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Cell Growth & Differentiation Vol. 12, 505-516, October 2001
© 2001 American Association for Cancer Research

Cell Cycle Attenuation by p120E4F Is Accompanied by Increased Mitotic Dysfunction1

Robert J. Rooney2

Department of Genetics, Duke University Medical Center, Durham, North Carolina 27710


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
In addition to their well-established roles at the G1-S checkpoint, recent reports support a role for universal cyclin-dependent kinase (CDK) inhibitors in the control of G2-M and suggest that their induction may stimulate the occurrence of endomitosis or polyploidy in a number of physiological settings. In this report, the stable expression of the p120E4F transcription factor, which attenuates G1-S progression by elevating p21WAF1 and p27KIP1 protein levels, was shown to also interfere with the regulation of G2-M and cytokinesis. Exponentially growing cultures of p120E4F-expressing fibroblast cell lines had reduced levels of CDC2 kinase activity, elevated levels of Cyclin B1 protein, and continuously generated a subpopulation of tetraploid cells and elevated numbers of multinucleated cells. Coexpression of activated Ras, which stimulates Cyclin D1 expression and G1-S-specific cyclin-CDK kinase activities, alleviated these effects without reducing p21WAF1 or p27KIP1 protein levels; p120E4F/ras-expressing cell lines contained reduced levels of Cyclin B1 protein, a restoration of Cyclin B-CDC2 kinase activity to control levels, and exhibited no increase of tetraploid or multinucleated cells. Interestingly, changes in the expression of Cyclin B1 and, to a lesser extent, CDC2 were primarily regulated by post-transcriptional mechanisms. The results indicate that mechanisms which moderately elevate CDK inhibitor levels can reduce CDC2 kinase activity to the point of impeding normal G2-M function and suggest that two molecular determinants commonly associated with the induction of polyploidy in a number of tissues, i.e., elevated levels of universal CDK inhibitors and sustained CDK2 kinase activity, may be solely sufficient to initiate endomitosis.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Ectopic expression of p120E4F, a ubiquitous cellular transcription factor that is down-regulated in response to the adenovirus E1A oncoprotein (1 , 2) , can cause cell cycle arrest at the G1-S transition through mechanisms that include the post-transcriptional elevation of the universal CDK 3 inhibitors p21WAF1 and p27KIP1 (3) . Recent evidence showed that G1 arrest mediated by ectopic p120E4F depends on the presence of wild-type p53 and RB tumor suppressor proteins, involving the independent physical interaction of p120E4F with p53 and RB (4 , 5) . Thus, although the precise cellular signals which regulate endogenous p120E4F have yet to be determined, the data with ectopic expression suggest that p120E4F may regulate cell cycle progression in response to a number of physiological and/or pathological signals.

It is also of interest that ectopic p120E4F expression has a graded effect on cell cycle progression. Whereas cells in which p120E4F is transiently expressed at high levels (e.g., from an inducible construct or by plasmid microinjection) rapidly undergo G1 arrest (3, 4, 5) , stable cell lines in which p120E4F is expressed at more physiologically relevant levels (~2–8-fold greater than endogenous levels) continue to grow albeit at a slow rate, exhibiting an attenuated G1-S transition and reduced but still detectable levels of G1-S-specific CDK activities (Cyclin D-CDK4/6, Cyclin E-CDK2, and Cyclin A-CDK2; Ref. 3 ). Although these effects are overcome by the coexpression of activated Ras, attributable to Ras-stimulated transcription of the Cyclin D1 gene and the resulting elevation of G1-S-specific CDK activities (3) , the ability of relatively low levels of ectopic p120E4F to attenuate the G1-S transition in nononcogenic cells suggests that physiological levels of endogenous p120E4F are sufficient to influence cell cycle progression under normal growth conditions.

Emerging evidence suggests that, in addition to regulating the G1-S transition, mechanisms that regulate universal CDK inhibitors may also induce changes in mitosis that contribute to important physiological and pathological processes. This has been suggested to occur during megakaryocytic differentiation, as the induction and/or redistribution of p21WAF1 and p27KIP1 coincides with the loss of Cyclin B-CDC2 kinase activity and the occurrence of endomitosis and polyploidy, both in vivo and in vitro (6, 7, 8, 9) , and ectopic expression of p21WAF1 or p27KIP1 induces polyploidization in undifferentiated megakaryoblastic cell lines (6 , 7) . Similarly, the polyploidization of trophoblast giant cells at the site of placental implantation (10 , 11) has been linked to the periodic induction of p57KIP2 (12) . A more sporadic effect is observed in aging or diseased human liver, where a reduction of regenerative ability that is associated with elevated p21WAF1 levels is also accompanied by an increased percentage of nonproliferative polyploid cells (13, 14, 15, 16, 17) . Likewise, elevated p21WAF1 levels in rat liver correlate with reduced hepatocyte proliferation and increased polyploidy after partial hepatectomy, particularly in older rats (18 , 19) , and targeted expression of a Waf1 transgene in the liver of mice significantly reduced hepatocyte proliferation after partial hepatectomy and increased the appearance of polyploid cells at a relatively young age (20) . A similar occurrence is also observed in damaged or hypertrophic cardiac and vascular tissue (21, 22, 23, 24, 25, 26, 27) , where increased polyploidy has been linked to the action of transforming growth factor-ß and angiotensin II, and their regulation of p21WAF1, p27KIP1, and other CDK inhibitors (28, 29, 30, 31) . Evidence for mitotic dysfunction and polyploidy is also found in at least six other normal and damaged tissues (32, 33, 34, 35, 36, 37) , although a link with universal CDK inhibitors has yet to be evaluated.

The disruption of normal G2-M function, which is required for the induction of endomitosis and polyploidy, often occurs through the suppression of CDC2 kinase activity (38, 39, 40, 41) , which may be achieved by universal CDK inhibitors in several ways. Many studies indicate that the inhibition of G1-S-specific CDKs can indirectly suppress CDC2 activity by reducing activation of the dual-specificity phosphatases CDC25C and CDC25B, which are required for CDC2 activation and G2 entry, and by delaying the transcriptional activation of the genes encoding CDC2, Cyclin B1, Cyclin A, and CDC25C (42, 43, 44, 45, 46, 47, 48) . Consistent with an indirect mechanism, in vitro experiments show that p21WAF1 and p27KIP1 have a significantly greater Ki for G1-S-specific cyclin-CDK complexes than for G2-M-specific Cyclin B-CDC2 (49 , 50) . Nevertheless, there are suggestions that CDC2 may also be directly inhibited through physical association, e.g., after its peak accumulation at the G1-S transition during a normal cell cycle, p21WAF1 protein reaccumulates in the nucleus at the G2-M transition to delay entry into mitosis, causing a transient buildup of premitotic cells that contain elevated levels of Cyclin B proteins (51) . Extension of this mitotic delay under conditions that significantly elevate universal CDK inhibitor levels is thought to serve as one of several redundant G2 checkpoint mechanisms (52) , and indeed, increased physical association of p21WAF1 or p27KIP1 with Cyclin B-CDC2 has been detected in three cases where cells undergo G2 attenuation or arrest as levels of p21WAF1 or p27KIP1 increase (53, 54, 55) . The elevation of Cyclin B protein levels in premitotic cycling cells (51) is also indicative of CDC2 inhibition by p21WAF1, as reduction of CDC2 activity during G2-M reduces activation of the APC/cyclosome and subsequent Cyclin B proteolysis; the inhibition of APC-mediated proteolysis may additionally impede progression through mitosis and cytokinesis (56, 57, 58) .

