RESUMEN
Despite considerable advances in understanding the molecular nature of cancer, many biophysical aspects of malignant development are still unclear. Here we study physical alterations of the surface of human cervical epithelial cells during stepwise in vitro development of cancer (from normal to immortal (premalignant), to malignant). We use atomic force microscopy to demonstrate that development of cancer is associated with emergence of simple fractal geometry on the cell surface. Contrary to the previously expected correlation between cancer and fractals, we find that fractal geometry occurs only at a limited period of development when immortal cells become cancerous; further cancer progression demonstrates deviation from fractal. Because of the connection between fractal behaviour and chaos (or far from equilibrium behaviour), these results suggest that chaotic behaviour coincides with the cancer transformation of the immortalization stage of cancer development, whereas further cancer progression recovers determinism of processes responsible for cell surface formation.
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BACKGROUND: There is no universally accepted, precise definition, nor standardisation in terminology and classification of halitosis. OBJECTIVE: To propose a new definition, free from subjective descriptions (faecal, fish odour, etc), one-time sulphide detector readings and organoleptic estimation of odour levels, and excludes temporary exogenous odours (for example, from dietary sources). Some terms previously used in the literature are revised. RESULTS: A new aetiologic classification is proposed, dividing pathologic halitosis into Type 1 (oral), Type 2 (airway), Type 3 (gastroesophageal), Type 4 (blood-borne) and Type 5 (subjective). In reality, any halitosis complaint is potentially the sum of these types in any combination, superimposed on the Type 0 (physiologic odour) present in health. CONCLUSION: This system allows for multiple diagnoses in the same patient, reflecting the multifactorial nature of the complaint. It represents the most accurate model to understand halitosis and forms an efficient and logical basis for clinical management of the complaint.
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Halitosis/clasificación , Esófago/fisiopatología , Halitosis/diagnóstico , Halitosis/etiología , Halitosis/fisiopatología , Halitosis/psicología , Humanos , Boca/fisiopatología , Odorantes , Sistema Respiratorio/fisiopatología , Estómago/fisiopatología , Terminología como AsuntoRESUMEN
We report on the first functional use of recently introduced ultrabright fluorescent mesoporous silica nanoparticles, which are functionalized with folic acid, to distinguish cancerous and precancerous cervical epithelial cells from normal cells. The high brightness of the particles is advantageous for fast and reliable identification of both precancerous and cancerous cells. Normal and cancer cells were isolated from three healthy women and three cancer patients. Three precancerous cell lines were derived by immortalization of primary cultures of normal cells with human papillomavirus type-16 (HPV-16) DNA. We observed substantially different particle internalization by normal and cancerous/precancerous cells after a short incubation time of 15 minutes. Compared to HPV-DNA and cell pathology tests, which are currently used for prescreening of cervical cancer, we demonstrated that the specificity of our method was similar (94-95%), whereas its sensitivity was significantly better (95-97%) than the sensitivity of those currently used tests (30-80%). FROM THE CLINICAL EDITOR: This team of investigators reports on the development of a new screening test for cervical cancer using ultrabright fluorescent mesoporous silica nanoparticles functionalized with folic acid, enabling significantly better sensitivity (95-97% vs. 30-80%) and maintained specificity (94-95%) compared with current clinical tests. This test should find a way to clinical use in the near future.
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Cuello del Útero/patología , Colorantes Fluorescentes , Ácido Fólico , Nanopartículas , Dióxido de Silicio , Neoplasias del Cuello Uterino/diagnóstico , Detección Precoz del Cáncer , Femenino , Colorantes Fluorescentes/química , Ácido Fólico/química , Humanos , Nanopartículas/química , Porosidad , Dióxido de Silicio/química , Células Tumorales Cultivadas , Neoplasias del Cuello Uterino/patologíaRESUMEN
Halitosis is a symptom and not a diagnosis. Rather, the topic represents a spectrum of disorders, including intra-oral, otorhinolaryngological, metabolic, systemic, pulmonary, psychological and neurological conditions. Halitosis may be the third most common trigger for patients to seek dental care and can cause significant impact on patient quality of life. About 10% of all genuine halitosis cases are attributed to extra-oral processes. Some authorities have reported that the nasal cavity and the oropharynx are the most common sites of origin of extra-oral halitosis. However, recent evidence appears to suggest that blood borne halitosis may be the most common subtype of extra-oral halitosis. Tangerman and Winkel report that dimethyl sulphide was the main volatile implicated in extra-oral blood borne halitosis. They proposed a hitherto unknown metabolic condition by way of explanation for this finding, resulting in systemic presence of dimethyl sulphide in blood and alveolar breath. This paper reviews the knowledge base regarding the behaviour of dimethyl sulphide in physiological systems and those disorders in which blood borne halitosis secondary to dimethylsulphidemia is thought to have an aetiopathological role.
