SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Functions of p53 during mitosis reportedly include prevention of polyploidy and transmission of aberrant chromosomes. However, whether p53 plays these roles during genomic surveillance in vivo and, if so, whether this is done via direct or indirect means remain unknown. The ability of normal, mature hepatocytes to respond to stimuli, reenter the cell cycle, and regenerate liver mass offers an ideal setting to assess mitosis in vivo. In quiescent liver, normally high ploidy levels in adult mice increased with loss of p53. Following partial hepatectomy, p53−/− hepatocytes exhibited early entry into the cell cycle and prolonged proliferation with an increased number of polyploid mitoses. Ploidy levels increased during regeneration of both wild-type (WT) and p53−/− hepatocytes, but only WT hepatocytes were able to dynamically resolve ploidy levels and return to normal by the end of regeneration. We identified multiple cell cycle and mitotic regulators, including Foxm1, Aurka, Lats2, Plk2, and Plk4, as directly regulated by chromatin interactions of p53 in vivo. Over a time course of regeneration, direct and indirect regulation of expression by p53 is mediated in a gene-specific manner. Conclusion: Our results show that p53 plays a role in mitotic fidelity and ploidy resolution in hepatocytes of normal and regenerative liver. (HEPATOLOGY 2013)

Chromosomal polyploidy presents a considerable challenge to the orderly process of mitosis. There are normal tissues and cells in both vertebrates and invertebrates that display polyploidy during development or as fully differentiated tissues. How mitotic fidelity is maintained in these cells is a question of considerable interest. Recent studies in Drosophila establish that polyploid chromosomes of larval rectal cells are faithfully duplicated and segregated through multiple cell cycles during the course of normal development.1 Although the division of these polyploid cells progresses through normal, recognizable stages, the time course of each is extended, and the process is highly error-prone. Genome instability and aneuploidy may be one cost of maintenance and proliferation of polyploid cells, as a substantial number of chromosomal abnormalities arise in these cells.

Hepatocytes of the mammalian liver develop polyploidy and aneuploidy over the life span of the organism. Hepatocytes can be mononucleated or binucleated, and each nucleus can have diploid, tetraploid, octaploid, or higher nuclear content.2 Polyploidization occurs via failed cytokinesis or endoreduplication.2 Moreover, proliferating polyploid hepatocytes undergo chromosome segregation errors, generating a high degree of aneuploidy. Approximately 60% of adult wild-type (WT) mouse hepatocytes are aneuploid, and 30% to 90% of hepatocytes in humans are aneuploid.3, 4 Hepatocytes are highly tolerant of nuclear alterations, undergoing cycles of ploidy expansion, ploidy reversal, and aneuploidy, described as the “ploidy conveyor.”3 Hepatocyte polyploidy may be further expanded during liver regeneration induced by a two-thirds partial hepatectomy (PH) in mice.5, 6 Given that a polyploid mitotic division may lead to increased aneuploidy and possibly tumor development,7, 8 it remains unclear how these hepatocytes remain mitotically active and accumulate chromosomal instability without becoming tumorigenic.

Genome integrity is protected in normal cells by various means, including cell cycle checkpoints, DNA repair mechanisms and the induction of apoptosis. A critical player in each of these processes is the p53 tumor suppressor, which provides surveillance against cellular insults of many types and may induce G1 arrest and cellular senescence in response to tetraploidy or missegregated chromosomes.9, 10 p53 and its close relative, p73, are also linked to the mitotic spindle assembly checkpoint, as both proteins interact with kinetochore and spindle checkpoint proteins.7, 11, 12 In fact, combined loss of p53 and p73 leads to increased polyploidy and aneuploidy in primary cultured cells9 and results in a higher incidence of tumor development in mouse liver.13

