Characterization of the p53-dependent postmitotic checkpoint following spindle disruption

Abstract

The p53 tumor suppressor gene product is known to act as part of a cell cycle checkpoint in G1 following DNA damage. In order to investigate a proposed novel role for p53 as a checkpoint at mitosis following disruption of the mitotic spindle, we have used time-lapse videomicroscopy to show that both p53+/+ and p53-/- murine fibroblasts treated with the spindle drug nocodazole undergo transient arrest at mitosis for the same length of time. Thus, p53 does not participate in checkpoint function at mitosis. However, p53 does play a critical role in nocodazole-treated cells which have exited mitotic arrest without undergoing cytokinesis and have thereby adapted. We have determined that in nocodazole-treated, adapted cells, p53 is required during a specific time window to prevent cells from reentering the cell cycle and initiating another round of DNA synthesis. Despite having 4N DNA content, adapted cells are similar to G1 cells in that they have upregulated cyclin E expression and hypophosphorylated Rb protein. The mechanism of the p53-dependent arrest in nocodazole-treated adapted cells requires the cyclin-dependent kinase inhibitor p21, as p21-/- fibroblasts fail to arrest in response to nocodazole treatment and become polyploid. Moreover, p21 is required to a similar extent to maintain cell cycle arrest after either nocodazole treatment or irradiation. Thus, the p53-dependent checkpoint following spindle disruption functionally overlaps with the p53-dependent checkpoint following DNA damage.

FIG. 1

p53 is required for cell cycle arrest following nocodazole treatment. (A) FACS profiles of wild-type and p53^−/− MEFs untreated or treated with nocodazole for 24 h. DNA content is represented on the x axis; number of cells counted is represented on the y axis. Data shown are representative of multiple experiments performed on two different wild-type and p53^−/− clones. (B) Immunofluorescent staining of wild-type and p53^−/− MEFs. MEFs were treated with nocodazole for 24 h, pulsed with BrdU in the presence of nocodazole for an additional 4 h, and fixed. Immunofluorescence was performed to detect BrdU incorporation (α-BrdU) and nuclear staining (DAPI). Immunofluorescence was performed on two different wild-type and p53^−/− clones.

FIG. 2

Time-lapse videomicroscopy of wild-type and p53^−/− MEFs at mitosis in the presence or absence of nocodazole. (A) Control p53^−/− MEF that underwent mitosis in normal media. Each photograph lists the time when the video recording occurred. The cell entered mitosis at 5:21 PM (arrowhead), cytokinesed at 5:32 PM (two arrowheads), and completed division by 5:40 PM. The cell shown is representative of over 30 cells (wild-type and p53^−/−) observed undergoing normal mitosis. (B) Two wild-type MEFs that initiated mitosis in the presence of nocodazole. Each cell entered mitosis (1:14 PM and 1:43 PM), remained arrested at mitosis for several hours (3:33 PM and 4:31 PM), and then adapted (5:33 PM and 6:02 PM). Black and white arrowheads indicate the two different cells. (C) p53^−/− MEF that entered mitosis in the presence of nocodazole (10:08 AM, arrowhead). It arrested at mitosis for several hours (11:00 AM and 12:17 PM) and eventually adapted (1:47 PM and 2:13 PM). (D) Quantitation of length of time that individual wild-type and p53^−/− MEFs spent at mitotic arrest. Length of mitotic arrest was determined morphologically by time-lapse videomicroscopy, beginning when a cell first became rounded and refractile and ending when it flattened back onto the coverslip. Wild-type MEFs spent an average of 4.4 ± 2.4 h at mitotic arrest, while p53^−/− MEFs spent an average of 4.6 ± 2.2 h at mitotic arrest. At least 60 cells were observed for each genotype.

