RESUMO
BACKGROUND: Primary radiotherapy (RT) is a mainstay of treatment for laryngeal squamous cell carcinoma (LSCC). Although the cure rates for early (T1) vocal cord tumours are high, RT proves ineffective in up to a third of T3 carcinomas. Moreover, RT is associated with debilitating early- and late-treatment-related toxicity, thus finding means to de-escalate therapy, while retaining/augmenting therapeutic effectiveness, is highly desirable. p53 is a key mediator of radiation responses; we therefore investigated whether Nutlin-3, a small-molecule inhibitor of MDM2 (mouse double minute 2; an essential negative regulator of p53), might radiosensitise LSCC cells. METHODS: We performed clonogenic assays to measure radiosensitivity in a panel of LSCC cell lines (for which we determined p53 mutational status) in the presence and absence of Nutlin-3. RESULTS: LSCC cells harbouring wild-type p53 were significantly radiosensitised by Nutlin-3 (P<0.0001; log-rank scale), and displayed increased cell cycle arrest and significantly increased senescence (P<0.001) in the absence of increased apoptosis; thus, our data suggest that senescence may mediate this increased radiosensitivity. CONCLUSION: This is the first study showing Nutlin-3 as an effective radiosensitiser in LSCC cells that retain wild-type p53. The clinical application of Nutlin-3 might improve local recurrence rates or allow treatment de-escalation in these patients.
Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Senescência Celular/efeitos dos fármacos , Genes p53 , Imidazóis/análise , Imidazóis/farmacologia , Neoplasias Laríngeas/tratamento farmacológico , Piperazinas/análise , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Tolerância a Radiação/efeitos dos fármacos , Radiossensibilizantes/farmacologia , Animais , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/radioterapia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Laríngeas/genética , Neoplasias Laríngeas/radioterapiaRESUMO
The p53 tumour suppressor functions as a sensor of genotoxic stress and, once activated, induces cell growth arrest or apoptosis. The precise intranuclear localization of latent p53 protein in non-stressed cells is unknown. Such information is essential in order to understand how relatively few molecules of p53 can detect and respond to DNA damage. Here we present the first detailed supramolecular localization of p53 in the nuclei of cells under normal conditions of growth. We show that soluble, non-bound p53 is released by permeabilization, leaving structurally bound p53 in both the nucleus and nucleolus. In situ biochemical studies reveal (i) that nuclear-bound p53 is tethered by RNA (directly or indirectly) and (ii) that a sub-population of nuclear-bound p53 co-localizes with sites of RNA synthesis. Transcriptional co-localization appeared to be independent of p53 conformation but dependent upon its quaternary structure. In the nucleolus p53 was observed at sites of rRNA synthesis and also adjacent to such sites. In contrast, nucleolar hdm-2 (shown by others to complex p53 and 5S RNA) was excluded from sites of rRNA synthesis. Our discovery that p53 is physically linked with sites of transcription may explain how relatively few p53 protein molecules can monitor genetic stress and respond preferentially to damage of actively transcribed genes.
Assuntos
Nucléolo Celular/química , Núcleo Celular/química , Transcrição Gênica , Proteína Supressora de Tumor p53/análise , Animais , Células Cultivadas , DNA/análise , Humanos , Camundongos , Mutação , RNA Mensageiro/biossínteseRESUMO
The tumour suppressor p53 is a multifunctional protein important for the maintenance of genomic integrity. It is able to form molecular complexes with different DNA targets and also with cellular proteins involved in DNA transcription and DNA repair. In mammalian cells the biochemical processing of DNA occurs on a nuclear sub-structure termed the nuclear matrix. Previously Deppert and co-workers have identified p53 in association with the nuclear matrix in viral- and non-viral transformed cell lines. In the present study we demonstrate, for the first time, that p53 is bound to the nuclear matrix in primary cultures of normal mammalian cells and that this binding increases following DNA damage. Analysis of cell lines expressing structural mutants of p53 revealed that association with the nuclear matrix is independent of the tertiary and quaternary structure of p53. However, the proline-rich domain towards the N-terminus of p53 (residues 67 to 98) appeared important for binding to the nuclear matrix. This was demonstrated by TET-ON regulated expression of p53-derived constructs in p53(-/-) murine embryonic fibroblasts (MEF p53(-/-)). The proline-rich domain of p53 has potential for SH3 protein-protein interaction, and has a role in p53-mediated apoptosis and possibly base excision repair of DNA damage. We discuss our observations in relation to the ability of p53 to facilitate DNA repair and also review evidence indicating that matrix-bound p53 in SV40-transformed cells may facilitate the transforming potential of SV40 large T antigen.
