Cell cycle regulatory functions of the human oncoprotein MDM2

The protein (MDM2) coded by the mouse double minute-2 (mdm2) gene or its human homologue is well known as an oncoprotein. Malignant human tumors particularly breast tumors and soft tissue sarcomas frequently overexpress MDM2. Artificial amplification of mdm2 gene derived from a transformed murine cell line enhances tumorigenic potential of murine cells. Consistent with its tumorigenic property, mouse or human MDM2 can inactivate several functions of the tumor suppressor p53 and can degrade p53. The protein also interacts with other tumor suppressors, and these interactions may contribute to its tumorigenic property. In spite of its oncogenic role, mouse or human MDM2 induces G(1) arrest in normal human or murine cells. Some cell lines bearing known genetic mutations are insensitive to MDM2-mediated growth arrest. This review is aimed to collect available information on the functions of MDM2 that could potentially regulate cell cycle and to discuss how this information may fit in one model that could explain the two apparently opposite G(1) arrest and oncogenic function of MDM2.     

The human oncoprotein MDM2 arrests the cell cycle: elimination of its cell-cycle-inhibitory function induces tumorigenesis.

The human oncoprotein MDM2 (hMDM2) overexpresses in various human tumors. If amplified, the mdm2 gene can enhance the tumorigenic potential of murine cells. Here, we present evidence to show that the full-length human or mouse MDM2 expressed from their respective cDNA can inhibit the G0/G1-S phase transition of NIH 3T3 and normal human diploid cells. The protein harbors more than one cell-cycle-inhibitory domain that does not overlap with the p53-interaction domain. Deletion mutants of hMDM2 that lack the cell-cycle-inhibitory domains can be stably expressed in NIH 3T3 cells, enhancing their tumorigenic potential. The tumorigenic domain of hMDM2 overlaps with the p53-interaction domain. Some tumor-derived cells, such as Saos-2, H1299 or U-2OS, are relatively insensitive to the growth-inhibitory effects of hMDM2. These observations suggest that hMDM2 overexpression in response to oncogenic stimuli would induce growth arrest in normal cells. Elimination or inactivation of the hMDM2-induced G0/G1 arrest may contribute to one of the steps of tumorigenesis.     

Aberrant expression of nucleostemin activates p53 and induces cell cycle arrest via inhibition of MDM2

The nucleolar protein nucleostemin (NS) is essential for cell proliferation and early embryogenesis. Both depletion and overexpression of NS reduce cell proliferation. However, the mechanisms underlying this regulation are still unclear. Here, we show that NS regulates p53 activity through the inhibition of MDM2. NS binds to the central acidic domain of MDM2 and inhibits MDM2-mediated p53 ubiquitylation and degradation. Consequently, ectopic overexpression of NS activates p53, induces G(1) cell cycle arrest, and inhibits cell proliferation. Interestingly, the knockdown of NS by small interfering RNA also activates p53 and induces G(1) arrest. These effects require the ribosomal proteins L5 and L11, since the depletion of NS enhanced their interactions with MDM2 and the knockdown of L5 or L11 abrogated the NS depletion-induced p53 activation and cell cycle arrest. These results suggest that a p53-dependent cell cycle checkpoint monitors changes of cellular NS levels via the impediment of MDM2 function.     

Role of the F-box protein Skp2 in adhesion-dependent cell cycle progression.

Cell adhesion to the extracellular matrix (ECM) is a requirement for proliferation that is typically lost in malignant cells. In the absence of adhesion, nontransformed cells arrest in G1 with increased levels of the cyclin-dependent kinase inhibitor p27. We have reported previously that the degradation of p27 requires its phosphorylation on Thr-187 and is mediated by Skp2, an F-box protein that associates with Skp1, Cul1, and Roc1/Rbx1 to form the SCF(Skp2) ubiquitin ligase complex. Here, we show that the accumulation of Skp2 protein is dependent on both cell adhesion and growth factors but that the induction of Skp2 mRNA is exclusively dependent on cell adhesion to the ECM. Conversely, the expression of the other three subunits of the SCF(Skp2) complex is independent of cell anchorage. Phosphorylation of p27 on Thr-187 is also not affected significantly by the loss of cell adhesion, demonstrating that increased p27 stability is not dependent on p27 dephosphorylation. Significantly, ectopic expression of Skp2 in nonadherent G1 cells resulted in p27 downregulation, entry into S phase, and cell division. The ability to induce adhesion-independent cell cycle progression was potentiated by coexpressing Skp2 with cyclin D1 but not with cyclin E, indicating that Skp2 and cyclin D1 cooperate to rescue proliferation in suspension cells. Our study shows that Skp2 is a key target of ECM signaling that controls cell proliferation.     