The striking tolerance of the liver for altered ploidy leads to consideration of whether a tetraploid checkpoint exists in hepatocytes. We addressed this question by analysis of checkpoint mediator p53, and characterized the synchronized process of cellular proliferation and growth that occurs to regenerate the liver in response to PH in both WT and p53-null mice. Our results reveal that p53 alters levels of hepatocyte ploidy during liver regeneration and aging. Although chromosome segregation errors are common in WT hepatocytes expressing p53, these errors (e.g., abnormal mitotic figures and lagging chromosomes) are even more frequent in hepatocytes deficient for p53. Since p53′s effects may be mediated by context-specific, mitotic regulators,14 we examined whether p53 regulated expression of mediators of hepatic cell division in normal and regenerating liver. We identified Aurora kinase A (Aurka), Forkhead-box transcription factor Foxm1, regulator of cytokinesis Lats2, and Polo-like kinases (Plk2 and Plk4) as directly regulated by p53 in quiescent liver, a mitotic transcription program that is altered during liver regeneration. Thus, our findings suggest that p53 plays a role in controlling levels of hepatic polyploidy/aneuploidy by direct transcription regulation of multiple downstream effectors.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Liver Regeneration in Mice.

PH to remove 70% of total liver tissue, or Sham surgery was performed using isoflurane anesthesia, as described.15 5-7 C57Bl6/Sv129 F1 mice, WT or p53−/−, 2 months of age, were used for each experimental condition according to The University of Texas MD Anderson Cancer Center Institutional Animal Care and Use Committee guidelines. p53 knockout mice were sacrificed 0.5, 1, 1.5, 2, 3, 3.5, 4, or 7 days following PH and sham surgery; remnant liver tissue was harvested, flash-frozen, and processed for RNA, immunoblot, and chromatin immunoprecipitation (ChIP) analyses. Liver/body weight ratios were calculated to determine the recovery of liver mass.

ChIP and RNA Analyses.

ChIP analyses were performed as described.15 Briefly, chromatin lysate was precleared and incubated overnight with the following specific antibodies for ChIP: p53 (Novocastra) and normal sheep immunoglobulin G (Upstate/Millipore). To analyze protein-bound DNA, primers for real-time quantitative polymerase chain reaction (PCR) were used (Supporting Table). The percentage of the input that was bound was calculated using the ΔCt method and averaged for at least three biological replicates.

Primers and reverse-transcription PCR determinations of RNA expression were performed as described.15 Briefly, total RNA from homogenized mouse liver was extracted with TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Complementary DNA was obtained by reverse transcription of 2 μg of RNA using the SuperScript system (Invitrogen). Real-time PCR was performed using primers for the indicated genes (Supporting Table).

Histology and Immunohistochemistry.

Collected liver samples were fixed in 10% neutral buffered formalin (Fisher), embedded in wax paraffin, and sectioned at 5-6 mm by the University of Texas MD Anderson Cancer Center Department of Veterinary Medicine and Surgery. Sections were rehydrated and stained with hematoxylin and eosin or with anti-Ki67 (Abcam) marker and then counterstained with hematoxylin following the manufacturers' recommended protocols. Antigen retrieval and immunodetection was performed using solutions from Vector Labs according to the manufacturer's instructions (Vector Laboratories). Endogenous peroxidase activity was quenched by incubating slides in a methanol/H2O2 solution.

Western Blot.

Immunoblotting was performed using standard sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blot methodology.15 Protein extracts from liver tissue collected at 0, 12, 24, 48, 72, 84, 96, and 168 hours following PH and 12 hours following sham surgery were prepared using T-PER buffer (Pierce/Thermoscientific) according to the manufacturer's instructions.

Flow Cytometry Analysis.

Hepatocytes were isolated from adult 129 p53+/+, p53+/−, and p53−/− littermates via collagenase perfusion.16 Live hepatocytes were loaded with Hoechst33342 (Invitrogen) and DNA content was determined with an InFlux flow cytometer (Beckton Dickinson) using a 150-μm nozzle as described.3 The Institutional Animal Care and Use Committee of Oregon Health & Science University approved the experiments.

To quantify DNA content (ploidy profiling), hepatocytes were isolated from livers after PH and sham surgery via collagenase perfusion.16 Cells were fixed by gentle vortexing in ice-cold 80% (vol/vol) ethanol and stained with 50 μg/mL propidium iodide in 0.2% Tween in phosphate-buffered saline supplemented with 1 μg/mL (wt/vol) RNAse A (Molecular Probes). Cytofluorometric acquisitions were performed on a FACSCalibur flow cytometer (BD Biosciences). Statistical analysis was performed with CellQuest software (BD Biosciences), upon gating on the events characterized by normal forward scatter and side scatter parameters.

