RESUMEN
In this review, we described human small DNA viruses discovered on the cusp of the 20th and 21st centuries as a result of cutting-edge technologies established in molecular biology. The problems of obtaining an evidence of the etiological role of new viruses in human diseases have been considered.
RESUMEN
Cervical cancers are characterized by the persistence of human papilloma virus (HPV) genome that is found in tissue samples starting from the early stages of tumor progression. Just like in other tumors, the activation of telomerase was observed in cervical carcinomas, but information about its expression was controversial. The aim of this study is to find possible correlations between the presence of HPV sequences, activity of telomerase and expression of different spliced forms of hTERT RNA in cervical intraepithelial neoplasias (CIN). The results show that HPV DNA is present in 60% of normal tissue adjacent to CIN lesions and up to 84% in CIN samples. Telomerase activity was found in 28% of adjacent normal tissue and in 68% of CIN II-III. hTERT RNA that encodes an active enzyme was present almost in all CIN samples. Variations in levels of telomerase activity are possibly not regulated by the splicing forms of hTERT mRNA with deletions.
Asunto(s)
Empalme del ARN , Telomerasa/genética , Displasia del Cuello del Útero/genética , Neoplasias del Cuello Uterino/genética , ADN Viral/genética , Femenino , Interacciones Huésped-Patógeno , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/fisiología , Humanos , Infecciones por Papillomavirus/virología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Telomerasa/metabolismo , Neoplasias del Cuello Uterino/enzimología , Neoplasias del Cuello Uterino/virología , Displasia del Cuello del Útero/enzimología , Displasia del Cuello del Útero/virologíaRESUMEN
Our aim was to investigate how replication protein A (RPA) in a wide range of concentration can regulate the activity of human telomerase. We used an in vitro system based on human cell extracts with or without RPA. It has been shown that removal of RPA leads to loss of telomerase activity and addition of RPA restores telomerase activity and at the same time promotes telomerase processivity. However, high excess of RPA inhibited telomerase processivity and caused the synthesis of relatively short DNA fragments (about 50-100 nucleotides). We assume that, together with other telomere-binding proteins, RPA may take part in activation of telomere overhang elongation by telomerase at a certain stage of a cell cycle as well as in regulation of telomere length.
Asunto(s)
Proteína de Replicación A/metabolismo , Telomerasa/metabolismo , Ciclo Celular , Células HeLa , Humanos , Telomerasa/genética , Telómero/metabolismoRESUMEN
Expression of the STAT1 gene belonging to the group of interferon-regulated genes was analyzed in cervical tumors and cell lines harboring the genome of human papilloma viruses (HPV) of so-called high risk group. Expression of this gene in invasive carcinomas was maintained on a definite level that was not significantly distinct from that in adjacent normal (control) tissue. Tumors from different patients differ from each other by expression level of the STAT1 gene. These variations can be attributed to the heterogeneity of tumor cell population and different ratio between normal and tumor cells, as well as to putative persistence of intra-individual variability of STAT1 expression in normal cell population. It was demonstrated that viral genome status (episomal or integrative) did not influence STAT1 gene transcription. In conclusion, these data demonstrate that the STAT1 gene is expressed in an individual and specific manner both in HPV-positive cervical tumors and cell lines harboring transforming genes of these viruses.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Carcinoma de Células Escamosas/metabolismo , Factor de Transcripción STAT1/metabolismo , Neoplasias del Cuello Uterino/metabolismo , Carcinoma de Células Escamosas/virología , Línea Celular Tumoral , Femenino , Genoma Viral , Papillomavirus Humano 16/genética , Humanos , Neoplasias del Cuello Uterino/virologíaRESUMEN
We explored the expression of four genes encoding for subunits of AP-3 in cervical tumors and cancer cell lines. Using RT-PCR we demonstrated more than twofold decrease in the levels of mRNA of AP3D1, AP3B1, AP3M1, and AP3S1 in 32, 28, 23, and 26% tumors in comparison with normal tissues of uterine cervix, respectively. The level of mRNA of at least one subunit was decreased in 28 out of 47 (60%) of tumors and in four out of five cancer cell lines in comparison to tissues adjacent to tumors. The suppression of expression of any of the subunits was revealed in 15 out of 28 cases (54%). The expression of two and more subunits was decreased simultaneously in different combinations in 13 cases (46%). This fact testifies to the lack of a common mechanism of downregulation of four subunits in tumors. There is a tendency to more frequent suppression of AP-3A expression in tumors associated with lymphatic node metastases as compared with tumors without metastases (P = 0.034). Thus, here we demonstrate for the first time the decrease in expression of genes encoding for AP-3A subunits in tumors.
