RESUMEN
Over the last decades, cancer research has focused on tumor suppressor genes and oncogenes. Genes in other cellular pathways has received less attention. Between 0.5% to 1% of the mammalian genome encodes for proteins that are tethered on the cell membrane via a glycosylphosphatidylinositol (GPI)-anchor. The GPI modification pathway is complex and not completely understood. Prion (PrP), a GPI-anchored protein, is infamous for being the only normal protein that when misfolded can cause and transmit a deadly disease. Though widely expressed and highly conserved, little is known about the functions of PrP. Pancreatic cancer and melanoma cell lines express PrP. However, in these cell lines the PrP exists as a pro-PrP as defined by retaining its GPI anchor peptide signal sequence (GPI-PSS). Unexpectedly, the GPI-PSS of PrP has a filamin A (FLNA) binding motif and binds FLNA. FLNA is a cytolinker protein, and an integrator of cell mechanics and signaling. Binding of pro-PrP to FLNA disrupts the normal FLNA functions. Although normal pancreatic ductal cells lack PrP, about 40% of patients with pancreatic ductal cell adenocarcinoma express PrP in their cancers. These patients have significantly shorter survival time compared with patients whose cancers lack PrP. Pro-PrP is also detected in melanoma in situ but is undetectable in normal melanocyte, and invasive melanoma expresses more pro-PrP. In this review, we will discuss the underlying mechanisms by which binding of pro-PrP to FLNA disrupts normal cellular physiology and contributes to tumorigenesis, and the potential mechanisms that cause the accumulation of pro-PrP in cancer cells.
Asunto(s)
Proteínas Contráctiles/metabolismo , Glicosilfosfatidilinositoles/metabolismo , Proteínas de Microfilamentos/metabolismo , Priones/metabolismo , Precursores de Proteínas/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Filaminas , Humanos , Melanoma/metabolismo , Melanoma/mortalidad , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/mortalidad , Priones/genética , Unión Proteica , Transducción de SeñalRESUMEN
BACKGROUND: Upper gastrointestinal tract intestinal metaplasia (IM) is termed Barrett's oesophagus (BO) or gastric intestinal metaplasia (GIM), depending on its location. BO and GIM are associated with chemical exposure resulting from gastro-oesophageal reflux and chronic Helicobacter pylori infection, respectively. Paneth cells (PCs), characterised by cytoplasmic eosinophilic granules, are found in a subset of IM at these sites, but histology may not accurately detect them. AIM: To determine human defensin 5 (HD5; an antimicrobial peptide produced by PCs) expression in BO and GIM, and to investigate its association with H pylori infection. METHODS: Endoscopic biopsies from 33 patients with BO and 51 with GIM, and control tissues, were examined by routine histology and for H pylori infection and HD5 mRNA and protein expression. RESULTS: In normal tissues, HD5 expression was specific for PCs in the small intestine. Five patients with BE and 42 with GIM expressed HD5, but few HD5 expressing cells in IM had the characteristic histological features of PCs. Most HD5 positive specimens were H pylori infected and most HD5 negative specimens were not infected. CONCLUSIONS: HD5 immunohistochemistry was often positive in IM when PCs were absent by conventional histology. Thus, HD5 immunohistochemistry may be superior to histology for identifying metaplastic PCs and distinguishing GIM from BO. The higher frequency of HD5 expression in GIM than in BO is associated with a higher frequency of H pylori infection, suggesting that in IM PCs may form part of the mucosal antibacterial response.
Asunto(s)
Esófago de Barrett/metabolismo , Defensinas/metabolismo , Mucosa Gástrica/metabolismo , Adulto , Anciano , Esófago de Barrett/microbiología , Western Blotting/métodos , Defensinas/genética , Defensinas/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Unión Esofagogástrica/metabolismo , Unión Esofagogástrica/patología , Femenino , Mucosa Gástrica/patología , Expresión Génica , Infecciones por Helicobacter/complicaciones , Infecciones por Helicobacter/metabolismo , Helicobacter pylori , Humanos , Masculino , Metaplasia/metabolismo , Metaplasia/microbiología , Persona de Mediana Edad , Células de Paneth/metabolismo , Células de Paneth/patología , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
In recent years major outbreaks of prion disease linked to oral exposure of the prion agent have occurred in animal and human populations. These disorders are associated with a conformational change of a normal protein, PrP(C) (prion protein cellular), to a toxic and infectious form, PrP(Sc) (prion protein scrapie). None of the prionoses currently have an effective treatment. A limited number of active immunization approaches have been shown to slightly prolong the incubation period of prion infection. Active immunization in wild-type animals is hampered by auto-tolerance to PrP and potential toxicity. Here we report that mucosal vaccination with an attenuated Salmonella vaccine strain expressing the mouse PrP, is effective at overcoming tolerance to PrP and leads to a significant delay or prevention of prion disease in mice later exposed orally to the 139A scrapie strain. This mucosal vaccine induced gut anti-PrP immunoglobulin (Ig)A and systemic anti-PrP IgG. No toxicity was evident with this vaccination approach. This promising finding suggests that mucosal vaccination may be a useful method for overcoming tolerance to PrP and preventing prion infection among animal and potentially human populations at risk.
