RESUMO
INTRODUCCIÓN: El virus del papiloma humano de alto riesgo (VPH-AR) es responsable del cáncer de cuello uterino y sus lesiones preneoplásicas. Los genotipos VPH16 y VPH18 son los más frecuentes en este cáncer. La integración del VPH-AR en el genoma de la célula hospedera es crucial en la carcinogénesis cervical, pero la etapa en que ocurre en la población chilena es incierta. OBJETIVO: Evaluar la integración de VPH16 y VPH18 en lesiones pre-neoplásicas de cuello uterino. MÉTODOS: Se analizaron 108 muestras de raspados cervicales. El VPH se genotipificó mediante reacción de polimerasa en cadena (RPC) e hibridación no radiactiva. La integración de VPH16 y VPH18 se determinó por presencia del gen E2 mediante RPC. RESULTADOS: VPH16 y VPH18 se detectaron en 36,1% y 12,0% de las muestras, respectivamente. El VPH16 se integró en 23,1% de los casos de VPH16, mientras que VPH18 se integró en 100% de las muestras positivas para este genotipo. CONCLUSIONES: La integración VPH-AR es un evento temprano en la carcinogénesis cervical que ocurre en casi la mitad de las lesiones pre-neoplásicas y es más frecuente en VPH18 que en VPH16. La evaluación de la integración VPH-AR puede ser una herramienta útil para detectar el virus en la población chilena.
BACKGROUND: High-risk Human Papillomaviruses (HR-HPVs) are the etiological agents of cervical cancer and its preneoplastic lesions. HPV16 and 18 are the most frequent HR-HPV genotypes detected in cervical cancer. HR-HPV genome integration into the host cell is an important event in the carcinogenic process. However, it remains uncertain which stage of cervical carcinogenesis HPV16 and 18 integration occurs in the Chilean population. AIM: The goal of this study was to evaluate HPV16 and HPV18 integration in preneoplastic lesions of the cervix. METHODS: DNA was extracted from 108 cervical scrape samples with preneoplastic lesions. HPV was genotyped using PCR and non-radioactive hybridization. The integration status of HPV16 and HPV 18 was determined by evaluating the E2 gene presence through PCR. RESULTS: HPV16 and HPV18 tested positive in 36.1% and 12.0% of samples, respectively. HPV16 was found integrated in 23.1% of HPV 16 cases, while HPV 18 in 100% of samples positive for this viral genotype. CONCLUSIONS: HR-HPV integration is an early event in cervical carcinogenesis, occurring in nearly half of preneoplastic lesions and being more frequent in HPV18 than in HPV16. The evaluation of HR-HPV integration can be utilized as a complementary tool for detecting HPV in the Chilean population.
Assuntos
Humanos , Feminino , Adolescente , Adulto , Pessoa de Meia-Idade , Adulto Jovem , Lesões Pré-Cancerosas/virologia , Colo do Útero/virologia , Integração Viral/genética , Papillomaviridae/isolamento & purificação , Papillomaviridae/genética , Lesões Pré-Cancerosas/genética , DNA Viral/genética , Colo do Útero/patologia , Chile , Reação em Cadeia da Polimerase , Estudos Transversais , Papillomavirus Humano 16/isolamento & purificação , Papillomavirus Humano 16/genética , Papillomavirus Humano 18/isolamento & purificação , Papillomavirus Humano 18/genética , Técnicas de Genotipagem , GenótipoRESUMO
Human papillomavirus (HPV) 16 displays substantial sequence variation; four HPV16 lineages (A, B, C, and D) have been described as well as multiple sublineages. To identify molecular events associated with HPV16 carcinogenesis, we evaluated viral variation, the integration of HPV16, and somatic mutation in 96 cervical cancer samples from Guatemala. A total of 65% (62/96) of the samples had integrated HPV16 sequences and integration was associated with an earlier age of diagnosis and premenopausal disease. HPV16 integration sites were broadly distributed in the genome, but in one tumor, HPV16 integrated into the promoter of the IFN regulatory factor 4 (IRF4) gene, which plays an important role in the regulation of the IFN response to viral infection. The HPV16 D2 and D3 sublineages were found in 23% and 30% of the tumors, respectively, and were significantly associated with adenocarcinoma. D2-positive tumors had a higher rate of integration, earlier age of diagnosis, and a lower rate of somatic mutation, whereas D3-positive tumors were less likely to integrate, had later age of diagnosis, and exhibited a higher rate of somatic mutation. In conclusion, Guatemalan cervical tumors have a high frequency of very high-risk HPV16 D2 and D3 sublineages harboring distinct histology, which may help guide future therapeutic strategies to target the tumor and reduce recurrence. SIGNIFICANCE: This study details the biological and molecular properties of the most pathogenic forms of HPV16, the cause of the majority of cervical cancers.
