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This corrects the article DOI: 10.1038/bjc.2012.109.
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BACKGROUND: We assessed the development in the number of new base of tongue squamous-cell carcinoma (BSCC) cases per year in eastern Denmark from 2000 to 2010 and whether HPV may explain any observable increased incidence. METHODS: We performed HPV DNA PCR and p16 immunohistochemistry analysis for all (n=210) BSCCs registered in the Danish Head and Neck Cancer Group (DAHANCA) and the Danish Pathology Data Bank, and genotyped all HPV-positive specimens with amplicon-based next-generation sequencing. RESULTS: The overall crude incidence of BSCCs increased significantly (5.4% per year) during the study period. This was explained by a significant increase in the number of HPV-positive BSCCs (8.1% per year), whereas the number of HPV-negative BSCCs did not increase significantly. The overall HPV prevalence was 51%, with HPV16 as the predominant HPV type. CONCLUSIONS: The increased number of HPV-positive BSCCs may explain the increasing incidence of BSCCs in eastern Denmark, 2000-2010.
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Alphapapillomavirus/aislamiento & purificación , Neoplasias de la Lengua/epidemiología , Alphapapillomavirus/genética , Dinamarca/epidemiología , Humanos , Incidencia , Neoplasias de la Lengua/virologíaRESUMEN
BACKGROUND: A significant proportion of squamous cell carcinomas of the oropharynx (OP-SCC) are related to human papillomavirus (HPV) infection and p16 overexpression. This subgroup proves better prognosis and survival but no evidence exists on the correlation between HPV and p16 overexpression based on diagnostic measures and definition of p16 overexpression. We evaluated means of p16 and HPV diagnostics, and quantified overexpression of p16 in HPV-positive and -negative OP-SCCs by mode of immunohistochemical staining of carcinoma cells. METHODS: PubMed, Embase, and the Cochrane Library were searched from 1980 until October 2012. We applied the following inclusion criteria: a minimum of 20 cases of site-specific OP-SCCs, and HPV and p16 results present. Studies were categorised into three groups based on their definition of p16 overexpression: verbal definition, nuclear and cytoplasmatic staining between 5 and 69%, and ≥70% staining. RESULTS: We identified 39 studies with available outcome data (n=3926): 22 studies (n=1980) used PCR, 6 studies (n=688) used ISH, and 11 studies (n=1258) used both PCR and ISH for HPV diagnostics. The methods showed similar HPV-positive results. Overall, 52.5% of the cases (n=2062) were HPV positive. As to p16 overexpression, 17 studies (n=1684) used a minimum of 5-69% staining, and 7 studies (n=764) used ≥70% staining. Fifteen studies (n=1478) referred to a verbal definition. Studies showed high heterogeneity in diagnostics of HPV and definition of p16. The correlation between HPV positivity and p16 overexpression proved best numerically in the group applying ≥70% staining for p16 overexpression. The group with verbal definitions had a significantly lower false-positive rate, but along with the group applying 5-69% staining showed a worse sensitivity compared with ≥70% staining. CONCLUSIONS: There are substantial differences in how studies diagnose HPV and define p16 overexpression. Numerically, p16 staining is better to predict the presence of HPV (i.e. larger sensitivity), when the cutoff is set at ≥70% of cytoplasmatic and nuclear staining.
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Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/virología , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/virología , Proteínas de Neoplasias/biosíntesis , Neoplasias Orofaríngeas/metabolismo , Neoplasias Orofaríngeas/virología , Papillomaviridae/aislamiento & purificación , Infecciones por Papillomavirus/metabolismo , Infecciones por Papillomavirus/virología , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Femenino , Genes p16 , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/patología , Humanos , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/genética , Neoplasias Orofaríngeas/genética , Neoplasias Orofaríngeas/patología , Papillomaviridae/genética , Infecciones por Papillomavirus/genética , Infecciones por Papillomavirus/patología , Pronóstico , Factores de Riesgo , Carcinoma de Células Escamosas de Cabeza y Cuello , Adulto JovenRESUMEN
BACKGROUND: Several environmental factors have been associated with increased risks for cervical cancer. We examined whether reproductive history, contraceptive use, or sexual behaviour increase the risk for cervical intraepithelial neoplasia grade 3 or worse (CIN3+) among women with persistent human papillomavirus (HPV) infection. METHODS: A population-based cohort of women participated in a personal interview and underwent a gynaecological examination at which cervical specimens were obtained for HPV DNA testing. Follow-up information (~13 years) on cervical lesions was obtained from the Danish Pathology Data Bank. Women who had a high-risk HPV infection comprised the overall study population (n=1353). A subgroup of women with persistent high-risk HPV infection (n=312) was identified. Hazard ratios (HRs) for a diagnosis of CIN3+ and the corresponding 95% confidence intervals (CIs) were calculated. RESULTS: Women with persistent HPV infection who had given birth had a significantly increased risk for CIN3+ (HR=1.78; 95% CI: 1.07-2.94). No association was found with pregnancy, use of intrauterine devices, or sexual behaviour. Based on small numbers, women with persistent HPV infection had a decreased risk for CIN3+ with any use of oral contraceptives (HR=0.54; 95% CI: 0.29-1.00). CONCLUSION: Childbirth increases the risk for subsequent CIN3+ among women with persistent HPV infection.