Assuntos
Dano ao DNA , Matriz Nuclear/metabolismo , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo , Animais , Antígenos Transformantes de Poliomavirus/fisiologia , Ciclo Celular , Linhagem Celular Transformada , Células Cultivadas , Deleção de Genes , Humanos , Camundongos , Mutação , Prolina/química , Estresse Fisiológico , Proteína Supressora de Tumor p53/genéticaRESUMO
Activated T cells can release lymphokines selectively towards the site of contact with the target cell. In this way the specificity of the target-effector cell interaction can be maintained in spite of signalling being mediated by soluble factors (Mosmann, 1988, Immunol. Today 9, 306). However, this polarised phenotype is not expressed in resting T cells; rather it appears to be induced in the first minutes following T cell activation. In order to analyse single molecules for their ability to induce T cell polarisation, we devised a technique based on targeting different T cell surface molecules with specific antibodies immobilised on to immunomagnetic beads. The polarised phenotype was determined from observation of the microtubule organising centre being oriented towards the site of interaction with the bead. When applied to T cell lines, the technique permitted the classification of CD3 as a polarisation-inducing molecule, while no polarisation was found when targeting CD2, CD6 and CD8 molecules. This technique has a number of potential applications since it can, in principle, be applied to any cell surface molecule or cell type. Technical details and the sensitivity of the procedure are discussed.
Assuntos
Antígenos de Superfície/análise , Antígenos de Superfície/fisiologia , Polaridade Celular/imunologia , Separação Imunomagnética/métodos , Proteínas de Insetos , Linfócitos T/imunologia , Antígenos CD/análise , Adesão Celular/imunologia , Vidro , Humanos , Leucemia-Linfoma de Células T do Adulto , Ativação Linfocitária/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/imunologia , Células Tumorais CultivadasRESUMO
Experiments have been carried out to demonstrate that, using antibody coated-Dynabeads as a model system for density labelling MOLT-4 T cells, the overall density of cells can be increased such that the cells that bind particles can be separated on isopycnic isotonic density gradients from cells that bind fewer particles. The increase in density is dependent on the cell volume and the number of particles bound. After centrifugation, cells with bound particles were found at positions in the gradient that reflected their increased density. Observed density ranges for cells with particular numbers of particles bound coincided closely with calculated expected density ranges. These results indicate the potential for separation of different subpopulations of cells on the basis of the immunological identity of the surface of cells using density perturbation methods involving antibody coated-density particles.
Assuntos
Separação Celular/métodos , Centrifugação Isopícnica/métodos , Linfócitos T/citologia , Tamanho Celular , Sobrevivência Celular , Humanos , Microesferas , Linfócitos T/imunologia , Células Tumorais CultivadasRESUMO
We have studied the attachment of immunomagnetic beads to different cells, with particular interest in cells that did not, as expected, appear to bind antibody-coated beads. Through the use of immunofluorescence and laser scanning confocal microscopy it was possible to demonstrate that beads can detach significant amounts of antigen from the surface of cells. This results in the appearance of antigen-depleted yet viable cells. Moreover, the detached antigen is found to be bound to beads and is associated with fragments of cell membrane which can also carry other (non-bead binding) cell surface proteins. After reculturing, antigen-depleted cells can recover their normal levels of surface antigen. Our results demonstrate the existence of an immunobead-induced cell membrane detachment phenomenon that can lead to the removal of all of a specific surface antigen without killing the cells, as judged by both vital staining and reculturing. An important aspect of this phenomenon is that immunoidentification of immunobead-selected populations of cells will give erroneous results. This may thus be of significance for the immunobead-based cell depletion methods that are used in medicine.