Assuntos
Adenocarcinoma/genética , Papillomavirus Humano 16/genética , Fatores Reguladores de Interferon/genética , Infecções por Papillomavirus/genética , Neoplasias do Colo do Útero/genética , Integração Viral/genética , Adenocarcinoma/virologia , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Classe I de Fosfatidilinositol 3-Quinases/genética , DNA Viral/análise , DNA Viral/genética , Feminino , Genoma Viral , Guatemala , Papillomavirus Humano 16/classificação , Humanos , Pessoa de Meia-Idade , Mutação , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/virologia , Lesões Pré-Cancerosas/complicações , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/virologia , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologiaRESUMO
HPV73 is classified as possibly oncogenic. It is neither routinely evaluated in HPV screening, nor covered by any of the prophylactic vaccines. We sought to investigate the carcinogenic characteristics of HPV73. Molecular studies were performed on eight cervix cancer biopsy specimens containing HPV73 from a cross-sectional cancer cohort of 590 women referred to the National Cancer Institute in Rio de Janeiro, Brazil. Transcriptional activity of HPV73 was evaluated by detection of spliced transcripts of E6/E6* and E1^E4 in cDNA created from RNA isolated from fresh tissue. Disruption of viral E1 and E2 genes in the tumor DNA was assessed by overlapping PCR amplification. Evaluation of viral integration was performed using a customized capture panel and next-generation sequencing, and an in-house bioinformatic pipeline. HPV73 E6/E6* transcripts were found in 7/7 specimens with available RNA, and three also had HPV73 E1^E4 transcripts. Disruption of E1 and E2 genes was observed in 4/8 specimens. Integration of HPV73 sequences into the cancer cell genomes was identified in all cervix cancer tissues. These results provide evidence that HPV73 is an oncogenic virus that can cause invasive cervix cancer. With current molecular screening and HPV vaccination, not all cervix cancers will be prevented.
Assuntos
Infecções por Papillomavirus/complicações , Neoplasias do Colo do Útero/etiologia , Neoplasias do Colo do Útero/virologia , Adulto , Idoso , Brasil , Estudos Transversais , DNA de Neoplasias/genética , Feminino , Humanos , Pessoa de Meia-Idade , Proteínas Oncogênicas Virais/genética , Papillomaviridae/genética , Reação em Cadeia da Polimerase/métodos , RNA Viral/genética , Integração Viral/genéticaRESUMO
Cervical cancer is one of the main causes of female cancer death worldwide, and human papilloma virus (HPV) its causal agent. To investigate viral oncogenesis several studies have focused on the effects of HPV oncoproteins E6 and E7 and the mechanisms by which these proteins stimulate the cellular transformation process. However, phenomena such as the physical state of the viral genome (episomal or integrated) and the effects of this integration on cell proliferation contribute new clues to understand how HPV infection causes carcinogenesis. New molecular technologies are currently facilitating these discoveries. This paper reviews the tumor development process initiated by HPV, recent findings on the process of viral integration into the host genome, new methods to detect HPV integration, and derived associated effects.
