The nucleolus: at the stem of immortality.
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Animals↗
Biomedical subjects
Pier Paolo Pandolfi
Publications and source records attributed to Pier Paolo Pandolfi.
Does the ribosome translate cancer?
Ribosome biogenesis and translation control are essential cellular processes that are governed at numerous levels. Several tumour suppressors and proto-oncogenes have been found either to affect the formation of the mature ribosome or to regulate the activity of proteins known as translation factors. Disruption in one or more of the steps that control protein biosynthesis has been associated with alterations in the cell cycle and regulation of cell growth. Therefore, certain tumour suppressors and proto-oncogenes might regulate malignant progression by altering the protein synthesis machinery. Although many studies have correlated deregulation of protein biosynthesis with cancer, it remains to be established whether this translates directly into an increase in cancer susceptibility, and under what circumstances.
Cell Cycle Proteins↗
Promyelocytic leukemia protein mediates interferon-based anti-herpes simplex virus 1 effects.
Herpes simplex virus (HSV) 1 disaggregates the nuclear domain 10 (ND10) nuclear structures and disperses its organizing promyelocytic leukemia protein (PML). An earlier report showed that ectopic overexpression of PML precludes the disaggregation of ND10 but has no effect on viral replication. PML has been reported to mediate the effects of interferon (IFN) and viral mutants lacking ICP0 (Delta alpha 0 mutants). To test the hypothesis that HSV disaggregates ND10 structures and disperses PML to preclude IFN-mediated antiviral effects, we tested the accumulation of viral proteins and virus yields from murine PML(+/+) and PML(-/-) cells mock treated or exposed to IFN-alpha, IFN-gamma, or both and infected with the wild-type or Delta alpha 0 mutant virus. We report the following results. (i) The levels of growth of wild-type and mutant viruses and of accumulation of viral proteins were not significantly different in untreated PML(+/+) and PML(-/-) cells. (ii) Major effects of IFN-alpha and -gamma were observed in PML(+/+) cells infected with the Delta alpha 0 mutant virus, and more minor effects were observed in cells infected with the wild-type virus. The effects of the IFNs on either wild-type or the mutant virus in PML(-/-) cells were minimal. (iii) The mixture of IFN-alpha and -gamma was more effective than either IFN alone, but again, the effect was more drastic in PML(+/+) cells than in PML(-/-) cells. We concluded that the anti-HSV state induced by exogenous IFN is mediated by PML and that the virus targets the ND10 structures and disseminates PML in order to preclude the establishment of the antiviral state induced by IFNs.
Animals↗
As(2)O(3) enhances retroviral reverse transcription and counteracts Ref1 antiviral activity.
Potent drugs such as cyclosporine have provided effective probes of signal transduction pathways and, as well, of human immunodeficiency virus type 1 (HIV-1) replication mechanisms. Recently, it was reported that As(2)O(3), a drug used to treat acute promyelocytic leukemia (PML), stimulates HIV-1 replication. We found that As(2)O(3) accelerates the kinetics of a spreading HIV-1 infection in human T cells and increases the number of cells bearing HIV-1 provirus after a single round of infection. The stimulatory effect occurred after membrane fusion and resulted in increased steady-state levels of newly synthesized viral cDNA. Stimulation was independent of HIV-1 env and most viral accessory genes, and As(2)O(3) had no detectable effects on viral expression postintegration or virion assembly. Murine leukemia virus (MLV) transduction was enhanced by As(2)O(3) to the same extent as HIV-1 transduction, but As(2)O(3) had no additional effect on Fv1 restriction. In contrast, As(2)O(3) largely overcame the specific block to N-tropic MLV reverse transcription posed by human Ref1. As(2)O(3) disrupts PML bodies, nuclear structures named for a major component, the PML protein. We observed no changes in PML bodies in response to HIV-1 infection. Experiments with PML-null target cells indicated that PML has no effect on HIV-1 infectivity and is dispensable for the stimulatory effect of As(2)O(3). As(2)O(3) caused cell death in uninfected cells at the same concentrations which stimulate HIV-1 replication. Among four additional apoptosis-inducing agents, a boost in HIV-1 infectivity was observed only with carbonyl cyanide m-chlorophenylhydrazone, a compound which, like As(2)O(3), disrupts the mitochondrial transmembrane potential. In summary, As(2)O(3) stimulates retroviral reverse transcription, perhaps via effects on mitochondria, and provides a useful tool for characterizing Ref1.