Statistical Analysis.

Results are expressed as the mean ± SEM. Statistical analyses were performed by Student t test; P < 0.05 was considered significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Lack of p53 Disrupts Ploidy Control in Proliferating Hepatocytes.

The ploidy of murine hepatocytes increases during development, liver disease, aging, and regeneration.6, 17 This tolerance of polyploidy suggests that specific checkpoints, which either maintain a diploid state and/or eliminate cells that exhibit altered ploidy, may be lacking in hepatic tissue. Although p53 is implicated in mitotic surveillance of cultured immortalized and tumor-derived cells,18, 19 this has not been assessed during normal development or under conditions of induced cellular proliferation and tissue regeneration. To address this, we determined the ploidy status of live WT (p53+/+) and p53-null hepatocytes during normal development by flow cytometry and analysis of DNA content (Fig. 1A). Consistent with previous studies of p53+/+ mouse liver, we observed that 60% of total hepatocytes in quiescent, 4- to 5-month-old p53+/+ liver were tetrapoloid (4c), with a second, major population of diploid cells (2c, ∼30%) and a smaller fraction of octaploid cells (8c, ∼10%). However, in quiescent p53−/− mouse liver of the same age, less than 50% of hepatocytes were tetraploid, and many were octaploid (>30%). Concomitantly, there was a significantly reduced number of diploid cells. This distribution in ploidy was dependent on p53 dosage, as indicated by an intermediate ploidy phenotype in heterozygous, p53+/− hepatocytes. These data suggest that hepatocyte ploidy, during normal growth and development of the liver, is monitored by a p53-dependent process.

thumbnail image

Figure 1. p53 controls parenchymal cell ploidy, size, and cell number in the liver. (A) Distribution of hepatic ploidy populations in p53+/+, p53+/−, and p53−/− mice. The frequency of 2c, 4c, and 8c populations was determined in live hepatocytes from p53+/+(n = 3), p53+/− (n = 9), and p53−/− (n = 4) littermates aged 4-5 months via fluorescence-activated cell sorting analysis. (B) Ki67(+) hepatocytes with nuclei that failed to form completely separated nuclear envelopes were counted in 10 fields at 48 hours after PH. The percentage of binucleated hepatocytes was significantly higher in p53−/− compared with WT liver sections, as determined by Student t test. *P < 0.05. (C) The frequency of hepatocytes with 16c nuclear content was determined in fixed cells isolated from livers of p53+/+ and p53−/− mice at 72 hours and 7 days after PH. Significant difference in number of 16c hepatocytes was determined by Student t test. ***P < 0.001. (D) Hematoxylin and eosin staining of liver tissue sections collected at 7 days following PH in p53+/+ and p53−/− mice was used to count hepatocyte nuclei. Ten fields per sample were counted, and the difference in the number of hepatocyte nuclei in view between p53+/+ and p53−/− was determined via Student t test. **P < 0.01. Representative photographs of hematoxylin and eosin–stained p53+/+ and p53−/− liver tissue at 7 days after PH were taken at 20× and 40× magnification. The similar size of p53−/− hepatocytes at 20× and of p53+/+ hepatocytes at 40× magnification suggests that p53−/− hepatocytes have at least 2× larger nuclei.