Assuntos
Interações Hospedeiro-Patógeno/genética , Papillomaviridae/genética , Infecções por Papillomavirus/virologia , Neoplasias do Colo do Útero/virologia , Integração Viral/genética , Progressão da Doença , Feminino , Humanos , Proteínas Oncogênicas Virais/genética , Papillomaviridae/patogenicidade , Infecções por Papillomavirus/patologia , Neoplasias do Colo do Útero/patologiaRESUMO
Although human papillomavirus was identified as an aetiological factor in cervical cancer, the key human gene drivers of this disease remain unknown. Here we apply an unbiased approach integrating gene expression and chromosomal aberration data. In an independent group of patients, we reconstruct and validate a gene regulatory meta-network, and identify cell cycle and antiviral genes that constitute two major subnetworks upregulated in tumour samples. These genes are located within the same regions as chromosomal amplifications, most frequently on 3q. We propose a model in which selected chromosomal gains drive activation of antiviral genes contributing to episomal virus elimination, which synergizes with cell cycle dysregulation. These findings may help to explain the paradox of episomal human papillomavirus decline in women with invasive cancer who were previously unable to clear the virus.
Assuntos
Antivirais/metabolismo , Ciclo Celular/genética , Redes Reguladoras de Genes/genética , Genes Neoplásicos/genética , Papillomaviridae/genética , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/virologia , Aberrações Cromossômicas , Cromossomos Humanos/genética , Bases de Dados Genéticas , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genoma Humano/genética , Instabilidade Genômica , Humanos , Proteínas de Membrana Lisossomal/metabolismo , Metanálise como Assunto , Proteínas de Neoplasias/metabolismo , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/virologia , Reprodutibilidade dos Testes , Neoplasias do Colo do Útero/patologia , Integração Viral/genéticaRESUMO
B-cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1) is a Polycomb group protein that is able to induce telomerase activity, enabling the immortalization of epithelial cells. Immortalized cells are more susceptible to double-strand breaks (DSB), which are subsequently repaired by homologous recombination (HR). BRCA1 is among the HR regulatory genes involved in the response to DNA damage associated with the RAD51 protein, which accumulates in DNA damage foci after signaling H2AX, another important marker of DNA damage. Topoisomerase IIIß (topoIIIß) removes HR intermediates before chromosomal segregation, preventing damage to cellular DNA structure. In breast carcinomas positive for BMI-1 the role of proteins involved in HR remains to be investigated. The aim of this study was to evaluate the association between BMI-1 and homologous recombination proteins. Using tissue microarrays containing 239 cases of primary breast tumors, the expression of Bmi-1, BRCA-1, H2AX, Rad51, p53, Ki-67, topoIIIß, estrogen receptors (ER), progesterone receptors (PR), and HER-2 was analyzed by immunohistochemistry. We observed high Bmi-1 expression in 66 cases (27.6%). Immunohistochemical overexpression of BMI-1 was related to ER (p=0.004), PR (p<0.001), Ki-67 (p<0.001), p53 (p=0.003), BRCA-1 (p= 0.003), H2AX (p=0.024) and topoIIIß (p<0,001). Our results show a relationship between the expression of BMI-1 and HR regulatory genes, suggesting that Bmi-1 overexpression might be an important event in HR regulation. However, further studies are necessary to understand the mechanisms in which Bmi-1 could regulate HR pathways in invasive ductal breast carcinomas.