Asunto(s)
Inmunoterapia , Membrana Mucosa/inmunología , Proteínas PrPC/inmunología , Enfermedades por Prión/inmunología , Enfermedades por Prión/prevención & control , Vacunación , Administración Oral , Análisis de Varianza , Animales , Western Blotting/métodos , Femenino , Regulación Viral de la Expresión Génica/fisiología , Inmunoglobulina A/metabolismo , Inmunoglobulina G/metabolismo , Ratones , Proteínas PrPC/química , Enfermedades por Prión/virología , Conformación Proteica , Factores de TiempoRESUMEN
When recombinant and cellular prion protein (PrP(C)) binds copper, it acquires properties resembling the scrapie isoform (PrP(Sc)), namely protease resistance, detergent insolubility and increased beta sheet content. However, whether the conformations of PrP(C) induced by copper and PrP(Sc) are similar has not been studied in great detail. Here, we use a panel of seven monoclonal antibodies to decipher the epitopes on full-length mouse PrP(C) that are affected by exogenous copper, and to compare the antigenicity of the copper-treated full-length PrP(C) with the full-length PrP(Sc) present in scrapie-infected mouse brains. In the presence of copper, we found that epitopes along residues 115-130 and 153-165 become more accessible on PrP(C). These regions correspond to the two beta sheet strands in recombinant PrP and they were proposed to be important for prion conversion. However, when we compared the antibody-binding patterns between full-length PrP(C) with full-length PrP(Sc) and between copper-treated full-length PrP(C) with full-length PrP(Sc), antibody binding to residues 143-155 and 175-185 was consistently increased on PrP(Sc). Collectively, our results suggest that copper-treated full-length PrP(C) does not resemble full-length PrP(Sc), despite acquiring PrP(Sc)-like properties. In addition, since each full-length protein reacts distinctively to some of the antibodies, this binding pattern could discriminate between PrP(C) and PrP(Sc).
Asunto(s)
Cobre/farmacología , Proteínas PrPC/efectos de los fármacos , Proteínas PrPC/inmunología , Proteínas PrPSc/inmunología , Animales , Anticuerpos Monoclonales , Sitios de Unión , Química Encefálica , Mapeo Epitopo , Técnicas In Vitro , Ratones , Ratones Noqueados , Proteínas PrPC/química , Proteínas PrPSc/química , Conformación Proteica/efectos de los fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/inmunologíaRESUMEN
Prion diseases are characterized by the conversion of the normal cellular prion protein (PrP(C)) into a pathogenic isoform (PrP(Sc)). PrP(C) binds copper, has superoxide dismutase (SOD)-like activity in vitro, and its expression aids in the cellular response to oxidative stress. However, the interplay between PrPs (PrP(C), PrP(Sc) and possibly other abnormal species), copper, anti-oxidation activity and pathogenesis of prion diseases remain unclear. In this study, we reported dramatic depression of SOD-like activity by the affinity-purified PrPs from scrapie-infected brains, and together with significant reduction of Cu/Zn-SOD activity, correlates with significant perturbations in the divalent metals contents. We also detected elevated levels of nitric oxide and superoxide in the infected brains, which could be escalating the oxidative modification of cellular proteins, reducing gluathione peroxidase activity and increasing the levels of lipid peroxidation markers. Taken together, our results suggest that brain metal imbalances, especially copper, in scrapie infection is likely to affect the anti-oxidation functions of PrP and SODs, which, together with other cellular dysfunctions, predispose the brains to oxidative impairment and eventual degeneration. To our knowledge, this is the first study documenting a physiological connection between brain metals imbalances, the anti-oxidation function of PrP, and aberrations in the cellular responses to oxidative stress, in scrapie infection.
