RESUMEN
Senescent cells accumulate in the host during the aging process and are associated with age-related pathogeneses, including cancer. Although persistent senescence seems to contribute to many aspects of cellular pathways and homeostasis, the role of senescence in virus-induced human cancer is not well understood. Merkel cell carcinoma (MCC) is an aggressive skin cancer induced by a life-long human infection of Merkel cell polyomavirus (MCPyV). Here, we show that MCPyV large T (LT) antigen expression in human skin fibroblasts causes a novel nucleolar stress response, followed by p21-dependent senescence and senescence-associated secretory phenotypes (SASPs), which are required for MCPyV genome maintenance. Senolytic and navitoclax treatments result in decreased senescence and MCPyV genome levels, suggesting a potential therapeutic for MCC prevention. Our results uncover the mechanism of a host stress response regulating human polyomavirus genome maintenance in viral persistency, which may lead to targeted intervention for MCC.
Asunto(s)
Carcinoma de Células de Merkel , Poliomavirus de Células de Merkel , Infecciones por Polyomavirus , Neoplasias Cutáneas , Infecciones Tumorales por Virus , Humanos , Infecciones por Polyomavirus/genética , Infecciones Tumorales por Virus/genética , Infecciones Tumorales por Virus/patología , Antígenos Virales de Tumores/genética , Poliomavirus de Células de Merkel/genética , Poliomavirus de Células de Merkel/metabolismo , Carcinoma de Células de Merkel/genética , Carcinoma de Células de Merkel/patología , Neoplasias Cutáneas/patología , Senescencia Celular , Genoma ViralRESUMEN
Nucleolar stress response is caused by perturbations in ribosome biogenesis, induced by the inhibition of ribosomal RNA processing and synthesis, as well as ribosome assembly. This response induces p53 stabilization and activation via ribosomal protein L11 (RPL11), suppressing tumor progression. However, anticancer agents that kill cells via this mechanism, and their relationship with the therapeutic efficiency of these agents, remain largely unknown. Here, we sought to investigate whether topoisomerase inhibitors can induce nucleolar stress response as they reportedly block ribosomal RNA transcription. Using rhabdomyosarcoma and rhabdoid tumor cell lines that are sensitive to the nucleolar stress response, we evaluated whether nucleolar stress response is associated with sensitivity to topoisomerase inhibitors ellipticine, doxorubicin, etoposide, topotecan, and anthracyclines. Cell proliferation assay indicated that small interfering RNA-mediated RPL11 depletion resulted in decreased sensitivity to topoisomerase inhibitors. Furthermore, the expression of p53 and its downstream target proteins via western blotting showed the suppression of p53 pathway activation upon RPL11 knockdown. These results suggest that the sensitivity of cancer cells to topoisomerase inhibitors is regulated by RPL11-mediated nucleolar stress responses. Thus, RPL11 expression may contribute to the prediction of the therapeutic efficacy of topoisomerase inhibitors and increase their therapeutic effect of topoisomerase inhibitors.
Asunto(s)
Neoplasias , Proteína p53 Supresora de Tumor , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Ribosómicas/metabolismo , Nucléolo Celular/metabolismo , Línea Celular Tumoral , Antibióticos Antineoplásicos/farmacología , ARN Ribosómico/genética , Inhibidores de Topoisomerasa II/farmacología , Inhibidores de Topoisomerasa II/metabolismo , Antraciclinas/farmacología , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Neoplasias/metabolismoRESUMEN
Approximately 20% of pediatric patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) relapse or are refractory to chemotherapy despite the low frequency of TP53 mutations. The nucleolar stress response is a P53-activating mechanism via MDM2 inhibition by ribosomal protein L11 (RPL11). We analyzed the role of the nucleolar stress response using BCP-ALL cell lines and patient samples by drug sensitivity tests, Western blotting, and reverse transcription polymerase chain reaction. We revealed that the nucleolar stress response works properly in TP53 wild-type human BCP-ALL cell lines. Next, we found that 6-mercaptopurine, methotrexate, daunorubicin, and cytarabine had anti-leukemic effects via the nucleolar stress response within BCP-ALL treatment. Comparing the samples at onset and relapse in children with BCP-ALL, RPL11 mRNA expression decreased at relapse in seven of nine cases. Furthermore, leukemia cells with relapse acquired resistance to these four drugs and suppressed P53 and RPL11 expression. Our findings suggest that the nucleolar stress response is a novel anti-leukemia mechanism in BCP-ALL. As these four drugs are key therapeutics for BCP-ALL treatment, dysfunction of the nucleolar stress response may be related to clinical relapse or refractoriness. Nucleolar stress response may be a target to predict and improve the chemotherapy effect for pediatric BCP-ALL.
