Regulation of p14ARF Through Subnuclear Compartmentalization

The p53-mediated pathway cell cycle arrest and apoptosis is central to cancer and an important point of focus for therapeutics development. The p14ARF ("ARF") tumor suppressor induces the p53 pathway in response to oncogene activation or DNA damage. However, ARF is predominantly nucleolar in localization and engages in several interactions with nucleolar proteins, whereas p53 is nucleoplasmic. This raises the question as to how ARF initiates its involvement in the p53 pathway. We have found that UV irradiation of cells disrupts the interaction of ARF with two of its nucleolar binding partners, B23(NPM, nucleophosmin, NO38, numatrin) and topoisomerase I, and promotes an immediate and transient subnuclear redistribution of ARF to the nucleoplasm, where it can engage the p53 pathway (Lee et al, Cancer Research 65:9834-42; 2005). The results support a model in which the nucleolus serves as a p53 upstream sensor of cellular stress, and add to a growing body of evidence that nucleolar sequestration of ARF prevents activation of p53. The results also have therapeutic implications for therapies based on exploiting p53 and other cellular stress response pathways to suppress cancer.     

Loss of Peter Pan protein is associated with cell cycle defects and apoptotic events

The nucleolus is a subnuclear compartment, which governs ribosome biogenesis. Moreover, it functions as hub in the stress response by orchestrating a variety of processes, such as regulation of cell cycle progression, senescence and apoptosis. Emerging evidence links the nucleolus also to the control of genomic stability and the development of human malignancies. Peter Pan (PPAN) is an essential ribosome biogenesis factor localized to nucleoli and mitochondria. We earlier showed that PPAN depletion triggers p53-independent nucleolar stress and apoptosis. In this study we investigated the precise localization of nucleolar PPAN during cell cycle and its function in cell cycle regulation. We show that PPAN knockdown impairs cell proliferation and induces G0/G1 as well as later G2/M cell cycle arrest in cancer cells. Although PPAN knockdown stabilizes the tumor suppressor p53 and induces CDKN1A/p21, the proliferation defects occur largely in a p53/p21-independent manner. We noticed a reduced number of knockdown cells entering cytokinesis and an elevation of binucleation. PPAN knockdown is also associated with increased H2A.X phosphorylation (γH2A.X) in cancer cells. We evaluated a potential signaling axis through the DNA damage response kinases ATM and ATR and alternatively apoptosis as a potent driver of γH2A.X. Interestingly, PPAN knockdown does not involve activation of ATM/ATR. Instead, γH2A.X is generated as a consequence of apoptosis induction in cancer cells. Strikingly, PPAN depletion in human fibroblasts did neither provoke apoptosis nor H2A.X phosphorylation, but recapitulated p53 stabilization. In summary, our data underline the notion that the PPAN-mediated, p53-independent nucleolar stress response has multiple facets.     

Disruption of the nucleolus mediates stabilization of p53 in response to DNA damage and other stresses

p53 protects against cancer through its capacity to induce cell cycle arrest or apoptosis under a large variety of cellular stresses. It is not known how such diversity of signals can be integrated by a single molecule. However, the literature reveals that a common denominator in all p53-inducing stresses is nucleolar disruption. We thus postulated that the impairment of nucleolar function might stabilize p53 by preventing its degradation. Using micropore irradiation, we demonstrate that large amounts of nuclear DNA damage fail to stabilize p53 unless the nucleolus is also disrupted. Forcing nucleolar disruption by anti-upstream binding factor (UBF) microinjection (in the absence of DNA damage) also causes p53 stabilization. We propose that the nucleolus is a stress sensor responsible for maintenance of low levels of p53, which are automatically elevated as soon as nucleolar function is impaired in response to stress. Our model integrates all known p53-inducing agents and also explains cell cycle-related variations in p53 levels which correlate with established phases of nucleolar assembly/disassembly through the cell cycle.     