Assuntos
Antígenos de Superfície/análise , Separação Imunomagnética , Antígenos CD/análise , Antígenos CD19 , Antígenos de Diferenciação de Linfócitos B/análise , Antígenos de Superfície/fisiologia , Separação Celular/métodos , Sobrevivência Celular/fisiologia , Fluoresceína , Fluoresceínas , Humanos , Microscopia/métodos , FenótipoRESUMO
Affinity chromatography on polymyxin B-Sepharose 4B is one of the most commonly used methods for the removal of contaminating lipopolysaccharides (LPS). However, the LPS of Brucella spp. do not bind to polymyxin B. An affinity chromatography method with an anti-O antigen of Brucella LPS monoclonal antibody as immunosorbent was developed. The method produced a 1000-fold reduction in the LPS content of the cytoplasmic fraction of B. abortus. The eluted proteins retained their antigenicity. The method, which uses mild physiological conditions, is simple, effective and reproducible.
Assuntos
Anticorpos Monoclonais/imunologia , Brucella abortus/química , Cromatografia de Afinidade/métodos , Imunoadsorventes/imunologia , Lipopolissacarídeos/isolamento & purificação , Testes de Aglutinação , Afinidade de Anticorpos , Brucella abortus/ultraestrutura , Citoplasma/química , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Immunoblotting , Imunodifusão , Lipopolissacarídeos/imunologia , Antígenos O , Polissacarídeos Bacterianos/imunologia , Reprodutibilidade dos TestesRESUMO
The fractionation of sub-populations of peripheral blood mononuclear cells has become an essential routine procedure and some of the main fractionation methods used today are immunomagnetic separations. We describe a less expensive method for the separation of subpopulations of mononuclear cells using density perturbation, which uses the binding of antibody-coated dense polystyrene beads to increase the density of specific sub-populations of cells. By incubating a total mononuclear fraction from human peripheral blood together with antibody-coated beads, in a commercially-available lymphocyte separation medium (Nycoprep 1.077), a depletion of 94.9 +/- 1.68% of the T cells could be obtained by this procedure; a depletion of 69.7 +/- 1.78% of the B cells was also achieved. These results indicate the potential for the separation of different sub-populations of peripheral blood mononuclear cells on the basis of the immunological identity of the surface of cells using density perturbation methods involving antibody-coated dense polystyrene beads.
Assuntos
Linfócitos B/fisiologia , Monócitos/fisiologia , Linfócitos T/fisiologia , Anticorpos Monoclonais , Separação Celular , Meios de Cultura , Técnica Indireta de Fluorescência para Anticorpo , Humanos , MicroesferasRESUMO
A variety of compounds which are known to cross-link proteins have been tested as possible cell fixatives for confocal microscopy. The criteria for good fixation that were used for this work were that the fixative should make the cells resistant to lysis and the loss of protein from the cell under hypotonic conditions. In addition, fixation should not change the shape nor the volume of the cell. Of the compounds tested, one compound, EDC, appeared to have an excellent potential as a cell fixative and has the additional advantage that it does not require amino groups for its crosslinking activity. Another compound, SPDP, appeared to have some potential as a readily reversible fixative by virtue of its disulfide bridge.
Assuntos
Reagentes de Ligações Cruzadas , Microscopia Confocal/métodos , Fixação de Tecidos/métodos , Tamanho Celular , Células Cultivadas , Imunofluorescência , Humanos , Soluções Hipotônicas , Processamento de Imagem Assistida por Computador , Coloração e RotulagemRESUMO
Results indicating that analysis of the immune humoral response of brucellosis patients by immunoblotting provides useful information for the characterization of antigenic fractions of possible diagnostic importance in human brucellosis are presented. Sera of 90 patients were obtained: 23 suffering from chronic brucellosis, 20 from acute brucellosis and 47 belonging to the group of serologically positive individuals (SPI) without clinical evidence of active infection at the time of examination, and 35 healthy volunteers. They were tested against three antigenic fractions: cytoplasmic (CYT), outer membrane (OM) and inner membrane (IM). These fractions, which include virtually all the bacterial protein components, were prepared from Brucella abortus 1119/3 strain by detergent solubilization, enzymatic digestion and ultracentrifugation. Results obtained with these fractions showed the existence of antigens that permit the detection of brucellosis patients and their differentiation from SPI patients, with very high sensitivity.