Animals↗
Targeting aberrant transcriptional repression in acute myeloid leukemia.
Histone acetyltransferase (HAT) and histone deacetylase (HDAC) activities determine the acetylation status of histones, and have the ability to regulate gene expression through chromatin remodeling. Aberrant histone acetylation is known to play a key role in leukemogenesis. A common biologic feature, shared by genetically heterogeneous acute myeloid leukemias (AML), is a block of hematopoietic differentiation by the fusion proteins produced by chromosomal translocations. In many cases, a DNA binding fusion protein, which abnormally interacts with transcriptional co-regulators and increases local concentration of HDAC complexes, imposes a transcriptional repressive state on target gene promoters, which may become refractory to physiologic stimuli. To target this transcriptional repression, HDAC inhibitors (HDACI) have been developed, which are thought to derepress a set of genes whose transcriptional activation induces cell-cycle arrest, apoptosis and cellular differentiation and thus anti-tumoral activity. Therefore, HDACI might be utilized as effective antileukemic agents, and are currently under clinical trials.
Acetyltransferases↗
Rabies virus P and small P products interact directly with PML and reorganize PML nuclear bodies.
The interferon-induced promyelocytic leukaemia (PML) protein localizes both in the nucleoplasm and in matrix-associated multi-protein complexes known as nuclear bodies (NBs). NBs are disorganized in acute promyelocytic leukaemia or during some viral infections, suggesting that PML NBs could be a part of cellular defense mechanism. Rabies virus, a member of the rhabdoviridae family, replicates in the cytoplasm. Rabies phosphoprotein P and four other amino-terminally truncated products (P2, P3, P4, P5) are all translated from P mRNA. P and P2 are located in the cytoplasm, whereas P3, P4 and P5 are found mostly in the nucleus. Infection with rabies virus reorganized PML NBs. PML NBs became larger and appeared as dense aggregates when analysed by confocal or electron microscopy, respectively. The expression of P sequesters PML in the cytoplasm where both proteins colocalize, whereas that of P3 results in an increase in PML body size, as observed in infected cells. The P and P3 interacted directly in vivo and in vitro with PML. The C-terminal domain of P and the PML RING finger seem to be involved in this binding. Moreover, PML-/- primary mouse embryonic fibroblasts expressed viral proteins at a higher level and produced 20 times more virus than wild-type cells, suggesting that the absence of all PML isoforms resulted in an increase in rabies virus replication.
Animals↗
Atypical t(15;17)(q13;q12) in a patient with all-trans retinoic acid refractory secondary acute promyelocytic leukemia: a case report and review of the literature.
A 69-year-old woman developed microgranular acute promyelocytic leukemia (APL-M3) 10 months after receiving adjuvant cyclophosphamide, doxorubicin, and paclitaxel for breast cancer. Replicate bone marrow aspirate karyotypes contained a translocation between the long arms of chromosomes 15 and 17, but not at breakpoints typical for APL. Fluorescence in situ hybridization paints and RARalpha/PML cosmid probes verified that the breakpoints on chromosomes 15 and 17 were proximal to both the PML and RARalpha genes; t(15;17)(q13;12). Although the patient received induction chemotherapy and a several month trial of all-trans retinoic acid (ATRA), there was no clinical improvement or hematological remission. We suspect that this patient developed postchemotherapy secondary APL with an atypical t(15;17), which rendered her leukemic cells unresponsive to ATRA therapy.
Aged↗
Maternally transmitted severe glucose 6-phosphate dehydrogenase deficiency is an embryonic lethal.
