RESUMO
Epigallocatechin-3-gallate (EGCG), the primary bioactive polyphenol in green tea, has been shown to inhibit the growth of human papilloma virus (HPV)-transformed keratinocytes. Here, we set out to examine the consequences of EGCG treatment on the growth of HPV18-immortalised foreskin keratinocytes (HFK-HPV18) and an authentic HPV18-positive vulvar intraepithelial neoplasia (VIN) clone, focusing on its ability to influence cell proliferation and differentiation and to impact on viral oncogene expression and virus replication. EGCG treatment was associated with degradation of the E6 and E7 oncoproteins and an upregulation of their associated tumour suppressor genes; consequently, keratinocyte proliferation was inhibited in both monolayer and organotypic raft culture. While EGCG exerted a profound effect on cell proliferation, it had little impact on keratinocyte differentiation. Expression of the late viral protein E4 was suppressed in the presence of EGCG, suggesting that EGCG was able to block productive viral replication in differentiating keratinocytes. Although EGCG did not alter the levels of E6 and E7 mRNA, it enhanced the turnover of the E6 and E7 proteins. The addition of MG132, a proteasome inhibitor, to EGCG-treated keratinocytes led to the accumulation of the E6/E7 proteins, showing that EGCG acts as an anti-viral, targeting the E6 and E7 proteins for proteasome-mediated degradation.
RESUMO
BACKGROUND: Necrotising fasciitis (NF) is a rapidly progressive, destructive soft tissue infection with high mortality. The primary aim of this study was to evaluate the incidence and mortality of NF amongst patients admitted to English National Health Service (NHS) hospitals. The secondary aims included the identification of risk factors for mortality and causative pathogens. METHODS: The Hospital Episodes Statistics database identified patients with NF admitted to English NHS Trusts from 1/1/2002 to 31/12/2017. Information on patient demographics, co-morbid conditions, microbiology specimens, surgical intervention and in-hospital mortality was collected. Uni- and multivariable analyses were performed to investigate factors related to in-hospital mortality. RESULTS: A total of 11,042 patients were diagnosed with NF. Age-standardised incidence rose from 9 per million in 2002 to 21 per million in 2017 (annual percentage change = 6.9%). Incidence increased with age and was higher in men. Age-standardised mortality rate remained at 16% over the study period, while in-hospital mortality declined. On multivariable analysis, the following factors were associated with increased risk of in-hospital mortality: emergency admission, female sex, history of congestive heart failure, peripheral vascular disease, chronic kidney disease and cancer. Admission year and diabetes, which was significantly prevalent at 27%, were not associated with increased risk of mortality. Gram-positive pathogens, particularly Staphylococci, decreased over the study period with a corresponding increase in Gram-negative pathogens, predominantly E. coli. CONCLUSION: The incidence of NF increased markedly from 2002 to 2017 although in-hospital mortality did not change. There was a gradual shift in the causative organisms from Gram-positive to Gram-negative.
Assuntos
Fasciite Necrosante/epidemiologia , Fasciite Necrosante/microbiologia , Insuficiência Cardíaca/epidemiologia , Neoplasias/epidemiologia , Doenças Vasculares Periféricas/epidemiologia , Insuficiência Renal Crônica/epidemiologia , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Criança , Comorbidade , Bases de Dados Factuais , Inglaterra/epidemiologia , Escherichia coli , Infecções por Escherichia coli/complicações , Fasciite Necrosante/diagnóstico , Fasciite Necrosante/mortalidade , Feminino , Mortalidade Hospitalar , Hospitalização/estatística & dados numéricos , Humanos , Incidência , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Fatores Sexuais , Infecções Estafilocócicas/complicações , Medicina Estatal , Adulto JovemRESUMO
Although the over-expression of angiogenic factors is reported in diffuse large B-cell lymphoma (DLBCL), the poor response to anti-VEGF drugs observed in clinical trials suggests that angiogenesis in these tumours might be driven by VEGF-independent pathways. We show that sphingosine kinase-1 (SPHK1), which generates the potent bioactive sphingolipid sphingosine-1-phosphate (S1P), is over-expressed in DLBCL. A meta-analysis of over 2000 cases revealed that genes correlated with SPHK1 mRNA expression in DLBCL were significantly enriched for tumour angiogenesis meta-signature genes; an effect evident in both major cell of origin (COO) and stromal subtypes. Moreover, we found that S1P induces angiogenic signalling and a gene expression programme that is present within the tumour vasculature of SPHK1-expressing DLBCL. Importantly, S1PR1 functional antagonists, including Siponimod, and the S1P neutralising antibody, Sphingomab, inhibited S1P signalling in DLBCL cells in vitro. Furthermore, Siponimod, also reduced angiogenesis and tumour growth in an S1P-producing mouse model of angiogenic DLBCL. Our data define a potential role for S1P signalling in driving an angiogenic gene expression programme in the tumour vasculature of DLBCL and suggest novel opportunities to target S1P-mediated angiogenesis in patients with DLBCL.