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Halitosis/etiología , Sulfuros/sangre , Halitosis/sangre , Halitosis/patología , Humanos , Prevalencia , Sulfuros/metabolismoRESUMEN
STUDY DESIGN: Survey. OBJECTIVES: Compare views of disability in able-bodied and spinal cord-injured individuals. SETTING: United States. METHODS: A group of able-bodied individuals were asked to imagine that they had sustained a spinal cord injury (SCI), then complete the Appraisals of DisAbility: primary and secondary scale (ADAPSS) as if they were injured. The mean scores of able-bodied individuals on each of the six Subscales was compared with the mean scores of real spinal cord-injured individuals using t-tests of independent means. RESULTS: Responses of able-bodied individuals was significantly different from real SCI individuals on five of the six Subscales of the ADAPSS. CONCLUSION: Able-bodied individuals' appraisals of disability after imagined SCI are much more negative than the actual appraisals of disability in real spinal cord-injured individuals.
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Personas con Discapacidad/psicología , Imaginación , Traumatismos de la Médula Espinal/complicaciones , Traumatismos de la Médula Espinal/psicología , Adolescente , Adulto , Anciano , Evaluación de la Discapacidad , Personas con Discapacidad/clasificación , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estados Unidos , Adulto JovenRESUMEN
A significant change of surface features of malignant cervical epithelial cells compared to normal cells has been previously reported. Here, we are studying the question at which progressive stage leading to cervical cancer the surface alteration happens. A non-traditional method to identify malignant cervical epithelial cells in vitro, which is based on physical (in contrast to specific biochemical) labelling of cells with fluorescent silica micron-size beads, is used here to examine cells at progressive stages leading to cervical cancer which include normal epithelial cells, cells infected with human papillomavirus type-16 (HPV-16), cells immortalized by HPV-16, and carcinoma cells. The study shows a statistically significant (at p < 0.01) difference between both immortal and cancer cells and a group consisting of normal and infected. There is no significant difference between normal and infected cells. Immortal cells demonstrate the signal which is closer to cancer cells than to either normal or infected cells. This implies that the cell surface, surface cellular brush changes substantially when cells become immortal. Physical labeling of the cell surface represents a substantial departure from the traditional biochemical labeling methods. The results presented show the potential significance of physical properties of the cell surface for development of clinical methods for early detection of cervical cancer, even at the stage of immortalized, premalignant cells.
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Transformación Celular Viral , Células Epiteliales/patología , Papillomavirus Humano 16 , Infecciones por Papillomavirus/patología , Neoplasias del Cuello Uterino/patología , Adhesión Celular , Células Epiteliales/virología , Femenino , Colorantes Fluorescentes/química , Humanos , Microscopía Fluorescente , Microesferas , Infecciones por Papillomavirus/virología , Rodaminas/química , Coloración y Etiquetado , Células Tumorales Cultivadas , Neoplasias del Cuello Uterino/virologíaRESUMEN
The aim of this interview study was to ask patients with head and neck cancer in the Mersey region about their need for financial benefits, the advice they were given about benefits and financial matters, and the financial burden of the disease. Stratified quota sampling was by employment status, whether work had been affected by the cancer, and by sex. Of 51 interviewees (mean (SD) age 61(8) years) 20 were retired, 11 were unemployed, 13 worked full-time, and 7 worked part-time. Cancer had affected the work status of 24. Since diagnosis 57% had suffered financially; this was particularly high in those who had retired (65%), and in those whose work had been affected by cancer (79%). Quality of life had decreased in 53% as a result of the financial impact of the disease. This was most common in the unemployed (64%), and in those whose work was affected by cancer (83%). Only a quarter had been given adequate help and information about finance; this was lowest in the unemployed (18%) and highest in those who were fully employed (39%). One third (17/51) had never claimed benefits. The most common benefits were Disability Living Allowance and Incapacity Benefit. Two-thirds (21/31) had applied for benefits after diagnosis, 18 of these were directly as a result of the disease. The median (IQR) weekly income from benefits was £88 (£60-170). Patients and carers need better access to financial advice. We suggest that each multidisciplinary team should have a designated benefits or financial advisor who is readily available to patients in the clinic and on the ward.