Mouse chimeras from embryonic stem cells in which the X-linked glucose 6-phosphate dehydrogenase (G6PD) gene had been targeted were crossed with normal females. First-generation (F(1)) G6PD(+/-) heterozygotes born from this cross were essentially normal; analysis of their tissues demonstrated strong selection for cells with the targeted G6PD allele on the inactive X chromosome. When these F(1) G6PD(+/-) females were bred to normal males, only normal G6PD mice were born, because: (i) hemizygous G6PD(-) male embryos died by E10.5 and their development was arrested from E7.5, the time of onset of blood circulation; (ii) heterozygous G6PD(+/-) females showed abnormalities from E8.5, and died by E11.5; and (iii) severe pathological changes were present in the placenta of both G6PD(-) and G6PD(+/-) embryos. Thus, G6PD is not indispensable for early embryo development; however, severe G6PD deficiency in the extraembryonic tissues (consequent on selective inactivation of the normal paternal G6PD allele) impairs the development of the placenta and causes death of the embryo. Most importantly, G6PD is indispensable for survival when the embryo is exposed to oxygen through its blood supply.
Alleles↗
The topoisomerase I-binding RING protein, topors, is associated with promyelocytic leukemia nuclear bodies.
We previously identified topors as a topoisomerase I-binding protein that localizes in punctate nuclear regions when expressed as a GFP fusion protein. We now demonstrate that both the GFP-topors fusion protein and endogenous topors are associated with promyelocytic leukemia (PML) nuclear bodies in exponentially growing HeLa cells. Studies using isogenic PML+/+ and PML-/- murine embryonic fibroblasts indicate that the punctate nuclear localization of topors is dependent on PML. A basic C-terminal region but not the N-terminal RING domain of topors is required for the punctate nuclear localization of this protein. Additional studies indicate that topors, but not PML, rapidly relocalizes from nuclear bodies to the nucleoplasm in cells exposed to the transcription inhibitor dichloro-1-beta-d-ribofuranolsylbenzimidazole or to the topoisomerase I-targeting drug camptothecin. These results identify topors as a new member of the group of proteins that associate dynamically with PML nuclear bodies and suggest that topors may be involved in the cellular response to camptothecin.
Amino Acid Sequence↗
Role of GITR in activation response of T lymphocytes.
In this study, we describe the generation and characterization of mice in which GITR gene (TNFRSF18 [tumor necrosis factor receptor superfamily 18]), a member of the TNFRSF expressed mainly on T lymphocytes, has been ablated (GITR(-/-) mice). Results indicate that GITR inactivation does not impair the normal development of the lymphoid organs but modulates T-cell activation. In fact, when GITR(-/-) T lymphocytes are activated by treatment with an anti-CD3 monoclonal antibody they proliferate more than wild-type cells. Moreover, activated GITR(-/-) T lymphocytes express higher levels of interleukin-2 receptor, produce larger amounts of interleukin-2, and are more sensitive to activation-induced cell death than controls. These results suggest that GITR is involved in the regulation of T-cell receptor/CD3-driven T-cell activation and programmed cell death.
Adjuvants, Immunologic↗
Bethesda proposals for classification of nonlymphoid hematopoietic neoplasms in mice.
The hematopathology subcommittee of the Mouse Models of Human Cancers Consortium recognized the need for a classification of murine hematopoietic neoplasms that would allow investigators to diagnose lesions as well-defined entities according to accepted criteria. Pathologists and investigators worked cooperatively to develop proposals for the classification of lymphoid and nonlymphoid hematopoietic neoplasms. It is proposed here that nonlymphoid hematopoietic neoplasms of mice be classified in 4 broad categories: nonlymphoid leukemias, nonlymphoid hematopoietic sarcomas, myeloid dysplasias, and myeloid proliferations (nonreactive). Criteria for diagnosis and subclassification of these lesions include peripheral blood findings, cytologic features of hematopoietic tissues, histopathology, immunophenotyping, genetic features, and clinical course. Differences between murine and human lesions are reflected in the terminology and methods used for classification. This classification will be of particular value to investigators seeking to develop, use, and communicate about mouse models of human hematopoietic neoplasms.