Assuntos
Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Lisofosfolipídeos/metabolismo , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Transdução de Sinais , Esfingosina/análogos & derivados , Transcriptoma , Animais , Linhagem Celular Tumoral , Biologia Computacional/métodos , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Linfoma Difuso de Grandes Células B/patologia , Lisofosfolipídeos/genética , Camundongos , RNA Mensageiro/genética , Esfingosina/genética , Esfingosina/metabolismoRESUMO
To assess the relationship of E2 gene disruption with viral gene expression and clinical outcome in human papillomavirus (HPV) positive head and neck squamous cell carcinoma, we evaluated 31 oropharyngeal and 17 non-oropharyngeal HPV16 positive carcinomas using two PCR-based methods to test for disruption of E2, followed by Sanger sequencing. Expression of HPV16 E6, E7 and E2 transcripts, along with cellular ARF and INK4A, were also assessed by RT-qPCR. Associations between E2 disruption, E2/E6/E7 expression, and clinical outcome were evaluated by Kaplan-Meier analysis for loco-regional recurrence and disease-specific survival. The majority (n = 21, 68%) of HPV16 positive oropharyngeal carcinomas had an intact E2 gene, whereas the majority of HPV16 positive non-oropharyngeal carcinomas (n = 10, 59%) had a disrupted E2 gene. Three of the oropharyngeal tumors and two of the non-oropharyngeal tumors had deletions within E2. Detection of an intact E2 gene was associated with a higher DNA viral load and increased E2/E6/E7, ARF and INK4A expression in oropharyngeal tumors. Oropharyngeal carcinomas with an intact E2 had a lower risk of loco-regional recurrence (log-rank p = 0.04) and improved disease-specific survival (p = 0.03) compared to tumors with disrupted E2. In addition, high E7 expression was associated with lower risk of loco-regional recurrence (p = 0.004) as was high E6 expression (p = 0.006). In summary, an intact E2 gene is more common in HPV16 positive oropharyngeal than non-oropharyngeal carcinomas; the presence of an intact E2 gene is associated with higher HPV viral load, higher viral oncogene expression, and improved clinical outcome compared to patients with a disrupted E2 gene in oropharyngeal cancer.
Assuntos
Alphapapillomavirus/isolamento & purificação , Carcinoma de Células Escamosas/terapia , Proteínas de Ligação a DNA/genética , Neoplasias de Cabeça e Pescoço/terapia , Proteínas Oncogênicas Virais/genética , Oncogenes , Carga Viral , Alphapapillomavirus/genética , Carcinoma de Células Escamosas/virologia , Feminino , Neoplasias de Cabeça e Pescoço/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , Reação em Cadeia da Polimerase , Carcinoma de Células Escamosas de Cabeça e PescoçoRESUMO
The ability to develop a comprehensive panel of treatment predicting factors would significantly improve our ability to stratify patients for cytotoxic or targeted therapies, and prevent patients receiving ineffective treatments. We have investigated if a recently developed genome-wide haploid genetic screen can be used to reveal the critical mediators of response to anticancer therapy. Pancreatic cancer is known to be highly resistant to systemic therapy. Recently epigenetic changes have been shown to be a key determinant in the maintenance of subpopulations of cancer cells with high-level resistance to cytotoxic therapy. We show that in human pancreatic cancer cell lines, treatment with the potent class I histone deacetylase inhibitor, entinostat, synergistically enhances gemcitabine-induced inhibition of cell proliferation and apoptosis. Using a genome-wide haploid genetic screen, we identified deoxycytidine kinase (DCK) as one of the genes with the highest degree of insertional enrichment following treatment with gemcitabine and entinostat; DCK is already known to be the rate-limiting activating enzyme for gemcitabine. Immunoblotting confirmed loss of DCK protein expression in the resistant KBM7 cells. CRISPR/Cas-9 inactivation of DCK in pancreatic cancer cell lines resulted in resistance to gemcitabine alone and in combination with entinostat. We have identified gemcitabine and entinostat as a potential new combination therapy in pancreatic cancer, and in this proof-of-principle study we have demonstrated that a recently developed haploid genetic screen can be used as a novel approach to identify the critical genes that determine treatment response.