RESUMEN
G protein coupled receptor kinase 5 (GRK5) is localized within the nucleus and moderates functions such as DNA transcription, in addition to its localization at the plasma membrane. In this report, we show that GRK5 modifies the nucleolar stress response activated by the DNA polymerase inhibitor, actinomycin D (ActD). We show an increased sensitivity to the apoptotic effects of ActD on cervical HeLa cells and the breast cancer cell line MDA MB 231 with reduced protein expression of GRK5. We also tested two types of breast cancer cells (MDA MB 231 and MCF7 cells) and found that the rate of response to ActD varied between them because they have innate differences in the protein expression of GRK5. We also found that GRK5 phosphorylates nucleophosmin (NPM1) at T199 before and during the early stages of ActD treatment. Phosphorylation at T199 increases the ability of NPM1 to interact with p14ARF in vitro, which may affect the protein expression levels of p14ARF. We found that the expression levels of p14ARF were lower in the cells transfected with the control shRNA, but higher in cells transfected with GRK5 shRNA. Collectively, this suggests that GRK5 modifies the nucleolar stress response associated with ActD.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Nucléolo Celular/patología , Dactinomicina/farmacología , Quinasa 5 del Receptor Acoplado a Proteína-G/metabolismo , Neoplasias/patología , Proteínas Nucleares/metabolismo , Apoptosis , Nucléolo Celular/efectos de los fármacos , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Quinasa 5 del Receptor Acoplado a Proteína-G/genética , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Nucleares/genética , Nucleofosmina , Fosforilación , Unión Proteica , Células Tumorales CultivadasRESUMEN
Senescence of smooth muscle cells (SMCs) has a crucial role in the pathogenesis of abdominal aortic aneurysm (AAA), a disease of vascular degeneration. Perturbation of cellular ribosomal DNA (rDNA) transcription triggers nucleolar stress response. Previously we demonstrated that induction of nucleolar stress in SMCs elicited cell cycle arrest via the ataxia-telangiectasia mutated (ATM)/ATM- and Rad3-related (ATR)-p53 axis. However, the specific roles of nucleolar stress in vascular degeneration remain unexplored. In the present study, we demonstrated for the first time that in both human and animal AAA tissues, there were non-coordinated changes in the expression of RNA polymerase I machinery components, including a downregulation of transcription initiation factor-IA (TIF-IA). Genetic deletion of TIF-IA in SMCs in mice (smTIF-IA-/-) caused spontaneous aneurysm-like lesions in the aorta. In vitro, induction of nucleolar stress triggered a non-canonical DNA damage response, leading to p53 phosphorylation and a senescence-like phenotype in SMCs. In human AAA tissues, there was increased nucleolar stress in medial cells, accompanied by localized DNA damage response within the nucleolar compartment. Our data suggest that perturbed rDNA transcription and induction of nucleolar stress contribute to the pathogenesis of AAA. Moreover, smTIF-IA-/- mice may be a novel animal model for studying spontaneous AAA-like vascular degenerations.
Asunto(s)
Nucléolo Celular/patología , Proliferación Celular , Senescencia Celular , Daño del ADN , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/patología , Animales , Aneurisma de la Aorta Abdominal/genética , Aneurisma de la Aorta Abdominal/metabolismo , Aneurisma de la Aorta Abdominal/patología , Puntos de Control del Ciclo Celular , Línea Celular , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , ADN Ribosómico/genética , ADN Ribosómico/metabolismo , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Fenotipo , Fosforilación , Proteínas del Complejo de Iniciación de Transcripción Pol1/genética , Proteínas del Complejo de Iniciación de Transcripción Pol1/metabolismo , Transducción de Señal , Transcripción Genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The translation of messenger RNA (mRNA) into protein is a multistep process by which genetic information transcribed into an mRNA is decoded to produce a specific polypeptide chain of amino acids. Ribosomes play a central role in translation by coordinately working with various translation regulatory factors and aminoacyl-transfer RNAs. Various stresses attenuate the ribosomal synthesis in the nucleolus as well as the translation rate in the cytosol. To efficiently reallocate cellular energy and resources, mammalian cells are endowed with mechanisms that directly link the suppression of translation-related processes to the activation of stress adaptation programmes. This review focuses on the integrated stress response (ISR) and the nucleolar stress response (NSR) both of which are activated by various stressors and selectively upregulate stress-responsive transcription factors. Emerging findings have delineated the detailed molecular mechanisms of the ISR and NSR and expanded their physiological and pathological significances.