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Costo de Enfermedad , Neoplasias de Cabeza y Cuello/economía , Necesidades y Demandas de Servicios de Salud/economía , Beneficios del Seguro/economía , Anciano , Empleo/economía , Empleo/estadística & datos numéricos , Femenino , Encuestas de Atención de la Salud , Humanos , Beneficios del Seguro/estadística & datos numéricos , Entrevistas como Asunto , Masculino , Persona de Mediana Edad , Calidad de Vida , Jubilación/economía , Jubilación/estadística & datos numéricos , Reino UnidoRESUMEN
The aim of this cross-sectional survey was to ask patients about the financial burden of having head and neck cancer, and to explore its relation with health-related quality of life (HRQoL). In the Mersey region 447/752 eligible patients (59%) responded to the questionnaire. There was no obvious response bias. The most notable financial costs of head and neck cancer that were a moderate or large burden to patients were petrol (25%, 112), home heating (24%, 108), change in the type of food (21%, 95), and loss of earnings (20%, 88). During the previous week 15% (63/423) had lost a moderate or large amount of income because of their medical condition. In terms of taking care of their financial needs, 10% (40) were moderately dissatisfied and 15% (61) very dissatisfied. Patients with worse physical and social emotional functioning experienced more notable financial burden, more difficult life circumstances in the past month and greater financial difficulty and loss in income due to their condition in the previous week, more dissatisfaction with how well they took care of their own financial needs and were more likely to have sought statutory benefits. Cancer of the head and neck has a serious impact on financial aspects of patients' lives and seems to be associated with a poor HRQoL. Multidisciplinary teams can do much more to address the cost of having treatment by recognising need earlier, and giving advice and access to appropriate benefits.
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Costo de Enfermedad , Neoplasias de Cabeza y Cuello/economía , Necesidades y Demandas de Servicios de Salud/economía , Renta/estadística & datos numéricos , Calidad de Vida , Anciano , Anciano de 80 o más Años , Empleo/economía , Femenino , Neoplasias de Cabeza y Cuello/psicología , Encuestas de Atención de la Salud , Humanos , Modelos Logísticos , Masculino , Persona de Mediana Edad , Jubilación/economía , Encuestas y Cuestionarios , Reino UnidoRESUMEN
Here we show that the surface of human cervical epithelial cells demonstrates substantially different fractal behavior when the cell becomes cancerous. Analyzing the adhesion maps of individual cervical cells, which were obtained using the atomic force microscopy operating in the HarmoniX mode, we found that cancerous cells demonstrate simple fractal behavior, whereas normal cells can only be approximated at best as multifractal. Tested on ~300 cells collected from 12 humans, the fractal dimensionality of cancerous cells is found to be unambiguously higher than that for normal cells.
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Cuello del Útero/citología , Cuello del Útero/patología , Fractales , Nanotecnología , Neoplasias del Cuello Uterino/patología , Adhesión Celular , Células Epiteliales/citología , Células Epiteliales/patología , Femenino , Humanos , Microscopía de Fuerza AtómicaRESUMEN
The atomic force microscope is broadly used to study the morphology of cells, but it can also probe the mechanics of cells. It is now known that cancerous cells may have different mechanical properties to those of normal cells, but the reasons for these differences are poorly understood. Here, we report quantitatively the differences between normal and cancerous human cervical epithelial cells by considering the brush layer on the cell surface. These brush layers, which consist mainly of microvilli, microridges and cilia, are important for interactions with the environment. Deformation force curves obtained from cells in vitro were processed according to the 'brush on soft cell model'. We found that normal cells have brushes of one length, whereas cancerous cells have mostly two brush lengths of significantly different densities. The observed differences suggest that brush layers should be taken into account when characterizing the cell surface by mechanical means.