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Infecciones por Papillomavirus/complicaciones , Paridad , Displasia del Cuello del Útero/virología , Neoplasias del Cuello Uterino/virología , Adolescente , Adulto , Anticoncepción , ADN Viral/análisis , Femenino , Humanos , Papillomaviridae , Conducta Sexual , Adulto JovenRESUMEN
BACKGROUND: Although the role of human papilloma virus (HPV) in cervical squamous cell carcinoma (CSCC) is well established, the role in head and neck SCC (HNSCC) is less clear. MicroRNAs (miRNAs) have a role in the cancer development, and HPV status may affect the miRNA expression pattern in HNSCC. To explore the influence of HPV in HNSCC, we made a comparative miRNA profile of HPV-positive (HPV+) and HPV-negative (HPV-) HNSCC against CSCC. METHODS: Fresh frozen and laser microdissected-paraffin-embedded samples obtained from patients with HPV+/HPV- HNSCC, CSCC and controls were used for microarray analysis. Differentially expressed miRNAs in the HPV+ and HPV- HNSCC samples were compared with the differentially expressed miRNAs in the CSCC samples. RESULTS: Human papilloma virus positive (+) HNSCC had a distinct miRNA profile compared with HPV- HNSCC. Significantly more similarity was seen between HPV+ HNSCC and CSCC than HPV- and CSCC. A set of HPV core miRNAs were identified. Of these especially the miR-15a/miR-16/miR195/miR-497 family, miR-143/miR-145 and the miR-106-363 cluster appear to be important within the known HPV pathogenesis. CONCLUSION: This study adds new knowledge to the known pathogenic pathways of HPV and substantiates the oncogenic role of HPV in subsets of HNSCCs.
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Biomarcadores de Tumor/genética , Carcinoma de Células Escamosas/genética , Neoplasias de Cabeza y Cuello/genética , MicroARNs/genética , Papillomaviridae/genética , Infecciones por Papillomavirus/genética , Adolescente , Biomarcadores de Tumor/metabolismo , Carcinoma de Células Escamosas/virología , Estudios de Casos y Controles , Niño , ADN Viral/genética , Femenino , Perfilación de la Expresión Génica , Neoplasias de Cabeza y Cuello/virología , Humanos , Captura por Microdisección con Láser , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Análisis de Secuencia por Matrices de Oligonucleótidos , Infecciones por Papillomavirus/virología , Pronóstico , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNAs, which regulate mRNA translation/decay, and may serve as biomarkers. We characterised the expression of miRNAs in clinically sampled oral and pharyngeal squamous cell carcinoma (OSCC and PSCC) and described the influence of human papilloma virus (HPV). METHODS: Biopsies obtained from 51 patients with OSCC/PSCC and 40 control patients were used for microarray analysis. The results were correlated to clinical data and HPV status. Supervised learning by support vector machines was employed to generate a diagnostic miRNA signature. RESULTS: One hundred and fourteen miRNAs were differentially expressed between OSCC and normal oral epithelium, with the downregulation of miR-375 and upregulation of miR-31 as the most significant aberrations. Pharyngeal squamous cell carcinoma exhibited 38 differentially expressed miRNAs compared with normal pharyngeal epithelium. Differences in the miRNA expression pattern of both normal epithelium and SCC were observed between the oral cavity compared with the pharynx. Human papilloma virus infection revealed perturbations of 21 miRNAs, most significantly in miR-127-3p and miR363. A molecular classifier including 61 miRNAs was generated for OSCC with an accuracy of 93%. CONCLUSION: MicroRNAs may serve as useful biomarkers in OSCC and PSCC. The influence of HPV on miRNA may provide a mechanism for the distinct clinical behaviour of HPV-infected tumours.