Assuntos
Antígenos de Bactérias/análise , Brucella abortus/imunologia , Brucelose/diagnóstico , Epitopos , Humanos , Immunoblotting , Sensibilidade e Especificidade , Testes SorológicosRESUMO
Nucleotide excision repair (NER) removes bulky DNA lesions and is thus crucial for the protection against environmental carcinogens and UV light exposure. Deficiencies in NER cause increased mutation rates and chromosomal aberrations. Current methods for studying NER are mostly based on either quantitation of lesion removal or detection of repair DNA synthesis. Both have their limitations: lesion removal is inaccurate at very short times post-lesion, where the fraction of removal is low. Repair synthesis is difficult to apply to normally cycling cells due to the need to discriminate repair from replicative DNA synthesis. To overcome these problems we developed a method for analysis of NER based on detection of transient single-stranded (ss) DNA stretches generated at the nucleotide excision step. Cells are metabolically labelled with BrdU, exposed to UV-irradiation and the ssDNA transients generated during excision repair are detected using an anti-BrdU antibody. The method allows single-cell microscopic analysis of the distribution of DNA repair sites as well as kinetic analysis of the DNA repair response. Studies using various DNA repair-deficient cell lines indicate that the detection method integrates a number of pre-synthesis nucleotide excision repair stages. Thus, assembled repair sites can be detected even when they may not lead to complete resolution of the DNA lesion. Using this approach, we show that repair helicase-deficient cells differ from endonuclease-deficient cells.
Assuntos
Reparo do DNA , DNA de Cadeia Simples/análise , Animais , Bromodesoxiuridina , Replicação do DNA , DNA de Neoplasias/efeitos da radiação , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Citometria de Fluxo , Imunofluorescência , Humanos , Camundongos , Microscopia Confocal , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/efeitos da radiação , Raios UltravioletaRESUMO
A preparation of Brucella abortus cytoplasmic proteins was depleted of lipopolysaccharide (LPS) by immunoadsorption with a monoclonal antibody (MAb), BC68, specific for the O antigen of B. abortus smooth LPS. Two enzyme-linked immunosorbent assay (ELISA) systems were developed and used in this study. The first system includes an LPS-free cytoplasmic protein preparation; the second one was based on antigen capture on MAb BC68. By using these systems, we have demonstrated that 94% (33 of 35) of the brucellosis patients studied showed immunoglobulin G antiprotein response and also that all of the patients showed a strong anti-LPS reactivity. Thirty-six serologically positive individuals with no active infection at the time of examination (SPI) were also included. No immunoglobulin G antibodies against proteins were detected in 34 of them (92%), whereas 31 SPI (86%) showed various degrees of anti-LPS reactivity. The use of the LPS-free protein extract in ELISAs made it possible to establish differential reactivity patterns between active and inactive brucellosis.
Assuntos
Anticorpos Antibacterianos/sangue , Brucelose/diagnóstico , Proteínas de Bactérias/imunologia , Brucella abortus/imunologia , Brucelose/imunologia , Diagnóstico Diferencial , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina G/sangue , Lipopolissacarídeos/imunologiaRESUMO
Se presentan resultados que muestan que el análisis por immunoblotting de la respuesta inmune humoral de pacientes brucelosos permite la caracterización de componentes antigénicso de posible utilidad para el diagnóstico de la enfermedad. Se analizó el suero de 90 pacientes: 20 brucelosos agudos, 23 crónicos y 47 individuos serológicamente positivos sin evidencias clínicas de infección activa al momento del examen (SPI); se utilizó además el suero de 35 personas sanas como control. Todos los sueros fueron ensayados frente a tres fracciones antigéneticas: citoplasmáticas (CYT), membrana externa (OM) y membrana interna (IM). Dichas fracciones, que incluyen virtualmente todas las proteínas bacterianas, fueron preparadas a partir de Brucella abortus 1119/3 por solubilización con detergentes, digestión enzimática y ultracentrifugación. Los resultados obtenidos revelan la existencia de antígenos que permiten detectar a pacientes brucelosos con alta sensibilidad, y diferenciar a éstos del grupo SPI