Download figure to PowerPoint

To determine whether p53 acts in mitotic surveillance during acute injury response, we used a model of surgically induced growth and replacement of liver tissue. We compared 2-month-old p53+/+ and p53−/− mice, which have fewer differences in ploidy at t = 0 than 4- to 5-month-old mice (data not shown and Fig. 1A). Two-thirds PH of mouse liver elicited a synchronized wave of cell cycle re-entry, proliferation, mitosis, and growth in the remnant liver, to regenerate and restore the size of the liver (liver/body weight ratio or liver index) to its presurgical set point (Supporting Fig. 1A).20 In situ staining of the DNA replication marker Ki67 revealed dividing hepatocytes at 48 hours after two-thirds PH in p53+/+ and p53−/− mice (Fig. 1B, left panel). Strikingly, binucleated Ki67(+) p53−/− hepatocytes were present at four-fold higher numbers than in p53+/+ liver (Fig. 1B, right panel), suggesting enhanced proliferation and/or cytokinesis failure. To examine ploidy, we analyzed nuclear content at various times following PH. Whereas nuclear content is equivalent to ploidy class in quiescent adult livers (e.g., 4c DNA = tetraploid cell),3 nuclear content in regenerating livers is complicated by ploidy class and cell cycle status. For instance, in proliferating hepatocytes, 4c DNA content indicates either a tetraploid cell in G0/G1 or a diploid cell in G2. Therefore, to focus exclusively on polyploid hepatocytes, we examined cells with nuclear content of 8c or higher. Compared with p53+/+ mice, the frequency of 8c hepatocytes was higher in p53−/− sham livers, and the population expanded by 48 hours after PH (Supporting Fig. 1B). Even more dramatic changes were seen in hepatocytes with 16c DNA content. In p53+/+ mice, regenerative proliferation after PH led to a small 16c population (Fig. 1C). However, the population of 16c hepatocytes in p53+/+ mice, which was clearly observed 72 hours after PH, diminished with restoration of liver mass (7 days after PH) (Fig. 1C). In contrast, the number of 16c hepatocytes in p53−/− regenerating liver continued to increase over time, resulting in a 50-fold increase in 16c hepatocytes compared with p53+/+ at the termination stage of liver regeneration (Fig. 1C). These data suggest that p53 regulates the formation and maintenance of polyploidy even in cells that are highly tolerant of polyploidy and aneuploidy.

To fully characterize cellular changes associated with hepatocyte proliferation, we also examined cellular and nuclear size. Analysis of sections of liver tissue isolated at the end of regeneration (day 7) revealed major differences between p53+/+ and p53−/− mice (Fig. 1D). p53−/− hepatocytes were significantly larger, resulting in fewer cells per field-of-view (Fig. 1D, left panel). Moreover, larger hepatic nuclei were observed in p53−/− mice (Fig. 1D, right panel), which is consistent with the high degree of polyploidy in p53−/− mice.

A lack of uniformity in increased cell size at day 7 after PH in p53−/− mice (Fig. 1D) suggests a possibility of liver overgrowth. However, in response to the challenges of cell division and growth after PH, both p53+/+ and p53−/− hepatocytes achieved a similar recovery of liver mass (Supporting Fig. 1A), despite their differences in cell size and ploidy (Fig. 1C,D). These data extend a recent report, showing the impact of increased hypertrophy of WT hepatocytes during liver regeneration,21 and link increases in ploidy to hypertrophy in p53−/− mice after PH.

p53 Regulates Mitotic Entry and Exit During Liver Regeneration.

To determine whether p53+/+ and p53−/− hepatocytes had comparable levels of proliferation after PH, we performed in situ staining of Ki67 over a time course of regeneration (Fig. 2A). Consistent with previous measurements by bromodeoxyuridine incorporation,20 p53+/+ mouse liver entered an initial period of cellular proliferation after 24 hours, reached a maximum at day 2 (48 hours), and engaged more than 80% of all remnant hepatocytes. In addition, we observed a second round of DNA synthesis that occurred at day 4 after PH, which involved 46% of hepatocytes in p53+/+ liver (Fig. 2A). In comparison, p53−/− liver had an earlier onset of cellular proliferation, less than 24 hours after PH, followed by a broadened span of proliferation over 2-3.5 days that involved only 63% of remnant hepatocytes. In p53−/− liver, a second, less distinct peak of proliferation occurred 12 hours earlier than the second proliferation wave in p53+/+ liver, followed by a significant number of p53−/− hepatocytes exiting mitosis at day 4 after PH (Fig. 2A).