Asunto(s)
Antioxidantes/metabolismo , Encéfalo/metabolismo , Metales/metabolismo , Proteínas PrPSc/metabolismo , Scrapie/metabolismo , Animales , Anticuerpos Monoclonales , Calcio/metabolismo , Cobre/metabolismo , Glutatión Peroxidasa/metabolismo , Hierro/metabolismo , Peroxidación de Lípido/fisiología , Magnesio/metabolismo , Ratones , Ratones Endogámicos , Nitritos/análisis , Estrés Oxidativo/fisiología , Proteínas PrPSc/análisis , Proteínas PrPSc/inmunología , Priones/análisis , Priones/inmunología , Priones/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Superóxido Dismutasa/metabolismo , Zinc/metabolismoRESUMEN
Human prion diseases are characterized by the conversion of the normal prion protein (PrP(C)) into a pathogenic isomer (PrP(Sc)). Distinct PrP(Sc) conformers are associated with different subtypes of prion diseases. PrP(C) binds copper and has antioxidation activity. Changes in metal-ion occupancy can lead to significant decline of the antioxidation activity and changes in conformation of the protein. We studied the trace element status of brains from patients with sporadic Creutzfeldt-Jakob disease (sCJD). We found a decrease of up to 50% of copper and an increase in manganese of approximately 10-fold in the brain tissues from sCJD subjects. We have also studied the metal occupancy of PrP in sCJD patients. We observed striking elevation of manganese and, to a lesser extent, of zinc accompanied by significant reduction of copper bound to purified PrP in all sCJD variants, determined by the PrP genotype and PrP(Sc) type, combined. Both zinc and manganese were undetectable in PrP(C) preparations from controls. Copper and manganese changes were pronounced in sCJD subjects homozygous for methionine at codon 129 and carrying PrP(Sc) type-1. Anti-oxidation activity of purified PrP was dramatically reduced by up to 85% in the sCJD variants, and correlated with increased in oxidative stress markers in sCJD brains. These results suggest that altered metal-ion occupancy of PrP plays a pivotal role in the pathogenesis of prion diseases. Since the metal changes differed in each sCJD variants, they may contribute to the diversity of PrP(Sc) and disease phenotype in sCJD. Finally, this study also presented two potential approaches in the diagnosis of CJD; the significant increase in brain manganese makes it potentially detectable by MRI, and the binding of manganese by PrP in sCJD might represent a novel diagnostic marker.
Asunto(s)
Metales/metabolismo , Enfermedades por Prión/metabolismo , Priones/metabolismo , Antioxidantes/metabolismo , Encéfalo/metabolismo , Humanos , Concentración Osmolar , Estrés Oxidativo/fisiología , Proteínas PrPSc/metabolismo , Superóxido Dismutasa/metabolismoRESUMEN
Prion diseases are not only genetic or sporadic neurodegenerative disorders, but more important, they are transmissible diseases. The etiological agent in these unprecedented diseases is believed to be prion protein (PrP), which undergoes post-translational conversion from the predominant a-helical conformation known as PrP(C), to a b-sheet rich abnormal isoform called scrapie PrP (PrP(Sc)). Accumulating evidence has shown PrP(C) to be a copper-binding antioxidant in vivo. The prevailing view that PrP binds copper weakly is based on in vitro observations using peptides or short fragment of recombinant PrP. However, recent in vitro evidence indicates human PrP has significantly higher affinity for copper, similar to other copper-binding proteins and copper uptake experiments show that PrP expressed by cells has a Km in the nanomolar range. Besides binding copper within the octarepeats region along the N-terminus, PrP can also binds copper at a second site further upstream. More importantly, PrP also binds other metals such as zinc and manganese at these two sites albeit at a lower affinity. This is important because there is evidence that native PrP in prion diseases binds not only copper, but also zinc. This abnormal metal binding probably resulted in the loss of its anti-oxidation function, and together with impairment in the cellular antioxidant mechanisms, contributed to the increased oxidative stress, and possibly trigger neurodegeneration.