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Cuello del Útero/citología , Microscopía de Fuerza Atómica/métodos , Microvellosidades/ultraestructura , Neoplasias del Cuello Uterino/ultraestructura , Interpretación Estadística de Datos , Células Epiteliales/ultraestructura , Femenino , Humanos , Fenómenos Mecánicos , Microscopía Electrónica de Transmisión , Propiedades de Superficie , Células Tumorales CultivadasRESUMEN
We describe a novel application of atomic force microscopy (AFM) to directly visualize cytoskeletal fibers in human foreskin epithelial cells. The nonionic detergent Triton X-100 in a low concentration was used to remove the membrane, soluble proteins, and organelles from the cell. The remaining cytoskeleton can then be directly visualized in either liquid or air-dried ambient conditions. These two types of scanning provide complimentary information. Scanning in liquid visualizes the surface filaments of the cytoskeleton, whereas scanning in air shows both the surface filaments and the total "volume" of the cytoskeletal fibers. The smallest fibers observed were ca. 50 nm in diameter. The lateral resolution of this technique was ca.20 nm, which can be increased to a single nanometer level by choosing sharper AFM tips. Because the AFM is a true 3D technique, we are able to quantify the observed cytoskeleton by its density and volume. The types of fibers can be identified by their size, similar to electron microscopy.
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Citoesqueleto/ultraestructura , Células Epiteliales/ultraestructura , Microscopía de Fuerza Atómica , Células Cultivadas , Humanos , Microscopía de Fuerza Atómica/métodos , Octoxinol , Manejo de EspecímenesRESUMEN
Infection with high-risk human papillomaviruses (HPV) is a major risk factor for development of cervical cancer. Expression of the HPV E6 and E7 oncoproteins increases in differentiating keratinocytes, resulting in inactivation of the p53 and retinoblastoma proteins, two important transcriptional regulators. We used cDNA microarrays to examine global alterations in gene expression in differentiating cervical keratinocytes after infection with retroviruses encoding HPV type 16 (HPV-16) E6 and E7. Expression of 80 cellular genes (approximately 4% of the genes on the array) was altered reproducibly by E6 and/or E7. Cluster analysis classified these genes into three functional groups: (i) interferon (IFN)-responsive genes, (ii) genes stimulated by NF-kappaB, and (iii) genes regulated in cell cycle progression and DNA synthesis. HPV-16 E6 or a dominant negative p53 protein downregulated multiple IFN-responsive genes. E6 decreased expression of IFN-alpha and -beta, downregulated nuclear STAT-1 protein, and decreased binding of STAT-1 to the IFN-stimulated response element. E7 alone was less effective; however, coexpression of E6 and E7 downregulated IFN-responsive genes more efficiently than E6. The HPV-16 E6 protein also stimulated expression of multiple genes known to be inducible by NF-kappaB and AP-1. E6 enhanced expression of functional components of the NF-kappaB signal pathway, including p50, NIK, and TRAF-interacting protein, and increased binding to NF-kappaB and AP-1 DNA consensus binding sites. Secretion of interleukin-8, RANTES, macrophage inflammatory protein 1alpha, and 10-kappaDa IFN-gamma-inducible protein were increased in differentiating keratinocytes by E6. Thus, high-level expression of the HPV-16 E6 protein in differentiating keratinocytes directly alters expression of genes that influence host resistance to infection and immune function.
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Regulación hacia Abajo , FN-kappa B/metabolismo , Proteínas Oncogénicas Virales/metabolismo , Papillomaviridae/metabolismo , Proteínas Represoras , Factores de Transcripción/metabolismo , Regulación hacia Arriba , Ciclo Celular , División Celular , Células Cultivadas , Cuello del Útero/citología , Citocinas/metabolismo , ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Expresión Génica , Humanos , Interferón Tipo I/farmacología , Interferón-alfa/genética , Interferón-alfa/farmacología , Interferón beta/genética , Interferón beta/farmacología , Interferón gamma/farmacología , Queratinocitos/citología , Queratinocitos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Oncogénicas Virales/genética , Oncogenes , Papillomaviridae/genética , Proteínas E7 de Papillomavirus , Proteínas Recombinantes , Elementos de Respuesta , Factor de Transcripción STAT1 , Transactivadores/genética , Transactivadores/metabolismo , Factor de Transcripción AP-1/metabolismo , Factores de Transcripción/genéticaRESUMEN
DNA damage has many cellular consequences including, in some cases, apoptosis. Expression of Gadd34 was shown to be increased by ionizing radiation only in cells that undergo rapid apoptosis following this treatment. The effects of various other apoptosis-inducing agents as well as apoptosis-inhibiting genes on regulation of Gadd34 were investigated. In many cell types, agents which have been reported to lead to increased intracellular ceramide levels led to an increase in Gadd34 transcript levels. These included TNFalpha, the ceramide analog C-2 ceramide, dimethyl sphingosine and anti-Fas antibody as well as ionizing radiation. Induction of Gadd34 by ionizing radiation was coincident with the onset of apoptosis and increased as apoptosis progressed. In a short-term transfection assay, more than 30% of Gadd34-transfected cells exhibited nuclear fragmentation by 48 hours. Apoptosis, as well as induction of Gadd34 by apoptotic stimuli, was attenuated by the apoptosis inhibitors, Bcl-2, cowpox virus CrmA and herpes simplex virus ICP34.5. Thus, activation of Gadd34 is a downstream event in apoptotic signaling pathways and may directly contribute to the apoptotic process.
