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
Proliferative verrucous leukoplakia (PVL) is a potentially premalignant lesion that undergoes malignant transformation in over 40% of cases. Its clinical homogeneity suggests that a single or a small number of molecular pathogenic pathways may exist. Using the Cochrane protocol for systematic reviews, we have looked at the reported evidence of the molecular aetiology and pathogenesis of PVL and compared it with that of conventional oral epithelial dysplasia (OED). Of the 43 papers studied, 19 met the inclusion criteria including 13 proteins assayed in 344 tissues, and genes investigated were TP53, p14ARF, and p16INK4A. In all studies the research objectives were defined and outcomes were clearly stated. This review has shown that the transformation of PVL does not follow the same pathway as that of OED. There was weak evidence to suggest possible correlations between DNA aneuploidy, loss of heterozygosity at locus 9p21, and specific expression of Mcm (mini chromosome maintenance) protein, to transformation of PVL. To show important or distinct pathways of this condition, further studies are needed to access the somatic genomic alterations that are found in malignancies.
Assuntos
Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Leucoplasia Oral/genética , Leucoplasia Oral/patologia , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/patologia , Humanos , Neoplasias Bucais/genética , Neoplasias Bucais/patologiaRESUMO
MDM2 is induced by p53 in response to cellular insults such as DNA damage and can have effects upon the cell cycle that are independent or downstream of p53. We used a yeast two-hybrid screen to identify proteins that bind to MDM2 and which therefore might be involved in these effects. We found that MDM2 can bind to the C-terminus of the catalytic subunit of DNA polymerase epsilon (DNA pol epsilon), to a region that is known to be essential in yeast. In an in vitro system we confirmed that MDM2 could bind to the homologous regions of both mouse and human DNA pol epsilon and to full-length human DNA pol epsilon. DNA pol epsilon co-immunoprecipitated with MDM2 from transfected H1299 cells and also from a HeLa cell nuclear extract. We show here that the DNA pol epsilon-interacting domain of MDM2 is located between amino acids 50 and 166. Our studies provide evidence that MDM2 interacts with a region of DNA pol epsilon that plays a critical role in the function of DNA pol epsilon.
Assuntos
DNA Polimerase II/metabolismo , Proteínas Nucleares , Proteínas Proto-Oncogênicas/metabolismo , Animais , Domínio Catalítico , Linhagem Celular , Clonagem Molecular , Humanos , Camundongos , Proteínas Proto-Oncogênicas c-mdm2 , Técnicas do Sistema de Duplo-Híbrido , LevedurasRESUMO
The frequency and location of prominent left ventricular trabeculations were studied in 474 autopsy specimens from subjects evenly distributed by sex and age. These structures were observed in 323 (68%) of the hearts, and their frequency was similar in male (72%) and female (65%) subjects. Neither the frequency nor the location varied appreciably with age. Among the 323 hearts with prominent left ventricular trabeculations, 172 (53%) exhibited 2 or more; thus, the total number of trabeculations was 582. Of these 582 trabeculations, 493 (85%) were septoparietal bundles that inserted into both the free wall and the septum. Trabeculations also were observed between two points on the ventricular septum in 37 (6%) of the hearts and between two points along the free wall in 36 (6%). Less common patterns included trabeculations between the ventricular septum and the posteromedial papillary muscle in 10 hearts (2%), the ventricular septum and the anterolateral papillary muscle in 2, the free wall and the posteromedial papillary muscle in 2, the two papillary muscles in 1 and the apex and the ventricular septum in 1. Accordingly, prominent left ventricular trabeculations are considered to be common variants of the normal human heart. Their size, shape and location may lead to their being misinterpreted, possibly as mural thrombi, by two-dimensional echocardiography.