Model predictions of MDM2 mediated cell regulation

In this work we present a mathematical approach to elucidate possible mechanisms involving mdm2 in the regulation of the cell cycle. It has been experimentally shown that the over-expression of MDM2 leads to decoupling of DNA synthesis with mitosis resulting in polyploidy cells with multiple copies of their genomes. The function of MDM2 that uncouples the DNA synthesis phase (S) and the Mitosis phase (M) is unclear. To answer this question, we first formulate a mathematical model of the dynamics of the cell cycle regulatory proteins during the DNA synthesis phase and mitosis. This model is then tested for bifurcation that produces period doubling cascades that we relate to the biological event of polyploidy. The model formulation, the underlying biology, and the bifurcation results to delineate the unknown function of MDM2 are presented. Based on reproducing known experimental result of polyploidy in MDM2 overexpressed cells, we propose several possible functions of mdm2, i.e., possible interactions with the other cell cycle regulating proteins that will result in uncoupling the S and M phases. We conclude that the most likely unknown function of MDM2 leading to the decoupling of the S and M phases is an obstruction of the activation of Cdc25C by MDM2.     

Protein kinase CK2 phosphorylates the cell cycle regulatory protein Geminin

Geminin contributes to cell cycle regulation by a timely inhibition of Cdt1p, the loading factor required for the assembly of pre-replication complexes. Geminin is expressed during S and G2 phase of the HeLa cell cycle and phosphorylated soon after its synthesis. We show here that Geminin is an excellent substrate for protein kinase CK2 in vitro; and that the highly specific CK2 inhibitor tetrabromobenzotriazole (TBB) blocks the phosphorylation of Geminin in HeLa protein extracts and HeLa cells in vivo. The sites of CK2 phosphorylation are located in the carboxyterminal region of Geminin, which carries several consensus sequence motifs for CK2. We also show that a minor phosphorylating activity in protein extracts can be attributed to glycogen synthase kinase 3 (GSK3), which most likely targets a central peptide in Geminin. Treatment of HeLa cells with TBB does not interfere with the ability of Geminin to interact with the loading factor Cdt1.     

Cyclin a-CDK phosphorylation regulates MDM2 protein interactions

The product of the MDM2 gene interacts with and regulates a number of proteins, in particular the tumor suppressor p53. The MDM2 protein is likely to be extensively modified in vivo, and such modification may regulate its functions in cells. We identified a potential cyclin-dependent kinase (CDK) site in murine MDM2, and found the protein to be efficiently phosphorylated in vitro by cyclin A-containing complexes (cyclin A-CDK2 and cyclin A-CDK1), but MDM2 was either weakly or not phosphorylated by other cyclin-containing complexes. Moreover, a peptide containing a putative MDM2 cyclin recognition motif specifically inhibited phosphorylation by cyclin A-CDK2. The site of cyclin A-CDK2 phosphorylation was identified as Thr-216 by two-dimensional phosphopeptide mapping and mutational analysis. Phosphorylation of MDM2 at Thr-216 both weakens its interaction with p53 and modestly augments its binding to p19(ARF). Interestingly, an MDM2-specific monoclonal antibody, SMP14, cannot recognize MDM2 phosphorylated at Thr-216. Changes in SMP14 reactivity of MDM2 in staged cell extracts indicate that phosphorylation of MDM2 at Thr-216 in vivo is most prevalent at the onset of S phase when cyclin A first becomes detectable.     

Expression and regulation of protein kinase CK2 during the cell cycle

There are indications from genetic, biochemical and cell biological studies that protein kinase CK2 (formerly casein kinase II) has a variety of functions at different stages in the cell cycle. To further characterize CK2 and its potential roles during cell cycle progression, one of the objectives of this study was to systematically examine the expression of all three subunits of CK2 at different stages in the cell cycle. To achieve this objective, we examined levels of CK2, CK2 and CK2 on immunoblots as well as CK2 activity in samples prepared from: (i) elutriated populations of MANCA (Burkitt lymphoma) cells, (ii) serum-stimulated GL30-92/R (primary human fibroblasts) cells and (iii) drug-arrested chicken bursal lymphoma BK3A cells. On immunoblots, we observed a significant and co-ordinate increase in the expression of CK2 and CK2 following serum stimulation of quiescent human fibroblasts. By comparison, no major fluctuations in CK2 activity were detected during any other stages during the cell cycle. Furthermore, we did not observe any dramatic differences between the relative levels of CK2 to CK2 during different stages in the cell cycle. However, we observed a significant increase in the amount of CK2 relative to CK2 in cells arrested with nocodazole. We also examined the activity of CK2 in extracts or in immunoprecipitates prepared from drug-arrested cells. Of particular interest is the observation that the activity of CK2 is not changed in nocodazole-arrested cells. Since CK2 is maximally phosphorylated in these cells, this result suggests that the phosphorylation of CK2 by p34cdc2 does not affect the catalytic activity of CK2. However, the activity of CK2 was increased by incubation with p34cdc2 in vitro. Since this activation was independent of ATP we speculate that p34cdc2 may have an associated factor that stimulates CK2 activity. Collectively, the observations that relative levels of CK2 increase in mitotic cells, that CK2 and CK2 are phosphorylated in mitotic cells and that p34cdc2 affects CK2 activity in vitro suggest that CK2 does have regulatory functions associated with cell division.     