RESUMO
High-risk human papillomavirus (HR-HPV) causes nearly 100% of cervical carcinoma. However, it remains unclear whether HPV can establish a latent infection, one which may be responsible for the second peak in incidence of cervical carcinoma seen in older women. Therefore, using Ventana in situ hybridisation (ISH), quantitative PCR assays and biomarkers of productive and transforming viral infection, we set out to provide the first robust estimate of the prevalence and characteristics of HPV genomes in FFPE tissue from the cervices of 99 women undergoing hysterectomy for reasons unrelated to epithelial abnormality. Our ISH assay detected HR-HPV in 42% of our study population. The majority of ISH positive samples also tested HPV16 positive using sensitive PCR based assays and were more likely to have a history of preceding cytological abnormality. Analysis of subsets of this population revealed HR-HPV to be transcriptionally inactive as there was no evidence of a productive or transforming infection. Critically, the E2 gene was always disrupted in those HPV16 positive cases which were assessed. These findings point to a reservoir of transcriptionally silent, disrupted HPV16 DNA in morphologically normal cervices, re-expression of which could explain the increase in incidence of cervical cancer observed in later life.
Assuntos
DNA Viral/genética , Proteínas de Ligação a DNA/deficiência , Papillomavirus Humano 16/fisiologia , Proteínas Oncogênicas Virais/deficiência , Infecções por Papillomavirus/virologia , Latência Viral , Adulto , Idoso , Doenças Assintomáticas , Transformação Celular Viral/genética , Colo do Útero/cirurgia , Colo do Útero/virologia , Estudos de Coortes , DNA Viral/isolamento & purificação , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Feminino , Fixadores , Formaldeído , Humanos , Histerectomia , Hibridização In Situ , Pessoa de Meia-Idade , Proteínas Oncogênicas Virais/química , Proteínas Oncogênicas Virais/genética , Infecções por Papillomavirus/diagnóstico , Reação em Cadeia da Polimerase , Risco , Fatores de Tempo , Inclusão do TecidoRESUMO
The current interest in epigenetic priming is underpinned by the belief that remodelling of the epigenetic landscape will sensitise tumours to subsequent therapy. In this pre-clinical study, paediatric AML cells expanded in culture and primary AML xenografts were treated with decitabine, a DNA demethylating agent, and cytarabine, a frontline cytotoxic agent used in the treatment of AML, either alone or in combination. Sequential treatment with decitabine and cytarabine was found to be more effective in reducing tumour burden than treatment with cytarabine alone suggesting that the sequential delivery of these agents may a have real clinical advantage in the treatment of paediatric AML. However we found no evidence to suggest that this outcome was dependent on priming with a hypomethylating agent, as the benefits observed were independent of the order in which these drugs were administered.
Assuntos
Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Azacitidina/análogos & derivados , Citarabina/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Animais , Antineoplásicos/administração & dosagem , Azacitidina/administração & dosagem , Azacitidina/uso terapêutico , Citarabina/administração & dosagem , Decitabina , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Epstein-Barr virus (EBV) is an oncogenic virus that is associated with the pathogenesis of several human lymphoid malignancies, including Hodgkin's lymphoma. Infection of normal resting B cells with EBV results in activation to lymphoblasts that are phenotypically similar to those generated by physiological stimulation with CD40L plus IL-4. One important difference is that infection leads to the establishment of permanently growing lymphoblastoid cell lines, whereas CD40L/IL-4 blasts have finite proliferation lifespans. To identify early events which might later determine why EBV infected blasts go on to establish transformed cell lines, we performed global transcriptome analyses on resting B cells and on EBV and CD40L/IL-4 blasts after 7 days culture. As anticipated there was considerable overlap in the transcriptomes of the two types of lymphoblasts when compared to the original resting B cells, reflecting common changes associated with lymphocyte activation and proliferation. Of interest to us was a subset of 255 genes that were differentially expressed between EBV and CD40L/IL-4 blasts. Genes which were more highly expressed in EBV blasts were substantially and significantly enriched for a set of interferon-stimulated genes which on further in silico analyses were found to be repressed by IL-4 in other cell contexts and to be up-regulated in micro-dissected malignant cells from Hodgkin's lymphoma biopsies when compared to their normal germinal center cell counterparts. We hypothesized that EBV and IL-4 were targeting and discordantly regulating a common set of genes. This was supported experimentally in our B cell model where IL-4 stimulation partially reversed transcriptional changes which follow EBV infection and it impaired the efficiency of EBV-induced B cell transformation. Taken together, these data suggest that the discordant regulation of interferon and IL-4 pathway genes by EBV that occurs early following infection of B cells has relevance to the development or maintenance of an EBV-associated malignancy.