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Apoptosis , Apoptosis/efectos de los fármacos , Proteínas/metabolismo , Proteínas/fisiología , Esfingosina/análogos & derivados , Regulación hacia Arriba , Antígenos de Diferenciación , Apoptosis/efectos de la radiación , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Proteínas de Ciclo Celular , División Celular/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Ceramidas/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , Fragmentación del ADN , Inhibidores Enzimáticos/farmacología , Colorantes Fluorescentes/farmacología , Humanos , Indoles/farmacología , Péptidos y Proteínas de Señalización Intracelular , Fenotipo , Plásmidos/metabolismo , Proteína Fosfatasa 1 , Proteínas Proto-Oncogénicas c-bcl-2/farmacología , Radiación Ionizante , Serpinas/farmacología , Transducción de Señal , Esfingosina/farmacología , Factores de Tiempo , Factor de Transcripción CHOP , Factores de Transcripción/metabolismo , Transfección , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/farmacología , Células U937 , Proteínas Virales/farmacología , Receptor fas/inmunología , Proteinas GADD45RESUMEN
The introduction of microarray technology has dramatically changed the way that researchers address many biomedical questions. DNA microarrays can measure expression of thousands of genes simultaneously, providing extensive information on gene interaction and function. Microarray technology is a powerful tool for identifying novel molecular drug targets and for elucidating mechanisms of drug action. Furthermore, microarrays can monitor the global profile of gene expression in response to specific pharmacologic agents, providing information on drug efficacy and toxicity. Over the last several years, dramatic advancements have occurred in array technology. In this review we describe basic aspects of microarray instrumentation and experimentation. Each of the major array formats including oligonucleotides arrays, spotted arrays, and macroarrays are examined, and advantages and options for using each format are presented. Important factors in the design and analysis of microarray experiments are also discussed. Most importantly, we explore recent developments in microarray technology that are relevant to pharmacogenomics and the discovery of gene function.
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Expresión Génica , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Farmacogenética , Animales , Perfilación de la Expresión Génica , Humanos , Estadística como AsuntoRESUMEN
The epidermal growth factor receptor (EGF-R) is frequently overexpressed in human papillomavirus (HPV)-associated dysplasias and carcinomas, implying that it is important for the progression of keratinocytes to malignancy. We used mice with a targeted disruption of the EGF-R gene to directly examine its role in cell immortalization and tumor development. Epidermal keratinocytes were cultured from EGF-R knockout, heterozygous, and wild-type mice, infected with retroviruses encoding HPV-16 E6 and E7 oncogenes, and grafted to nude mice. E6/E7 induced immortalization of EGF-R wild-type cells 5-fold more efficiently than null cells. Immortal EGF-R null cells grew more slowly, achieved a lower saturation density, and were more sensitive to apoptosis than the immortalized wild-type or heterozygous cells. Analyses using cDNA expression arrays showed that EGF-R null cells expressed increased levels of RNAs encoding p21waf and insulin-like growth factor-binding protein-2. EGF-R-positive immortal keratinocytes formed papillomas in 17% (15 of 90) of skin grafts, and seven grafts progressed to squamous carcinoma after 6-12 months. EGF-R null keratinocytes did not form papillomas, but 1 of 96 grafts progressed to a squamous carcinoma after 1 year. However, treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate induced tumors in 18 and 35% of grafts containing EGF-R null or EGF-R-positive cells, respectively. Transduction with an activated v-Ha-ras gene, which signals downstream of the EGF-R, induced rapidly growing carcinomas in all grafts regardless of EGF-R genotype. These results directly show that the EGF-R is important, but not essential, for immortalization by HPV and for progression of immortal cells to papillomas and carcinomas.