Asunto(s)
Complejo 3 de Proteína Adaptadora/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias del Cuello Uterino/genética , Línea Celular Tumoral , Regulación hacia Abajo/genética , Femenino , Células HeLa , Humanos , Subunidades de Proteína/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias del Cuello Uterino/patologíaAsunto(s)
Biomarcadores de Tumor/metabolismo , Carcinoma de Células Escamosas/enzimología , Telomerasa/metabolismo , Neoplasias del Cuello Uterino/enzimología , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/virología , Femenino , Células HeLa , Papillomavirus Humano 16 , Humanos , Telómero/metabolismo , Neoplasias del Cuello Uterino/diagnóstico , Neoplasias del Cuello Uterino/virologíaRESUMEN
DNA methylation plays an important role in the establishment and maintenance of the program of gene expression. Tumor cells are characterized by a paradoxical alteration of DNA methylation pattern: global DNA demethylation and local hypermethylation of certain genes. Hypermethylation and inactivation of tumor suppressor genes are well documented in tumors. The role of global genome demethylation in carcinogenesis is less studied. New data provide evidence for independence of DNA hypo- and hypermethylation processes in tumor cells. These processes alter expression of genes that have different functions in malignant transformation. Recent studies have demonstrated that global decrease in the level of DNA methylation is related to hypomethylation of repeated sequences, increase in genetic instability, hypomethylation and activation of certain genes that favor tumor growth, and increase in their metastatic and invasive potential. The recent data on the role of DNA demethylation in carcinogenesis are discussed in this review. The understanding of relationships between hypo- and hypermethylation in tumor cells is extremely important due to reversibility of DNA methylation and attempts to utilize for anti-tumor therapy the drugs that modify DNA methylation pattern.
Asunto(s)
Metilación de ADN , ADN de Neoplasias/genética , ADN de Neoplasias/fisiología , Regulación Neoplásica de la Expresión Génica , Neoplasias/genética , Animales , Humanos , Neoplasias/metabolismo , Análisis de Secuencia de ADNRESUMEN
METHODS: The activities of cathepsin L and its endogenous inhibitors were analyzed in rat embryo fibroblasts, immortalized and transformed by different genes. RESULTS: Regardless of the transfecting agent used (DNA of adenovirus SA7 or polyomavirus LT gene), the immortal cells showed an increase in the cathepsin L activity (in both lysates and conditioned media) compared to primary fibroblasts. Transformed cells exhibited either an increase (with c-Ha-ras gene) or decrease (with E7 HPV gene) in cathepsin L activity in lysates as opposed to immortal cells. CONCLUSIONS: The data are suggestive of alterations in the trafficking of cathepsin L upon fibroblast transfection with polyomavirus LT gene and E7 HPV gene. An endogenous inhibitor(s) of cysteine proteinase was found in conditioned media, but not in lysates, of all cell cultures studied and its activity in normal fibroblasts was higher than in media of immortal and transformed cells.
Asunto(s)
Catepsinas/antagonistas & inhibidores , Catepsinas/biosíntesis , ADN/genética , Fibroblastos/metabolismo , Proteínas E1A de Adenovirus/genética , Adenovirus de los Simios/genética , Animales , Catepsina L , Catepsinas/genética , Línea Celular Transformada/citología , Línea Celular Transformada/metabolismo , Cisteína Endopeptidasas , Inhibidores de Cisteína Proteinasa/metabolismo , Embrión de Mamíferos/citología , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Fibroblastos/citología , Genes ras/genética , Ratas , TransfecciónRESUMEN
The latest experimental data on the role of viruses in the origin of human tumors are discussed. This group of viruses consists of T-cell leukemia virus type 1 (HTLV 1), herpes viruses (HHV 8 and Epstein-Barr virus), hepatitis B virus, and human papilloma viruses. The most typical feature of this group of viruses is a very long latent period from the initial infection to the development of the disease that varies between 10 and 40 years. The mechanism of malignant cell conversion is specific for each viral type but is mainly associated with a disruption of functions of cellular genes participating in the control of cell division and proliferation. It can be a direct inactivation of tumor suppressor genes by their interaction with viral gene products (papilloma viruses), or a trans-activation of cellular genes modulating cell proliferation by viral gene products (hepatitis B virus and HTLV 1). Viruses play an initiative role and additional genetic changes in the genome of infected cells are necessary for complete expression of the oncogenic potential of the viral genes. Only these cells will give rise to a monoclonal cell population with uncontrolled proliferation. New approaches for the creation of vaccines against cancers associated with hepatitis B virus and papilloma viruses (hepatocellular carcinomas and cervical tumors, respectively) are in progress. These vaccines have been found to be effective in prevention of the disease in the experimental models and are now beginning to be used for human vaccination.