Asunto(s)
Metales/metabolismo , Enfermedades por Prión/etiología , Animales , Encéfalo/metabolismo , Cobre/metabolismo , Humanos , Enfermedades por Prión/metabolismo , Priones/metabolismo , Unión ProteicaRESUMEN
Normal cellular prion protein, a necessary protagonist in fatal neurodegenerative prion diseases, was mapped in rodent cerebellum to establish its cellular and ultrastuctural localization. Existing morphological data about native prion protein distribution in brain tissues remain, indeed, contradictory and do not fit with biochemical and cell biological results. Using ultrastructural preembedding immunocytochemistry and a monoclonal anti-mouse prion protein antibody, this report shows that cellular prion protein is present in all cortico-cerebellar and deep nuclei neuronal cell types, as well as in all glial cell types. The heaviest expression appears on parallel fibres and astrocytic processes. The protein is exclusively located on the outer cell membrane and in Golgi and endosomal intracytoplasmic organelles, with no cytoplasmic or synaptic vesicle labelling. Most important, and in contrast with previous ultrastructural data, cellular prion protein is shown to be distributed on all portions of neurons, without any preferential synaptic targeting. The present morphological report shows, for the first time in vivo, that the cellular prion protein is present on the entire cell surface membrane of all neuronal and glial cell types of the rat cerebellum. This ubiquitous presence supports the notion that prion protein has a generalized cellular function in brain tissue rather than a specialized role restricted to synaptic transmission.
Asunto(s)
Membrana Celular/metabolismo , Cerebelo/metabolismo , Neuroglía/metabolismo , Neuronas/efectos de los fármacos , Orgánulos/metabolismo , Proteínas PrPC/metabolismo , Animales , Anticuerpos/farmacología , Membrana Celular/ultraestructura , Cerebelo/ultraestructura , Cricetinae , Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados/anatomía & histología , Ratones Noqueados/metabolismo , Microscopía Electrónica , Neuroglía/ultraestructura , Neuronas/metabolismo , Orgánulos/ultraestructura , Proteínas PrPC/genética , Proteínas PrPC/inmunología , Ratas , Ratas Wistar , Sinapsis/metabolismo , Sinapsis/ultraestructuraRESUMEN
The phenotype of human sporadic prion diseases is affected by patient genotype at codon 129 of the prion protein (PrP) gene, the site of a common methionine/valine polymorphism, and by the type of the scrapie PrP (PrP(Sc)), which likely reflects the prion strain. However, two distinct disease phenotypes, identified as sporadic Creutzfeldt-Jakob disease (M/M2 sCJD) and sporadic fatal insomnia (sFI), share methionine homozygosity at codon 129 and PrP(Sc) type 2. One-dimensional gel electrophoresis and immunoblotting reveal no difference between the M/M2 sCJD and sFI species of PrP(Sc) in gel mobility and glycoform ratio. In contrast, the two-dimensional immunoblot demonstrates that in M/M2 sCJD the full-length PrP(Sc) form is overrepresented and carries glycans that are different from those present in the PrP(Sc) of sFI. Because the altered glycans are detectable only in the PrP(Sc) and not in the normal or cellular PrP (PrP(C)), they are likely to result from preferential conversion to PrP(Sc) of rare PrP(C) glycoforms. This is the first evidence that a qualitative difference in glycans contributes to prion diversity.
Asunto(s)
Electroforesis en Gel Bidimensional/métodos , Priones/química , Humanos , Immunoblotting , Priones/clasificaciónRESUMEN
Creutzfeldt-Jakob disease (CJD), believed to be caused by a protease-resistant isoform of prion protein (PrP(Sc)), usually manifests itself as a clinically distinctive age-related dementia because of its rapid progression, occasionally accompanied by cerebellar ataxia. Recently, a variant CJD (vCJD) has been described, which has prominent early psychiatric symptoms and an earlier age of death. Although cerebrospinal fluid (CSF) is part of the extracellular fluid of the central nervous system (CNS), the bulk of its proteins are derived from the plasma and there is increasing concern about possible transmission of prion disease by blood. As investigation of CSF has played a significant role in the diagnosis and management of several neurological diseases, it was decided to characterize PrP present in the CSF of CJD individuals. Significant variation was observed in the level of PrP in the CSF from both non-CJD and CJD (including vCJD) patients, and the detected PrP forms are protease-sensitive. Using a conformation-dependent immunoassay, it was further demonstrated that the PrP detected in the CSF from CJD patients was broadly similar in conformation to that found in non-CJD patients. Taken together, the results of this study fail to demonstrate any correlation between the presence of protease-resistant PrP isoform (PrP(Sc)) in the CSF and disease manifestation.