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Carcinoma de Células Escamosas/prevención & control , Receptores ErbB/fisiología , Queratinocitos/virología , Papiloma/prevención & control , Papillomaviridae/genética , Proteínas Represoras , Neoplasias Cutáneas/prevención & control , Animales , Carcinógenos , Carcinoma de Células Escamosas/inducido químicamente , Carcinoma de Células Escamosas/virología , Línea Celular Transformada , Transformación Celular Viral , Cocarcinogénesis , Progresión de la Enfermedad , Receptores ErbB/genética , Expresión Génica , Genes ras/genética , Genotipo , Humanos , Queratinocitos/patología , Queratinocitos/trasplante , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Mutación , Proteínas Oncogénicas Virales/genética , Papiloma/inducido químicamente , Papiloma/virología , Proteínas E7 de Papillomavirus , Neoplasias Cutáneas/virología , Acetato de TetradecanoilforbolRESUMEN
Infection with high-risk human papillomaviruses (HPVs) represents a major risk factor for the development of cervical cancer. The HPV-16 E6 and E7 proteins are highly expressed in differentiating keratinocytes, where they inactivate the p53 and retinoblastoma (pRb) proteins, two important transcriptional regulators. We have used cDNA expression arrays to identify global alterations in gene expression induced by E6 and E7 in differentiating cultures of human cervical keratinocytes. We show that E6 and E7 decrease expression of TGF-beta2 mRNA and alter expression of multiple TGF-beta-responsive genes involved in cell cycle regulation, apoptosis, and tissue remodeling. E6 and E7 inhibited expression of TGF-beta2 RNA 7-fold (relative effectiveness, E6/ E7 > E6 > E7 > control) and decreased secretion of biologically active TGF-beta2 by 70-80% (reduced from 70 to 10 pg/10(6) cells/24 h). Downregulation occurred through p53- and pRb-dependent pathways. In contrast, E6 and E7 did not alter expression of TGF-beta1 and TGF-beta3. Down-regulation of TGF-beta2 was biologically relevant because the addition of recombinant cytokine (10-200 pg/ml) to E6/E7-expressing cells restored expression of TGF-P-responsive genes, inhibited growth of keratinocytes, and decreased immortalization by E6 and E7. These results suggest that TGF-32- and TGF-3-responsive genes are important targets for the HPV-16 E6 and E7 oncoproteins in differentiating cervical keratinocytes.
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Cuello del Útero/virología , Queratinocitos/virología , Proteínas Oncogénicas Virales/fisiología , Papillomaviridae , Proteínas Represoras , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Northern Blotting , Diferenciación Celular/fisiología , División Celular/fisiología , Transformación Celular Viral , Células Cultivadas , Cuello del Útero/citología , Cuello del Útero/metabolismo , Regulación hacia Abajo/fisiología , Femenino , Silenciador del Gen , Genes de Retinoblastoma , Genes p53 , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Proteínas Oncogénicas Virales/genética , Proteínas Oncogénicas Virales/metabolismo , Papillomaviridae/genética , Proteínas E7 de Papillomavirus , Retroviridae/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Crecimiento Transformador beta/biosíntesis , Factor de Crecimiento Transformador beta/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Tumor necrosis factor-alpha (TNF-alpha) inhibits growth of normal cervical keratinocytes but stimulates proliferation of human papillomavirus (HPV)-immortalized and cervical carcinoma-derived cell lines when mitogens such as epidermal growth factor (EGF) or serum are depleted. Current work identifies the mechanism of growth stimulation. TNF-alpha promoted cell cycle progression by increasing expression of HPV-16 E6/E7 RNAs and enhancing activity of cyclin-dependent kinase (cdk)2 and cdc2 after 3 d. Increased kinase activity was mediated by upregulation of cyclins A and B and decreases in cdk inhibitors p21(waf) and p27(kip). TNF-alpha stimulated these changes in part by increasing transcription and stabilization of RNA for amphiregulin, an EGF receptor ligand, and amphiregulin directly increased HPV-16 E6/E7 and cyclin A RNAs. To define which components of the EGF receptor signaling pathway were important, HPV-immortalized cells were transfected with activated or dominant negative mutants of Ha-ras, raf, or MAPKK. Expression of activated Ha-ras maintained HPV-16 and cyclin gene expression and promoted rapid growth in the absence of EGF. Furthermore, ras activation was necessary for TNF-alpha mitogenesis as transfection with a dominant negative ras mutant (Asn-17) strongly inhibited growth. Thus, activation of ras promotes expression of HPV-16 E6/E7 RNAs, induces cyclins A and B, and mediates growth stimulation of immortal keratinocytes by TNF-alpha. These studies define a pathway by which ras mutations, which occur in a subset of cervical cancers, may contribute to pathogenesis. Mol. Carcinog. 27:97-109, 2000. Published by Wiley-Liss, Inc.