DNA damage, p14ARF, Nucleophosmin (NPM/B23), and cancer

The p53/p14ARF/mdm2 stress response pathway plays a central role in mediating cellular responses to oncogene activation, genome instability, and therapy-induced DNA damage. Abrogation of the pathway occurs in most if not all cancers, and may be essential for tumor development. The high frequency with which the pathway is disabled in cancer and the fact that the pathway appears to be incompatible with tumor cell growth, has made it an important point of focus in cancer research and therapeutics development. Recently, Nucleophosmin (NPM, B23, NO38 and numatrin), a multifunctional nucleolar protein, has emerged as a p14ARF binding protein and regulator of p53. While complex formation between ARF and NPM retains ARF in the nucleolus and prevents ARF from activating p53, DNA damaging treatments promote a transient subnuclear redistribution of ARF to the nucleoplasm, where it interacts with mdm2 and promotes p53 activation. The results add support to a recently proposed model in which the nucleolus serves as a p53-uspstream sensor of stress, and where ARF links nucleolar stress signals to nucleoplasmic effectors of the stress response. A better understanding of ARF’s nucleolar interactions could further elucidate the regulation of the p53 pathway and suggest new therapeutic approaches to restore p53 function.     

Nucleolar Stress Induces Ubiquitination-independent Proteasomal Degradation of PICT1 Protein

The nucleolar protein PICT1 regulates tumor suppressor p53 by tethering ribosomal protein L11 within the nucleolus to repress the binding of L11 to the E3 ligase MDM2. PICT1 depletion results in the release of L11 to the nucleoplasm to inhibit MDM2, leading to p53 activation. Here, we demonstrate that nucleolar stress induces proteasome-mediated degradation of PICT1 in a ubiquitin-independent manner. Treatment of H1299 cells with nucleolar stress inducers, such as actinomycin D, 5-fluorouridine, or doxorubicin, induced the degradation of PICT1 protein. The proteasome inhibitors MG132, lactacystin, and epoxomicin blocked PICT1 degradation, whereas the inhibition of E1 ubiquitin-activating enzyme by a specific inhibitor and genetic inactivation fail to repress PICT1 degradation. In addition, the 20 S proteasome was able to degrade purified PICT1 protein in vitro. We also found a PICT1 mutant showing nucleoplasmic localization did not undergo nucleolar stress-induced degradation, although the same mutant underwent in vitro degradation by the 20 S proteasome, suggesting that nucleolar localization is indispensable for the stress-induced PICT1 degradation. These results suggest that PICT1 employs atypical proteasome-mediated degradation machinery to sense nucleolar stress within the nucleolus.     

Curcumin homing to the nucleolus: mechanism for initiation of an apoptotic program

Curcumin is a plant-derived polyphenol that displays antitumor properties. Incubation of cultured SF-767 glioma cells with curcumin gave rise to intense intranuclear foci of curcumin fluorescence. In vitro studies revealed that nuclear homing by curcumin is not a result of DNA/chromatin binding. On the other hand, curcumin fluorescence colocalized with nucleophosmin, a nucleolus marker protein. To determine the temporal relationship between curcumin-induced apoptosis and nucleolar homing, confocal live cell imaging was performed. The data show that curcumin localization to the nucleolus occurs prior to cell surface exposure of phosphatidylserine. In studies of the mechanism of curcumin-induced apoptosis in SF-767 cells, its effect on the subcellular location of p14^ARF was determined. Whereas p14^ARF was confined to the nucleolus in untreated cells, 2 h following incubation with curcumin, it displayed a diffuse nuclear distribution. Given the role of nuclear p14^ARF in binding the E3 ubiquitin ligase, mouse double minute 2 homolog (MDM2), the effect of curcumin treatment on cellular levels of the tumor suppressor protein, p53, was examined. Between 2 and 4 h following curcumin treatment, p53 levels increased with maximum levels reached by 8 h. Thus, curcumin homing to the nucleolus induces redistribution of p14^ARF to the nucleoplasm where interaction with MDM2 leads to stabilization of p53, with subsequent initiation of apoptosis.     

Silencing of Ribosomal Protein S9 Elicits a Multitude of Cellular Responses Inhibiting the Growth of Cancer Cells Subsequent to p53 Activation

Background

Disruption of the nucleolus often leads to activation of the p53 tumor suppressor pathway through inhibition of MDM2 that is mediated by a limited set of ribosomal proteins including RPL11 and RPL5. The effects of ribosomal protein loss in cultured mammalian cells have not been thoroughly investigated. Here we characterize the cellular stress response caused by depletion of ribosomal protein S9 (RPS9).