Animals↗
The t(8;21) fusion protein, AML1 ETO, specifically represses the transcription of the p14(ARF) tumor suppressor in acute myeloid leukemia.
The t(8;21) is one of the most frequent chromosomal translocations associated with acute leukemia. This translocation creates a fusion protein consisting of the acute myeloid leukemia-1 transcription factor and the eight-twenty-one corepressor (AML1 ETO), which represses transcription through AML1 (RUNX1) DNA binding sites and immortalizes hematopoietic progenitor cells. We have identified the p14(ARF) tumor suppressor, a mediator of the p53 oncogene checkpoint, as a direct transcriptional target of AML1 ETO. AML1 ETO repressed the p14(ARF) promoter and reduced endogenous levels of p14(ARF) expression in multiple cell types. In contrast, AML1 stimulated p14(ARF) expression and induced phenotypes consistent with cellular senescence. Chromatin immunoprecipitation assays demonstrated that AML1 ETO was specifically bound to the p14(ARF) promoter. In acute myeloid leukemia samples containing the t(8;21), levels of p14(ARF) mRNA were markedly lower when compared with other acute myeloid leukemias lacking this translocation. Repression of p14(ARF) may explain why p53 is not mutated in t(8;21)-containing leukemias and suggests that p14(ARF) is an important tumor suppressor in a large number of human leukemias.
Antigens, CD↗
Nuclear and cytoplasmic shuttling of TRADD induces apoptosis via different mechanisms.
The adapter protein tumor necrosis factor receptor (TNFR)1-associated death domain (TRADD) plays an essential role in recruiting signaling molecules to the TNFRI receptor complex at the cell membrane. Here we show that TRADD contains a nuclear export and import sequence that allow shuttling between the nucleus and the cytoplasm. In the absence of export, TRADD is found within nuclear structures that are associated with promyelocytic leukemia protein (PML) nuclear bodies. In these structures, the TRADD death domain (TRADD-DD) can activate an apoptosis pathway that is mechanistically distinct from its action at the membrane-bound TNFR1 complex. Apoptosis by nuclear TRADD-DD is promyelocytic leukemia protein dependent, involves p53, and is inhibited by Bcl-xL but not by caspase inhibitors or dominant negative FADD (FADD-DN). Conversely, apoptosis induced by TRADD in the cytoplasm is resistant to Bcl-xL, but sensitive to caspase inhibitors and FADD-DN. These data indicate that nucleocytoplasmic shuttling of TRADD leads to the activation of distinct apoptosis mechanisms that connect the death receptor apparatus to nuclear events.
Amino Acid Sequence↗
Modelling haematopoietic malignancies in the mouse and therapeutical implications.
Modelling human disease in the mouse has become an essential activity in biomedical research in order to unravel molecular mechanisms underlying pathological conditions as well as to determine in vivo the consequences of aberrant gene function. The mouse is by far the most accessible mammalian system physiologically similar to humans. Furthermore, the development of novel techniques for manipulating the murine genome, which allow the in vivo modification of virtually any genomic region in a time and/or tissue specific manner, renders the mouse an ideal model system to study human pathological conditions. Modelling human diseases in mice has reached an even greater relevance in the field of haematological malignancies, due to the already advanced characterization of the molecular basis of many haematological disorders. In this review, we describe the most important technological developments that made it possible to reproduce in the mouse the genetic lesions that characterize human haematological malignancies, thus often generating faithful mouse models of the human condition. We provide specific examples of the advantages and limitations of the various genetic approaches utilized to model leukaemia and lymphoma in the mouse. Finally, we discuss the power of mouse modelling in developing and testing novel therapeutic modalities in pre-clinical studies.
Animals↗
Downstream of kinase, p62(dok), is a mediator of Fc gamma IIB inhibition of Fc epsilon RI signaling.