Normally, the concurrent abilities of universal CDK inhibitors to prevent G1-S progression and CDC2 function serve to coordinate mitotic events with DNA synthesis. However, in the contexts cited above, or when G2-M arrest is triggered by DNA damage or mitotic spindle dysfunction in cells where the RB-mediated G1-S checkpoint is mutated (e.g., cancer cells), DNA synthesis can proceed without completion of an intervening mitosis (i.e., endomitosis or endoreduplication; Refs. 52 and 59, 60, 61, 62 ). In fact, ectopic expression of p21WAF1 or p27KIP1 in RB-null cells has been shown to actually promote the occurrence of endomitosis in the absence of DNA damage or mitotic spindle dysfunction (52) . Thus, it appears that both physiological and pathological circumstances exist where the G1-S and G2-M functions of p21WAF1, p27KIP1, and p57KIP2 can be uncoupled or differentially manifested to induce endomitosis. However, in the absence of somatic mutation, it has not been determined if the induction of endomitosis by CDK inhibitors is restricted to specific cell types and regulatory pathways, as cited in the examples above, or is instead a more general outcome associated with elevated CDK inhibitor levels.

p120E4F-expressing cell lines provide an opportunity to examine the relationship between a mechanism that elevates CDK inhibitor levels and the alterations in mitotic function that can lead to endomitosis in a setting that has an intact RB checkpoint pathway and is distinct from the cell types, signaling pathways, and differentiation programs normally associated with the induction of endomitosis and tissue polyploidization. This report shows that in addition to an attenuated G1-S transition and slow growth, p120E4F- expressing cell lines also continuously generate tetraploid cells that have a limited proliferative capacity and exhibit apparent defects in cytokinesis. Both properties coincide with elevated levels of Cyclin B1 protein and reduced Cyclin B-CDC2 activity, all of which are alleviated by coexpression of activated Ras. The results suggest that with moderate elevation of p21WAF1 and p27KIP1 levels by p120E4F, a balance can be established between CDC2 inhibition and residual CDK2 activity that is sufficient to induce the generation of tetraploid cells.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Representative p120E4F-expressing cell lines (E4F2.5K/3T3) were shown previously to contain elevated levels of p21WAF1, p27KIP1, and Cyclin E proteins, and coexpression of activated Ras with p120E4F (E4F2.5K/ras) was shown to increase the expression of Cyclin D1 but had little effect on p21WAF1, p27KIP1, and Cyclin E levels (3) ; an example of these altered protein levels is shown in Fig. 1ACitation . In addition, G1-S-specific CDK activities were shown to be reduced in E4F2.5K/3T3 cells and significantly elevated in E4F2.5K/ras cells (3) . Accordingly, all 11 independently isolated E4F2.5K/3T3 cell lines have an average GDT ~3-fold longer than that of four E4F2.5K/ras cell lines and various control cell lines, including parental NIH 3T3, two matched neo R-control lines, two Ras-expressing cell lines, and two cell lines that overexpress p50E4F (E4F262/3T3), an NH2-terminal fragment of p120E4F that does not induce cell cycle arrest (Ref. 3 ; Fig. 1BCitation ).



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Fig. 1. The growth rates of p120E4F-expressing fibroblast cell lines reflect the expression levels of p21WAF1, p27KIP1, and Cyclin D1 proteins. A, Western blot analysis of G1-S cell cycle regulatory proteins in representative cell lines that express p120E4F (E4F2.5K/3T3–7), p120E4F, and activated Ras (E4F2.5K/ras-1) and in an NIH 3T3 control cell line (3T3/neo 23-1). Whole cell extracts (50 µg of protein/lane) were separated by SDS-10%PAGE or SDS-12%PAGE and probed after Western blotting with antibodies to p21WAF1, p27KIP1, p120E4F, ß-actin, and the specified cyclins. Ectopically expressed p120E4F protein was detected by precipitation with S protein-agarose (from 100 µg of protein) and Western blotting with {alpha}-E4F-Nterm antisera after separation by SDS-10%PAGE. Ras proteins were detected by Western blotting using a pan anti-Ras monoclonal antibody after separation of membrane-enriched extracts (25 µg of protein/lane) by SDS-15%PAGE. Antibodies to ß-actin were used as a control for protein loading. B, mean GDTs of 11 p120E4F-expressing cell lines (E4F2.5K/3T3–1, -2, -4, -5, -6, -7, -9, -10, -11, -12, and -13), four p120E4F/ras-expressing cell lines (E4F2.5K/ras-1, -3, -7, and -8), four Ras control cell lines (3T3/ras 17-1 and 17-2 and E4F262/ras 19-1 and 19-2), two p50E4F-expressing cell lines (E4F262/3T3 25-1 and 25-2), and three NIH 3T3 control cell lines (NIH 3T3 and 3T3/neo 23-1 and 23-2). Cells were plated in duplicate at 1 x 104/35-mm diameter well and counted at 24-h intervals for 5–7 days. GDTs were calculated using the equations described in "Materials and Methods." The GDT of E4F2.5K/3T3–1, -2, -6, -9, -10, -11, -12, and -13 cell lines were determined from one experiment. The GDT of E4F2.5K/3T3–4, -5, and -7 cell lines, and all other cell lines, were determined from two to three experiments. Black bars, SDs.

 
A second phenotypic change, as revealed by FACS analysis of representative cell lines, is that asynchronously growing E4F2.5K/3T3 cell lines, but not E4F2.5K/ras or other control lines, contain an abnormally high percentage of cells with an apparent G2-M or 4C DNA content (Table 1Citation and Fig. 2Citation ); the three E4F2.5K/3T3 cell lines used for this analysis cover the range of p120E4F expression levels (E4F2.5K/3T3-4 > E4F2.5K/3T3-7 > E4F2.5K/3T3-5) found in all 11 E4F2.5K/3T3 cell lines (3) . This was also observed in cell cycle synchronization experiments, where a high percentage of cells with an apparent G2-M or 4C DNA content persisted in serum-starved E4F2.5K/3T3 cells but not in serum-starved parental NIH 3T3 cells (Fig. 3Citation ; 0 h poststimulation). Although this anomaly could simply reflect a block or attenuation in G2-M (in addition to the G1-S delay), three lines of evidence suggest it is attributable to the existence of a subpopulation of tetraploid cells: (a) in addition to the aberrantly large 4C peak, FACS histograms of E4F2.5K/3T3 cell lines show a small but distinct peak of cells with an 8C DNA content, indicative of tetraploid cells in G2-M or octaploid cells in G1 (Fig. 2)Citation ; E4F2.5K/ras and other control cell lines do not show this feature; (b) on serum stimulation of quiescent E4F2.5K/3T3 cells, the decrease of cells with a 4C DNA content mirrored the decrease of cells with a 2C DNA content as they entered S phase, which is consistent with the G1 to S phase progression of a tetraploid subpopulation (Fig. 3BCitation ; 30–33 h poststimulation); and (c) during serum stimulation, the time it took for the 2C peak to increase from its minimum in S phase to its maximum in the next G1 phase (6–7 h) was about the same for both E4F2.5K/3T3 and parental NIH 3T3 cells (Fig. 3, A and B)Citation , indicating that the G2-M phase for the majority of diploid E4F2.5K/3T3 cells was of similar duration to that of control NIH 3T3 cells and thus not generally blocked or attenuated.


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Table 1 Apparent cell cycle distribution (DNA content) of p120E4F-expressing cell lines

 


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Fig. 2. E4F2.5K/3T3 cells contain an increased percentage of cells with 4C and 8C DNA content. Asynchronous, exponentially growing cultures of representative cell lines that express p120E4F (E4F2.5K/3T3-5), p120E4F and activated Ras (E4F2.5K/ras-7), activated Ras alone (3T3/ras 17-1), p50E4F (E4F262/3T3 25-2), and an NIH 3T3 control line (3T3/neo 23-1) were stained with PI and analyzed by FACS. The positions of cells with 2C, 4C, and 8C DNA content are indicated in each histogram; the average percentages are shown in Table 1Citation .

 


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Fig. 3. Quiescent E4F2.5K/3T3 cells with a 4C DNA content behave as tetraploid cells in G0-G1 after serum stimulation. E4F2.5K/3T3-7 cells (bottom panel) and control NIH 3T3 cells (top panel) were grown to 30% confluency, synchronized in G0 by a 48-h incubation in media containing 0.1% serum, and stimulated to reenter the cell cycle by the addition of serum to 10%. At the indicated times, the distribution of cells with 2C (G0-G1; {diamondsuit}), intermediate (S; {circ}), and 4C (G2-M; {blacksquare}) DNA content was determined by FACS analysis after staining with PI. FACS profiles were determined for three serum stimulation experiments; values from a representative experiment are shown.