cdc2-related protein kinases and cell cycle control in trypanosomatids

The molecular mechanisms that control the cell cycle have been studied extensively in yeast and higher eukaryotes. Investigations have centred on the cyclin-dependent kinase family of serine/threonine protein kinases, the best characterized of which is cdc2, a key regulatory element in the control of mitosis. Cell cycle control plays an important role in trypanosomes and Leishmania, not only in cellular proliferation, but also in the developmental system that controls the transfer of the parasite between hosts. In this review, Jeremy Mottram compares the family of trypanosome cdc2-related kinases with that of yeast and the higher eukaryotes.     

Inhibiting the apoptosis pathway using MDM2 in mammalian cell cultures

The ability to regulate apoptosis in mammalian cell cultures represents one approach to developing more economical and efficient processes. Genetic modification of cells using anti-apoptotic genes is one method that may be used to improve cellular performance. This study investigates a method to inhibit upstream apoptosis pathways through the overexpression of MDM2, an E3 ubiquitin ligase for p53. Both 293 and CHO cells expressing MDM2 were examined under both batch and spent media conditions. For batch cultures, MDM2 overexpression increased viable cell densities and viabilities over control cells with the largest enhancements observed in CHO cells. When CHO cells were passaged without medium exchange, cells expressing MDM2 reached a viable cell density that was nearly double the control and survived for an extra day in culture. When exposed to spent media initially, both 293-MDM2 and CHO-MDM2 cells continued to grow for 2 days while the control cells stopped growing after the first day. DNA analysis using flow cytometry confirmed that while CHO controls were found to be undergoing DNA fragmentation, CHO-MDM2 cells exhibit DNA degradation at a much slower rate. When compared to Bcl-2-expressing cells, MDM2 expression showed greater protection against apoptosis in passaged culture, spent medium, and following transient p53 overexpression. However, expression of the RING sequence of MDM2 responsible for E3 ligase activity without the other components of the protein was found to be toxic to 293 cells in culture. These results suggest that the overexpression of heterologous MDM2 represents a promising method to delay apoptosis in mammalian cell cultures.     

Regulation of the cell cycle by protein phosphatase 2A in Saccharomyces cerevisiae.

Protein phosphatase 2A (PP2A) has long been implicated in cell cycle regulation in many different organisms. In the yeast Saccharomyces cerevisiae, PP2A controls cell cycle progression mainly through modulation of cyclin-dependent kinase (CDK) at the G(2)/M transition. However, CDK does not appear to be a direct target of PP2A. PP2A affects CDK activity through its roles in checkpoint controls. Inactivation of PP2A downregulates CDK by activating the morphogenesis checkpoint and, consequently, delays mitotic entry. Defects in PP2A also compromise the spindle checkpoint and predispose the cell to an error-prone mitotic exit. In addition, PP2A is involved in controlling the G(1)/S transition and cytokinesis. These findings suggest that PP2A functions in many stages of the cell cycle and its effect on cell cycle progression is pleiotropic.     

MDM2 promotes proteasome-dependent ubiquitin-independent degradation of retinoblastoma protein

Inactivation of retinoblastoma protein (Rb) plays a critical role in the development of human malignancies. It has been shown that Rb is degraded through a proteasome-dependent pathway, yet the mechanism is largely unclear. MDM2 is frequently found amplified and overexpressed in a variety of human tumors. In this study, we find that MDM2 promotes Rb degradation in a proteasome-dependent and ubiquitin-independent manner. We show that Rb, MDM2, and the C8 subunit of the 20S proteasome interact in vitro and in vivo and that MDM2 promotes Rb-C8 interaction. Expression of wild-type MDM2, but not the mutant MDM2 defective either in Rb interaction or in RING finger domain, promotes cell cycle S phase entry independent of p53. Furthermore, MDM2 ablation results in Rb accumulation and inhibition of DNA synthesis. Taken together, these findings demonstrate that MDM2 is a critical negative regulator for Rb and suggest that MDM2 overexpression contributes to cancer development by destabilizing Rb.     