thumbnail image

Figure 2. Proliferation of hepatocytes during liver regeneration is controlled by p53. (A) Tissue sections from livers collected at the indicated time points following PH and sham surgeries were stained with Ki67 antibody, followed by hematoxylin staining to reveal nonproliferating nuclei. Positive Ki67(+) staining was determined by immunohistochemistry. Ki67(+) hepatocyte nuclei and (hematoxylin only–stained) hepatocyte nuclei were counted in 10 fields for each time point. A significant difference between Ki67(+) hepatocytes between p53+/+ and p53−/− was determined via Student t test separately at each time point. *P < 0.05. **P < 0.01. ***P < 0.001. Sham livers did not show any Ki67(+) cells. (B) Representative photographs of mitotic figures in p53+/+ and p53−/− regenerating livers collected at the indicated time points after PH. Hematoxylin and eosin staining of liver tissue sections collected at 48, 72, and 96 hours after PH was used to count mitotic figures. Ten fields per sample were counted, and lagging chromosomes and multipolar mitotic spindles were determined visually based on criteria discussed elsewhere3, 10 and shown as a percentage of the total mitotic figures in the field. The increase in the number of dividing cells with lagging chromosomes in p53−/− livers compared with p53+/+ was statistically significant at 48 hours (P < 0.01) and 72 hours (P < 0.05); the number of p53−/− hepatocytes with multipolar spindles was significantly higher at 48 hours (P < 0.05).

Download figure to PowerPoint

To assess whether p53−/− hepatocytes were progressing through mitosis over a time course of regeneration or, alternatively, undergoing endoreduplication where cells undergo multiple rounds of DNA replication without entering mitosis, which may lead to formation of polyploidy,22 we determined gain or loss of specific phosphorylation sites that mark activated cyclin-dependent kinase 1 during mitotic transition.2 This analysis revealed similar profiles in WT and p53−/− liver, supporting S-phase replication followed by mitotic entry/transition, and not endoreduplication, during regeneration (Supporting Fig. 2A).

p53 Deficiency Leads to Increased Mitotic Errors.

We next compared the number and appearance of mitotic figures in regenerating livers from p53+/+ and p53−/− livers. As reported, p53+/+ hepatocytes display multipolar spindles and lagging chromosomes during regenerative proliferation.3 During normal growth and in response to PH, approximately 95% of multipolar spindles resolve into bipolar spindles in polyploid WT hepatocytes, generating daughter cells with ploidy levels equivalent to the parental cell. Furthermore, cell divisions by polyploid hepatocytes can generate daughter cells with reduced ploidy3, 5 (Fig. 1). Similar to WT livers, regenerating p53−/− livers also had abnormal mitotic figures and lagging chromosomes, but the frequency of these events was higher (Fig. 2B and Supporting Fig. 2B). Together, these data indicate that increases in nuclear segregation errors by p53−/− hepatocytes correlate with the altered ploidy levels seen in p53−/− livers.

p53 Binds to Consensus Motifs in Mitotic Targets and Regulates Their Expression in Liver.

The majority of p53 functions are attributed to its ability to regulate transcription of target genes. p53 has transcriptional activity in quiescent liver,23, 24 but direct target genes involved in hepatic cell division are unknown. Using a previously determined consensus DNA sequence for p53 binding (p53 response element [p53RE]),15 we assessed genes implicated in the regulation of mitotic progression and fidelity for potential p53 binding sites within 10 kilobases upstream and downstream of transcription start sites. We identified p53REs in seven genes encoding major mitotic regulators: Aurora kinase A (Aurka), Forkhead-box transcription factor Foxm1, regulator of cytokinesis Lats2, and Polo-like kinases (Plk1, Plk2, Plk4, and Plk5) (Fig. 3A). ChIP with a p53 antibody detected significant binding of p53 to five p53REs of these genes in WT liver: Aurka, Foxm1, Lats2, Plk2, and Plk4 (Fig. 3B). Interestingly, motif analysis of the p53REs of these genes revealed general but not perfect agreement with the “canonical” consensus of p53-bound response elements, derived primarily from in vitro studies15 (Supporting Fig. 3A).