 
The expression of Cyclin E protein, which is specific for late G1 and early S phase, and Cyclin B1 protein, which is specific for G2-M phase, was used to determine whether E4F2.5K/3T3 cell lines do, in fact, contain a tetraploid subpopulation. Cells from a representative E4F2.5K/3T3 line and a control line were stained with the DNA intercalating dye Hoechst 33342, sterile sorted by FACS into fractions with 2C DNA content or 4C DNA content (Fig. 4A)Citation , and analyzed by Western blotting for the presence of Cyclin E and Cyclin B1 proteins (Fig. 4B)Citation . Similar amounts of Cyclin E were present in both the 2C and 4C peaks from E4F2.5K/3T3 cells, whereas it was present only in the 2C peak from the control line. Cyclin B1 was detected only in the 4C peak (Fig. 4B)Citation . These results indicate that the 4C peak of E4F2.5K/3T3 cells is a mix of diploid cells in G2-M and tetraploid cells in G0/G1.



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Fig. 4. E4F2.5K/3T3 cell lines continuously generate tetraploid cells. A, isolation of E4F2.5K/3T3 and NIH 3T3 cells with 2C and 4C DNA content. Asynchronous, exponentially growing cultures of E4F2.5K/3T3-7 and NIH 3T3 cells were sterile sorted by FACS after incubation with Hoechst dye No. 33342, as described in "Materials and Methods." The gates used to sort populations of cells with 2C (A) and 4C (B) DNA content are indicated in the FACS histogram; only the histogram for E4F2.5K/3T3-7 cells is shown. Total number of collected cells with 2C DNA content: 5.4 x 106 cells (E4F2.5K/3T3-7) and 2.9 x 106 cells (NIH 3T3); total number of collected cells with 4C DNA content: 4.6 x 106 cells (E4F2.5K/3T3-7) and 1.5 x 106 cells (NIH 3T3). B, Western blot analysis of Cyclin E and Cyclin B1 proteins in E4F2.5K/3T3 and NIH 3T3 cells with 2C and 4C DNA content. Whole cell extracts were prepared from the sorted populations of E4F2.5K/3T3-7 and NIH 3T3 cells, as indicated in A, and probed with antibodies to Cyclin E or antisera to Cyclin B1 after separation by SDS-10%PAGE (25 µg of protein/lane) and Western blotting; the positions of Cyclin E and Cyclin B1 proteins are marked by arrows. A faint band for Cyclin B1 protein was detected in extracts of NIH 3T3 cells with 4C DNA content (data not shown). C, FACS histogram of a sorted population of E4F2.5K/3T3-7 cells with 2C DNA content. A total of 3.25 x 106 cells with 2C DNA content was collected for replating and passaging. D, the rate of appearance of tetraploid cells in cultures of E4F2.5K/3T3-7 cells isolated with 2C DNA content. Sorted E4F2.5K/3T3-7 cells with 2C DNA content (C) were plated in culture (day 0) and passaged 1:3 every 7 days when the cells were ~70% confluent. At the times indicated, cells were harvested, stained with PI, and analyzed by FACS to determine the percentage of cells with 4C and 8C DNA content.

 
Moreover, when E4F2.5K/3T3 cells isolated from the 2C peak (Fig. 4C)Citation were grown and passaged in culture, the same high percentage of cells with 4C and 8C DNA content that was seen in the asynchronously growing cultures reappeared over a 4-week period (Fig. 4D)Citation , indicating that a tetraploid subpopulation was being continuously generated. By subtracting the initial percentage of cells with 4C DNA content at day 2 from that seen after 4 weeks, the tetraploid population was estimated to comprise ~30% of the asynchronously growing culture. This coincides with the percentage of serum-starved cells with 4C DNA content (Fig. 3BCitation ; 0 h poststimulation). However, the percentage of E4F2.5K/3T3 cells with an 8C DNA content was never greater than one-tenth of the percentage of cells with a 4C DNA content, suggesting that the tetraploid cells had a lower proliferative rate than the diploid cells. This was confirmed by FACS analysis of asynchronously growing E4F2.5K/3T3 cells that had been labeled for 5 h with BrdUrd, which showed that the tetraploid subpopulation (cells with >4C to 8C DNA content) comprised only 12% of cells undergoing DNA synthesis (data not shown). The lack of BrdUrd incorporation by the majority of tetraploid cells is presumably attributable to replicative senescence rather than apoptosis, because E4F2.5K/3T3 cell lines appear to have the same viability as control cell lines (3) .

Morphological changes also distinguished E4F2.5K/3T3 cell lines from E4F2.5K/ras and control lines. E4F2.5K/3T3 cells appeared large and flattened (Fig. 5, D–F)Citation in comparison to parental or control fibroblasts (Fig. 5, A–C)Citation . Analysis of all 11 E4F2.5K/3T3 cell lines showed that the average surface area covered by an individual cell ranged from 3–8-fold greater than that covered by individual control fibroblasts (data not shown). Of particular interest was a noticeable increase in the number of cells with multiple nuclei in E4F2.5K/3T3 cell lines (Fig. 5, D and F)Citation . Analysis of all 11 E4F2.5K/3T3 cell lines revealed that the percentage of multinucleated cells in E4F2.5K/3T3 cell lines, although variable, was significantly higher than in control lines (Table 2)Citation , suggesting that some change elicited by p120E4F was interfering with cytokinesis. By contrast, E4F2.5K/ras cells (Fig. 6, C and D)Citation appeared similar in size to control fibroblasts (Fig. 6A)Citation , displayed the spindle shape and refractile appearance typical of Ras-transformed fibroblasts, and did not exhibit an increased percentage of multinucleated cells (Table 2)Citation . Thus, similar to its effect on growth rate and DNA ploidy, coexpression of activated Ras also alleviated the morphological changes elicited by p120E4F.



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Fig. 5. E4F2.5K/3T3 cell lines exhibit a flattened morphology and a greater percentage of multinucleated cells. Light micrographs of exponentially growing cultures of NIH 3T3 (A), 3T3/neo 23-1 (B), and 3T3/neo 23-2 (C) control cell lines or E4F2.5K/3T3-4 (D), E4F2.5K/3T3-5 (E), and E4F2.5K/3T3-7 (F) cell lines. All cultures were photographed at the same magnification using a x20 objective lens. Examples of binucleated cells can be observed in B, D, and F. The percentages of multinucleated cells found in all cell lines examined in this study are shown in Table 2Citation .

 

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Table 2 Percentage of multinucleated cells in p120E4F-expressing cell lines

 


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Fig. 6. Coexpression of activated ras alleviates p120E4F-induced morphological changes. Light micrographs of exponentially growing cultures of 3T3/neo 23-1 (a), E4F2.5K/3T3-4 (b), E4F2.5K/ras-1, and E4F2.5K/ras-3 (d) cell lines. All cultures were photographed at the same magnification using a x20 objective lens.

 
Abnormalities in DNA ploidy and cytokinesis suggested that in addition to the decreased activities of G1-S-specific cyclin-CDK complexes, Cyclin B-CDC2 activity might also be altered in E4F2.5K/3T3 cell lines. Immunoprecipitates from extracts of E4F2.5K/3T3, E4F2.5K/ras, and control cell lines were collected using antibodies against Cyclin B1 and CDC2, as well as with antibodies against Cyclin E, Cyclin A, CDK2, and CDK4/6, and their relative kinase activities were measured in vitro (Fig. 7A)Citation . Similar to what was observed for G1-S-specific cyclin-CDKs, the kinase activities associated with Cyclin B1 and CDC2 in E4F2.5K/3T3 cell extracts were reduced relative to control extracts; Cyclin B1-associated kinase activity and total CDC2 activity (combined activities of both Cyclin A-CDC2 and Cyclin B-CDC2) were both reduced 3-fold. Conversely, total CDC2 activity in E4F2.5K/ras cell extracts increased 2.5–3-fold relative to control extracts, similar to the increase of CDK2 activity. However, Cyclin B1-associated kinase activity in E4F2.5K/ras cell extracts was only marginally higher than that in control extracts, indicating that Cyclin A-CDC2 was mostly responsible for the elevation of CDC2 activity.