Asunto(s)
Síndrome de Creutzfeldt-Jakob/líquido cefalorraquídeo , Priones/líquido cefalorraquídeo , Electroforesis en Gel de Poliacrilamida , Endopeptidasa K/farmacología , Ensayo de Inmunoadsorción Enzimática , Mapeo Epitopo , Humanos , Priones/efectos de los fármacos , Priones/inmunología , Conformación Proteica , Isoformas de Proteínas/líquido cefalorraquídeoRESUMEN
Ectopic expression of the doppel (Dpl) protein, a homologue of the prion protein (PrP), was recently associated with cerebellar Purkinje cell degeneration observed in two aging prion protein knock-out (Prnp(0/0)) mouse lines. We investigated the possible role of Dpl in oxidative metabolism. Two Prnp(0/0) mouse lines of similar genetic background were studied. One line expresses Dpl in the brain and displays Dpl-associated cerebellar abnormalities. The other has no elevated expression of Dpl and no cerebellar abnormalities. We observed a correlation between Dpl expression and the induction of both heme oxygenase 1 (HO-1) and nitric oxide synthase systems (nNOS and iNOS). These responses are suggestive of increased oxidative stress in the brains of the Dpl-expressing Prnp(0/0) mice. No induction was observed with Hsp-60, indicating a specific response by the HO/NOS system. We proposed that Dpl expression exacerbates oxidative damage that is antagonistic to the protective function of wild-type PrP.
Asunto(s)
Hemo Oxigenasa (Desciclizante)/genética , Óxido Nítrico Sintasa/genética , Priones/genética , Priones/metabolismo , Células de Purkinje/enzimología , Animales , Chaperonina 60/genética , Proteínas Ligadas a GPI , Regulación Enzimológica de la Expresión Génica , Hemo-Oxigenasa 1 , Peroxidación de Lípido/fisiología , Proteínas de la Membrana , Ratones , Ratones Noqueados , Óxido Nítrico Sintasa de Tipo I , Óxido Nítrico Sintasa de Tipo II , Nitritos/metabolismo , Estrés Oxidativo/fisiologíaRESUMEN
Satin (sa) homozygous mice have a silky coat with high sheen arising from structurally abnormal medulla cells and defects in differentiation of the hair shaft. We demonstrate that the winged helix/forkhead transcription factor, Foxq1 (Forkhead box, subclass q, member 1) is mutant in sa mice. An intragenic deletion was identified in the radiation-induced satin mutant of the SB/Le inbred strain; a second allele, identified by an N-ethyl-N-nitrosourea (ENU) mutagenesis screen, has a missense mutation in the conserved winged helix DNA-binding domain. Homozygous mutants of the two alleles are indistinguishable. We show that Foxq1 is expressed during embryogenesis and exhibits a tissue-restricted expression pattern in adult tissues. The hair defects appear to be restricted to the inner structures of the hair; consequently, Foxq1 has a unique and distinct function involved in differentiation and development of the hair shaft. Despite an otherwise healthy appearance, satin mice have been reported to exhibit suppressed NK-cell function and alloimmune cytotoxic T-cell function. We show instead that the immune defects are attributable to genetic background differences.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Folículo Piloso/citología , Queratinas/genética , Queratinas/ultraestructura , Transactivadores/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Diferenciación Celular , Mapeo Cromosómico , Cromosomas Humanos Par 6 , Clonación Molecular , Femenino , Factores de Transcripción Forkhead , Regulación de la Expresión Génica , Folículo Piloso/embriología , Folículo Piloso/ultraestructura , Humanos , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C3H , Datos de Secuencia Molecular , Ratas , Receptores Notch , Factores de TranscripciónRESUMEN
Scrapie is a naturally occurring prion (PrP) disease causing a fatal neurodegenerative disorder in sheep and goats. Previous studies suggest that scrapie is transmitted naturally through exposure to the scrapie agent in wasted placentas of infected ewes. This study determined the distribution and biochemical properties of PrP cellular (PrP-C) and the distribution of PrP scrapie (PrP-Sc) in reproductive, placental, and selected fetal tissues and fetal fluids in sheep. Glycosylated, N-terminally truncated, proteinase K-sensitive PrP-C with apparent molecular masses of 23-37 kDa was present in reproductive, placental, and fetal tissues and fetal fluids. PrP-C was low or undetectable in intercotyledonary chorioallantois, amnion, urachus, amniotic fluid, and fetal urine. In pregnant ewes, cotyledonary chorioallantois, allantoic fluid, and caruncular endometrium contained higher levels of PrP-C than did intercaruncular endometrium, myometrium, oviduct, ovary, fetal bladder, or fetal kidney. Caruncular endometrial PrP-C was up-regulated during pregnancy. Despite the wide distribution of PrP-C in reproductive, placental, and selected fetal tissues and fetal fluid, PrP-Sc was detected only in caruncular endometrium and cotyledonary chorioallantois of pregnant scrapie-infected ewes. The embryo/fetus may not be exposed to scrapie in utero because it is separated physically from PrP-positive allantois and chorioallantois by PrP-negative amnion.