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Carcinoma de Células Escamosas/genética , MicroARNs/biosíntesis , Neoplasias de la Boca/genética , Neoplasias Faríngeas/genética , Femenino , Expresión Génica , Humanos , Masculino , Análisis por Micromatrices , Persona de Mediana Edad , Estudios ProspectivosRESUMEN
High-risk human papillomavirus (HPV) is a major causative agent of cervical cancer and the E6 and E7 genes encode the major HPV oncoproteins. The E7 protein from high-risk HPV types alters cell cycle progression and represses genes encoding components of the antigen-presentation pathway, suggesting a role for E7 in tumour immune evasion. We show that knockdown of E7 expression in HPV16- and HPV18-transformed cervical carcinoma cells by RNA interference increased expression of major histocompatibility complex (MHC) class I at the cell surface and reduced susceptibility of these cells to natural killer (NK) cells. Tetracycline-regulated induction of HPV16 E7 resulted in reduced expression of cell surface MHC class I molecules and increased NK cell killing. Our results suggest that, for HPV-associated malignancies, reduced MHC class I expression is the result of an active immune evasion strategy that has evolved to assist viral replication.
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Antígenos de Histocompatibilidad Clase I/metabolismo , Papillomavirus Humano 16/inmunología , Papillomavirus Humano 18/inmunología , Células Asesinas Naturales/inmunología , Proteínas Oncogénicas Virales/biosíntesis , Proteínas Oncogénicas Virales/genética , Infecciones por Papillomavirus/inmunología , Células Cultivadas , Femenino , Células HeLa , Papillomavirus Humano 16/metabolismo , Humanos , Células Asesinas Naturales/metabolismo , Proteínas Oncogénicas Virales/fisiología , Proteínas E7 de Papillomavirus , Infecciones por Papillomavirus/metabolismo , Escape del Tumor/inmunología , Neoplasias del Cuello Uterino/inmunología , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/virologíaRESUMEN
Several members of the S100 family of Ca(2+) binding proteins are at present known to be secreted and to have extracellular activities. We have investigated the neurite inducing potential of extracellularly added S100A12. Human recombinant S100A12 was found to dramatically induce neuritogenesis of hippocampal cells isolated from 17 to 19 days old rat embryos. The response to S100A12 was dependent on the dose in a bell-shaped manner. A 10-fold increase in neurite outgrowth was observed upon treatment with S100A12 in concentrations between 0.1 and 2.0 microM already after 24 h. Exposure to S100A12 for only 15 min was enough to induce neuritogenesis when measured after 24 h, but to obtain a maximal response, S100A12 had to be present in the culture for at least 4 h. The response to S100A12 was abolished by inhibitors of phospholipase C (PLC), protein kinase C (PKC), Ca(2+) flux, Ca(2+)/calmodulin dependent kinase II (CaMKII) or mitogen-activated protein kinase kinase (MEK). Therefore, we suggest that extracellular S100A12 triggers intracellular signal transduction in neurons, involving the classical mitogen-activated protein (MAP) kinase pathway and a phospholipase C-generated second messenger pathway leading to an increase in intracellular Ca(2+) and activation of PKC, ultimately resulting in neuronal differentiation.
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Proteínas de Unión al Calcio/farmacología , Neuritas/efectos de los fármacos , Neuronas/efectos de los fármacos , Proteínas Recombinantes/farmacología , Proteínas S100 , Animales , Calcio/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Hipocampo/citología , Hipocampo/efectos de los fármacos , Hipocampo/embriología , Humanos , Neuritas/ultraestructura , Neuronas/citología , Neuronas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Ratas , Ratas Wistar , Proteína S100A12 , Factores de Tiempo , Fosfolipasas de Tipo C/antagonistas & inhibidores , Fosfolipasas de Tipo C/metabolismoRESUMEN
The present investigation evaluated the relationship between dysplasia of the uterine cervix and telomerase activity, expression of p53, MIB-1 and PCNA. Telomerase activity was measured on cervical cytobrush material from 126 women suspected of having dysplasia and 61 controls using the telomeric repeat amplification protocol. Immunohistochemistry was used to detect the tumor suppressor protein p53 and cell proliferation, the latter by MIB-1 and PCNA expression. Infection with human papillomavirus 16 was detected by PCR amplification and Southern blot hybridization of DNA extracted from the same brush material. Positive telomerase activity was found in 5 of 43 (11.6%) normal samples, 12 of 57 (21.1%) samples with inflammation or koilocytosis, 7 of 17 (41.2%) CIN 1 (cervical intraepithelial neoplasia, grade 1), 8 of 20 (40.0%) CIN 2, and 25 of 42 (59.5%) CIN 3/ CIS. Telomerase activity was significantly related to the level of dysplasia (p<0.001) and proliferation measured by MIB-1 (p=0.019), but not to the level of PCNA (p=0.445), HPV 16 status (p=0.098) or staining for p53 (p=0.271). Dysplasia was also related to PCNA, MIB1, p53, and presence of HPV 16. A sequential increase in the examined parameters, paralleling the progression of abnormality, was observed. PCNA and telomerase showed an increase in CIN 1, MIB-1 and HPV16 in CIN 2, and finally p53 in CIN 3/CIS.