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Quinasas Ciclina-Dependientes/metabolismo , Queratinocitos/enzimología , Proteínas Oncogénicas Virales/genética , Papillomaviridae/genética , ARN Viral/biosíntesis , Proteínas Represoras , Transducción de Señal/genética , Factor de Necrosis Tumoral alfa/fisiología , Proteínas ras/fisiología , Ciclo Celular/genética , Línea Celular Transformada , Activación Enzimática/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Queratinocitos/virología , Proteínas Oncogénicas Virales/metabolismo , Papillomaviridae/fisiología , Proteínas E7 de Papillomavirus , Receptores del Factor de Necrosis Tumoral/biosíntesis , Receptores del Factor de Necrosis Tumoral/metabolismoRESUMEN
In the cellular response to genotoxic stress, cell cycle checkpoint and apoptosis are considered to be two of the major biological events in maintaining genomic stability. The tumor suppressor p53 has been shown to play critical roles in these stress-induced cellular responses at least in part through the activation of its down-stream genes, such as p21CIP1/WAF1, GADD45 and BAX. In addition, p53 has been found to down-regulate the expression of BCL-2, which is able to block apoptosis induced by both p53-dependent and independent signaling events. In this report, we have found that increased expression of Bcl-2 protein in the human Burkitt's lymphoma WMN cell line suppressed apoptosis induced by different DNA-damaging agents. The induction of p53-regulated genes including GADD45, p21CIP1/WAF1 and BAX by genotoxic stress was substantially reduced in cells expressing high levels of Bcl-2 protein. Furthermore, Bcl-2 protein was shown to specifically suppress the p53-mediated transactivation of p21CIP1/WAF1 and PG13-CAT, which is a typical p53-binding-site reporter construct. Similarly, the inhibitory effect of Bcl-2 protein was seen in a GADD45 promoter reporter construct after treatment with methylmethane sulfonate or UV-radiation. These results indicate that in addition to its apoptosis-suppressing activity, Bcl-2 protein is able to inhibit transactivation of p53-regulated genes, which function in multiple important cellular responses to genotoxic stress, including the control of cell cycle checkpoints, cell growth suppression and DNA repair.
Asunto(s)
Metilmetanosulfonato/farmacología , Mutágenos/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Activación Transcripcional , Proteína p53 Supresora de Tumor/metabolismo , Apoptosis , Daño del ADN , Regulación de la Expresión Génica , Humanos , Células Tumorales CultivadasRESUMEN
Our aim was to establish an effective non-surgical treatment for cervical intraepithelial neoplasia (CIN) through inactivation of human papillomavirus (HPV), the major etiological agent for this disease. We show that vidarabine, a DNA polymerase inhibitor, suppressed growth and HPV gene expression in human cervical keratinocytes immortalized by HPV or in cervical cancer cell lines. Expression of HPV-16 E6 and E7 proteins in normal cervical keratinocytes sensitized cells to apoptosis in the presence of podophyllin or vidarabine. We applied vidarabine ointment and/or podophyllin to cervical epithelium in 28 cases of CIN I-II to evaluate the therapeutic effectiveness of these agents. Co-application of vidarabine and podophyllin in six treatments caused regression of lesions cytologically and histologically, and disappearance of HPV-16 or -18 DNA in 17 of 21 (81%) women. Our results suggest that the combination of vidarabine and podophyllin therapy is an effective non-surgical treatment for HPV-positive CIN.