Methodology/Principal Findings

Depletion of RPS9 impaired production of 18S ribosomal RNA and induced p53 activity. It promoted p53-dependent morphological differentiation of U343MGa Cl2:6 glioma cells as evidenced by intensified expression of glial fibrillary acidic protein and profound changes in cell shape. U2OS osteosarcoma cells displayed a limited senescence response with increased expression of DNA damage response markers, whereas HeLa cervical carcinoma cells underwent cell death by apoptosis. Knockdown of RPL11 impaired p53-dependent phenotypes in the different RPS9 depleted cell cultures. Importantly, knockdown of RPS9 or RPL11 also markedly inhibited cell proliferation through p53-independent mechanisms. RPL11 binding to MDM2 was retained despite decreased levels of RPL11 protein following nucleolar stress. In these settings, RPL11 was critical for maintaining p53 protein stability but was not strictly required for p53 protein synthesis.

Conclusions

p53 plays an important role in the initial restriction of cell proliferation that occurs in response to decreased level of RPS9. Our results do not exclude the possibility that other nucleolar stress sensing molecules act upstream or in parallel to RPL11 to activate p53. Inhibiting the expression of certain ribosomal proteins, such as RPS9, could be one efficient way to reinitiate differentiation processes or to induce senescence or apoptosis in rapidly proliferating tumor cells.     

Nucleolar Disruption Ensures Nuclear Accumulation of p21 upon DNA Damage

p21^cip1 is a protein with a dual function in oncogenesis depending mainly on its intracellular localization: tumor suppressor in the nucleus and oncogenic in the cytoplasm. After DNA damage, p21^cip1 increases and accumulates in the nucleus to ensure cell cycle arrest. We show here that the nuclear accumulation of p21^cip1 is not only a consequence of its increased levels but to a DNA damage cellular response, which is ataxia telangiectasia and Rad3 related (ATR)/ataxia telangiectasia mutated (ATM) and p53 independent. Furthermore, after DNA damage, p21^cip1 not only accumulates in the nucleoplasm but also in the disrupted nucleolus. Inside the nucleolus, it is found in spherical structures, which are not a protrusion of the nucleoplasm. The steady‐state distribution of p21^cip1 in the nucleolus resulted from a highly dynamic equilibrium between nucleoplasmic and nucleolar p21^cip1 and correlated with the inhibition of p21^cip1 nuclear export. Most interestingly, inhibition of ribosomal export after expressing a dominant‐negative mutant of nucleophosmin induced p21^cip1 accumulation in the nucleus and the nucleolus in the absence of DNA damage. This proved the existence of a nucleolar export route to the cytoplasm for p21^cip1 in control conditions that would be inhibited upon DNA damage leading to nuclear and nucleolar accumulation of p21^cip1.     

Perturbation of RNA Polymerase I transcription machinery by ablation of HEATR1 triggers the RPL5/RPL11-MDM2-p53 ribosome biogenesis stress checkpoint pathway in human cells

Ribosome biogenesis is an energy consuming process which takes place mainly in the nucleolus. By producing ribosomes to fuel protein synthesis, it is tightly connected with cell growth and cell cycle control. Perturbation of ribosome biogenesis leads to the activation of p53 tumor suppressor protein promoting processes like cell cycle arrest, apoptosis or senescence. This ribosome biogenesis stress pathway activates p53 through sequestration of MDM2 by a subset of ribosomal proteins (RPs), thereby stabilizing p53. Here, we identify human HEATR1, as a nucleolar protein which positively regulates ribosomal RNA (rRNA) synthesis. Downregulation of HEATR1 resulted in cell cycle arrest in a manner dependent on p53. Moreover, depletion of HEATR1 also caused disruption of nucleolar structure and activated the ribosomal biogenesis stress pathway – RPL5 / RPL11 dependent stabilization and activation of p53. These findings reveal an important role for HEATR1 in ribosome biogenesis and further support the concept that perturbation of ribosome biosynthesis results in p53-dependent cell cycle checkpoint activation, with implications for human pathologies including cancer.     

The Nucleolar Protein Myb-binding Protein 1A (MYBBP1A) Enhances p53 Tetramerization and Acetylation in Response to Nucleolar Disruption

Tetramerization of p53 is crucial to exert its biological activity, and nucleolar disruption is sufficient to activate p53. We previously demonstrated that nucleolar stress induces translocation of the nucleolar protein MYBBP1A from the nucleolus to the nucleoplasm and enhances p53 activity. However, whether and how MYBBP1A regulates p53 tetramerization in response to nucleolar stress remain unclear. In this study, we demonstrated that MYBBP1A enhances p53 tetramerization, followed by acetylation under nucleolar stress. We found that MYBBP1A has two regions that directly bind to lysine residues of the p53 C-terminal regulatory domain. MYBBP1A formed a self-assembled complex that provided a molecular platform for p53 tetramerization and enhanced p300-mediated acetylation of the p53 tetramer. Moreover, our results show that MYBBP1A functions to enhance p53 tetramerization that is necessary for p53 activation, followed by cell death with actinomycin D treatment. Thus, we suggest that MYBBP1A plays a pivotal role in the cellular stress response.     