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Fig. 7. E4F2.5K/3T3 cell lines contain reduced CDC2 activity and elevated levels of Cyclin B1 protein. A, relative cyclin-CDK kinase activities present in p120E4F, p120E4F/ras, and control cell lines. Antiserum to Cyclin A, Cyclin B1, Cyclin E, CDC2, CDK2, and a mix of antisera to CDK4 and CDK6 were used to immunoprecipitate kinase activities from extracts of two control cell lines (NIH 3T3 and 3T3/neo 23-1), two p120E4F cell lines (E4F2.5K/3T3-4 and E4F2.5K/3T3-7), and two p120E4F/ras cell lines (E4F2.5K/ras-1 and E4F2.5K/ras-3). In vitro phosphorylation reactions containing Cyclin A, Cyclin E, and CDK2 used histone H1 and GST-RB proteins as substrates; Cyclin B1 and CDC2 kinase reactions used histone H1; CDK4/6 reactions used GST-RB. Kinase activities were measured by PhosphorImager analysis of 32P-labeled substrates from two to four experiments, averaged together, and then normalized to the averaged activities from control cell extracts; Thin black bars, SDs. B, Western blot analysis of Cyclin B1 and CDC2 proteins in extracts from two control cell lines (NIH 3T3 and 3T3/neo 23-1), two p120E4F cell lines (E4F2.5K/3T3-4 and -7), and two p120E4F/ras cell lines (E4F2.5K/ras-1 and -3). Extracts (50 µg of protein/lane) were probed with antisera against mouse Cyclin B1 or mouse CDC2 after separation by SDS-12% PAGE and Western blotting. C, Northern blot analysis of poly(A)+ RNA (2 µg of RNA/lane) isolated from two control cell lines (NIH 3T3 and 3T3/neo 23-1), two p120E4F cell lines (E4F2.5K/3T3-4 and -7), and two p120E4F/ras cell lines (E4F2.5K/ras-1 and -3). Blots were hybridized with 32P-labeled cDNA probes for Cyclin B1, CDC2, and glyceraldehyde-3-phosphate dehydrogenase (used as a control for RNA concentration and loading).

 
Cyclin B1 and CDC2 protein levels were determined by immunoblot analysis (Fig. 7B)Citation . Relative to control cell lines, Cyclin B1 protein levels were significantly elevated in E4F2.5K/3T3 cell lysates and reduced in E4F2.5K/ras cell lysates, whereas CDC2 protein levels remained unaltered in E4F2.5K/3T3 cell lysates but were greatly elevated in E4F2.5K/ras cell lysates. Northern blot analysis revealed that Cyclin B1 mRNA levels were unaltered in both E4F2.5K/3T3 and E4F2.5K/ras cell lines. CDC2 mRNA levels were unaltered in E4F2.5K/3T3 cell lines and slightly elevated (~2–3-fold by densitometry) in E4F2.5K/ras cell lines (Fig. 7C)Citation . Thus, changes in Cyclin B1 protein levels appear to be regulated post-transcriptionally in asynchronously growing E4F2.5K/3T3 cells and E4F2.5K/ras cells. The elevated levels of CDC2 protein in asynchronously growing E4F2.5K/ras cells may, in part, reflect an increase of CDC2 mRNA but, as with Cyclin B1, also appear to be regulated mostly at the post-transcriptional level.


    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Previous observations showed that stable, low-level expression of ectopic p120E4F in NIH 3T3 fibroblasts caused the elevation of p21WAF1, p27KIP1, and Cyclin E protein levels, suppression of CDK2 and CDK4/6 kinase activities, and attenuation of the G1-S transition (3, 4, 5) . Here it is shown that p120E4F-expressing cell lines also exhibit a number of changes that relate to the regulation of G2-M, including reduced CDC2 kinase activity, elevated levels of Cyclin B1 protein, significant numbers of multinucleated cells, and the continuous generation of tetraploid cells. These changes are concordant with evidence emerging from other experimental systems that suggest CDK inhibitors regulate events in G2-M, in addition to their role at the G1 checkpoint, and that p21WAF1 and p27KIP1 levels may regulate the incidence of endomitosis and polyploidy in several types of differentiated tissue. The results presented here not only support the involvement of CDK inhibitors in the regulation of G2-M-related processes but that they can do so independent of the differentiated cell types and mutational backgrounds in which such effects have been normally observed.

Although the data does not entirely rule out a direct effect by p120E4F on G2-M, e.g., by altering the expression of a key mitotic protein, the ability of activated ras to alleviate all of the G2-M-related changes induced by p120E4F strongly supports the contention that the induction of p21WAF1 and p27KIP1 is responsible for them. In NIH 3T3 cells, increased levels of Cyclin D1-CDK4/6 complexes that result from Ras-stimulated transcription of the Cyclin D1 gene have been shown to titrate p21WAF1 and p27KIP1 away from other cyclin-CDK complexes, leading to increased CDK activities and accelerated cell cycle progression (63 , 64) . Coexpression of activated Ras with p120E4F not only stimulated Cyclin D1 expression, all G1-S CDK kinase activities, and G1-S progression (Ref. 3 and Fig. 1BCitation ), but it also restored CDC2 kinase activity to normal levels and alleviated all of the other G2-M-related changes (Figs. 2Citation 3Citation 4Citation , 6Citation , and 7Citation ; Tables 1Citation and 2Citation ). Moreover, the continued elevation of p21WAF1 and p27KIP1 protein levels in E4F2.5K/ras cells (Ref. 3 and Fig. 1ACitation ) suggests this occurred without functional inactivation of p120E4F. Stable expression of p120E4F in cell lines harboring a naturally occurring RasK mutation also failed to produce any noticeable changes in G2-M function.4

The reduction of CDC2 activity in E4F2.5/3T3 cells is likely responsible for the other G2-M-related phenotypic changes observed in these cells. As mentioned earlier, the induction of G2-M arrest by suppression of CDC2 activity, e.g., in response to DNA damage or mitotic spindle dysfunction, will cause endomitosis and polyploidy in cells that cannot undergo G1 arrest. Here, E4F2.5K/3T3 cells exhibited significantly reduced levels of CDC2 activity (~30% relative to control cells) while still maintaining detectable levels of CDK2 activity (likely driven by the elevated levels of Cyclin E protein). Although the level of CDK2 activity was also reduced relative to control cells, the incorporation of BrdUrd into both diploid and tetraploid DNA indicates it was sufficient to drive both normal and endomitotic DNA synthesis. Reports also show that the suppression of CDC2 activity leads to reduced activation of the APC/cyclosome and decreased Cyclin B proteolysis (56 , 57) and that the prevention of APC-dependent proteolysis, e.g., by proteosome inhibitors or by expression of dominant-negative mutants, will cause late mitotic arrest and/or aberrant cytokinesis (58 , 65, 66, 67) . The elevated levels of Cyclin B1 protein found in E4F2.5K/3T3 cells coupled with the lack of a corresponding increase in Cyclin B1 mRNA levels (Fig. 7, B and C)Citation strongly suggests that Cyclin B1 proteolysis and, hence, APC activity were also reduced in these cells, which would account for the increased percentage of multinucleated cells (in addition to possibly contributing to endomitosis by inhibiting G2-M progression). Thus, endomitosis and aberrant cytokinesis, two processes that can arise with the suppression of CDC2 activity, most likely produced the tetraploid and octaploid cells detected by FACS analysis and the multinucleated cells observed by microscopy, respectively. It is possible that a third process in which cells escape from prolonged G2-M arrest, called checkpoint adaptation, might also have contributed to the increase of polyploid cells (as detected by FACS analysis); however, the synchrony with which both diploid and tetraploid cells progressed through the cell cycle after serum stimulation (Fig. 3B)Citation is more consistent with endomitosis.

In considering these effects on G2-M function, it is important to point out that tetraploid cells comprised only a limited subpopulation (~30%) of E4F2.5K/3T3 cell lines, multinucleated cells comprised an even smaller subpopulation (~10%), and that tetraploid cells accumulated from FACS-sorted diploid cells at a relatively slow rate (Fig. 4D)Citation , all of which indicates that only a fraction of the cells experience significant G2-M dysfunction and implies that the reduced level of CDC2 activity measured in a particular E4F2.5K/3T3 cell line (and the degree to which G2-M functions were affected by it) actually reflects a range of reduced activities in individual cells. This suggests that within that range, there exists a window in which CDC2 activity can be reduced to the point of impeding G2-M functions, whereas the reduction of CDK2 activity is still not sufficient for full engagement of the G1-S checkpoint, thereby allowing the generation of tetraploid or multinucleated cells. In this scenario, cells with lower levels of p21WAF1 and p27KIP1 would have minimal reductions of CDC2 activity, little delay in G2-M, and would remain diploid, whereas those with intermediate CDK inhibitor levels would be expected to have moderate reductions in both CDC2 and CDK2 activities and undergo endomitosis or aberrant cytokinesis; cells with higher CDK inhibitor levels would have more severe reductions in CDC2 activity but also a greater inhibition of CDK2 and undergo G1 arrest. Thus, as observed in E4F2.5K/3T3 cell lines, only a fraction of the population would generate tetraploid cells. Moreover, those tetraploid cells would have an inherently slower proliferative rate attributable to their higher levels of p21WAF1 and p27KIP1, which is consistent with the lower incorporation of BrdUrd measured in tetraploid E4F2.5K/3T3 cells. The lower percentage of multinucleated cells in E4F2.5K/3T3 cell lines suggests that defects arising from aberrant cytokinesis may be even less well tolerated than endomitosis and act to additionally elevate CDK inhibitor levels to inhibit the proliferation of multinucleated cells. The heterogeneity of multinucleated cells found among these cell lines (Table 2)Citation , however, may reflect a variety of factors, including variations in CDC2 activity, APC activity, and the degree to which they must be inhibited to affect cytokinesis.