Asunto(s)
Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Estrés Fisiológico , Factores de Transcripción/metabolismo , Animales , Humanos , ARN Mensajero/genéticaRESUMEN
The nucleolus is a dynamically changing organelle that is central to a number of important cellular functions. Not only is it important for ribosome biogenesis, but it also reacts to stress by instigating a nucleolar stress response and is further involved in regulating the cell cycle. Several studies report nucleolar dysfunction in Alzheimer's disease (AD). Studies have reported a decrease in both total nucleolar volume and transcriptional activity of the nucleolar organizing regions. Ribosomes appear to be targeted by oxidation and reduced protein translation has been reported. In addition, several nucleolar proteins are dysregulated and some of these appear to be implicated in classical AD pathology. Some studies also suggest that the nucleolar stress response may be activated in AD, albeit this latter research is rather limited and requires further investigation. The purpose of this review is to draw the connections of all these studies together and signify that there are clear changes in the nucleolus and the ribosomes in AD. The nucleolus is therefore an organelle that requires more attention than previously given in relation to understanding the biological mechanisms underlying the disease.
RESUMEN
The mammalian Sirt1 deacetylase is generally thought to be a nuclear protein, but some pilot studies have suggested that Sirt1 may also be involved in orchestrating nucleolar functions. Here, we show that nucleolar stress response is a ubiquitous cellular reaction that can be induced by different types of stress conditions, and Sirt1 is an endogenous suppressor of nucleolar stress response. Using stable isotope labeling by amino acids in cell culture approach, we have identified a physical interaction of between Sirt1 and the nucleolar protein nucleophosmin, and this protein-protein interaction appears to be necessary for Sirt1 inhibition on nucleolar stress, whereas the deacetylase activity of Sirt1 is not strictly required. Based on the reported prerequisite role of nucleolar stress response in stress-induced p53 protein accumulation, we have also provided evidence suggesting that Sirt1-mediated inhibition on nucleolar stress response may represent a novel mechanism by which Sirt1 can modulate intracellular p53 accumulation independent of lysine deacetylation. This process may represent an alternative mechanism by which Sirt1 regulates functions of the p53 pathway.
Asunto(s)
Nucléolo Celular/metabolismo , Sirtuina 1/metabolismo , Estrés Fisiológico , Proteína p53 Supresora de Tumor/metabolismo , Acetilación , Células HeLa , Humanos , Imagen ÓpticaRESUMEN
Ribosomes, which synthesize the proteins of a cell, comprise ribosomal RNA and ribosomal proteins, which coassemble hierarchically during a process termed ribosome biogenesis. Historically, biochemical and molecular biology approaches have revealed how preribosomal particles form and mature in consecutive steps, starting in the nucleolus and terminating after nuclear export into the cytoplasm. However, only recently, due to the revolution in cryo-electron microscopy, could pseudoatomic structures of different preribosomal particles be obtained. Together with in vitro maturation assays, these findings shed light on how nascent ribosomes progress stepwise along a dynamic biogenesis pathway. Preribosomes assemble gradually, chaperoned by a myriad of assembly factors and small nucleolar RNAs, before they reach maturity and enter translation. This information will lead to a better understanding of how ribosome synthesis is linked to other cellular pathways in humans and how it can cause diseases, including cancer, if disturbed.
Asunto(s)
Eucariontes/metabolismo , ARN Ribosómico/metabolismo , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Nucléolo Celular/metabolismo , Microscopía por Crioelectrón , Humanos , Biogénesis de Organelos , Multimerización de ProteínaRESUMEN
Alkaloids isolated from the Amaryllidaceae plants have potential as therapeutics for treating human diseases. Haemanthamine has been studied as a novel anticancer agent due to its ability to overcome cancer cell resistance to apoptosis. Biochemical experiments have suggested that hemanthamine targets the ribosome. However, a structural characterization of its mechanism has been missing. Here we present the 3.1 Å resolution X-ray structure of haemanthamine bound to the Saccharomyces cerevisiae 80S ribosome. This structure reveals that haemanthamine targets the A-site cleft on the large ribosomal subunit rearranging rRNA to halt the elongation phase of translation. Furthermore, we provide evidence that haemanthamine and other Amaryllidaceae alkaloids also inhibit specifically ribosome biogenesis, triggering nucleolar stress response and leading to p53 stabilization in cancer cells. Together with a computer-aided interpretation of existing structure-activity relationships of Amaryllidaceae alkaloids congeners, we provide a rationale for designing molecules with enhanced potencies and reduced toxicities.
