track individual cells for 12 hours. Key and definition are the same as given in Fig. 2A.
- Figure 5.
Mitotic catastrophe can be promoted by delaying mitotic exit. A, downregulation of CDC20 and p31comet. HeLa cells were transfected with control or siRNAs against CDC20 or p31comet. Lysates were prepared and analyzed by immunoblotting (asterisks, phosphorylated forms of BUBR1 and CDC27). B, depletion of CDC20 inhibits mitotic exit. HeLa cells were transfected with either control or CDC20 siRNA. After 6 hours, the cells were irradiated, grown for 16 hours, and treated with UCN-01. After 12 hours, DNA contents were analyzed with flow cytometry. C, downregulation of CDC20 or p31comet enhances mitotic catastrophe. HeLa cells expressing histone H2B-GFP and mRFP1-APC/C biosensor were transfected with control siRNA (n = 50), CDC20 siRNA (n = 68), or p31comet siRNA (n = 74). The cells were irradiated, grown for 16 hours, and treated with UCN-01. Individual cells were tracked with time-lapse microscopy for 12 hours. Key and definition are the same as given in Fig. 2A.
- Figure 6.
Different cell lines display different susceptibility to mitotic catastrophe. A, the unperturbed mitotic length of different cell lines. The indicated cell lines, which expressed histone H2B-GFP, were subjected to time-lapse microscopy. Mitosis length in individual cells was analyzed (n = 50, mean ± 90% CI). B, mitotic length after checkpoint abrogation. The indicated histone H2B-GFP-expressing cell lines were irradiated (15 Gy), grown for 16 or 24 hours (for U2OS and NIH3T3), and treated with UCN-01. Individual cells were tracked with time-lapse microscopy (n = 50) to analyze the duration of mitosis (mean ± 90% CI). C, different cell lines exhibit different susceptibility to mitotic catastrophe. The indicated histone H2B-GFP–expressing cell lines were transfected with control or CDC20 siRNA. The cells were irradiated with either 15 or 40 Gy, grown for 16 or 24 hours (for U2OS and NIH3T3), and treated with UCN-01. Mitotic catastrophe was analyzed by time-lapse microscopy (12 hours, n = 50).
Additional Files
Supplementary Data, On, et al.
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Volume 10, Issue 5
