|Home | About | Journals | Submit | Contact Us | Français|
The cellular homologues Mdm2 and MdmX play critical roles in regulating the activity of the p53 tumor suppressor in damaged and non-damaged cells and during development in mice. Recently, we have utilized genetically defined primary cells and mice to reveal that endogenous levels of MdmX can also suppress multipolar mitosis and transformation in hyperploid p53-deficient cells and tumorigenesis in p53-deficient mice. These MdmX functions are not shared by Mdm2, and are distinct from the well-established ability of MdmX to complex with and inhibit p53 activity. Here we discuss some of the ramifications of MdmX loss in p53-deficient cells and mice, and we explore further the fate of MdmX/p53-double null embryonic fibroblasts undergoing multi-polar cell division using time-lapse video microscopy. We also discuss the relationship between chromosomal loss, cell proliferation, and the tumorigenic potential of p53-deficient cells lacking MdmX.
The p53 transcription factor is activated by inappropriate cell growth stimulation or by certain types of DNA damage and it regulates the expression of other genes involved in cell growth and death, DNA repair, and chromosomal stability.1 The importance of these p53 functions in preventing tumorigenesis is underscored by findings that mice deleted for p53 form spontaneous tumors with 100% penetrance and that mutation of the p53 gene or the p53-signaling pathway is the most common genetic alteration identified in human cancer.2 The ability of p53 to arrest cell growth and induce cell death following genetic or metabolic insult or during development and in normal cell homeostasis is kept tightly in check by cellular proteins that bind with p53 and downregulate p53 activity. Chief among these p53 inhibitors are the cellular homologues Mdm2 and MdmX.
Mdm2 was initially cloned as one of several genes present on a mouse double minute chromosome found in a spontaneously transformed derivative of a NIH3T3 cells. Subsequently, Mdm2 was found to complex with p53 and negatively regulate p53-induced transcription of several target genes, including the Mdm2 gene itself.3 Although Mdm2 is ubiquitously expressed at a low level, p53 strongly upregulates Mdm2 gene expression following DNA damage by binding to a p53 response element within the first intron of the Mdm2 gene.4 Induction of increased Mdm2 protein levels leads to an increase in Mdm2-p53 complex formation that interferes with the ability of p53 to transactivate Mdm2 expression. Thus, Mdm2 is autoregulated through the ability of Mdm2 to negatively regulate p53.5 Mdm2 interferes with the ability of p53 to transactivate target genes by binding to the N-terminal activation domain of the p53 protein,6,7 or by promoting p53 protein modifications that inhibit p53 transcriptional activity.8 In addition, Mdm2 induces shuttling of p53 from the nucleus into the cytoplasm.9,10 Importantly, Mdm2 can also function as an E3 ligase to ubiquitinate and induce the degradation of p53 in the 26S proteosome.11–14 The ability of Mdm2 to negatively regulate p53 activity is best illustrated by mouse studies wherein the early embryonic lethal phenotype of Mdm2-null mice was fully rescued by co-deletion of p53.15,16
The Mdm2 homologue, MdmX, is also a ubiquitously expressed gene that encodes a p53-binding protein that inhibits p53 transcriptional activation.17 However, MdmX expression does not appear to be regulated by p53, and MdmX does not function as an E3 ligase to direct the ubiquitination and destabilization of p53. Although there are distinct differences in the manner by which Mdm2 and MdmX inhibit p53 activity,18 deletion of MdmX can also induce an embryonic lethal phenotype in mice that can be rescued by either the concomitant deletion of p53 or by overexpression of Mdm2.19–22 These results indicate that MdmX, like Mdm2, is a key regulator of p53 activity in development. Subsequent analyses of various mouse models bearing conditional alleles of Mdm2 or MdmX has further demonstrated that these MDM proteins play key roles in regulating p53 activity in organogenesis during later stages of development and in tissue homeostasis in adult mice.23–29
Given that the MDM proteins are key negative regulators of the p53 tumor suppressor, it is not surprising that Mdm2 and MdmX have strong oncogenic potential. MDM2 is overexpressed in a wide variety of human tumors, and the MDM2 gene is amplified in approximately one-third of human sarcomas and in roughly 10% of all human cancers.30,31 Likewise, MDMX is amplified or overexpressed in 10–20% of all human cancers, and upregulation of MDMX was recently found to be an important mechanistic step in the formation of retinoblastoma.32
While there is a large amount of evidence to substantiate that overexpression of Mdm2 or MdmX induces tumorigenesis through the ability of these oncoproteins to suppress p53 activity, reports have also suggested that MDM proteins might have additional, p53-independent, roles in cell growth control and in tumorigenesis. Human tumors have been identified that overexpress MDM2 and yet lack functional p53, a seemingly redundant set of mutations.33,34 Furthermore, patients that present with sarcomas or bladder cancers containing both mutations have a worse prognosis than patients with only one or the other mutation.33,35 Other genetic evidence for a secondary role for Mdm2 in tumorigenesis (in addition to p53 inhibition) has been provided by transgenic mouse studies. Overexpression of the Mdm2 gene alters the spectrum of spontaneous tumors that arise in p53-deficient mice,36 and expression of MDM2 cDNA in the mammary epithelium of transgenic mice was found to inhibit mammary gland morphogenesis and uncouple DNA synthesis from mitosis in the presence or absence of p53.37 In addition, Mdm2 splice variants lacking the p53-binding domain have been isolated from human or mouse tumors that overexpress Mdm2. Expression of these Mdm2 isoforms has been reported to promote proliferation, inhibit apoptosis, and induce transformation in p53-deficient cells in culture, and promote tumorigenesis in vivo in mice.38–40