Although the precise way by which CDC2 activity was regulated was not fully addressed in this study, it is noteworthy that significant changes in CDC2, Cyclin B1, and Cyclin A mRNA levels were not contributing mechanisms in either E4F2.5K/3T3 or E4F2.5K/ras cells (see Northern blot analyses in Ref. 3 and in Fig. 7CCitation ). A number of reports indicate that E2F- and CDF-1 (CBF/cdc2)-regulated transcription plays a major role in controlling the cell cycle-dependent expression of CDC2, Cyclin B1, and Cyclin A (44, 45, 46, 47, 48) . Given that E4F2.5K/3T3 and E4F2.5K/ras cells actively progress through the cell cycle, one thus might have expected CDC2, Cyclin B1, and Cyclin A mRNA levels to mirror the differences in CDC2, Cyclin B1, and Cyclin A protein levels or CDC2 kinase activity that were observed in each cell type. However, very little change in Cyclin B1 or CDC2 mRNA levels accompanied the fairly dramatic changes in Cyclin B1 and CDC2 protein levels and CDC2 activity. It is particularly notable that in E4F2.5K/ras cells, the reduction of Cyclin B1 protein levels seemed to counterbalance the elevation of CDC2 protein levels, as CDC2 kinase activity was maintained close to that in control cells. This strongly implies that in an actively cycling cell population, as opposed to quiescent or arrested cells stimulated to reenter the cell cycle, post-transcriptional mechanisms (i.e., translation and degradation) may be the predominant means for controlling the levels of Cyclin B1 and CDC2 proteins, with transcriptional regulation perhaps being more important for controlling the timing rather than the magnitude of their expression.

On the basis of the current literature, mechanisms that have been implicated in the polyploidization of tissues in response to either physiological stimuli or stress have similar characteristics with the molecular events observed in p120E4F-expressing fibroblasts. Several mechanisms that elevate CDK inhibitor levels have been linked to polyploidization in differentiating megakaryocytes, regenerating liver, and hypertrophic cardiovascular tissue (7 , 18 , 28) , with enforced expression of some CDK inhibitors stimulating polyploidization in some of these tissues (6 , 7 , 20) . The initial induction of polyploidization in megakaryoblastic cell lines has been additionally linked to increased levels of Cyclin B protein and the loss of Cyclin B-CDC2 kinase activity, as well as to increased levels of Cyclin E protein and the maintenance of CDK2 activity (9 , 68 , 69) . Moreover, a marked increase in the number of polyploid cells is also seen in various tissues of Skp2-/- knockout mice, where p27KIP1 and Cyclin E levels are elevated because of the loss of Skp2-dependent ubiquitination and proteolysis (70) . Although additional mechanisms to spur DNA synthesis and inhibit mitotic functions clearly come into play in some specialized cases, such as megakaryocyte or trophoblast giant cell polyploidization (69 , 71, 72, 73, 74) , all of the physiological contexts cited above appear to share the same pattern of events found in E4F2.5K/3T3 cells. It will thus be of interest to determine whether p120E4F, or the genes regulated by it, are common participants in the polyploidization of these or other tissues.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cell Culture.
All cell lines were maintained in DMEM containing 10% FBS. Eleven p120E4F-expressing cell lines (E4F2.5K/3T3–1, -2, -4, -5, -6, -7, -9, -10, -11, -12, and -13) were generated as described (3) ; one additional line (E4F2.5K/3T3–8) was tetraploid and not included in the analysis. Briefly, NIH 3T3 cells were transfected with pCMVs-E4F2.5K, which expresses p120E4F tagged at the NH2 terminus with the S-peptide (Novagen), and pßA-Pr-neo (75) as a selection marker using LipofectAMINE (Life Technologies, Inc.) and selected in media containing 400 µg/ml G418. p50E4F-expressing cell lines were obtained using pCMVs-E4F262 and pßA-Pr-neo. Four p120E4F/ras-expressing cell lines (E4F2.5K/ras-1, -3, -7, and -8) were obtained using pCMVs-E4F2.5K, pßA-Pr-neo, and pSP72-ras, which expresses the T-24-derived activated H-ras mutant (76) . Control lines (3T3/neo) or Ras-expressing lines (3T3/ras) were obtained using pßA-Pr-neo or pSP72-ras and pßA-Pr-neo, respectively. Cells were synchronized in G0 by a 48-h incubation in DMEM containing 0.1% FBS and stimulated to reenter the cell cycle by the addition of FBS to a 10% final concentration.

Cell Cycle Analysis, Cell Sorting, and Growth Rates.
Analysis of cell cycle distribution and DNA content by FACS was performed as described previously (77 , 78) . Briefly, cells from asynchronously growing or serum-stimulated synchronized cultures (0.4–1 x 106 cells/sample) were harvested with trypsin, washed in PBS containing 10 µg/ml soybean trypsin inhibitor (Boehringer Mannheim), and resuspended in PI staining solution (0.05 mg/ml PI, 0.1% sodium citrate, 0.1% Triton X-100). Fluorescence emitted from PI-stained nuclei was measured with a FACScan flow cytometer and CellQuest software (Becton Dickinson). The percentage of cells within G0-G1, S, and G2-M cell cycle phases or with 2C, 4C, and 8C DNA content was determined using the ModFit program (Verity Software House). The extent of DNA synthesis in diploid and tetraploid subpopulations of asynchronously growing p120E4F-expressing cells was determined after a 5-h incubation with 10 µM BrdUrd (Sigma Chemical Co.); the cells were fixed and permeabilized, stained with PI and anti-BrdUrd FITC-conjugated monoclonal antibody (Becton Dickinson) according to the manufacturer, and analyzed by FACScan using CellQuest and ModFit software. Sterile sorting of cells with 2C and 4C DNA content was accomplished by incubating asynchronously growing cultures with Hoechst dye No. 33342 (Hoechst AG) at a final concentration of 10 µM for 60–90 min, after which the cells were harvested in PBS, placed on ice, and collected after sorting using a FACSvantage flow cytometer and CellQuest software (Becton Dickinson; Ref. 78 ). On isolation, the cells were immediately plated in DMEM/10%FBS or pelleted by centrifugation and lysed for Western blot analysis. Cell growth rates were determined by plating 1 x 104 cells in duplicate wells of six-well trays (Falcon) in DMEM containing 10% FBS and counting cells using a hemocytometer every 24 h for 5–7 days. Cell counts obtained during periods of exponential growth were used to calculate the number of cell doublings per day (k) for each cell line with the following equation: k = [log2(Xf/Xi)]/t, where t = time of growth in days, Xf = final cell count, and Xi = initial cell count; the GDT = 24 h/k. Cell viability was determined by trypan blue dye exclusion.

Microscopic Analysis.
Multinucleated cells were identified visually in subconfluent cultures plated at least 3 days before inspection. All cells in a visual field were scored as containing either one or two or more nuclei, and the percentage of multinucleated cells was derived after scoring 100-1000 cells for each cell line. Cells were scored as multinucleated if they contained two or more distinct and separate nuclei; the majority of multinucleated cells were binucleated, although some cells appeared to contain three or four distinct nuclear structures. Scoring was performed on one to three independently plated cultures for each cell line, and multiple fields were scored for each culture. Cells were photographed on a Nikon TMS microscope using a x20 objective lens and Polaroid 667 film.