Asunto(s)
Carcinoma/virología , Papillomaviridae , Neoplasias del Cuello Uterino/virología , Vacunas contra el Cáncer/uso terapéutico , Transformación Celular Neoplásica , Femenino , Regulación Viral de la Expresión Génica , Humanos , Papillomaviridae/genética , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/prevención & controlRESUMEN
We have explored a possible role of an activated N-ras oncogene in aberrant methylation of CpG clusters in DNAs of transformed cells. Using three lines of hamster cells transformed by Rous sarcoma virus (RSV) and the method of detection of CpG islands as clustered sites for methylation-sensitive restriction enzymes we have demonstrated that in each cell line the transcribed RSV proviruses are integrated in the vicinity of sequence containing the cluster of unmethylated CpG dinucleotides. Two out of three examined CpG clusters had hypermethylation patterns in N-ras-neo- but not in neo-transfected variants of the cell lines. De novo methylation of CpG dinucleotides correlated with transcriptional inactivation of adjacent RSV proviruses that was related neither to the lack of transcriptional factors binding RSV long terminal repeat (LTR) nor to the transcriptional incompetence of the LTR, as measured by reporter gene assays with the LTR cloned from DNA of these cells. These data suggest that activation of N-ras signal transduction pathway in transformed cells may be relevant to long-term inactivation of selective genes by hypermethylation of their CpG islands.
Asunto(s)
Transformación Celular Viral/genética , Islas de CpG/genética , Metilación de ADN , Genes ras/fisiología , Transcripción Genética , Animales , Virus del Sarcoma Aviar/fisiología , Línea Celular Transformada , Embrión de Pollo , Cricetinae , ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN Viral/química , ADN Viral/genética , Fibroblastos/virología , Vectores Genéticos , Provirus/genéticaRESUMEN
The presence of human papillomavirus (HPV) sequences in 21 biopsies from cervical carcinomas, 11 specimens of tissues adjacent to tumours, 2 specimens of cervical tissues with radiation fibrosis from patients after radiation therapy of cervical cancer and 7 normal epithelial tissues from the patients with other genital tumours were examined by polymerase chain reaction (PCR) and Southern-blot analysis. All tumours were HPV-positive by type-specific PCR and 86% by Southern-blot analysis. In normal epithelial and adjacent tissues, HPV sequences were detected in 20% of samples by Southern-blot analysis and in 70% of samples by PCR, including 2 cases of tissues after radiation therapy. HPV16 was the most prevalent type in tumours (18/21) as well as in normal epithelial tissues (5/7). One HPV-positive tumour contained HPV18 DNA and 2 were doubly infected with HPVs 16 and 18 (2/21). The persistence of exclusively episomal HPV16 DNA was observed in 5 out of 11 tumours examined: 3 cases of squamous-cell carcinomas on the early stage of tumour progression and 2 advanced tumours (squamous-cell carcinoma and adenocarcinoma). The integration of HPV16 genome was detected in 6 out of 11 tumours, but most of them contained episomal forms of viral DNA simultaneously (5 out of 6). The integrative HPV18 genome was found in 2 tumours examined, and the persistence of episomal forms was also observed in one of them. Our data demonstrate that cervical tumours are associated invariably with high-risk types of HPV in Russia.