Death of p53-defective cells triggered by forced mitotic entry in the presence of DNA damage is not uniquely dependent on Caspase-2 or the PIDDosome

Much effort has been put in the discovery of ways to selectively kill p53-deficient tumor cells and targeting cell cycle checkpoint pathways has revealed promising candidates. Studies in zebrafish and human cell lines suggested that the DNA damage response kinase, checkpoint kinase 1 (Chk1), not only regulates onset of mitosis but also cell death in response to DNA damage in the absence of p53. This effect reportedly relies on ataxia telangiectasia mutated (ATM)-dependent and PIDDosome-mediated activation of Caspase-2. However, we show that genetic ablation of PIDDosome components in mice does not affect cell death in response to γ-irradiation. Furthermore, Chk1 inhibition largely failed to sensitize normal and malignant cells from p53^−/− mice toward DNA damaging agents, and p53 status did not affect the death-inducing activity of DNA damage after Chk1 inhibition in human cancer cells. These observations argue against cross-species conservation of a Chk1-controlled cell survival pathway demanding further investigation of the molecular machinery responsible for cell death elicited by forced mitotic entry in the presence of DNA damage in different cell types and model organisms.     

Nucleophosmin (B23) targets ARF to nucleoli and inhibits its function

The ARF tumor suppressor is a nucleolar protein that activates p53-dependent checkpoints by binding Mdm2, a p53 antagonist. Despite persuasive evidence that ARF can bind and inactivate Mdm2 in the nucleoplasm, the prevailing view is that ARF exerts its growth-inhibitory activities from within the nucleolus. We suggest ARF primarily functions outside the nucleolus and provide evidence that it is sequestered and held inactive in that compartment by a nucleolar phosphoprotein, nucleophosmin (NPM). Most cellular ARF is bound to NPM regardless of whether cells are proliferating or growth arrested, indicating that ARF-NPM association does not correlate with growth suppression. Notably, ARF binds NPM through the same domains that mediate nucleolar localization and Mdm2 binding, suggesting that NPM could control ARF localization and compete with Mdm2 for ARF association. Indeed, NPM knockdown markedly enhanced ARF-Mdm2 association and diminished ARF nucleolar localization. Those events correlated with greater ARF-mediated growth suppression and p53 activation. Conversely, NPM overexpression antagonized ARF function while increasing its nucleolar localization. These data suggest that NPM inhibits ARF's p53-dependent activity by targeting it to nucleoli and impairing ARF-Mdm2 association.     

Nuclear DNA helicase II (RNA helicase A) binds to an F-actin containing shell that surrounds the nucleolus

Nuclear DNA helicase II (NDH II), alternatively named RNA helicase A (RHA), is an F-actin binding protein that is particularly enriched in the nucleolus of mouse cells. Here, we show that the nucleolar localization of NDH II of murine 3T3 cells depended on an ongoing rRNA synthesis. NDH II migrated out of the nucleolus after administration of 0.05 microg/ml actinomycin D, while nucleolin and the upstream binding factor (UBF) remained there. In S phase-arrested mouse cells, NDH II was frequently found at the nucleolar periphery, where it was accompanied by newly synthesized nucleolar RNA. Human NDH II was mainly distributed through the whole nucleoplasm and not enriched in the nucleoli. However, in the human breast carcinoma cell line MCF-7, NDH II was also found at the nucleolar periphery, together with the tumor suppressor protein p53. Both NDH II and p53 were apparently attached to the F-actin-based filamentous network that surrounded the nucleoli. Accordingly, this subnuclear structure was sensitive to F-actin depolymerizing agents. Depolymerization with gelsolin led to a striking accumulation of NDH II in the nucleoli of MCF-7 cells. This effect was abolished by RNase, which extensively released nucleolus-bound NDH II when added together with gelsolin. Taken together, these results support the idea that an actin-based filamentous network may anchor NDH II at the nucleolar periphery for pre-ribosomal RNA processing, ribosome assembly, and/or transport.