Report |
Address correspondence to Greenfield Sluder, University of Massachusetts Medical School, Biotech 4, 3rd floor, 377 Plantation St., Worcester, MA 01605. Tel.: (508) 856-8651. Fax: (508) 856-8774. email: Greenfield.Sluder{at}umassmed.edu
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Abstract |
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Key Words: cell cycle; checkpoint; cleavage; cytokinesis; tetraploidy
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Introduction |
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Given the perceived dangers of cleavage failure, it has been of interest to determine if there are mechanisms that block the proliferation of cells that fail cleavage and become tetraploid. The notion that cells have a p53-dependent checkpoint that blocks the propagation of cells that failed to divide originated with findings that continuous treatment of normal cells with cytochalasin to block cleavage leads to a cell cycle arrest (Carter, 1967; Wright and Hayflick, 1972; Hirano and Kurimura, 1974; Lohez et al., 2003). In addition, cells treated with microtubule inhibitors eventually adapt to spindle assembly checkpoint, exit mitosis without dividing, and arrest in G1 (Minn et al., 1996; Lanni and Jacks, 1998). Similar experiments on cells with a compromised p53 pathway revealed that they continue cycling (Minn et al., 1996; Lanni and Jacks, 1998; Andreassen et al., 2001). The most clear and most explicit demonstration of this checkpoint came from a report by Andreassen et al. (2001) that used dihydrocytochalasin B to inhibit cytokinesis in REF52 cells, a primary rat fibroblast cell line. After drug removal, they found that the tetraploid cells arrested in G1, whereas many of the mononucleate cells in the same preparations continued cycling. By temporally separating the G1 arrest from the action of the drug, these workers provided evidence that the arrest was specific to the binucleate condition. Further indications that this arrest was due to failed cleavage came from observations that expression of a dominant-negative mutant p53 allowed the binucleates to undergo DNA synthesis.
The notion that normal mammalian somatic cells have a "tetraploidy checkpoint" that arrests binucleate cells in G1 after cleavage failure has been intensely attractive for many, including ourselves, because it provides a logical way for an organism to deal with a potentially dangerous and intractable situation (for review see Margolis et al., 2003). Not surprisingly, this proposed checkpoint has received considerable interest and has been reviewed as an established mechanism. However, the universal applicability of this checkpoint is brought into question by evidence that liver regrowth in living humans and rodents is due in part to the proliferation of multinucleate hepatocytes (for review see Fausto and Campbell, 2003).
Our interest in this checkpoint led us to initiate a series of experiments with telomerase-immortalized normal human cells (hTERT-RPE1) to determine what event or condition this checkpoint monitors. Using cytochalasin D at the concentration used by Andreassen et al. (2001) for dihydrocytochalasin B, we essentially reproduced their results. However, our finding that a significant percentage of the binucleates synthesized DNA prompted us to reexamine the link between cleavage failure and G1 arrest using lower drug concentrations and modification of substrate characteristics.
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Results and discussion |
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We note that for 20 of the binucleate cells of this last dataset, we initiated time-lapse observations of individual mitotic cells just after addition of the cytochalasin at the start of the experiments. After 12 h, the cells under observation were circled on the coverslip with a diamond scribe and the drug was washed out before time-lapse observations resumed. We found that none of the 20 cells showed any signs of cleavage furrow formation (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200403014/DC1), yet 13 of these binucleates proceeded into mitosis. These observations indicate that the tetraploidy checkpoint does not monitor the formation of a cleavage furrow.
Next, we lowered the concentration of cytochalasin D to 0.5 µM, the minimum concentration able to inhibit cytokinesis in this cell line, and grew the cells on bare glass. Observations of cells undergoing mitosis in the presence of this lower drug concentration revealed that cleavage furrows often formed, but later regressed (Video 1). We found that 92.0% of mononucleate cells and 77.0% of binucleated cells incorporated BrdU at 18 h (Fig. 1 C and Table I). Time-lapse video analysis revealed that 40 of 41 mononucleate cells proceeded through normal mitosis within 36 h, as expected. Strikingly, 13 of 14 binucleate cells completed mitosis to form two daughter cells within this period (Fig. 1 D and Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200403014/DC1). We note that a higher proportion of binucleates entered mitosis (13/14) by 36 h than incorporated BrdU (77%) at 18 h after cytochalasin removal. We think that this is due to some binucleates being slow to come into S phase and thus not being counted as BrdU positive by the time the coverslips were fixed. The time-lapse records were run twice as long, and this extra time presumably allowed the slower binucleates to go through S phase and enter mitosis by the time the cine records were terminated. For BrdU incorporation assays, coverslips were fixed at 18 h because thereafter the fastest cycling binucleates undergo mitosis to form mononucleate cells. Together, these results indicate that the ability of binucleate cells to progress through interphase is influenced by the concentration of cytochalasin used to block cleavage, and progression through G1 is not dependent on the presence of fibronectin on the substrate.
Inhibition of cleavage by blebbistatin
As an alternative agent to block cleavage, we used the myosin II inhibitor ()-blebbistatin (Straight et al., 2003). Asynchronous RPE1 cells, grown on bare glass, were treated with 100 µM ()-blebbistatin and individual mitotic cells were observed by time-lapse cinematography for the 45-min duration of the treatment. After each cell was circled on the coverslip with a diamond scribe, the drug was washed out and time-lapse observations were resumed. None of the cells exiting mitosis in the presence of the drug showed any furrowing activity (Video 1). Nevertheless, all 20 binucleates followed went through mitosis by 18 h after drug removal (Fig. 1 E and Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200403014/DC1). These results indicate that the previously observed progression of binucleate RPE1 cells through G1 was not due to unexpected side effects of cytochalasin that could putatively abrogate the tetraploidy checkpoint.