thumbnail image

Figure 3. p53 binds the p53REs of mitotic target genes and regulates their expression in normal quiescent liver. (A) p53-DNA binding motifs (p53 response elements, p53REs) were found upstream of the transcription start site of seven target genes using an algorithm.37 Two half-sites are shown in uppercase letters, with conserved core (CA/TG) in bold, and a spacer sequence is in lowercase letters (if present). Locations of the identified p53REs are shown as a distance from the transcription start site (TSS). (B) p53 binds to the p53RE of Aurka, Foxm1, Lats2, Plk2, and Plk4 in quiescent liver. ChIP from adult mouse liver tissue was performed using antibodies against p53. Primers used for consequent ChIP experiments were specific to the p53RE sites indicated in panel A. Binding of p53 to the previously identified p53RE of Foxo3 served as a positive control (data not shown). The nonspecific region located 2 kb away from Plk1 p53RE was used as a negative control. Average of at least three independent ChIPs is shown as percentage of input bound. *P < 0.05, **P < 0.01 (p53 binding to p53REs versus the nonspecific site). (C) Aurka, Foxm1, Lats2, Plk2, and Plk4 messenger RNA levels in p53+/+ and p53−/− mice were compared using real-time PCR. (B and C) *P < 0.05, **P < 0.01 and ***P < 0.001 (p53+/+ versus p53−/−).

Download figure to PowerPoint

Because p53 may regulate transcription of target genes as either a direct repressor or activator,25 we compared expression of the p53-bound genes in p53+/+ and p53−/− liver (Fig. 3C). Expression of Aurka was up-regulated six-fold in p53−/− liver compared with p53+/+, suggesting that p53 repressed Aurka expression in normal quiescent liver. In contrast, Foxm1, Lats2, and Plk4 were strongly down-regulated in p53−/− mice, indicating that p53 activated expression of these genes in normal quiescent liver. Plk2 messenger RNA levels did not change significantly in p53−/− livers compared with p53+/+, indicating that p53 binding to Plk2 p53RE may not regulate basal expression of this gene in the quiescent liver.

To determine whether p53 regulates each of these genes during the process of liver regeneration, we examined expression at time points after PH (Fig. 4). First, expression of Aurka in the WT liver dramatically increased during the first round of mitosis at 24 and 48 hours after PH, whereas p53 was still bound to the Aurka p53RE (Fig. 4A,B). These results suggest that p53 deficiency could lead to elevated Aurka expression (i.e., loss of p53-mediated Aurka inhibition) during regeneration. However, we found reduced Aurka levels in p53−/− livers compared with WT at 24 and 48 hours after PH, indicating that Aurka expression is independent of p53 through the first round of mitosis (Fig. 4B). Interestingly, expression of Aurka was significantly up-regulated at the end of liver regeneration in p53−/− liver (day 7; Fig. 4B). This finding suggests that p53-mediated repression of Aurka expression, observed in normal quiescent liver (Fig. 3), was re-established with cessation of liver regeneration. Indeed, we observed an increase in p53 binding to the Aurka p53RE at the later time points after PH (72-96 hours; Fig. 4A).

thumbnail image

Figure 4. p53 regulates mitotic genes during liver regeneration. (A) Levels of p53 binding to the target genes during the proliferative phase of liver regeneration (24, 48, 72, and 96 hours) compared with sham (as described in Fig. 3). (B-F) Expression of Aurka (B), Foxm1 (C), Plk2 (D), Plk4 (E), and Lats2 (F) in p53+/+ and p53−/− regenerating livers. RNA was isolated from mouse liver tissue at the indicated time points after PH and sham surgeries. Messenger RNA levels were measured by real-time PCR and calculated as a fold change over sham for each time point. *P < 0.05, **P < 0.01, ***P < 0.001 (p53+/+ versus p53−/− regenerating livers).

Download figure to PowerPoint

Second, expression analysis of Foxm1, which has been reported as a critical regulator of the G2-M transition in regenerating liver,26 showed p53-dependent and -independent changes over a time course of regeneration (Fig. 4A,C). p53-independent, compensatory mechanisms activated expression of Foxm1 during the onset of the first cell cycle. At the onset of the second wave of hepatic proliferation, these mechanisms did not provide compensation, and significantly decreased Foxm1 expression occurred in p53−/− liver (Fig. 4C). We observed binding of p53 to the Foxm1 p53RE at 72 hours after PH (Fig. 4A), together with high levels of expression of Foxm1 at 72-84 hours after PH (second round of mitosis). The increase in Foxm1 expression was not detected in p53−/− liver after the first round of mitosis (Fig. 4C), when hepatocyte proliferation was significantly lower compared with WT mice (Fig. 2A). Thus, p53-mediated activation of Foxm1 may be necessary for the onset of the second cell cycle during liver regeneration.