Assuntos
Neoplasias da Mama/genética , Carcinoma Ductal de Mama/genética , Complexo Repressor Polycomb 1/genética , Proteínas Proto-Oncogênicas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Linfócitos B/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patologia , Progressão da Doença , Feminino , Humanos , Imuno-Histoquímica , Camundongos , Pessoa de Meia-Idade , Vírus da Leucemia Murina de Moloney/patogenicidade , Complexo Repressor Polycomb 1/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Reparo de DNA por Recombinação/genética , Integração Viral/genéticaRESUMO
OBJETIVO: Caracterizar el ambiente genómico de las secuencias adyacentes al virus linfotrópico humano de células T tipo 1 (HTLV-1) en pacientes con paraparesia espßstica tropical y mielopatía asociada a la infección con HTLV-1 (PET/MAH) de diferentes regiones de Colombia y del Japón. MÉTODOS: Se enfrentaron 71 clones recombinantes con secuencias del genoma humano adyacentes al 5'-LTR de pacientes con PET/MAH, a las bases de datos del Genome Browser y del Gen-Bank. Se identificaron y analizaron estadísticamente 16 variables genómicas estructurales y composicionales mediante el programa informßtico R, versión 2.8.1, en una ventana de 0,5 Mb. RESULTADOS: El 43,0 por ciento de los provirus se localizaron en los cromosomas del grupo C; 74 por ciento de las secuencias se ubicaron en regiones teloméricas y subteloméricas (P < 0,05). Un anßlisis de conglomerados permitió establecer las relaciones jerßrquicas entre las características genómicas incluidas en el estudio; el anßlisis de componentes principales identificó las componentes que definieron los ambientes genómicos preferidos para la integración proviral en casos de PET/MAH. CONCLUSIONES: El HTLV-1 se integró con mayor frecuencia en regiones de la cromatina ricas en islas de citocina fosfato guanina (CpG), de alta densidad de genes y de repeticiones tipo LINE (elemento disperso largo [long interspersed element]) y transposones de ADN que, en conjunto, conformarían los ambientes genómicos blanco de integración. Este nuevo escenario promoverß cambios sustanciales en el campo de la salud pública y en el manejo epidemiológico de las enfermedades infecciosas, y permitirß desarrollar potentes herramientas para incrementar la eficiencia de la vigilancia epidemiológica.
OBJECTIVE: Characterize the genomic environment of the sequences adjacent to human T-cell lymphotropic virus type 1 (HTLV-1) in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in different regions of Colombia and Japan. METHODS: A total of 71 recombinant clones with human genome sequences adjacent to 5' LTR in patients with HAM/TSP were compared to the Genome Browser and GenBank databases. Sixteen structural and compositional genome variables were identified, and statistical analysis was conducted in the R computer program, version 2.8.1, in a 0.5 Mb window. RESULTS: A total of 43.0 percent of the proviruses were located in the group C chromosomes; 74 percent of the sequences were located in the telomeric and subtelomeric regions (P < 0.05). A cluster analysis was used to establish the hierarchical relations between the genome characteristics included in the study. The analysis of principal components identified the components that defined the preferred genome environments for proviral integration in cases of HAM/TSP. CONCLUSIONS: HTLV-1 was integrated more often in chromatin regions rich in CpG islands with a high density of genes and LINE type repetitions, and DNA transposons which, overall, would form the genomic environments targeted for integration. This new scenario will promote substantial changes in the field of public health and in epidemiological management of infectious diseases. It will also foster the development of powerful tools for increasing the efficiency of epidemiological surveillance.
Assuntos
Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Idoso , Genoma Humano , Vírus Linfotrópico T Tipo 1 Humano/genética , Paraparesia Espástica Tropical/genética , Provírus/genética , Sequências Repetidas Terminais/genética , Integração Viral/genética , Mapeamento Cromossômico , Cromossomos Humanos/genética , Colômbia/epidemiologia , Ilhas de CpG , DNA Recombinante/genética , Paraparesia Espástica Tropical/epidemiologia , Paraparesia Espástica Tropical/virologia , Retroelementos/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência do Ácido NucleicoRESUMO
OBJECTIVE: Characterize the genomic environment of the sequences adjacent to human T-cell lymphotropic virus type 1 (HTLV-1) in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in different regions of Colombia and Japan. METHODS: A total of 71 recombinant clones with human genome sequences adjacent to 5' LTR in patients with HAM/TSP were compared to the Genome Browser and GenBank databases. Sixteen structural and compositional genome variables were identified, and statistical analysis was conducted in the R computer program, version 2.8.1, in a 0.5 Mb window. RESULTS: A total of 43.0% of the proviruses were located in the group C chromosomes; 74% of the sequences were located in the telomeric and subtelomeric regions (P < 0.05). A cluster analysis was used to establish the hierarchical relations between the genome characteristics included in the study. The analysis of principal components identified the components that defined the preferred genome environments for proviral integration in cases of HAM/TSP. CONCLUSIONS: HTLV-1 was integrated more often in chromatin regions rich in CpG islands with a high density of genes and LINE type repetitions, and DNA transposons which, overall, would form the genomic environments targeted for integration. This new scenario will promote substantial changes in the field of public health and in epidemiological management of infectious diseases. It will also foster the development of powerful tools for increasing the efficiency of epidemiological surveillance.