FIG. 3

Timing of S phase entry in p53^−/− MEFs treated with nocodazole. (A) Time-lapse videomicroscopy and subsequent immunofluorescence of representative p53^−/− MEF that arrested at mitosis in the presence of nocodazole. The cell initiated mitotic arrest starting shortly after 12 AM and then adapted at 5:30 AM (top panels and lower left panel). At 11 AM, the BrdU label was added to the media; the cell was then recorded for an additional 4 h and fixed. Immunofluorescence was performed to detect BrdU incorporation (lower right panel). As indicated by arrowheads, the same cell was identified during video recording and immunofluorescence by its position on a gridded coverslip. (B) Measurement of time of S phase entry relative to time of adaptation from mitotic arrest. Ten p53^−/− MEFs on gridded coverslips were treated with nocodazole, monitored by time-lapse videomicroscopy, and pulsed with BrdU for 4 h at various times following adaptation. For each cell, the horizontal line indicates the period during which BrdU was present (measured in hours) relative to the time elapsed since the cell underwent adaptation. The + or − indicates whether the cell stained positive for BrdU incorporation by immunofluorescence.

FIG. 4

Requirement for p21 in cell cycle arrest following nocodazole treatment. (A) Immunoblot analysis for p21 protein on extracts from wild-type and p53^−/− MEFs treated with nocodazole. Unsynchronized cells were treated with nocodazole for times indicated and harvested for protein analysis. (B) FACS profiles of p21^−/− MEFs untreated or treated with nocodazole for 24 h. DNA content is represented on the x axis; number of cells counted is represented on the y axis. Data shown are representative of three experiments performed on two different p21^−/− clones. (C) Immunofluorescent staining of p21^−/− MEFs. Cells were treated with nocodazole for 24 h, pulsed with BrdU in the presence of nocodazole for an additional 4 h, and fixed. Immunofluorescence was performed to detect BrdU incorporation (α-BrdU) and nuclear staining (DAPI). Immunofluorescence was performed on two different p21^−/− clones. (D) Quantitation of number of cells in S phase in wild-type, p21^−/−, and p53^−/− MEFs following nocodazole treatment. Cells were treated with nocodazole, and immunofluorescence was performed as described for panel C. Data shown are the averages from three different experiments, with standard deviations as indicated. In each experiment, 100 cells of each genotype were examined and the number of BrdU-positive nuclei was counted to determine the percentage of S phase cells. Two different clones were tested for each genotype.

FIG. 5

Mitotic arrest and adaptation occur in synchronized, nocodazole-treated NIH 3T3 cells. (A) Untreated NIH 3T3 cells. Cells were synchronized in low serum for 48 h and then released into high serum and monitored for 32 h. At each time point, cells were photographed (left panels) and then prepared for FACS analysis for DNA content (right panels). Time points are measured in hours postrelease from serum starvation. Cells were initially in G₀ (0 h), entered S phase at 14 h, and completed cell division by 26 h. (B) Nocodazole-treated NIH 3T3 cells. Cells were synchronized in low serum for 48 h and then released into high serum plus nocodazole and monitored for 32 h. Progression of cells through cell cycle was monitored as above. Cells exited G₀ and entered S phase by 14 h, remained arrested at mitosis through 26 h, and adapted by 32 h with a 4N DNA content.

FIG. 6

Expression of cyclin E protein is upregulated in nocodazole-treated, adapted cells. Immunoblot analysis of cyclin B1 and cyclin E expression. NIH 3T3 cells were synchronized in low serum for 48 h and released into high serum plus nocodazole. Extracts were prepared at 8-h time points for the next 48 h and analyzed by immunoblotting for cyclin B1 and cyclin E expression. Cyclin E expression was highest in cells entering S phase (16 h) and in cells that had adapted (40 and 48 h), while cyclin B1 expression was highest in mitotic cells (24 h) and declined thereafter. The asterisk indicates a cross-reacting protein detected by cyclin E antibody that serves as an internal loading control.

FIG. 7

RB protein is hypophosphorylated in nocodazole-treated, adapted cells. (A) Immunoblot analysis of pRB. NIH 3T3 cells were synchronized in low serum for 48 h and released into high serum plus nocodazole. Extracts were prepared at time points for the next 48 h and analyzed by immunoblotting for pRB. Hypophosphorylated pRB (pRB) and hyperphosphorylated pRB (pRB*) are indicated by arrows. (B) DNA content of NIH 3T3 cells. Cells were treated as described in panel A. At each time point, a duplicate plate was collected and prepared for FACS analysis of DNA content. Cells were initially synchronized in G₀ (0 h), progressed to S phase (16 h), and arrested at mitosis (24 h).