Assuntos
Ventrículos do Coração/anatomia & histologia , Coração/anatomia & histologia , Adolescente , Adulto , Idoso , Criança , Ecocardiografia , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
RNA-directed DNA polymerase (reverse transcriptase) activity was detected in platelets from 4/4 patients with primary proliferative polycythaemia (PPP), 7/7 patients with essential thrombocythaemia (ET), 1/4 patients with relative "stress" polycythaemia, 0/2 patients with secondary polycythaemia, and 0/3 normal subjects. The activity appeared to be particle-associated and was detected under conditions not appropriate for any known cellular enzymes. Active particulate fractions from 1 patient with PPP and 1 patient with ET were examined by electron microscopy and revealed objects with the features of retroviruses. Similar retrovirus-like particles were observed in 2/2 further patients with PPP and 2/2 further patients with ET but in 0/3 controls.
Assuntos
Plaquetas/enzimologia , Policitemia Vera/enzimologia , DNA Polimerase Dirigida por RNA/análise , Retroviridae/enzimologia , Trombocitemia Essencial/enzimologia , Plaquetas/ultraestrutura , Humanos , Microscopia Eletrônica , Mitocôndrias/enzimologia , Policitemia Vera/sangue , Policitemia Vera/microbiologia , Retroviridae/ultraestrutura , Especificidade por Substrato , Moldes Genéticos , Trombocitemia Essencial/sangue , Trombocitemia Essencial/microbiologia , Transferases/análiseRESUMO
The MDM2 protein, through its interaction with p53, plays an important role in the regulation of the G(1) checkpoint of the cell cycle. In addition to binding to and inhibiting the transcriptional activation function of the p53 protein, MDM2 binds, inter alia, to RB and the E2F-1.DP-1 complex and in so doing may promote progression of cells into S phase. Mice transgenic for Mdm2 possess cells that have cell cycle regulation defects and develop an altered tumor profile independent of their p53 status. MDM2 also blocks the growth inhibitory effects of transforming growth factor-beta1 in a p53-independent manner. We show here that a novel growth regulatory molecule is also the target of MDM2-mediated inhibition. Using a yeast two-hybrid screen, we have identified a gene that encodes a novel cellular protein (MTBP) that binds to MDM2. MTBP can induce G(1) arrest, which in turn can be blocked by MDM2. Our results suggest the existence of another growth control pathway that may be regulated, at least in part, by MDM2.
Assuntos
Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Fase G1 , Proteínas Nucleares , Proteínas Proto-Oncogênicas/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Proteínas de Transporte/genética , Divisão Celular , Citometria de Fluxo , Perfilação da Expressão Gênica , Inibidores do Crescimento/antagonistas & inibidores , Inibidores do Crescimento/química , Inibidores do Crescimento/genética , Inibidores do Crescimento/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-mdm2 , Alinhamento de Sequência , Transfecção , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/fisiologia , Técnicas do Sistema de Duplo-HíbridoRESUMO
We have previously reported that particles resembling retroviral particles and possessing an RNA-directed DNA polymerase activity can be prepared from platelets. Furthermore, we and others have shown that these particles are present at higher levels in patients with essential thrombocythemia and polycythemia vera. We show here that these particles package RNA molecules that encode HERV-K-related pol genes. A subset of the RNA molecules that are packaged are likely to encode the RNA directed DNA polymerase activity and, because these RNAs possess long/full-length open reading frames for the reverse transcriptase and RNaseH (also for part of the integrase domains in genomic clones) of HERV-K, we propose that these transcripts are indeed strong candidates for encoding the enzyme activity found in these particles. Moreover, by using a modification of the polymerase chain reaction-based reverse transcriptase assay in which activated DNA is added during cDNA synthesis to suppress DNA polymerase-mediated RNA-directed DNA synthesis, we have found that the particle-associated enzyme behaves like a retroviral reverse transcriptase, further supporting the conclusion that retrovirus-like, perhaps HERV-K sequences, encode this enzyme activity.