Western Blot Analyses.
Detection of E4F protein by S protein-agarose precipitation followed by Western blot was done as described previously (1) using {alpha}-E4F-Nterm polyclonal antisera, horseradish peroxidase-conjugated donkey antirabbit IgG (Life Technologies, Inc.), and ECL detection systems (Pierce). Ras proteins were detected in 50 µg of protein/lane of membrane-containing fractions from sonicated cells after separation by SDS-15% PAGE, electroblotting to nitrocellulose (Schleicher and Schuell), and probing with a pan anti-Ras monoclonal antibody (Santa Cruz Biotechnology) and ECL. Cyclins, CDKs, and CDK inhibitor proteins were detected in 25–50 µg of whole cell lysate with antibodies against p21WAF1 and Cyclin E (Santa Cruz Biotechnology), p27KIP1 (Transduction Laboratories), Cyclin A and Cyclin B1 (PharMingen), Cyclin D1 and CDC2 (C. J. Sherr, St. Jude Children’s Research Hospital, Memphis, TN), and visualized by ECL.

In Vitro Kinase Assays.
Immunoprecipitation and analysis of kinase activities from whole cell extracts of E4F, E4F/ras, and control cell lines were performed essentially as described (3) . CDKs were immunoprecipitated from 100 µg of extract with rabbit antisera or purified antibodies against CDC2, CDK2, Cyclin B1, a combination of antisera against CDK4 and CDK6 (C. J. Sherr), Cyclin E, (Santa Cruz Biotechnology), and Cyclin A (PharMingen), respectively. All reactions were performed at 30°C in 20 µl of kinase buffer [50 mM HEPES (pH 7.5), 10 mM MgCl2, 1 mM DTT] containing 20 µM ATP and 10 µCi of [{gamma}- 32P]-ATP (6000 Ci/mmol; New England Nuclear). CDK4/6 complexes were tested in reactions that contained 50 ng of GST-RB (379-928) protein as substrate, CDC2 and Cyclin B1 complexes were tested in reactions that contained 1 µg of histone H1 (Sigma Chemical Co.), and CDK2, Cyclin E, and Cyclin A complexes were tested on both substrates. Phosphorylation of substrate proteins was analyzed by SDS-10% PAGE of boiled immune complex kinase reactions and quantitated by Storm PhosphorImager (Molecular Dynamics).

Northern Blot Analysis.
Poly(A)+ mRNA was prepared using RNeasy and Oligotex mRNA isolation kits (Qiagen). Poly(A)+ RNA (2 µg) was electrophoresed through a 1.2% formaldehyde-agarose gel (79) in parallel with RNA size markers (Life Technologies, Inc.) and transferred to nitrocellulose. Probes were gel-purified plasmid inserts and 32P-labeled using the Megaprime DNA labeling system (Amersham). Individual probes for mouse Cyclin B1 and mouse CDC2 were obtained from Joseph R. Nevins (Duke University, Durham, NC).


    Acknowledgments
 
We thank Dr. Elma R. Fernandes for the creation and initial characterization of the cell lines used in this study, Dr. Joseph R. Nevins for mouse Cyclin B1 and CDC2 cDNAs, Dr. Richard Ashmun, Sam Lucas, and Ed Wingfield at St. Jude Children’s Research Hospital for FACS analyses, Dr. Charles Sherr for antisera against CDK2, CDK4, CDK6, CDC2, Cyclin B1, and Cyclin D1, Drs. Scott Hiebert and Gerard Zambetti for critical reading of the manuscript, and Ruby Tharp for excellent technical assistance.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by National Institute of General Medical Sciences Grant R01 GM51299. Back

2 To whom requests for reprints should be addressed, at Department of Genetics, Duke University Medical Center, Box 3054, Durham, NC 27710. Phone: (919) 684-4262; Fax: (919) 684-2790; Back

3 The abbreviations used are: CDK, cyclin-dependent kinase; RB, retinoblastoma tumor suppressor protein; APC, anaphase promoting complex; FACS, fluorescence-activated cell sorting; BrdUrd, bromodeoxyuridine; FBS, fetal bovine serum; PI, propidium iodide; GDT, generation doubling time; ECL, enhanced chemiluminescence; poly(A)+ RNA, polyadenylated RNA. Back

4 R. J. Rooney, unpublished observations. Back

Received for publication 2/28/01. Revision received 8/29/01. Accepted for publication 9/ 4/01.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