Asunto(s)
Priones , Scrapie , Animales , Femenino , Intercambio Materno-Fetal , Embarazo , OvinosRESUMEN
The normal cellular prion protein (PrP(C)) plays an essential role in the development of prion diseases. Indirect evidence has suggested that different PrP(C) glycoforms may be expressed in different brain regions and perform distinct functions. However, due to a lack of monoclonal antibodies (Mabs) that are specific for mouse PrP(C), the expression of PrP(C) in the mouse brain has not been studied in great detail. We used Mabs specific for either the N-terminus or the C-terminus of the mouse PrP(C) to study its expression in the mouse brain by immunoblotting and immunohistochemistry. Immunoblotting studies demonstrated that the expression of PrP(C) differed quantitatively as well as qualitatively in different regions of the brain. The anti-C-terminus Mabs reacted with all three molecular weight bands of PrP(C); the anti-N-terminus Mabs only reacted with the 39-42 kDa PrP(C). The results from immunohistochemical staining revealed the spatial distribution of PrP(C) in the mouse brain, which were consistent with that from immunoblotting. Although expression of PrP(C) has been reported to be required for long-term survival of Purkinje cells, we were unable to detect PrP(C) in the Purkinje cell layer in the cerebellum with multiple anti-PrP Mabs. Our findings suggest that PrP(C) variants, i.e. various glycoforms and truncated forms, might be specifically expressed in different regions of mouse brain and might have different functions.
Asunto(s)
Anticuerpos Monoclonales , Química Encefálica/fisiología , Priones , Animales , Especificidad de Anticuerpos , Western Blotting , Femenino , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos , Ratones Noqueados , Enfermedades por Prión/fisiopatología , Priones/análisis , Priones/genética , Priones/inmunología , Células de Purkinje/química , Células de Purkinje/fisiologíaRESUMEN
Originating from a post-switch memory B cell or plasma cell compartment in peripheral lymphoid tissues, malignant myeloma cells accumulate in the bone marrow of patients with multiple myeloma. In this favorable microenvironment their growth and survival are dependent upon both soluble factors and physical cell-to-cell and cell-to-extracellular matrix contacts. In this report we show that hyaluronan (HA), a major nonprotein glycosaminoglycan component of the extracellular matrix in mammalian bone marrow, is a survival and proliferation factor for human myeloma cells. The effect of HA is mainly mediated through a gp 80-interleukin 6 (IL-6) receptor pathway by a CD44-independent mechanism, suggesting that HA retains and concentrates IL-6 close to its site of secretion, thus favoring its autocrine activity. In addition, we show that HA-mediated survival and proliferation of myeloma cells is associated with a down-regulation in the expression of p27(kip1) cyclin-dependent kinase inhibitor and a hyperphosphorylation of the retinoblastoma protein (pRb). These data suggest that HA could be an important component in the myeloma cell physiopathology in vivo by potentiating autocrine and/or paracrine IL-6 activities.
Asunto(s)
División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ácido Hialurónico/farmacología , Interleucina-6/metabolismo , Mieloma Múltiple/patología , Proteína de Retinoblastoma/metabolismo , Apoptosis/efectos de los fármacos , Humanos , Receptores de Hialuranos/metabolismo , Mieloma Múltiple/metabolismo , Fosforilación , Células Tumorales CultivadasRESUMEN
We studied the expression of normal cellular prion protein (PrP(C)) in mouse lymphoid tissues with newly developed mAbs to PrP(C). Most of the mature T and B cells in the peripheral lymphoid organs do not express PrP(C). In contrast, most thymocytes are PrP(C+). In the bone marrow, erythroid cells and maturing granulocytes are PrP(C+). Approximately 50% of the cells in the region of small lymphocytes and progenitor cells also express PrP(C). Most of these PrP(C+) cells are CD43(+), but B220(-), surface IgM(-) (sIgM(-)), and IL-7R(-), a phenotype that belongs to cells not yet committed to the B cell lineage. Another small group of the PrP(C+) cell are B220(+), and some of these are also sIgM(+). The majority of the B220(+) cells, however, are PrP(C-). Therefore, PrP(C) is preferentially expressed in early bone marrow progenitor cells and subsets of maturing B cells. Supporting this interpretation is our observation that stimulation of bone marrow cells in vitro with PMA results in a decrease in the number of PrP(C+)B220(-) cells with a corresponding increase of sIgM(+)B220(high) mature B cells. This result suggests that the PrP(C+)B220(-) cells are potential progenitors. Furthermore, in the bone marrow of Rag-1(-/-) mice, there are an increased number of PrP(C+)B220(-) cells, and most of the developmentally arrested pro-B cells in these mice are PrP(C+). Collectively, these results suggest that PrP(C) is expressed preferentially in immature T cells in the thymus and early progenitor cells in the bone marrow, and the expression of PrP(C) is regulated during hemopoietic differentiation.