Assuntos
Linfócitos B/metabolismo , Linfócitos B/virologia , Transformação Celular Viral/genética , Regulação da Expressão Gênica , Herpesvirus Humano 4/fisiologia , Fatores Reguladores de Interferon/genética , Interleucina-4/metabolismo , Linfócitos B/patologia , Ligante de CD40/imunologia , Ligante de CD40/farmacologia , Linhagem Celular Tumoral , Transformação Celular Viral/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Doença de Hodgkin/genética , Doença de Hodgkin/metabolismo , Humanos , Interferons/metabolismo , Interleucina-4/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Transdução de Sinais , Transcriptoma , Proteínas da Matriz Viral/metabolismoRESUMO
Hodgkin's lymphoma is unusual among B cell lymphomas, in so far as the malignant Hodgkin/Reed-Sternberg (HRS) cells lack a functional B cell receptor (BCR), as well as many of the required downstream signalling components. In Epstein-Barr virus (EBV)-positive cases of Hodgkin's lymphoma, HRS cells express the viral latent membrane proteins (LMP)-1 and -2A. LMP2A is thought to contribute to the pathogenesis of Hodgkin's lymphoma by providing a surrogate BCR-like survival signal. However, LMP2A has also been shown to induce the virus-replicative cycle in B cells, an event presumably incompatible with lymphomagenesis. In an attempt to resolve this apparent paradox, we compared the transcriptional changes observed in primary HRS cells with those induced by LMP2A and by BCR activation in primary human germinal centre (GC) B cells, the presumed progenitors of HRS cells. We found a subset of genes that were up-regulated by both LMP2A expression and BCR activation but which were down-regulated in primary HRS cells. These genes included EGR1, an immediate-early gene that is required for BCR-induced entry to the virus-replicative cycle. We present data supporting a model for the pathogenesis of EBV-positive Hodgkin's lymphoma in which LMP2A-expressing HRS cells lacking BCR signalling functions cannot induce EGR1 and are consequently protected from entry to the virus lytic cycle. The primary microarray data are available from GEO (http://www.ncbi.nlm.nih.gov/geo/) under series Accession No 46143.
Assuntos
Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Herpesvirus Humano 4/metabolismo , Doença de Hodgkin/metabolismo , Células de Reed-Sternberg/metabolismo , Proteínas da Matriz Viral/metabolismo , Linfócitos B/metabolismo , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patologia , Linfoma de Burkitt/virologia , Linhagem Celular Tumoral , Efeito Citopatogênico Viral , Proteína 1 de Resposta de Crescimento Precoce/genética , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/patogenicidade , Doença de Hodgkin/genética , Doença de Hodgkin/patologia , Doença de Hodgkin/virologia , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Receptores de Antígenos de Linfócitos B/metabolismo , Células de Reed-Sternberg/patologia , Células de Reed-Sternberg/virologia , Transativadores/metabolismo , Transfecção , Regulação para Cima , Proteínas da Matriz Viral/genéticaRESUMO
Previous studies have reported that the tumour cells of nasopharyngeal carcinoma (NPC) exhibit recurrent chromosome abnormalities. These genetic changes are broadly assumed to lead to changes in gene expression which are important for the pathogenesis of this tumour. However, this assumption has yet to be formally tested at a global level. Therefore a genome wide analysis of chromosome copy number and gene expression was performed in tumour cells micro-dissected from the same NPC biopsies. Cellular tumour suppressor and tumour-promoting genes (TSG, TPG) and Epstein-Barr Virus (EBV)-encoded oncogenes were examined. The EBV-encoded genome maintenance protein EBNA1, along with the putative oncogenes LMP1, LMP2 and BARF1 were expressed in the majority of NPCs that were analysed. Significant downregulation of expression in an average of 76 cellular TSGs per tumour was found, whilst a per-tumour average of 88 significantly upregulated, TPGs occurred. The expression of around 60% of putative TPGs and TSGs was both up-and down-regulated in different types of cancer, suggesting that the simplistic classification of genes as TSGs or TPGs may not be entirely appropriate and that the concept of context-dependent onco-suppressors may be more extensive than previously recognised. No significant enrichment of TPGs within regions of frequent genomic gain was seen but TSGs were significantly enriched within regions of frequent genomic loss. It is suggested that loss of the FHIT gene may be a driver of NPC tumourigenesis. Notwithstanding the association of TSGs with regions of genomic loss, on a gene by gene basis and excepting homozygous deletions and high-level amplification, there is very little correlation between chromosomal copy number aberrations and expression levels of TSGs and TPGs in NPC.