Assuntos
Produtos do Gene pol/genética , DNA Polimerase Dirigida por RNA/genética , Retroviridae/genética , Trombocitopenia/enzimologia , Trombocitopenia/genética , Adulto , Idoso , Sequência de Aminoácidos , Sequência de Bases , Feminino , Produtos do Gene pol/isolamento & purificação , Genoma Humano , Genoma Viral , Humanos , Dados de Sequência Molecular , DNA Polimerase Dirigida por RNA/isolamento & purificação , Trombocitopenia/virologiaRESUMO
A 932-bp retrovirus sequence was cloned by reverse transcriptase PCR from salivary gland tissue of a patient with Sjögren's syndrome. The sequence is related to that of type B and type D retroviruses and was present in a sucrose density gradient fraction corresponding to that of an enveloped retrovirus particle. Sequences amplified from tissues of eight individuals with or without Sjögren's syndrome had over 90% similarity and were present at a level of less than one copy per 10(3) cells. The sequence was not detectable in human genomic DNA by PCR or by Southern hybridization. These data indicate that the sequence represents an infectiously acquired genome, provisionally called human retrovirus 5.
Assuntos
DNA Viral/análise , Retroviridae/genética , Síndrome de Sjogren/virologia , Proteínas Virais/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Técnicas de Cocultura , Células HeLa , Humanos , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , Retroviridae/classificação , Retroviridae/isolamento & purificação , Glândulas Salivares/virologia , Homologia de Sequência de Aminoácidos , Síndrome de Sjogren/patologia , Células Tumorais CultivadasRESUMO
To investigate the hypothesis that the human endogenous sequence ERV-3 has a function, we have cloned and expressed the transmembrane region of its envelope gene and raised specific antibodies to the fusion protein and to a synthetic peptide. These antibodies reacted with a 65-kDa polypeptide which constituted approximately 0.1% of the cellular protein in syncytiotrophoblasts in placenta. The evolutionary conservation and abundant expression of this endogenous retroviral protein in a specific cell type support the concept of a biological function. The similarity of a domain of ERV-3 env to putative immunosuppressive p15E sequences suggests that ERV-3 might form part of the placental immunosuppressive barrier between mother and foetus.
Assuntos
Placenta/metabolismo , Proteínas dos Retroviridae/biossíntese , Trofoblastos/metabolismo , Proteínas do Envelope Viral/biossíntese , Sequência de Aminoácidos , Sequência de Bases , Células Cultivadas , Primers do DNA , Feminino , Imunofluorescência , Humanos , Dados de Sequência Molecular , Placenta/citologia , Placenta/virologia , Proteínas dos Retroviridae/genética , Proteínas dos Retroviridae/fisiologia , Trofoblastos/virologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/fisiologiaRESUMO
Four chronic heroin addicts had renal amyloidosis and the nephrotic syndrome. Amyloidosis had the secondary type of tissue distribution and developed after many years of addiction. That chronic immunologic stimulation by one or more exogenous antigens or multiple acute inflammatory episodes were important factors in the pathogenesis of amyloidosis in these patients is postulated. Amyloidosis, although uncommon, must be considered among the different types of renal involvement that are known to occur in drug addicts, especially those with the nephrotic syndrome, and amyloid should be specifically searched for in their renal biopsies.
Assuntos
Amiloidose/complicações , Dependência de Heroína/complicações , Nefropatias/complicações , Síndrome Nefrótica/etiologia , Adulto , Amiloidose/etiologia , Amiloidose/patologia , Autopsia , Biópsia , Doença Crônica , Feminino , Humanos , Nefropatias/etiologia , Nefropatias/patologia , Masculino , Pessoa de Meia-IdadeRESUMO
The single copy endogenous retrovirus locus ERV-3 is known to be primarily expressed in the placenta. The absence of expression of this gene in choriocarcinoma cell lines has led to speculation that this may be a defect associated with this abnormality. We show here that ERV-3 is not normally expressed in the cytotrophoblast from which these tumour cells are derived but is expressed in normal syncytiotrophoblast. The conservation of the ERV-3 open reading frame for env in ape and old world monkey species and its tight regulation and site of expression suggest a functional role for this gene in this tissue.