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Biomarcadores de Tumor/aislamiento & purificación , Displasia del Cuello del Útero/diagnóstico , Neoplasias del Cuello Uterino/diagnóstico , Adolescente , Adulto , Antígenos Nucleares , Femenino , Humanos , Antígeno Ki-67 , Persona de Mediana Edad , Proteínas Nucleares/aislamiento & purificación , Papillomaviridae/aislamiento & purificación , Antígeno Nuclear de Célula en Proliferación/aislamiento & purificación , Reproducibilidad de los Resultados , Telomerasa/aislamiento & purificación , Proteína p53 Supresora de Tumor/aislamiento & purificaciónRESUMEN
Archival, formalin-fixed, paraffin-embedded cervical cancer specimens from 53 Alaska natives, 32 Greenland natives and 34 Danish Caucasians were analyzed for human papillomavirus (HPV) genotypes 16, 18, 31, 33, 35 and 45 and unidentified genotypes (HPV X) using PCR. The specimens were from the time period 1980-1989. No significant differences were observed in the overall HPV detection rates among cases from Alaska (98.1%), Greenland (84.4%) and Denmark (85.3%). HPV genotype 16 was the most prevalent type: 78.8% in Alaska natives, 96.3% in Greenland natives and 82.8% in Danish Caucasians. A prevalence of 21.2% HPV 31 and 30.8% HPV 33 was found in Alaska natives, of which most were coinfections with HPV 16. Only 3.7% HPV 31 and 3.7% HPV 33 were found in Greenland natives and no HPV 31 and 6.9% HPV 33 were found in Danish Caucasians. HPV 18 was only detected in Alaska natives and HPV 35 and 45 were not detected in any of the three populations. Infections with multiple genotypes were prevalent in Alaskan (36.5%) but not in Greenland natives (3. 7%) and Danish Caucasians (6.9%). The Eskimo subgroup of the Alaska native population has a significantly higher prevalence of HPV genotypes 31 and 33 associated with mixed infections in invasive cancer than the two other native subgroups (P = 0.04) and Greenland and Danish populations, reflecting genotype distributions in dysplasia and normal cervical cytology. The reason for HPV genotype diversity, although unknown, may be relevant to the current development of HPV vaccines.
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Carcinoma de Células Escamosas/virología , Papillomaviridae/genética , Infecciones por Papillomavirus/virología , Infecciones Tumorales por Virus/virología , Neoplasias del Cuello Uterino/etnología , Neoplasias del Cuello Uterino/virología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Alaska/epidemiología , Carcinoma de Células Escamosas/etnología , Dinamarca/epidemiología , Femenino , Genotipo , Groenlandia/epidemiología , Humanos , Persona de Mediana Edad , Papillomaviridae/clasificación , Infecciones por Papillomavirus/etnología , Reacción en Cadena de la Polimerasa/métodos , Prevalencia , Infecciones Tumorales por Virus/etnología , Población BlancaRESUMEN
Human papillomavirus type 16 (HPV-16) is the dominant risk factor for the development of cervical cancer. The virus encodes three oncoproteins, of which the E7 oncoprotein is the major protein involved in cell immortalization and transformation. E7 is a multi-functional protein. It binds the retinoblastoma tumour-suppressor protein (pRb), which regulates progression through the G(1) restriction point in the cell cycle. The E7 protein interacts with transcription-regulatory proteins such as the TATA box-binding protein and with proteins of the AP1 transcription factor family. To identify additional proteins regulated by E7, differential-display PCR was used to identify differentially expressed mRNAs in cells containing an inducible E7 protein. It is reported that E7 expression leads to regulation of the genes encoding the calcium-binding protein S100P and the mitochondrial ADP/ATP carrier protein. These data identify new functions of the E7 protein and thus expand the number of routes by which HPV-16 influences cell growth control, although the function of S100P has still to be elucidated.