  1. Fernandes E. R., Rooney R. J. The adenovirus E1A-regulated transcription factor E4F is generated from the human homolog of nuclear factor phiAP3. Mol. Cell. Biol., 17: 1890-1903, 1997.[Abstract/Free Full Text]
  2. Rooney R. J., Daniels R. R., Jenkins N. A., Gilbert D. J., Rothammer K., Morris S. W., Higgs D. R., Copeland N. G. Chromosomal location and tissue expression of the gene encoding the adenovirus E1A-regulated transcription factor E4F in humans and mice. Mamm. Genome, 9: 320-323, 1998.[Medline]
  3. Fernandes E. R., Zhang J. Y., Rooney R. J. Adenovirus E1A-regulated transcription factor p120E4F inhibits cell growth and induces the stabilization of the cdk inhibitor p21WAF1. Mol. Cell. Biol., 18: 459-467, 1998.[Abstract/Free Full Text]
  4. Fajas L., Paul C., Zugasti O., Le Cam L., Polanowska J., Fabbrizio E., Medema R., Vignais M. L., Sardet C. pRB binds to and modulates the transrepressing activity of the E1A-regulated transcription factor p120E4F. Proc. Natl. Acad. Sci. USA, 97: 7738-7743, 2000.[Abstract/Free Full Text]
  5. Sandy P., Gostissa M., Fogal V., Cecco L. D., Szalay K., Rooney R. J., Schneider C., Del Sal G. p53 is involved in the p120E4F-mediated growth arrest. Oncogene, 19: 188-199, 2000.[Medline]
  6. Kikuchi J., Furukawa Y., Iwase S., Terui Y., Nakamura M., Kitagawa M., Komatsu N., Miura Y. Polyploidization and functional maturation are two distinct processes during megakaryocytic differentiation: involvement of cyclin-dependent kinase inhibitor p21 in polyploidization. Blood, 89: 3980-3990, 1997.[Abstract/Free Full Text]
  7. Matsumura I., Ishikawa J., Nakajima K., Oritani K., Tomiyama Y., Miyagawa J., Kato T., Miyazaki H., Matsuzawa Y., Kanakura Y. Thrombopoietin-induced differentiation of a human megakaryoblastic leukemia cell line, CMK, involves transcriptional activation of p21(WAF1/Cip1) by STAT5. Mol. Cell. Biol., 17: 2933-2943, 1997.[Abstract/Free Full Text]
  8. Taniguchi T., Endo H., Chikatsu N., Uchimaru K., Asano S., Fujita T., Nakahata T., Motokura T. Expression of p21(Cip1/Waf1/Sdi1) and p27(Kip1) cyclin-dependent kinase inhibitors during human hematopoiesis. Blood, 93: 4167-4178, 1999.[Abstract/Free Full Text]
  9. Garcia P., Cales C. Endoreplication in megakaryoblastic cell lines is accompanied by sustained expression of G1/S cyclins and downregulation of cdc25C. Oncogene, 13: 695-703, 1996.[Medline]
  10. Sarto G. E., Stubblefield P. A., Therman E. Endomitosis in human trophoblast. Hum. Genet., 62: 228-232, 1982.[Medline]
  11. MacAuley A., Cross J. C., Werb Z. Reprogramming the cell cycle for endoreduplication in rodent trophoblast cells. Mol. Biol. Cell, 9: 795-807, 1998.[Abstract/Free Full Text]
  12. Hattori N., Davies T. C., Anson-Cartwright L., Cross J. C. Periodic expression of the cyclin-dependent kinase inhibitor p57(Kip2) in trophoblast giant cells defines a G2-like gap phase of the endocycle. Mol. Biol. Cell, 11: 1037-1045, 2000.[Abstract/Free Full Text]
  13. Kudryavtsev B. N., Kudryavtseva M. V., Sakuta G. A., Stein G. I. Human hepatocyte polyploidization kinetics in the course of life cycle. Virchows Arch. B Cell Pathol. Incl. Mol. Pathol., 64: 387-393, 1993.[Medline]
  14. Watanabe T., Tanaka Y. Age-related alterations in the size of human hepatocytes. A study of mononuclear and binucleate cells. Virchows Arch. B Cell Pathol. Incl. Mol. Pathol., 39: 9-20, 1982.[Medline]
  15. Koike Y., Kamijyo K., Suzuki Y., Kiyosawa K., Nagata A., Furuta S., Nagata T. DNA content of hepatocytes in various stages of liver cirrhosis. Liver, 5: 156-161, 1985.[Medline]
  16. Crary G. S., Albrecht J. H. Expression of cyclin-dependent kinase inhibitor p21 in human liver. Hepatology, 28: 738-743, 1998.[Medline]
  17. Sigal S. H., Rajvanshi P., Gorla G. R., Sokhi R. P., Saxena R., Gebhard D. R., Jr, Reid L. M., Gupta S. Partial hepatectomy-induced polyploidy attenuates hepatocyte replication and activates cell aging events. Am. J. Physiol., 276: G1260-G1272, 1999.[Abstract/Free Full Text]
  18. Timchenko N. A., Wilde M., Kosai K. I., Heydari A., Bilyeu T. A., Finegold M. J., Mohamedali K., Richardson A., Darlington G. J. Regenerating livers of old rats contain high levels of C/EBP{alpha} that correlate with altered expression of cell cycle associated proteins. Nucleic Acids Res., 26: 3293-3299, 1998.[Abstract/Free Full Text]
  19. Albrecht J. H., Meyer A. H., Hu M. Y. Regulation of cyclin-dependent kinase inhibitor p21(WAF1/Cip1/Sdi1) gene expression in hepatic regeneration. Hepatology, 25: 557-563, 1997.[Medline]
  20. Wu H., Wade M., Krall L., Grisham J., Xiong Y., Van Dyke T. Targeted in vivo expression of the cyclin-dependent kinase inhibitor p21 halts hepatocyte cell-cycle progression, postnatal liver development and regeneration. Genes Dev., 10: 245-260, 1996.[Abstract/Free Full Text]
  21. Vranes D., Cooper M. E., Dilley R. J. Cellular mechanisms of diabetic vascular hypertrophy. Microvasc. Res., 57: 8-18, 1999.[Medline]
  22. Chobanian A. V., Prescott M. F., Haudenschild C. C. Recent advances in molecular pathology. The effects of hypertension on the arterial wall. Exp. Mol. Pathol., 41: 153-169, 1984.[Medline]
  23. Yan S. M., Finato N., Artico D., Di Loreto C., Cataldi P., Bussani R., Silvestri F., Beltrami C. A. DNA content, apoptosis and mitosis in transplanted human hearts. Adv. Clin. Path., 2: 205-219, 1998.[Medline]
  24. Beltrami C. A., Di Loreto C., Finato N., Yan S. M. DNA content in end-stage heart failure. Adv. Clin. Path., 1: 59-73, 1997.[Medline]
  25. Herget G. W., Neuburger M., Plagwitz R., Adler C. P. DNA content, ploidy level and number of nuclei in the human heart after myocardial infarction. Cardiovasc. Res., 36: 45-51, 1997.[Abstract/Free Full Text]
  26. Dominiczak A. F., Devlin A. M., Lee W. K., Anderson N. H., Bohr D. F., Reid J. L. Vascular smooth muscle polyploidy and cardiac hypertrophy in genetic hypertension. Hypertension, 27: 752-759, 1996.[Abstract/Free Full Text]
  27. Vliegen H. W., Eulderink F., Bruschke A. V., van der Laarse A., Cornelisse C. J. Polyploidy of myocyte nuclei in pressure overloaded human hearts: a flow cytometric study in left and right ventricular myocardium. Am. J. Cardiovasc. Pathol., 5: 27-31, 1995.[Medline]
  28. Agrotis A., Saltis J., Dilley R., Bray P., Bobik A. Transforming growth factor-ß 1 and the development of vascular hypertrophy in hypertension. Blood Press. Suppl., 2: 43-48, 1995.[Medline]
  29. Devlin A. M., Davidson A. O., Gordon J. F., Campbell A. M., Morton J. J., Reid J. L., Dominiczak A. F. Vascular smooth muscle polyploidy in genetic hypertension: the role of angiotensin II. J. Hum. Hypertens., 9: 497-500, 1995.[Medline]
  30. Servant M. J., Coulombe P., Turgeon B., Meloche S. Differential regulation of p27(Kip1) expression by mitogenic and hypertrophic factors: involvement of transcriptional and posttranscriptional mechanisms. J. Cell Biol., 148: 543-556, 2000.[Abstract/Free Full Text]
  31. Braun-Dullaeus R. C., Mann M. J., Ziegler A., von der Leyen H. E., Dzau V. J. A novel role for the cyclin-dependent kinase inhibitor p27(Kip1) in angiotensin II-stimulated vascular smooth muscle cell hypertrophy. J. Clin. Investig., 104: 815-823, 1999.[Medline]
  32. Auer G. U., Backdahl M., Forsslund G. M., Askensten U. G. Ploidy levels in nonneoplastic and neoplastic thyroid cells. Anal. Quant. Cytol. Histol., 7: 97-106, 1985.[Medline]
  33. Neal J. V., Potten C. S. Polyploidy in the murine colonic pericryptal fibroblast sheath. Cell Tissue Kinet., 14: 527-536, 1981.[Medline]
  34. Winkelmann M., Pfitzer P., Schneider W. Significance of polyploidy in megakaryocytes and other cells in health and tumor disease. Klin. Wochenschr., 65: 1115-1131, 1987.[Medline]
  35. Ikebe H., Takamatsu T., Itoi M., Fujita S. Changes in nuclear DNA content and cell size of injured human corneal endothelium. Exp. Eye Res., 47: 205-215, 1988.[Medline]
  36. Ikebe H., Takamatsu T., Itoi M., Fujita S. Age-dependent changes in nuclear DNA content and cell size of presumably normal human corneal endothelium. Exp. Eye Res., 43: 251-258, 1986.[Medline]
  37. Biesterfeld S., Gerres K., Fischer-Wein G., Bocking A. Polyploidy in non-neoplastic tissues. J. Clin. Pathol., 47: 38-42, 1994.[Abstract/Free Full Text]
  38. Jackman M. R., Pines J. N. Cyclins and the G2/M transition. Cancer Surv., 29: 47-73, 1997.[Medline]
  39. O’Connor P. M. Mammalian G1 and G2 phase checkpoints. Cancer Surv., 29: 151-182, 1997.[Medline]