Assuntos
Genoma Humano , Vírus Linfotrópico T Tipo 1 Humano/genética , Paraparesia Espástica Tropical/genética , Provírus/genética , Sequências Repetidas Terminais/genética , Integração Viral/genética , Adulto , Idoso , Mapeamento Cromossômico , Cromossomos Humanos/genética , Colômbia/epidemiologia , Ilhas de CpG , DNA Recombinante/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Paraparesia Espástica Tropical/epidemiologia , Paraparesia Espástica Tropical/virologia , Retroelementos/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência do Ácido NucleicoRESUMO
The aim of the present study is to determine the presence and molecular integrity of high-risk HPV types in colorectal adenocarcinomas and to assess whether viral DNA is related to common proto-oncogene alterations, such as k-ras mutations and c-myc gene amplification, in colorectal cancer. Seventy-five colorectal adenocarcinomas were screened for HPV infection using nested-PCR (MY09/11-GP5+/6+). HPV typing was performed by type-specific PCR for HPV 16 and HPV 18 DNA. Unidentified samples were subsequently sequenced to determine the viral genotype. The physical status of HPV was determined by a nested PCR approach for type-specific E2 sequences. C-myc amplification was assessed by co-amplification with ß-globin as control locus, and mutation in k-ras codons 12 and 13 by ARMS-PCR. Overall, HPV was detected in thirty-three colorectal specimens (44%). HPV 16 was the prevalent type (16/75), followed by HPV 18 (15/75), HPV 31 (1/75) and HPV 66 (1/75). E2 disruption was detected in 56.3% of HPV 16 and in 40% of HPV 18 positive tumors. C-myc amplification was detected in 29.4% of cases, while k-ras mutations in 30.7%. There was no significant trend for HPV infection in tumors harboring either k-ras or c-myc alterations. This study demonstrates HPV DNA and viral integration in colorectal tumors, suggesting a potential role of this virus in colorectal carcinogenesis. There was no concurrence, however, of k-ras and c-myc activation with viral infection.
Assuntos
Adenocarcinoma/genética , Adenocarcinoma/virologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/virologia , Infecções por Papillomavirus/genética , DNA Viral/análise , DNA Viral/genética , Feminino , Amplificação de Genes , Genes myc , Genes ras , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Oncogenes , Papillomaviridae/genética , Reação em Cadeia da Polimerase , Proto-Oncogene Mas , Integração Viral/genéticaRESUMO
INTRODUCTION: Although the integration of human T-cell lymphotropic virus type I into the T-cells is not a random process, the mechanistic details are not understood. OBJECTIVES: The characteristics of the flanking host chromatin were evaluated at the integration sites in adult T-cell leukaemia/lymphoma (ATLL) patients infected with the virus. MATERIALS AND METHODS: From seven leukemic Colombian patients positive for the human T-cell lymphotropic virus type I (HTLV-I), lymphocyte DNA samples were extracted and amplified by inverse polymerase chain reaction (IPCR). Clonal expansion and human genome nucleotide composition in an extension of 50 bp was determined. To establish the characteristics of the human genome flanking provirus, 61 IPCR sequences from Colombian and Japanese ATLL patients, were analyzed in silico to obtain insights about the genomic structure, functions and nature of associated chromatin. RESULTS: The clonal expansion of cell clones was predominantly oligoclonal. From 61 IPCR sequences, 155 alignments with homology higher than 95% (e-value < 0.05) were screened. Seventy-five percent of those sequences corresponded to non coding elements that include repetitive and non-repetitive DNA. Fifty percent of the proviral integrations were associated with chromosomes of A and B groups. Viral DNA integration tended to favor exons of genes that replicated early, controlled the cell cycle, or were involved in signal transduction. CONCLUSIONS: The results indicated that HTLV-I integration was preferentially directed towards genomic environments with high C:G content, and toward genes that replicate early, regulate cell cycle or involved with signal transduction.