REF52 cells
Next, we wanted to address the theoretical concern that the hTERT-RPE1 cell line has a diminished response to cleavage failure or has somehow acquired properties that could conceivably abrogate the proposed tetraploidy checkpoint. Thus, we turned to the REF52 cell line originally used by Andreassen et al. (2001). Asynchronous REF52 cells were treated with 0.5 µM cytochalasin D for 412 h and were fixed for analysis of BrdU incorporation at 24 h after cytochalasin removal; others were observed by continuous time-lapse microscopy. When cultured on bare glass, 84.7% of mononucleate cells and 25.0% of binucleate cells incorporated BrdU (Fig. 2 A and Table I). Time-lapse observations revealed that 8 of 11 mononucleate cells and 0 of 6 binucleate cells went through mitosis within 72 h. However, our results were quite different when the cytochalasin-treated cells were grown on fibronectin-coated glass. 93.0% of the mononucleate cells and now 85.3% of the binucleate cells incorporated BrdU by 24 h after cytochalasin removal (Fig. 2 B and Table I). Also, 23 of 25 mononucleate cells and all but one (12/13) of the binucleate cells completed mitosis within 48 h (Fig. 2 C, Table I; Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200403014/DC1). As before, we think that a 48-h duration of the time-lapse records reveals the interphase progression of binucleates that were slow to enter S phase and thus were not counted as BrdU positive. Nine of the binucleates divided into two, and three divided into three daughters. The finding that almost all binucleate REF52 cells, under such conditions, enter mitosis indicates that rat cells and RPE1 cells have an equivalent response to cleavage failure.
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Possible causes for binucleate cells arresting in G1
Our results with lowered cytochalasin D concentrations suggest that the G1 arrest in binucleate cells observed by a number of investigators (Carter, 1967; Wright and Hayflick, 1972; Hirano and Kurimura, 1974; Andreassen et al., 2001; Lohez et al., 2003), and ourselves for higher drug doses, may be due to subtle disorganization of the actin cytoskeleton that persists even when the drug is washed out. Such disorganization may induce the p38 and the SAPK/JNK stress-activated protein kinase pathways (Ailenberg and Silverman, 2003). The observation that the G1 progression of mononucleate cells is seemingly less sensitive than that of binucleate cells to higher doses of cytochalasin is a possibly informative phenomenon for which we do not have a definitive explanation. Whatever the difference may be, it must be subtle because immunofluorescence did not reveal any obvious differences in F-actin distribution between mononucleate and binucleate cells (unpublished data). Also, vinculin and phosphotyrosine immunofluorescence did not reveal any obvious differences in the number per unit area, size, or distribution of focal adhesions between mononucleate and binucleate cells (unpublished data).
Summary
Our results provide a functional demonstration that normal human somatic cells and rat cells do not possess a tetraploidy checkpoint that arrests the cell cycle in G1 after a mitosis in which cleavage fails. Thus, cleavage failure is still, in principle, a potentially dangerous event for the organism. Given the perceived dangers of cleavage failure, it is interesting that neoplastic transformation does not occur at a higher rate than it does during the many mitoses that take place during development, growth, and adult life of humans. This suggests that in the living organism, cleavage failure is either essentially nonexistent or not sufficient by itself to cause problems.
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Materials and methods |
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BrdU incorporation
After cytochalasin D removal, BrdU (Sigma-Aldrich) was added to a final concentration of 5 µg/ml. After fixation in 20°C methanol, the cells were treated with 2N HCL at 22°C for 30 min, incubated with 1:500 mouse anti-BrdU antibody (Becton Dickinson), and labeled with 1:1,000 goat FITC-mouse antibody (Molecular Probes, Inc.). Observations were made with a microscope (DMR series; Leica) equipped for phase contrast and fluorescence. Images were recorded with a camera (Retiga 1300; Qimaging Corp.) and with QCapture software (Qimaging Corp.).
Time-lapse video analysis
Coverslips bearing cells were assembled into chambers (Hinchcliffe et al., 2001) containing cytochalasin D or ()-blebbistatin at the indicated concentrations. Individual binucleate cells were circled on the coverslip with a diamond scribe and then followed at 37°C with Universal (Carl Zeiss MicroImaging, Inc.) or BH-2 (Olympus) microscopes equipped with phase-contrast or differential interference contrast optics. After 4 h cytochalasin D or 45 min ()-blebbistatin exposure, the coverslips were washed with medium and time-lapse recordings were resumed. Some time-lapse observations began just after removal of the cytochalasin D. Images were recorded with Orca ER, Orca 100 (Hamamatsu Corporation), and Retiga EX or EXi (Qimaging Corp.) cameras; sequences were written to the hard drives of PC computers using C-imaging software (Compix, Inc.) or SlideBook software (Intelligent Imaging Innovations) and were exported as QuickTime movies.
Online supplemental material
Time-lapse sequences of cells failing cleavage and binucleate cells progressing through mitosis are available at http://www.jcb.org/cgi/content/full/jcb.200403014/DC1.
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Acknowledgments |
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This work is supported by National Institutes of Health grant GM 30758 (to G. Sluder).
Submitted: 2 March 2004
Accepted: 3 May 2004
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References |
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