Asunto(s)
Antígenos CD , Células Madre Hematopoyéticas/metabolismo , Tejido Linfoide/metabolismo , Proteínas PrPC/biosíntesis , Animales , Subgrupos de Linfocitos B/citología , Subgrupos de Linfocitos B/metabolismo , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Diferenciación Celular/inmunología , División Celular/efectos de los fármacos , División Celular/inmunología , Genes RAG-1/inmunología , Granulocitos/citología , Granulocitos/inmunología , Granulocitos/metabolismo , Células Madre Hematopoyéticas/citología , Inmunoglobulina M/biosíntesis , Antígenos Comunes de Leucocito/biosíntesis , Leucosialina , Activación de Linfocitos/efectos de los fármacos , Tejido Linfoide/citología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas PrPC/deficiencia , Proteínas PrPC/genética , Receptores de Antígenos de Linfocitos B/biosíntesis , Receptores de Interleucina-7/biosíntesis , Sialoglicoproteínas/biosíntesis , Bazo/citología , Bazo/inmunología , Bazo/metabolismo , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Timo/citología , Timo/inmunología , Timo/metabolismoRESUMEN
We examined expression of the normal cellular prion protein (PrP(C)) in human peripheral blood mononuclear cells (PBMC) and in transfected neuroblastoma cells with a panel of six monoclonal antibodies (Mabs). While all six of the Mabs reacted strongly with the neuroblastoma cells, only four of the Mabs reacted with PrP(C) expressed by human PBMC. PrP(C) is expressed at high levels in human T cells, B cells, monocytes, and dendritic cells, but not in red blood cells. Immunoblotting studies revealed that the PrP(C) glycoforms and the composition of the N-linked glycans on PrP(C) in human PBMC are different from those of the brain or the neuroblastoma cells. In human PBMC and the neuroblastoma cell lines the N-terminal portion of the PrP(C) is hypersensitive to proteolytic digestion, suggesting that the N-terminus of the PrP(C) on the surface of a living cell lacks secondary structure. We found that the level of PrP(C) expressed on the surface of human T lymphocytes was up-regulated as a consequence of cellular activation. Accordingly, memory T cells express more PrP(C) than naïve T cells. In addition, the proliferation of human T lymphocytes stimulated with an anti-CD3 Mab was inhibited by anti-PrP(C) Mabs. Collectively, these results suggest that PrP(C) can participate in signal transduction in human T lymphocytes.
Asunto(s)
Activación de Linfocitos/inmunología , Linfocitos/inmunología , Proteínas PrPC/fisiología , Anticuerpos Monoclonales/inmunología , Complejo CD3/inmunología , División Celular , Endopeptidasa K/metabolismo , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito B/metabolismo , Expresión Génica , Glicosilación , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/inmunología , Proteínas PrPC/genética , Proteínas PrPC/inmunología , Linfocitos T , Transfección , Tripsina/metabolismo , Células Tumorales Cultivadas , Regulación hacia ArribaRESUMEN
Although minor abnormalities have been reported in prion protein (PrP) knock-out (Prnp-/-) mice, the normal physiological function of PrP, the causative agent implicated in transmissible spongiform encephalopathies (TSE), remains unresolved. Since there are increasing correlations between oxidative stress and amyloidoses, we decided to investigate whether PrP plays a role in oxidative modulation. We found higher levels of oxidative damage to proteins and lipids in the brain lysates of Prnp-/- as compared to wild-type (WT) mice of the same genetic background. These two indicators, protein oxidation and lipid peroxidation, are hallmarks of cellular oxidative damage. Elevated levels of ubiquitin-protein conjugates were also observed in Prnp-/- mice, a probable consequence of cellular attempts to remove the damaged proteins as indicated by increased proteasome activity. Taken together, these findings are indicative of a role for PrP in oxidative homeostasis in vivo.