Asunto(s)
Adenocarcinoma/virología , Carcinoma de Células Escamosas/virología , Cuello del Útero/virología , Papillomaviridae/aislamiento & purificación , Infecciones por Papillomavirus/epidemiología , Reacción en Cadena de la Polimerasa , Infecciones Tumorales por Virus/epidemiología , Neoplasias del Cuello Uterino/virología , Adenocarcinoma/patología , Secuencia de Bases , Southern Blotting , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/terapia , Línea Celular , Cartilla de ADN , ADN Viral/análisis , Epitelio/virología , Femenino , Humanos , Datos de Secuencia Molecular , Estadificación de Neoplasias , Infecciones por Papillomavirus/patología , Infecciones por Papillomavirus/virología , Prevalencia , Valores de Referencia , Federación de Rusia/epidemiología , Infecciones Tumorales por Virus/patología , Infecciones Tumorales por Virus/virología , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/terapiaRESUMEN
Aspartyl and cysteine proteinases at distinct stages of carcinogenesis were analyzed in rat embryo fibroblasts, sequentially immortalized and transformed by 2 different genes: the early region of simian adenovirus SA7 and c-Ha-ras oncogene. The dynamics of expression and distribution of proteinases throughout the transformation process were examined. It was shown that in immortalized and transformed cells the activities of the aspartyl and cysteine proteinases were expressed to a variable degree and that the expression was dependent on cell-propagation time in vitro. The increase in activity both of cathepsin-D-like aspartyl proteinase and of cathepsin-L- and -B-like cysteine proteinases in cell lysates was correlated with the stages of fibroblast transformation (immortalization and tumorigenic transformation). In all cell types the major part of cysteine proteinases was localized inside the cell, while the cathepsin-D-like proteinase was apparently predominant among secreted proteinases. The cathepsin-L-like proteinase accounts for the major part of the cysteine-proteinase activity as measured by Z-Phe-Arg-MCA hydrolysis. We suggest that considerable portions of the cathepsin-D- and -L-like proteinases in all cell lines studied are secreted as a complex with inhibitor(s) and that inhibitor expression plays an important role in regulating the activity of cathepsin-D-like proteinase at different stages of transformation. Cathepsin-L-like proteinase is probably secreted in the precursor form.
Asunto(s)
Ácido Aspártico Endopeptidasas/metabolismo , Transformación Celular Neoplásica/metabolismo , Cisteína Endopeptidasas/metabolismo , Fibroblastos/enzimología , Secuencia de Aminoácidos , Animales , Catepsina L , Catepsinas/biosíntesis , Células Cultivadas/enzimología , Embrión de Mamíferos , Activación Enzimática , Precursores Enzimáticos/biosíntesis , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Genes Inmediatos-Precoces , Genes ras , Hemólisis , Datos de Secuencia Molecular , RatasRESUMEN
The percentages of cells with different types of cell surface relief were determined in cell suspensions derived from monolayer cultures. Primary cultures of rat embryo fibroblasts (REF) and cell lines REF (LT) and REF-1, immortalized cells of which preserved normal phenotypic characteristics of the initial primary culture REF, as well as morphologically transformed tumorigenic lines REF (LT) ras and REF-2EJ were studied. In REF suspensions the cells with the blebbed type of surface relief were shown to be predominant as compared with those with microvillus relief whereas cell suspensions derived from both immortalized and fully transformed cultures display the reverse ratio of cells with those types of surface relief. Therefore, the pattern of cell surface relief in cell suspensions derived from fibroblastic monolayer cultures may serve as a morphological marker of the initial stage of neoplastic transformation-immortalization when typical morphological signs of cell transformation are not yet manifested in monolayer cultures.
Asunto(s)
Transformación Celular Neoplásica/patología , Fibroblastos/patología , Adenoviridae/genética , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/fisiología , Animales , Antígenos Transformadores de Poliomavirus/genética , Antígenos Transformadores de Poliomavirus/fisiología , Muerte Celular , Transformación Celular Viral , Ratones , Ratones Desnudos , Microscopía Electrónica de Rastreo , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/fisiología , Poliomavirus/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/fisiología , Ratas , Ratas Endogámicas F344/embriología , Transfección , Células Tumorales Cultivadas/ultraestructuraRESUMEN
Rous sarcoma virus-33 (RSV-33) was obtained from a sample of chicken Rous sarcoma which had been dried and stored in 1933. RSV-33, like the RSV-29, has the minimal number of passages beyond its isolation from chicken tumour No. 1. Our experiments demonstrated that the Rous sarcoma virus-33 was replication non-defective and was pathogenic for rats. Established rat tumorigenic cell lines express the viral genome. All three species of viral RNA were detected and v-src proteins and gag polyproteins were identified as well in cells of R9 and R74 lines. The virus can be rescued from cells of R9 and R74 lines, thus indicating that the cells are virogenic. The cells of a permanent tumorigenic line RT1 are infected but not transformed by RSV-33. Although they contain a complete proviral genome, they do not express detectable virus-specific RNA. The virus is not rescuable from RT1 cells under in vivo conditions. Proviral DNA analysis showed that the RSV-33 contained a full-length genome, including the env gene, in contrast to the RSV-29 which was found replication defective.