Third, Polo-like kinases are known positive regulators of proliferation at all stages of the cell cycle.27 p53 binding to the Plk2 and Plk4 p53REs at 24, 48, 72, and 96 hours after PH was measurable (Fig. 4A), but Plk2 and Plk4 levels changed inconsistently with p53 deficiency, suggesting that p53 is not a primary driver of hepatic Plk2 and Plk4 expression during liver regeneration (Fig. 4D).

Finally, Lats2 is a negative regulator of cell growth and a tissue-specific inhibitor of cell size.28-30 Similar to Foxm1, p53 remained bound to a Lats2 p53RE at 24 hours after PH (Fig. 4A). However, p53-independent mechanisms compensate for loss of p53 regulation of Lats2 during regeneration of the liver (Fig. 4F). In quiescent liver, the data indicate that both mitotic activators and negative regulators of cell growth are directly regulated by p53, as either a gene-specific activator or repressor of transcription. During liver regeneration, however, regulation of specific p53 target genes and mitotic regulators, cyclin B and Aurora kinase B that lack p53REs (Supporting Fig. 3B), is complex and p53-dependent or -independent at specific time points.

Our results demonstrate that p53 directly binds the p53REs of genes regulating the cell cycle in regenerating mouse liver. While Plk2, Plk4, and Lats2 may be additionally regulated by a p53-independent mechanism, expression of Aurka and Foxm1 is directly regulated by p53 at specific time points following PH. Importantly, p53-mediated activation of Foxm1 occurs at the onset of the first as well as the second mitotic division of hepatocytes after PH. p53-mediated repression of Aurka in quiescent hepatocytes is diminished at the peak of proliferative stage, and partially regained at the cessation of liver regeneration.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Aneuploidy and polyploidy are detected in multiple types of cancer, and they are clearly linked to neoplasia and tumor development.7 However, recent studies show that evolutionarily conserved, but poorly understood, mechanisms allow replication of polyploid chromosomes without generation of genomic instability in “ploidy-tolerant” cells.1, 3 Such cells are found in significant numbers, which increase with age, in normal liver and exist in a polyploid state without progression to a dysplasia. Polyploid hepatocytes use dynamic aneuploidization and polyploidization to support homeostasis or overcome environmental stress by a process termed the ploidy conveyor.3 Likely candidates for regulation of the ploidy conveyor are members of the p53 family, such as p53 and p73 isoforms, which can prevent propagation of aneuploid and polyploid cultured cells.7, 9 We found that p53 impacts ploidy levels in the liver: normal ratios of diploid, tetraploid and octaploid hepatocytes in adult mouse liver were significantly skewed toward octaploidy in the absence of p53, at the expense of diploid and tetraploid hepatocyte populations. Intriguingly, PH-induced liver regeneration, which challenges the normally quiescent, polyploid hepatocytes to undergo cell cycle re-entry, mitosis, and growth, accelerates the ability of oncogenes to induce HCC and may predispose liver to cancer development.31 Despite known deletions and mutations of p53 in hepatocellular carcinoma (HCC),32 it was previously unknown whether p53 played any role in liver regeneration linked to genomic instability. Our findings demonstrate that absence of p53 reduces ploidy resolution and regeneration of p53−/− liver relies more on increased cell size, due to overrepresentation of polyploid hepatocytes. Thus, loss of p53 functions and accumulative effects on ploidy during cycles of regenerative repair may accelerate liver tumorigenesis and decrease time of progression to HCC.

Polyploid WT hepatocytes form multipolar spindles and have lagging chromosomes during mitotic divisions Here, we show that this process involves p53-dependent regulation of transcription during normal liver development and regeneration. In response to regenerative signaling, multipolar spindles and lagging chromosomes were seen in both WT and p53−/− hepatocytes, but these abnormal mitotic figures were observed in higher numbers in p53−/− mice. We speculate that elevated frequency of nuclear segregation errors in p53−/− hepatocytes contributed to cytokinesis failure and, therefore, enhanced polyploidization.10 Our observation of an orderly progression of mitosis, as marked by comparable activation of Cdk1/cdc2 in WT and p53−/− hepatocytes, suggests that endoreduplication does not contribute to higher polyploidy with p53 deficiency.