Asunto(s)
Química Encefálica , Encéfalo/metabolismo , Estrés Oxidativo/fisiología , Proteínas PrPC/deficiencia , Aldehídos/análisis , Animales , Biomarcadores/análisis , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/metabolismo , Cetonas/análisis , Peroxidación de Lípido , Lípidos/química , Sustancias Macromoleculares , Malondialdehído/análisis , Ratones , Ratones Noqueados , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Oxidación-Reducción , Proteínas PrPC/genética , Complejo de la Endopetidasa Proteasomal , Proteínas/química , Ubiquitina/químicaRESUMEN
BACKGROUND AND OBJECTIVES: Transmissible spongiform encephalopathies (TSEs) including sheep scrapie are characterized by the conversion of a normal, cellular prion protein (PrPc) to an abnormal protease-resistant form (PrPSc). Like human peripheral blood, the peripheral blood of scrapie-infected sheep remains one possible source of disease transmission. As a first step in understanding the disease requirements in the natural scrapie host, the presence of PrPc was evaluated in peripheral blood cells from five normal and five scrapie-infected Suffolk sheep. DESIGN AND METHODS: Live peripheral blood cells from normal and scrapie-infected sheep were analyzed for the presence of PrP using flow cytometry and reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: PrP mRNA was detected in peripheral blood mononuclear cells (PBMC) but not in platelets or granulocytes. Consistent with PrP mRNA expression, cell-surface expressed PrP was detected on PBMC, but was not detected on granulocytes, platelets, or erythrocytes. Two-color flow cytometric analysis of PBMC specific phenotypes revealed that regardless of scrapie-status, expression of PrP was significantly higher on B2 positive B-lymphocytes than on CD4, CD8, WC1 positive T-lymphocytes or CD14 positive monocytes. In addition, PrP expressed on PBMC from normal and scrapie-infected sheep was sensitive to proteinase K (PK)and phosphatidylinositol-specific phospholipase C (PIPLC). INTERPRETATION AND CONCLUSIONS: Regardless of the scrapie-status of the sheep, resting PBMC transcribe PrPc and express PrPc as a cell-surface protein sensitive to both PK and PIPLC. Because of the abundance of PrPc on PBMC, future diagnostic tests using PK and PIPLC to discriminate between protease sensitive and resistant PrP must be carefully evaluated.
Asunto(s)
Leucocitos Mononucleares/metabolismo , Enfermedades por Prión/etiología , Priones/metabolismo , Enfermedades de las Ovejas/transmisión , Animales , Plaquetas/metabolismo , Endopeptidasa K/metabolismo , Femenino , Proteínas de la Membrana/metabolismo , Fosfatidilinositol Diacilglicerol-Liasa , Fosfoinositido Fosfolipasa C , Proteínas PrPSc/metabolismo , Enfermedades por Prión/sangre , Enfermedades por Prión/veterinaria , Ovinos/sangre , Enfermedades de las Ovejas/sangre , Enfermedades de las Ovejas/etiología , Fosfolipasas de Tipo C/metabolismoRESUMEN
Some tumor cells have deficits in class I MHC antigen processing, suggesting that T cells exert selective pressure on tumor cells. Previous studies have not revealed increased tumor incidence in mice with deficits in T-cell immunity, including mice lacking TAP1 (a subunit of the transporter for antigen presentation) or LMP2 (a regulated subunit of the 20S proteasome). The incidence of spontaneous tumors in these mice, however, is too low to assess differences in host resistance to tumors. To increase tumor incidence and better assess the role of systemic expression of TAP1 and LMP2 in responses to tumors, TAP1-/- and LMP2-/- mice were bred with p53-/- mice to create TAP1-/-p53-/- and LMP2-/-p53-/- double knockout mice. Lymphomas and sarcomas (malignant fibrous histiocytoma and angiosarcoma) occurred with high incidence in all p53-deficient populations. Tumor incidence and death rate were similar in TAP1-/-p53-/- mice and closely matched control TAP1+/+p53-/- mice. Tumor incidence and death rate were slightly accelerated in LMP2-/-p53-/- mice relative to control LMP2+/+p53-/- mice, but the biological significance of this difference was unclear. The relative incidence of lymphomas vs. sarcomas was not significantly altered by variation in TAP1 or LMP2. In conclusion, systemic absence of TAP1 did not alter tumor incidence, while absence of LMP2 was associated with only a slight acceleration of tumor incidence of uncertain significance. These observations are consistent with other evidence that normal T-cell responses do not effectively limit tumorigenesis. Even though T cells can attack some tumor cells, the ability of tumors to alter their immunogenicity and evade T-cell surveillance may render the native immune system ineffective at providing a rate-limiting barrier to tumorigenesis and preventing cancer.