The low-affinity receptor for IgG, Fc gamma RIIB, is expressed widely in the immune system and functions to attenuate Ag-induced immune responses. In mast cells, coaggregation of Fc gamma RIIB with the high-affinity IgE receptor, Fc epsilon RI, leads to inhibition of Ag-induced degranulation and cytokine production. Fc gamma RIIB inhibitory activity requires a conserved motif within the Fc gamma RIIB cytoplasmic domain termed the immunoreceptor tyrosine-based inhibition motif. When coaggregated with an activating receptor (e.g., Fc epsilon RI, B cell Ag receptor), Fc gamma RIIB is rapidly phosphorylated on tyrosine and recruits the SH2 domain-containing inositol 5-phosphatase (SHIP). However, the mechanisms by which SHIP mediates Fc gamma RIIB inhibitory function in mast cells remain poorly defined. In this report we demonstrate that Fc gamma RIIB coaggregation with Fc epsilon RI stimulates enhanced SHIP tyrosine phosphorylation and association with Shc and p62(dok). Concurrently, enhanced p62(dok) tyrosine phosphorylation and association with RasGAP are observed, suggesting that SHIP may mediate Fc gamma RIIB inhibitory function in mast cells via recruitment of p62(dok) and RasGAP. Supporting this hypothesis, recruitment of p62(dok) to Fc epsilon RI is sufficient to inhibit Fc epsilon RI-induced calcium mobilization and extracellular signal-regulated kinase 1/2 activation. Interestingly, both the amino-terminal pleckstrin homology and phosphotyrosine binding domains and the carboxyl-terminal proline/tyrosine-rich region of p62(dok) can mediate inhibition, suggesting activation of parallel downstream signaling pathways that converge at extracellular signal-regulated kinase 1/2 activation. Finally, studies using gene-ablated mice indicate that p62(dok) is dispensable for Fc gamma RIIB inhibitory signaling in mast cells. Taken together, these data suggest a role for p62(dok) as a mediator of Fc gamma RIIB inhibition of Fc epsilon RI signal transduction in mast cells.
Adaptor Proteins, Signal Transducing↗
The role of PML in tumor suppression.
The PML gene, involved in the t(15;17) chromosomal translocation of acute promyelocytic leukemia (APL), encodes a protein which localizes to the PML-nuclear body, a subnuclear macromolecular structure. PML controls apoptosis, cell proliferation, and senescence. Here, we review the current understanding of its role in tumor suppression.
Adaptor Proteins, Signal Transducing↗
PTEN in neural precursor cells: regulation of migration, apoptosis, and proliferation.
PTEN is a lipid phosphatase, and PTEN mutations are associated with gliomas, macrocephaly, and mental deficiencies. We have used PTEN +/- mice to assess PTEN's role in subventricular zone (SVZ) precursor cells. For cultured SVZ neurosphere cells, haploinsufficiency for PTEN increases phosphorylation of Akt and forkhead transcription factor and slightly enhances proliferation. Based on a filter penetration assay, PTEN +/- cells are substantially more migratory and invasive than +/+ cells. The +/- cells also are more resistant to H(2)O(2)-induced apoptosis. Analysis of PTEN +/- and +/+ mice by BrdU labeling reveals no difference in the rate of cell proliferation in the SVZ. Exit of BrdU-labeled cells from the SVZ and radial migration to the outer layers of the olfactory bulb are more rapid for +/- cells. These observations indicate that PTEN regulates SVZ precursor cell function and is particularly important for migration and apoptosis in response to oxidative stress.
Animals↗
The ETS protein MEF plays a critical role in perforin gene expression and the development of natural killer and NK-T cells.
We utilized gene targeting by homologous recombination to define the role that MEF, a transcriptional activating member of the ETS family of transcription factors, plays in lymphopoiesis. MEF-/- mice have a profound reduction in the number of NK-T and NK cells. Purified MEF-/- NK cells cannot lyse tumor cell targets and secrete only minimal amounts of IFNgamma. Perforin protein expression is severely impaired in MEF-deficient NK cells, likely accounting for the lack of tumor cell cytotoxicity. Promoter studies and chromatin immunoprecipitation analyses demonstrate that MEF and not ETS-1 directly regulates transcription of the perforin gene in NK cells. Our results uncover a specific role of MEF in the development and function of NK cells and in innate immunity.
Animals↗