Transfection studies and biochemical analysis has indicated that Mdm2 can complex with a variety of cell growth regulatory proteins aside from p53, including the Rb tumor suppressor protein,41–44 the E2F1 and DP1 transcription factors,45 as well as with FOXO3a,46 E-cadherin,47 Numb,48 MTBP,49 SMADs,50 Tip60,51 and β-arrestin.52 In addition, Mdm2 has been recently reported to complex with Nbs1, a component of the Mre11/Rad50/ Nbs1 DNA repair complex and can inhibit DNA break repair in p53-deficient cells.53 Although these various findings indicate that there may be additional roles for Mdm2 in regulating cell growth or transformation that are distinct from its crucial role as a negative regulator of p53, it is interesting to note that these additional roles have been uncovered mainly through experiments investigating the effects of Mdm2 overexpression in cells or in mice. As noted previously, mice lacking both Mdm2 and p53 undergo normal development and display the same spontaneous tumor formation rate and tissue spectrum as mice lacking p53 alone,15,36 suggesting that any functions possessed by Mdm2 aside from its ability to regulate p53 are dispensable for normal growth and development. Thus, normal physiologic levels of Mdm2 primarily serve to regulate p53 activity. Furthermore, primary fibroblasts lacking both Mdm2 and p53 are indistinguishable from p53-null fibroblasts in their growth characteristics, their rate of immortalization, and in their response to metabolic insult or DNA damage. Therefore, it is possible that any p53-independent roles for Mdm2 are of biological significance only when Mdm2 is expressed above normal physiologic levels, such as during tumorigenesis in cells that lack the Mdm2-inhibitor p14ARF or that contain amplified copy numbers of the Mdm2 gene.
Similar to Mdm2, MdmX has been found to complex with and/ or inhibit the activity of growth regulatory transcription factors other than p53, including several of the Smad proteins and E2F1.50,54–56 However, these molecular targets have yet to be confirmed in vivo, and the contribution of these interactions with MdmX to cell growth control is uncertain. However, our lab has provided data using primary cells and mice to document a role for MdmX in cell growth and transformation distinct from its well-established ability to inhibit p53 activity. We have previously noted that mouse embryonic fibroblasts (MEFs) that lacked p21 or that overexpressed Mdm2 proliferated faster when MdmX was also deleted in these cells.22,57 This is an unusual set of findings if the only function of MdmX was to inhibit p53. Thus, in order to establish whether a p53-independent role truly exists for MdmX in cell growth or in tumorigenesis, we characterized the growth and transformation potential of p53-null primary cells that were deficient in MdmX or contained physiologic levels of MdmX.58
We have recently discovered that the presence or absence of MdmX did not alter the proliferation of low-passage p53-null MEFs, in agreement with data published previously by other groups.20 However, we noted that deletion of MdmX drastically increased the rate of cell proliferation when the cells were at higher passages.58 Differences in proliferation between MdmX/p53-null (DKO) cells and p53-null cells become apparent around the eighth or ninth passage, a time when wild type cells cultured under the same conditions typically enter senescence. MEFs lacking p53 often undergo centrosome amplification and display hyperploidy after several passages in culture, and both events were readily observed in our p53-null cells and in DKO cells. In addition to augmented growth at higher passages, p53-null MEFs co-deleted for MdmX had increased incidence of multipolar mitosis and reduced number of chromosomes relative to p53-null cells. We noted that these genetic events correspond to more rapid growth of the DKO cells during a 3T3 immortalization assay. Unlike p53-null MEFs at high passage, cells lacking both MdmX and p53 underwent a reduction in chromosome number and displayed a spontaneously transformed phenotype in culture. In agreement with these findings, mice deleted for both MdmX and p53 had a significantly increased rate of spontaneous tumorigenesis, though the spectrum of tumor types observed in the two populations of mice was unchanged. Tumor cells were subsequently isolated from the p53-null mice and from MdmX/p53-double null mice and grown in culture. The double-null tumor cells grew much faster than the p53-null tumor cells, and displayed reduced ploidy and increased incidence of multipolar mitosis. Conversely, transduction of an MdmX expression vector into the DKO tumor cells resulted in a decrease in the rate of cell growth and in the percentage of cells undergoing multipolar mitosis. Collectively, these results reveal that MdmX has an anti-proliferative, anti-transformation function and can promote bipolar mitosis and prevent chromosome loss in hyperploid p53-deficient cells (Fig. 1). We briefly discuss several aspects of this newly discovered MdmX functions.