Asunto(s)
Alcaloides de Amaryllidaceae/farmacología , Antineoplásicos/farmacología , Neoplasias del Colon/metabolismo , Fenantridinas/farmacología , Ribosomas/metabolismo , Saccharomyces cerevisiae/metabolismo , Alcaloides de Amaryllidaceae/química , Antineoplásicos/química , Sitios de Unión , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Cristalografía por Rayos X , Células HCT116 , Humanos , Modelos Moleculares , Conformación Molecular , Fenantridinas/química , ARN Ribosómico/química , ARN Ribosómico/metabolismo , Ribosomas/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Relación Estructura-Actividad , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Increasing attention has been paid to certain ribosomal or ribosome biosynthesis-related proteins involved in oncogenesis. Members of one group are classified as "tumor suppressive factors" represented by RPL5 and RPL11; loss of their functions leads to cancer predisposition. RPL5 and RPL11 prevent tumorigenesis by binding to and inhibiting the MDM2 ubiquitin ligase and thereby up-regulating p53. Many other candidate tumor suppressive ribosomal/nucleolar proteins have been suggested. However, it remains to be experimentally clarified whether many of these factors can actually prevent tumorigenesis and if so, how they do so. Conversely, some ribosomal/nucleolar proteins promote tumorigenesis. For example, PICT1 binds to and anchors RPL11 in nucleoli, down-regulating p53 and promoting tumorigenesis. GRWD1 was recently identified as another such factor. When overexpressed, GRWD1 suppresses p53 and transforms normal human cells, probably by binding to RPL11 and sequestrating it from MDM2. However, other pathways may also be involved.
Asunto(s)
Nucléolo Celular/metabolismo , Proteínas Nucleares/metabolismo , Ribosomas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Nucléolo Celular/genética , Humanos , Proteínas Nucleares/genética , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Ribosomas/genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The nucleolus is the most prominent nuclear domain, where the core processes of ribosome biogenesis occur vigorously. All these processes are finely orchestrated by many nucleolar factors to build precisely ribosome particles. In animal cells, perturbations of ribosome biogenesis, mostly accompanied by structural disorders of the nucleolus, cause a kind of cellular stress to induce cell cycle arrest, senescence, or apoptosis, which is called nucleolar stress response. The best-characterized pathway of this stress response involves p53 and MDM2 as key players. p53 is a crucial transcription factor that functions in response to not only nucleolar stress but also other cellular stresses such as DNA damage stress. These cellular stresses release p53 from the inhibition by MDM2, an E3 ubiquitin ligase targeting p53, in various ways, which leads to p53-dependent activation of a set of genes. In plants, genetic impairments of ribosome biogenesis factors or ribosome components have been shown to cause characteristic phenotypes, including a narrow and pointed leaf shape, implying a common signaling pathway connecting ribosomal perturbations and certain aspects of growth and development. Unlike animals, however, plants have neither p53 nor MDM2 family proteins. Then the question arises whether plant cells have a nucleolar stress response pathway. In recent years, it has been reported that several members of the plant-specific transcription factor family NAC play critical roles in the pathways responsive to various cellular stresses. In this mini review, we outline the plant cellular stress response pathways involving NAC transcription factors with reference to the p53-MDM2-dependent pathways of animal cells, and discuss the possible involvement of a plant-unique, NAC-mediated pathway in the nucleolar stress response in plants.
RESUMEN
RNA polymerase I (Pol I)-mediated transcription of the ribosomal RNA genes (rDNA) is confined to the nucleolus and is a rate-limiting step for cell growth and proliferation. Inhibition of Pol I by CX-5461 can selectively induce p53-mediated apoptosis of tumour cells in vivo. Currently, CX-5461 is in clinical trial for patients with advanced haematological malignancies (Peter Mac, Melbourne). Here we demonstrate that CX-5461 also induces p53-independent cell cycle checkpoints mediated by ATM/ATR signaling in the absence of DNA damage. Further, our data demonstrate that the combination of drugs targeting ATM/ATR signaling and CX-5461 leads to enhanced therapeutic benefit in treating p53-null tumours in vivo, which are normally refractory to each drug alone. Mechanistically, we show that CX-5461 induces an unusual chromatin structure in which transcriptionally competent relaxed rDNA repeats are devoid of transcribing Pol I leading to activation of ATM signaling within the nucleoli. Thus, we propose that acute inhibition of Pol transcription initiation by CX-5461 induces a novel nucleolar stress response that can be targeted to improve therapeutic efficacy.