  40. Nilsson I., Hoffmann I. Cell cycle regulation by the Cdc25 phosphatase family. Prog. Cell Cycle Res., 4: 107-114, 2000.[Medline]
  41. Weinert T. A DNA damage checkpoint meets the cell cycle engine. Science (Wash. DC), 277: 1450-1451, 1997.[Abstract/Free Full Text]
  42. Guadagno T. M., Newport J. W. Cdk2 kinase is required for entry into mitosis as a positive regulator of Cdc2-cyclin B kinase activity. Cell, 84: 73-82, 1996.[Medline]
  43. Poon R. Y. C., Jiang W., Toyoshima H., Hunter T. Cyclin-dependent kinases are inactivated by a combination of p21 and Thr-14/Tyr-15 phosphorylation after UV-induced DNA damage. J. Biol. Chem., 271: 13283-13291, 1996.[Abstract/Free Full Text]
  44. Tommasi S., Pfeifer G. P. In vivo structure of the human cdc2 promoter: release of a p130–E2F-4 complex from sequences immediately upstream of the transcription initiation site coincides with induction of cdc2 expression. Mol. Cell. Biol., 15: 6901-6913, 1995.[Abstract/Free Full Text]
  45. Liu N., Lucibello F. C., Engeland K., Muller R. A new model of cell cycle-regulated transcription: repression of the cyclin A promoter by CDF-1 and anti-repression by E2F. Oncogene, 16: 2957-2963, 1998.[Medline]
  46. Lucibello F. C., Liu N., Zwicker J., Gross C., Muller R. The differential binding of E2F and CDF repressor complexes contributes to the timing of cell cycle-regulated transcription. Nucleic Acids Res., 25: 4921-4925, 1997.[Abstract/Free Full Text]
  47. Zwicker J., Muller R. Cell cycle-regulated transcription in mammalian cells. Prog. Cell Cycle Res., 1: 91-99, 1995.[Medline]
  48. DeGregori J., Kowalik T., Nevins J. R. Cellular targets for activation by the E2F1 transcription factor include DNA synthesis-and G1/S-regulatory genes. Mol. Cell. Biol., 15: 4215-4224, 1995.[Abstract/Free Full Text]
  49. Harper J. W., Adami G. R., Wei N., Keyomarsi K., Elledge S. J. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell, 75: 805-816, 1993.[Medline]
  50. Toyoshima H., Hunter T. p27, a novel inhibitor of G1 cyclin-Cdk protein kinase activity, is related to p21. Cell, 78: 67-74, 1994.[Medline]
  51. Dulic V., Stein G. H., Far D. F., Reed S. I. Nuclear accumulation of p21Cip1 at the onset of mitosis: a role at the G2/M-phase transition. Mol. Cell. Biol., 18: 546-557, 1998.[Abstract/Free Full Text]
  52. Niculescu A. B., Chen X., Smeets M., Hengst L., Prives C., Reed S. I. Effects of p21(Cip1/Waf1) at both the G1/S and the G2/M cell cycle transitions: pRb is a critical determinant in blocking DNA replication and in preventing endoreduplication. Mol. Cell. Biol., 18: 629-643, 1998.[Abstract/Free Full Text]
  53. Barboule N., Lafon C., Chadebech P., Vidal S., Valette A. Involvement of p21 in the PKC-induced regulation of the G2/M cell cycle transition. FEBS Lett., 444: 32-37, 1999.[Medline]
  54. Baghdassarian N., Peiretti A., Devaux E., Bryon P. A., Ffrench M. Involvement of p27Kip1 in the G1- and S/G2-phase lengthening mediated by glucocorticoids in normal human lymphocytes. Cell Growth Differ., 10: 405-412, 1999.[Abstract/Free Full Text]
  55. Choi Y. H., Zhang L., Lee W. H., Park K. Y. Genistein-induced G2/M arrest is associated with the inhibition of cyclin B1 and the induction of p21 in human breast carcinoma cells. Int. J. Oncol., 13: 391-396, 1998.[Medline]
  56. King R. W., Deshaies R. J., Peters J., Kirschner M. W. How proteolysis drives the cell cycle. Science (Wash. DC), 274: 1652-1659, 1996.[Abstract/Free Full Text]
  57. Fang G., Yu H., Kirschner M. W. Control of mitotic transitions by the anaphase-promoting complex. Philos. Trans. R. Soc. Lond. B Biol. Sci., 354: 1583-1590, 1999.[Abstract/Free Full Text]
  58. Wheatley S. P., Hinchcliffe E. H., Glotzer M., Hyman A. A., Sluder G., Wang Yl. CDK1 inactivation regulates anaphase spindle dynamics and cytokinesis in vivo. J. Cell Biol., 138: 385-393, 1997.[Abstract/Free Full Text]
  59. Stewart Z. A., Leach S. D., Pietenpol J. A. p21(Waf1/Cip1) inhibition of cyclin E/Cdk2 activity prevents endoreduplication after mitotic spindle disruption. Mol. Cell. Biol., 19: 205-215, 1999.[Abstract/Free Full Text]
  60. Khan S. H., Wahl G. M. p53 and pRb prevent rereplication in response to microtubule inhibitors by mediating a reversible G1 arrest. Cancer Res., 58: 396-401, 1998.[Abstract/Free Full Text]
  61. Waldman T., Lengauer C., Kinzler K. W., Vogelstein B. Uncoupling of S phase and mitosis induced by anticancer agents in cells lacking p21. Nature (Lond.), 381: 713-716, 1996.[Medline]
  62. Bulavin D. V., Tararova N. D., Aksenov N. D., Pospelov V. A., Pospelova T. V. Deregulation of p53/p21Cip1/Waf1 pathway contributes to polyploidy and apoptosis of E1A+cHa-ras transformed cells after {gamma}- irradiation. Oncogene, 18: 5611-5619, 1999.[Medline]
  63. Winston J. T., Coats S. R., Wang Y. Z., Pledger W. J. Regulation of the cell cycle machinery by oncogenic ras. Oncogene, 12: 127-134, 1996.[Medline]
  64. Sherr C. J., Roberts J. M. CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev., 13: 1501-1512, 1999.[Free Full Text]
  65. Sherwood S. W., Kung A. L., Roitelman J., Simoni R. D., Schimke R. T. In vivo inhibition of cyclin B degradation and induction of cell-cycle arrest in mammalian cells by the neutral cysteine protease inhibitor N-acetylleucylleucylnorleucinal. Proc. Natl. Acad. Sci. USA, 90: 3353-3357, 1993.[Abstract/Free Full Text]
  66. Huchon D., Rime H., Jessus C., Ozon R. Control of metaphase I formation in Xenopus oocyte: effects of an indestructible cyclin B and of protein synthesis. Biol. Cell, 77: 133-141, 1993.[Medline]
  67. Townsley F. M., Aristarkhov A., Beck S., Hershko A., Ruderman J. V. Dominant-negative cyclin-selective ubiquitin carrier protein E2-C/UbcH10 blocks cells in metaphase. Proc. Natl. Acad. Sci. USA, 94: 2362-2367, 1997.[Abstract/Free Full Text]
  68. Garcia P., Frampton J., Ballester A., Cales C. Ectopic expression of cyclin E allows non-endomitotic megakaryoblastic K562 cells to establish re-replication cycles. Oncogene, 19: 1820-1833, 2000.[Medline]
  69. Datta N. S., Williams J. L., Caldwell J., Curry A. M., Ashcraft E. K., Long M. W. Novel alterations in CDK1/cyclin B1 kinase complex formation occur during the acquisition of a polyploid DNA content. Mol. Biol. Cell, 7: 209-223, 1996.[Abstract/Free Full Text]
  70. Nakayama K., Nagahama H., Minamishima Y. A., Matsumoto M., Nakamichi I., Kitagawa K., Shirane M., Tsunematsu R., Tsukiyama T., Ishida N., Kitagawa M., Hatakeyama S. Targeted disruption of Skp2 results in accumulation of cyclin E and p27(Kip1), polyploidy and centrosome overduplication. EMBO J., 19: 2069-2081, 2000.[Abstract]
  71. Zhang Y., Wang Z., Liu D. X., Pagano M., Ravid K. Ubiquitin-dependent degradation of cyclin B is accelerated in polyploid megakaryocytes. J. Biol. Chem., 273: 1387-1392, 1998.[Abstract/Free Full Text]
  72. Zimmet J. M., Ladd D., Jackson C. W., Stenberg P. E., Ravid K. A role for cyclin D3 in the endomitotic cell cycle. Mol. Cell. Biol., 17: 7248-7259, 1997.[Abstract/Free Full Text]
  73. Matsumura I., Tanaka H., Kawasaki A., Odajima J., Daino H., Hashimoto K., Wakao H., Nakajima K., Kato T., Miyazaki H., Kanakura Y. Increased D-type cyclin expression together with decreased cdc2 activity confers megakaryocytic differentiation of a human thrombopoietin-dependent hematopoietic cell line. J. Biol. Chem., 275: 5553-5559, 2000.[Abstract/Free Full Text]
  74. Palazon L. S., Davies T. J., Gardner R. L. Translational inhibition of cyclin B1 and appearance of cyclin D1 very early in the differentiation of mouse trophoblast giant cells. Mol. Hum. Reprod., 4: 1013-1020, 1998.[Abstract/Free Full Text]
  75. Gunning P., Leavitt J., Muscat G., Ng S-Y., Kedes L. A human ß-actin expression vector system directs high-level accumulation of antisense transcripts. Proc. Natl. Acad. Sci. USA, 84: 4831-4835, 1987.[Abstract/Free Full Text]
  76. Santos E., Tronick S. R., Aaronson S. A., Pulcianni S., Barbacid M. T24 human bladder carcinoma oncogene is an activated form of the normal human homologue of BALB and Harvey-MSV transforming genes. Nature (Lond.), 298: 343-347, 1982.[Medline]
  77. Krishan A. Rapid flow cytometric analysis of the mammalian cell cycle by propidium iodide staining. J. Cell Biol., 66: 188-193, 1975.[Abstract/Free Full Text]
  78. Shapiro H. eds. . Practical Flow Cytometry, Alan R. Liss, Inc. New York 1988.
  79. Sambrook J. Fritsch E. F. Maniatis T. eds. . Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory NY 1989.



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