Assuntos
Retroviridae/crescimento & desenvolvimento , Trofoblastos/microbiologia , Regulação para Cima , Sequência de Bases , Diferenciação Celular , Células Cultivadas , Coriocarcinoma/microbiologia , Feminino , Humanos , Dados de Sequência Molecular , Gravidez , Trofoblastos/citologia , Células Tumorais CultivadasRESUMO
We have examined the human mammary tumor cell line T47D and have found that these cells produce virus-like particles which band at the typical density for retroviral particles on a sucrose gradient, possess reverse transcriptase activity, and package HERV-K10-like sequences. Using this information and a bacterial expression system to identify long open reading frames, we have identified individual clones which have full-length open reading frames for reverse transcriptase and RNase H and which could encode the reverse transcriptase activity detected in these cells.
Assuntos
DNA Polimerase Dirigida por RNA/análise , Retroviridae/isolamento & purificação , Células Tumorais Cultivadas/virologia , Sequência de Bases , DNA Viral , Feminino , Humanos , Dados de Sequência Molecular , DNA Polimerase Dirigida por RNA/genética , Retroviridae/enzimologiaRESUMO
Specific point mutations which affect viral tropism have been identified in both the V3 loop and in the CD4-binding region of the human immunodeficiency virus type 1 surface glycoprotein gp120. Here we report that a single point mutation in the first variable region (V1) of human immunodeficiency virus type 1 strain JRCSF is responsible for a change in viral tropism.
Assuntos
Síndrome da Imunodeficiência Adquirida/genética , Variação Genética , Proteína gp120 do Envelope de HIV/genética , HIV-1/genética , Sequência de Aminoácidos , Sequência de Bases , Células Cultivadas , Leucócitos Mononucleares/microbiologia , Dados de Sequência Molecular , Mutação Puntual , Linfócitos T/microbiologia , Linfócitos T Reguladores/microbiologiaRESUMO
Neutropenia in the newborn is often associated with sepsis, maternal hypertension, or prematurity. We describe a 654-g infant born at 30 weeks' gestation by cesarean section due to severe maternal hypertension. His course was complicated by five episodes of sepsis, including three with group B streptococcus. The results of hematologic and immunologic studies were normal except that absolute neutrophil counts were low (less than 1 x 10(9)/L) with intermittent increases during sepsis. Human recombinant granulocyte colony-stimulating factor administered subcutaneously (10 micrograms/kg per day initially) resulted in an absolute neutrophil count of greater than 30 x 10(9)/L within 2 weeks. The dosage was lowered and the absolute neutrophil counts were maintained at 8 to 12 x 10(9)/L with no further septic episodes. The human recombinant granulocyte colony-stimulating factor therapy was discontinued after 7 months, and the patient remained healthy with an absolute neutrophil count of greater than 2 x 10(9)/L. Thus, treatment with human recombinant granulocyte colony-stimulating factor may be useful as a temporary measure for neonatal neutropenia associated with sepsis. A controlled, clinical trial is warranted.
Assuntos
Fator Estimulador de Colônias de Granulócitos/uso terapêutico , Doenças do Prematuro/terapia , Neutropenia/terapia , Humanos , Recém-Nascido , Contagem de Leucócitos , Masculino , Receptores de Fator Estimulador de Colônias de GranulócitosRESUMO
All primates studied to date produce retroviral-like particles in their placentae. We have purified these particles from two primate species, one Old World (human) and one New World (marmoset), and have identified the retroviral sequences which are packaged into these particles. Three families of sequences have been detected in these particles in human, all of which have the highest homology to B- and D-type retroviruses and to the human endogenous retrovirus HERV-K10. Previous studies have reported that the New World monkeys do not possess sequences with homology to HERV-K10. We have identified a new family of low-copy-number sequences which are present in New World monkeys and which possess 70% homology to the HERV-K family. Particles from both species possess reverse transcriptase activity and we have found that some of these retroviral particles package sequences which encode long open reading frames in pol, as revealed by expression cloning in Escherichia coli. These open reading frames could encode the reverse transcriptase enzyme activity found in the particles.