It has been known for some time that MEFs isolated from p53-deficient mice are genetically unstable and have amplified centrosomes and increased ploidy.59–61 Although several studies have explored p53 in control of centrosome number, the p53-dependent mechanisms that regulate these events have not been fully elucidated. One proposed mechanisms for p53 regulation of centrosomes involves the p53 transcriptional target gene, p21. Cdk2-cyclin E activity appears crucial for centrosome reproduction,62,63 and inhibition of Cdk2-cyclin E activity by p21,64 may provide coordination between centrosome duplication events and DNA replication. In addition, p53 has been proposed to inhibit centrosome amplification by regulating the spindle checkpoint protein BubR1, which can promote the death of cells with amplified centrosomes.65 A role for p53 that involves direct p53 centrosomal localization has also been suggested.66,67 Regardless of the precise mechanism involved in the control of centrosome number, loss of p53 leads to generation of supernumerary centrosomes that facilitate the formation of multiple mitotic spindles leading to genetic imbalance and chromosome instability.68–70
Imunofluorescence analysis of mitotic DKO MEFs revealed an increased incidence of multipolar spindles compared to p53-null MEFs at similar passages, suggesting that the loss of MdmX might further enhance centrosome amplification in p53-null cells.58 The representative image of cell undergoing multipolar mitosis is shown in Figure 2A, left panel. However, closer inspection of mitotic cells revealed that the bipolar spindles of p53-null MEFs frequently contained multiple centrosomes clustered at opposite poles (Fig. 2A, right), indicating that p53-null cells with amplified centrosomes can still form bipolar spindles, and that MdmX might not affect centrosome amplification in p53-null MEFs. To confirm these observations, we have analyzed interphase p53-null MEFs and DKO MEFs by immunostaining for γ-tubulin (Fig. 2B) and determined that the rate of cells with amplified centrosomes is very similar (around 20%) in DKO and p53-null populations. In contrast, spindle multipolarity is a much less frequent event in p53-null MEFs or in p53-null tumor cells that retain functional MdmX compared to the incidence of multipolarity observed in DKO MEFs or tumor cells.58 Therefore, we conclude that MdmX does not affect centrosomes amplification but rather the organization of amplified centrosomes, preventing formation of multipolar spindles and promoting bipolar mitosis and cell division.
Normally, an individual diploid (2N) cell becomes tetraploid (4N) prior to mitosis, with bipolar mitosis resulting in the generation of two separate (2N) daughter cells. Only hyperploid cell (>4N) undergoing multipolar mitosis can divide into more than two viable daughter cells. Our previous DAPI-staining of hyperploid p53-deficient cells and examination of telophase figures indicated that the multipolar spindles that formed in DKO cells often led to multi-directional chromosome segregation, suggesting that multipolar mitosis in DKO cells can be followed by multipolar cell division.58 As discussed previously, loss of p53 has been well established to promote centrosome amplification and hyperploidy in primary fibroblasts,60 and our own data demonstrate that more than half of p53-null MEFs display a greater than triploid DNA content at higher cell passages. However, 90% of all mitotic p53-null MEFs display bipolar spindles and undergo bipolar mitosis. In contrast, multipolar mitosis is apparent in more than 20% of mitotic DKO MEFs. To determine whether multipolar spindles allow generation of more than two daughter cells per division, we employed time-lapse photography to document the fate of cells undergoing multipolar mitosis.