Questions arise as to whether a mitotic checkpoint exists in hepatocytes, in light of this fluidity of ploidy numbers. Although the liver exhibits dynamic changes in levels of polyploidy during aging and regeneration, it is clear that p53 exercises some level of control over this process. We uncovered a network of ploidy determinants, which are direct gene targets of p53, regulated in quiescent liver and responsive in a gene-specific manner to regenerative signaling. To our knowledge, this report is the first description of p53 binding to these newly identified p53REs and, more specifically, to cell cycle regulators during mitotic division in vivo. Our results reveal p53-dependent differences in the expression of genes that regulate mitotic entry and progression (Aurka, Foxm1), division (Plk2, Plk4), and exit back to the G0 (Lats2). Importantly, we identified Foxm1 and Aurka genes as new direct transcriptional targets of p53. Our data demonstrate that binding of p53 to Foxm1 p53RE occurs specifically at the onset of the first and the second round of mitosis (24 and 72 hours after PH) resulting in the robust activation of the Foxm1 expression, which is essential for DNA replication and mitosis in regenerating hepatocytes.26 The direct repression of Aurka by p53 in quiescent liver may be necessary to suppress the tumor-promoting consequences of the overexpression of Aurora kinase A in liver.33, 34 The overall results of our p53 ChIP and target gene expression analyses demonstrate that transcriptional regulation by p53 is necessary, whether by direct or indirect means, for timely activation or repression of specific target genes at different stages of the cell cycle (Fig. 5).

thumbnail image

Figure 5. Modes of p53-dependent regulation of mitotic target genes and outcomes of p53 loss during regenerative proliferation of hepatocytes. Aurora A is frequently overexpressed in HCC in correlation with mutation of p53 and poor prognosis.33 Lats2 is associated with regulated cytokinesis: depletion of Lats2 induces accumulation of polyploid cells.28 The Foxm1 transcription factor is activated during liver regeneration26 to impact timely mitotic entry and accurate chromosome segregation.38 Plk4+/− liver has lower activation of p53, aberrant mitotic figures after PH, and develops HCC.39 Plk2 shows loss of heterozygocity in HCC and is a critical regulator of G1-S transition and mitosis during cell proliferation.27, 40 Aurora A, Foxm1, Lats2 and specific Plk's are regulators of mitosis that impact ploidy levels in a p53-dependent manner.

Download figure to PowerPoint

Cell division is a highly conserved process, but there are clearly tissue-specific modes of regulation. In other cell systems (i.e., nonhepatocytes), p53-mediated suppression of Aurka prevents premitotic arrest,34 and basal activation of Lats2 minimizes aneuploidy/polyploidy. In hepatocytes, cell division is complex, because polyploidy and aneuploidy are extremely high in p53+/+ livers from mice3 and humans.4 Nonetheless, disruption of normal Aurka and Lats2—and to a lesser extent Foxm1 and Plk4—expression partially accounts for enrichment in mitotic segregation errors and enhanced polyploidy seen in p53-deficient liver. After PH, transcriptional activity of p53 and how it contributes to activation or repression of mitotic or cell cycle regulators is more difficult to interpret. There may be a partial compensation by TA-p73, which has been shown to play a role in liver tumor suppression in combination with p53.35

A fully delineated story of how hepatocytes survive, and even thrive, in spite of high levels of polyploidy and aneuploidy is not yet clear. p53 and its downstream effectors contribute to polyploidization and mitotic fidelity, as shown here in vivo. Whether p53 regulation is connected to activation of the insulin receptor and AKT signaling, implicated in cytokinesis failure and formation of polyploid hepatic cells,36 is unknown. Further characterization of new hepato-specific cell cycle pathways and definition of regulatory mechanisms are critical to understanding development, homeostasis, regeneration, and pathology of the liver.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
HEP_26233_sm_SuppFig1.tif1361KSupporting Information Figure 1.
HEP_26233_sm_SuppFig2A.tif1024KSupporting Information Figure 2A.
HEP_26233_sm_SuppFig2B.tif4208KSupporting Information Figure 2B.
HEP_26233_sm_SuppFig3.tif5654KSupporting Information Figure 3.
HEP_26233_sm_SuppTab1.doc33KSupporting Information Table 1.

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.