MDM2--master regulator of the p53 tumor suppressor protein

MDM2 is an oncogene that mainly functions to modulate p53 tumor suppressor activity. In normal cells the MDM2 protein binds to the p53 protein and maintains p53 at low levels by increasing its susceptibility to proteolysis by the 26S proteosome. Immediately after the application of cellular stress, the ability of MDM2 to bind to p53 is blocked or altered in a fashion that prevents MDM2-mediated degradation. As a result, p53 levels rise, causing cell cycle arrest or apoptosis. In this review, we present evidence for the existence of three highly conserved regions (CRs) shared by MDM2 proteins and MDMX proteins of different species. These highly conserved regions encompass residues 42-94 (CR1), 301-329 (CR2), and 444-483 (CR3) on human MDM2. These three domains are respectively important for binding p53, for binding the retinoblastoma protein, and for transferring ubiquitin to p53. This review discusses the major milestones uncovered in MDM2 research during the past 12 years and potential uses of this knowledge in the fight against cancer.     

Ubiquitination and degradation of the anti-apoptotic protein ARC by MDM2

Current evidence shows that cardiomyocyte apoptosis plays a central role in the pathogenesis of myocardial disease and that reactive oxygen species is critically responsible for mediating cardiomyocyte apoptosis in both ischemia-reperfusion injury and dilated cardiomyopathy. ARC (Apoptosis Repressor with Caspase recruitment domain) is an anti-apoptotic protein that is found abundantly in terminally differentiated cells such as cardiomyocytes. The ARC knock-out mouse developed larger infarct in response to ischemia-reperfusion and transitioned more rapidly and severely to dilated cardiomyopathy following aortic constriction. In addition, ARC protein levels are decreased in human dilated cardiomyopathy and when cardiomyocytes are exposed to oxidative stress in vitro, but the mechanisms regulating ARC protein levels are not known. Here we show that degradation of ARC is dependent on the p53-induced ubiquitin E3 ligase, MDM2. Oxidative stress reduced ARC levels and up-regulated MDM2. MDM2 directly accelerated ARC protein turnover via ubiquitination and proteasomal-dependent degradation. This activity requires a functioning MDM2 ring finger domain because the MDM2(C464A) mutant was unable to direct ARC degradation. Furthermore, ARC degradation requires MDM2, because MDM2 knock-out fibroblasts showed defective ARC degradation that could be rescued by MDM2. Proteasomal inhibitors rescued both MDM2 and H(2)O(2)-induced degradation of ARC and inhibited cardiomyocyte apoptosis. Dilated cardiomyopathic hearts from mice that have undergone transverse aortic banding have increased MDM2 levels associated with decreased ARC levels. We conclude that MDM2 is a critical regulator of ARC levels in cardiomyocytes. Prevention of MDM2-induced degradation of ARC represents a potential therapeutic target to prevent cardiomyocyte apoptosis.     

Nucleostemin与MDM2在肺腺癌组织中的表达及意义

核干细胞因子(Nucleostemin,NS)是细胞增殖的调节因子,在肺腺癌组织中的表达及与鼠双微染色体2(murine double minute 2,MDM2)表达的相关性不清楚.通过反转录聚合酶链反应(RT-PCR)检测22对肺腺癌和癌旁正常组织NS mRNA的表达;应用免疫组织化学SP法检测45例肺腺癌和22例癌旁正常组织NS与MDM2蛋白的表达,分析其相关性及意义.研究发现,肺腺癌组织中NS mRNA的相对表达强度明显高于癌旁正常组织(P0.01).NS和MDM2蛋白在肺腺癌组织中的阳性表达率分别为73.3%(33/45)、57.8%(26/45),而癌旁正常组织中无阳性表达(P0.01).二者的阳性表达均与组织学分级相关(P0.05),并且表达呈正相关(P0.05),而与患者性别、年龄、肿瘤大小、TNM分期及淋巴结转移无关(P0.05).结果表明NS基因的高表达对肺腺癌细胞的恶性增殖发挥了重要作用,可能是通过p53通路对细胞周期影响所实现的.     

Regulation of the cell cycle by the cdk2 protein kinase in cultured human fibroblasts

In mammalian cells inhibition of the cdc2 function results in arrest in the G2-phase of the cell cycle. Several cdc2-related gene products have been identified recently and it has been hypothesized that they control earlier cell cycle events. Here we have studied the relationship between activation of one of these cdc2 homologs, the cdk2 protein kinase, and the progression through the cell cycle in cultured human fibroblasts. We found that cdk2 was activated and specifically localized to the nucleus during S phase and G2. Microinjection of affinity-purified anti-cdk2 antibodies but not of affinity-purified anti-cdc2 antibodies, during G1, inhibited entry into S phase. The specificity of these effects was demonstrated by the fact that a plasmid-driven cdk2 overexpression counteracted the inhibition. These results demonstrate that the cdk2 protein kinase is involved in the activation of DNA synthesis.