Multopolar mitosis was readily observed and filmed in DKO MEFs. A representative series of still pictures from the time-lapse video microscopy is presented in Figure 3. We have followed division of a single DKO cell into four viable daughter cells (Fig. 3, parts a and b), and subsequent division of one of the daughters (b; arrow) into three cells (c and d). Two of these three resulting cells continued to divide but generated only two daughter cells, and the third cell (d; arrow) become multinucleated (e) and eventually died (f). Thus, multipolar spindle formation and mitosis in DKO MEFs can result in the formation of multiple viable daughter cells. However, analysis of the time-lapse revealed that many of the resulting daughter cells die within one or two rounds of cell division. As the chromosomal content of the hyperploid DKO cells is rapidly reduced during cell passage,58 we conclude that the formation of multipolar spindles in p53-null cells co-deleted for MdmX increases genomic instability, and that the unequal chromosomal distribution or loss of chromosomes during multipolar mitosis compromises survival in fraction of progeny. Thus, although more than two daughter cells are generated from some DKO cells undergoing multipolar mitosis, many of these cells are lost in subsequent divisions. Consequently, increased proliferative capacity of DKO compared to p53-null cells does not simply reflect an increase in the number of daughter cells generated through the multipolar division.
The decreased ploidy and increased proliferation rate of p53-null cells when co-deleted for MdmX suggest that differences in the growth rate between p53-null and DKO cells may reflect prolonged duration of mitosis in p53-null cells since they have to coordinate centrosome clustering and properly organize their large chromosome complement throughout mitosis. To explore this possibility, we measured the duration of bipolar and multipolar mitosis in p53-null MEFs, and compared it to the duration of bipolar and multipolar mitosis in DKO MEFs using time-lapse video microscopy (Fig. 4). We found that the duration of either bipolar or multipolar mitosis measured from nuclear envelope breakdown (NEB) to the cleavage onset is not impacted by the presence or absence of MdmX. In addition, the duration of multipolar mitosis was nearly twice that of bipolar mitosis, regardless of genotype. As the frequency of multipolar mitosis is higher in DKO population, the duration of mitosis in DKO cells is unlikely to account for the faster growth rate of DKO cells compared to p53-null cells.
Following plating and passaging in culture, MEFs lacking p53 display supernumerary centosomes and hyperploidy regardless of MdmX status. However, unlike p53-null MEFs, DKO MEFs undergo a rapid reduction in ploidy, proliferate faster, and form numerous foci when plated in a cell transformation assay. Thus, MdmX suppress the transformation of immortalized p53-null cells. In agreement with these results, mice deficient for MdmX and p53 form tumors faster than p53-deficient mice. The increased transformation potential of DKO cells in vitro and in vivo is most likely a function of the chromosomal instability of DKO cells. Multipolar mitosis and aneuploidy have been tightly linked to tumorigenesis,71–75 and the reduction in chromosome number observed in hyperploid p53-deficient cells after prolonged culturing in vitro has been proposed to result in aneuploid, but more stable karyotype that confers growth advantage to the cell.76 This process resembles previously described genomic convergence that occurs during tumor progression in vivo.77
The more rapid loss of chromosomes in DKO MEFs might facilitate cell transformation by promoting the loss of growth regulatory genes that inhibit cell proliferation. Conversely, the more rapid proliferation of DKO cells in culture may simply reflect their “more transformed” state. However, tumor cells cultured from DKO mouse tumors displayed more rapid proliferation than tumor cells from p53-null mice, and transduction of MdmX into these DKO cells increased their chromosome numbers and reduced their rate of proliferation, even though these DKO tumor cells were already fully transformed. Therefore, it is unlikely that the increased proliferation of DKO cells simply reflects their more transformed phenotype or the loss of a specific gene(s) during chromosomal reduction in culture. Rather, it would appear that MdmX alters the proliferation of p53-null cells through a more specific mechanism, one that might involve interaction with other cell cycle regulatory proteins. Further experiments are needed to distinguish between the ability of MdmX to suppress the proliferation of p53-null cells and to suppress the transformation of p53-null cells in vitro and in vivo. Interestingly, increased reduction in chromosome number during passaging in culture is also observed in MdmX-null cells that retain p53 but are deleted for p21, as well as in MdmX/p53 DKO cells also lacking Mdm2.58 These initial findings suggest that the ability of MdmX to promote bipolar mitosis is independent of these p53 target genes. Therefore, elucidation of the precise roles of MdmX in promoting bipolar mitosis and in altering cell proliferation will require additional studies to identify p53-independent binding partners and functions for MdmX. Since p53-null mice deleted for MdmX display increased tumorigenesis, and since approximately half of all human cancers are mutated for p53, a clearer understanding of the p53-independent roles of MDMX in inhibiting transformation is needed.
We thank Kathleen Hoover for technical assistance and other members of the Jones lab for useful discussion. This work was supported by grant R01GM30758 and RO1CA77735 from the National Institutes of Health to GS and SNJ, respectively.