Asunto(s)
Virus del Sarcoma Aviar/fisiología , Animales , Virus del Sarcoma Aviar/crecimiento & desarrollo , Virus del Sarcoma Aviar/patogenicidad , Línea Celular , Pollos , Cromosomas , Genoma Viral , Provirus/genética , Ratas , Ratas Endogámicas Lew , Sarcoma Experimental/química , Sarcoma Experimental/microbiología , Sarcoma Experimental/patología , Células Tumorales Cultivadas , Proteínas Virales/metabolismoRESUMEN
c-src protein was found in 60% of lung carcinomas (20 of 33 cases or primary tumours) by immunoblotting with a monoclonal antibody (Mab 327) and immunohistochemistry with serum from rabbits bearing tumours induced by Rous sarcoma virus. src protein expression was assessed in 4 small cell lung carcinomas and in an atypical carcinoid of neuroendocrine origin. However, pp60c-src was also found in non-small cell lung carcinomas: in 60-80% of adenocarcinomas and bronchiolo-alveolar cancers and in 50% of squamous cell carcinomas. In the squamous cell carcinomas, src protein was expressed more frequently in poorly differentiated than in well and moderately differentiated carcinomas. Expression of pp60c-src was not found in epithelial cells of histologically unchanged lung tissues. These results show that pp60c-src may be activated in human lung carcinomas of different histopathological types.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/química , Carcinoma de Células Pequeñas/química , Neoplasias Pulmonares/química , Proteínas Proto-Oncogénicas pp60(c-src)/análisis , Adenocarcinoma/química , Carcinoma de Células Escamosas/química , Humanos , Immunoblotting , Técnicas para InmunoenzimasRESUMEN
Plasmids containing the E6 and E7 open reading frames of human papillomavirus type 18 transformed rat-embryo fibroblasts when expressed under the cytomegalovirus promoter. The fibroblasts had been previously immortalized with the large T-antigen gene of the polyomavirus to produce rat embryo fibroblast (large T-antigen) [REF(LT)] cells. REF(LT) cells were transformed by the E6 and E7 sequences to anchorage independence and tumourigenicity, but there were no significant morphological alterations. Transformation by these sequences of REF(LT) cells differed from that achieved by pEJras, in which case significant morphological changes and tumourigenicity in nude mice did occur.
Asunto(s)
Transformación Celular Neoplásica/genética , Fibroblastos/patología , Genes Virales , Papillomaviridae/genética , Animales , Antígenos Virales de Tumores/genética , Línea Celular Transformada , Embrión de Mamíferos , Humanos , Ratones , Ratones Desnudos , RatasRESUMEN
The expression of 9 oncogenes in primary tumors and in human tumors passaged in nude mice was tested: a total of 28 tumor types was analyzed. Oncogenes src, fps, and mos were not expressed in any of the tumors tested but oncogene myc was transcribed in most of the tumors and myc was over-expressed in 3 tumors passaged in nude mice (Ewing sarcoma, large intestine carcinoma and kidney carcinoma) and in primary fibrous histiocytoma. Enhanced transcription of ras and fos genes was observed nonspecifically in different tumors. Oncogene sis was activated specifically in metastases of different tumors in lymph nodes.
Asunto(s)
Neoplasias/genética , Oncogenes , ARN Neoplásico/análisis , Animales , Electroforesis , Femenino , Humanos , Masculino , Ratones , Ratones Desnudos , Metástasis de la Neoplasia , Neoplasias/patología , Hibridación de Ácido NucleicoRESUMEN
The injection of purified Rous sarcoma virus (RSV) (Prague strain) into Drosophila melanogaster (Oregon R line) eggs changes the fly phenotype in certain cases, and RSV-specific sequences can be identified in the Drosophila genome (ref. 1 and preceding paper). Here we have used Southern blotting to analyse in greater detail the proviral DNA present in several mutant lines of D. melanogaster produced by microinjection of intact RSV or plasmid DNA containing the viral insert. In certain populations of flies, RSV provirus was found to be incorporated into cellular DNA, and in one mutant family the unintegrated form of plasmid DNA was identified. Generally, the presence of injected genetic material in fly cells correlated with morphological changes in Drosophila.