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Proteínas de Unión al Calcio/genética , Proteínas de Neoplasias , Proteínas Oncogénicas Virales/genética , Papillomaviridae/genética , Infecciones por Papillomavirus/genética , Receptores Citoplasmáticos y Nucleares/genética , Infecciones Tumorales por Virus/genética , Regulación Viral de la Expresión Génica , Genes Virales , Humanos , Proteínas E7 de Papillomavirus , Reacción en Cadena de la PolimerasaRESUMEN
Because cell cultures are essential in biological research which involves the analysis of virus morphogenesis, this study focused on examining the significance of cell passages. Human embryonic lung fibroblasts (MRC-5) at passage (P) 27 were seeded twice a week to P 32, P 40, and P 48, when just at confluence and then infected with herpes simplex virus type 1 (HSV-1). The structure of the non-virus-infected (MOCK) and HSV-1 infected cells, the amount of cellular infectious virus particles and the capability to express HSV-1 glycoproteins C (gC-1) and D (gD-1) were investigated by phase-contrast and immunofluorescence light microscopy, immunogold cryosection EM, plaque assays, immunoblots, and total protein assays. Modified cell structure including fragmentation of tubulin fibers were visible in MOCK from P 38 onwards. The quantity of vimentin remained unchanged while actin accumulated and beta-tubulin decreased in HSV-1 infected late P cells compared to early P cultures. Cells of high P counts contained significantly fewer infectious virus particles, very likely of lower virulence, and their expression of gC-1 and gD-1 were concordantly reduced. These observations indicate that the number of cell P must be considered in order to reproduce results of cell biology and viral morphogenesis. The MRC-5 cells ought not to be passaged more than ten times beyond P 27 in the laboratory.
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Técnicas de Cultivo de Célula , Tamaño de la Célula , Senescencia Celular , Efecto Citopatogénico Viral , Herpesvirus Humano 1/fisiología , Línea Celular , Citoesqueleto/ultraestructura , Herpesvirus Humano 1/ultraestructura , Humanos , Immunoblotting , Inmunohistoquímica , Microscopía Electrónica/métodos , Microscopía Fluorescente , Orgánulos/ultraestructura , Tubulina (Proteína)/análisis , Proteínas del Envoltorio Viral/análisis , Ensayo de Placa ViralRESUMEN
The small hydrophobic E5 protein of Human Papillomavirus type 16 (HPV16) binds to the 16-kDa subunit of the V-H+-ATPase. This binding has been suggested to interfere with acidification of late endocytic structures. We here used video microscopy, ratio imaging and confocal microscopy of living C127 fibroblasts to study the effects of E5. Various endocytic markers including the pH-sensitive probe DM-NERF coupled to dextran, TransFluoSpheres and TRITC-concanavalin A, were applied. In E5-transfected cells, none of these markers colocalized with the membrane permeable probe LysoTracker Red, which accumulates in acidic, late endocytic structures, or with a green fluorescent version of the small GTPase Rab7 labeling late endocytic structures. Importantly, however, late endocytic structures accumulating LysoTracker were still present in the E5-transfected cells. It is therefore concluded that HPV16 E5 perturbs trafficking from early to late endocytic structures rather than acidification.
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Endocitosis , Endosomas/metabolismo , Proteínas Oncogénicas Virales/metabolismo , Actinas/metabolismo , Animales , Transporte Biológico , Biomarcadores/análisis , Línea Celular , Tamaño de la Célula , Citoesqueleto/química , Citoesqueleto/metabolismo , Endosomas/química , Fibroblastos , Colorantes Fluorescentes/metabolismo , Oro , Humanos , Concentración de Iones de Hidrógeno , Queratinocitos , Ratones , Microscopía Fluorescente , Microscopía por Video , Proteínas Oncogénicas Virales/genética , ARN Mensajero/análisis , ARN Mensajero/genética , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión a GTP rab7RESUMEN
It is known that the proper function of the vacuolar H(+)-ATPase is inhibited by bafilomycin A(1). In transfected cells the E5 protein interacts with the 16 kDa subunit of the vacuolar H(+)-ATPase. Thereby the pH gradient in endocytic structures is impaired. The present study demonstrates for the first time that the inhibition of the vacuolar H(+)-ATPase in NIH3T3 cells with bafilomycin A(1) or by transfection of cells with the HPV-16 E5 oncogene leads to a changed morphology and a reduced motility as shown by computer-assisted video recordings and image analysis. Bafilomycin A(1) potentiates the effect of the E5 protein on cell motility and this cooperative effect indicates that the E5 protein and bafilomycin A(1) either target the vacuolar H(+)-ATPase differently or that the E5 protein has additional targets in transfected cells. Our data therefore show that proper function of the vacuolar H(+)-ATPase is needed for normal cell locomotion.
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Antibacterianos/farmacología , Movimiento Celular/efectos de los fármacos , Macrólidos , Proteínas Oncogénicas Virales/farmacología , ATPasas de Translocación de Protón/antagonistas & inhibidores , Células 3T3 , Animales , Tamaño de la Célula/efectos de los fármacos , Sinergismo Farmacológico , Expresión Génica , Ratones , Proteínas Oncogénicas Virales/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
Transcription of the human papillomavirus type 16 (HPV-16) genome is controlled by several promoters; the P(97) promoter is considered to be the main one. An additional promoter has been identified within the E7 ORF as well as an antisense promoter just upstream of the L2 ORF. The significance of these promoters for early and late gene expression and their activity related to cell differentiation is not known in detail. Identification of two new, previously undescribed transcription start sites at nt 542 just upstream of the E7 ORF and at nt 611 within the E7 ORF is reported. The promoter responsible for the start site at nt 542 (P(542)) was active in SiHa, HeLa and C33A cells. Very low promoter activity was found upstream of the nt 611 start site. The E7 protein has previously been shown to be synthesized from a polycistronic mRNA encoding both the E6 and E7 proteins under the control of the P(97) promoter. The data reported in the present paper suggest that promoter P(542) may control synthesis of the E7 oncoprotein from a monocistronic mRNA.
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Proteínas Oncogénicas Virales/genética , Papillomaviridae/genética , Regiones Promotoras Genéticas , Transcripción Genética , Secuencia de Bases , Línea Celular , Clonación Molecular/métodos , ADN Complementario/genética , Elementos de Facilitación Genéticos , Regulación Viral de la Expresión Génica , Células HeLa , Humanos , Queratinocitos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Proteínas E7 de Papillomavirus , Mutación PuntualRESUMEN
Cell biology concerns the interactions between different cellular compartments and between the cell and the environment. The mechanisms of herpes simplex virus type-1 (HSV-1) envelopment and the transport of virus particles and HSV-1 glycoproteins have not been completely investigated. It is of interest to examine the formation of complete virus particles and the cellular distribution of viral glycoproteins correlated with microtubules. The illustration of these conditions by immunocytochemistry is best done by multiple labelling techniques in the same cell. Single-staining of neighbouring serial sections or two-face double-immunolabelling methods are not technically compatible with ultrathin cryosections. The results are reported here of a simultaneous, simple and reliable immunogold double-staining technique using primary antibodies of the same species in ultrathin cryosections. Compared to other inactivation procedures, phosphate-buffered 3% paraformaldehyde plus 2% glutaraldehyde for 2 h at room temperature is an excellent and gentle method to destroy free anti-IgG binding sites on the antibodies and to prevent cross-labelling, which has proven necessary for obtaining reproducible results on cellular distribution of tubulin and viral glycoproteins gD-1 and gC-1.
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Anticuerpos Monoclonales/metabolismo , Herpesvirus Humano 1/inmunología , Inmunohistoquímica/métodos , Microscopía Electrónica , Sitios de Unión , Línea Celular , Crioultramicrotomía , Estudios de Evaluación como Asunto , Humanos , Inmunoglobulina G/metabolismo , Pulmón/metabolismo , Pulmón/virología , Sondas Moleculares/metabolismo , Tubulina (Proteína)/metabolismo , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Herpes simplex virus type 1 (HSV-1) is a common human pathogen of skin and mucous membranes and is potentially dangerous when the infection is disseminated. Viral morphogenesis, especially the mechanism of viral envelopment and the exact pathway for processing and transport of HSV-1 glycoproteins, is still unclear. We report the results of optimized immunogold-labeled cryosection electron microscopy of HSV-1-infected cultured human fibroblasts (MRC-5). The simplified method presented has proved necessary to obtain reproducible results on cellular distribution of viral glycoproteins. It is now possible to demonstrate the viral glycoprotein gD-1, but not gC-1, in the nuclear membranes and to demonstrate gD-1- and gC-1-labeled viral particles in the perinuclear space, and to show the fate of the viral particles in the endoplasmic reticulum and Golgi area in infected cells.