Assuntos
Carcinoma/metabolismo , Carcinoma/virologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/virologia , Feminino , Deleção de Genes , Perfilação da Expressão Gênica , Regulação Viral da Expressão Gênica , Herpesvirus Humano 4/metabolismo , Homozigoto , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Transcrição Gênica , Regulação para Cima , Proteínas da Matriz Viral/biossíntese , Proteínas Virais/biossínteseRESUMO
The contribution of early virus-induced epigenetic changes to human papillomavirus (HPV)-associated carcinogenesis is poorly understood. Using genome-wide methylation array profiling and a cell-based model, which supports replication of HPV episomes, we found that transfection of primary human foreskin keratinocytes with episomal forms of high-risk HPV types was followed by upregulation of the DNA methyltransferases, DNMT1 and DNMT3B, and changes in the methylation status of cellular genes many of which are reported to be differentially methylated in cervical neoplasia. HPV16- and HPV18-associated changes were not randomly distributed across the genome, but clustered at specific chromosomal locations which mapped on to known HPV integration sites and to chromosomal regions lost and gained in high-grade cervical neoplasia. Methylation changes were directed in part by the same cis-acting factors that appear to direct methylation changes in cancer, the presence of a bivalent chromatin mark in human embryonic stem cells and promoter CpG content; these associations explain much of the ontological profile of genes found to have increased methylation following HPV16 transfection. We were also able to show, using sequential samples from a cohort of young women with incident HPV16 infections, that the detection in cervical samples of methylated forms of the tumour suppressor gene, RARB, often parallels the natural history of cervical HPV infection. Our findings suggest that further investigation of the distribution and determinants of early virus-induced epigenetic reprogramming will provide important insights into the pathogenesis of virus-associated malignancy.
Assuntos
Alphapapillomavirus/isolamento & purificação , Metilação de DNA , Infecções por Papillomavirus/virologia , Neoplasias do Colo do Útero/virologia , Feminino , Humanos , Infecções por Papillomavirus/patologia , Neoplasias do Colo do Útero/patologiaRESUMO
Although there is increasing evidence that aberrant expression of those enzymes which control protein arginine methylation contribute to carcinogenesis, their de-regulation by oncogenic viruses in primary cells has yet to be reported. We first show that the protein arginine methyltransferases, CARM1, PRMT1 and PRMT5 are strongly expressed in Hodgkin Reed-Sternberg (HRS) cells, and up-regulated in Hodgkin's lymphoma (HL) cell lines. Given that Epstein-Barr virus (EBV) can be detected in approximately 50% of primary HL, we next examined how EBV infection of germinal centre (GC) B cells, the presumptive precursors of HRS cells, modulated the expression of these proteins. EBV infection of GC B cells was followed by the up-regulation of CARM1, PRMT1 and PRMT5, and by the down-regulation of the arginine deiminase, PADI4. Latent membrane protein 1 (LMP1), the major EBV transforming gene was shown to induce PRMT1 in GC B cells and in a stably transfected B cell line. The recent development of compounds which inhibit PRMT-mediated reactions provides a compelling case for continuing to dissect the contribution of virus induced changes in these proteins to lymphomagenesis.
RESUMO
Although Epstein-Barr virus (EBV) usually establishes an asymptomatic lifelong infection, it is also implicated in the development of germinal center (GC) B-cell-derived malignancies, including Hodgkin's lymphoma (HL). Following primary infection, EBV remains latent in the memory B-cell population, where host-driven methylation of viral DNA contributes to the repression of viral gene expression. However, it is still unclear how EBV harnesses the cell's methylation machinery in B cells, how this contributes to viral persistence, and what impact this has on the methylation of cellular genes. We show that EBV infection of GC B cells is followed by upregulation of the DNA methyltransferase DNMT3A and downregulation of DNMT3B and DNMT1. We show that the EBV latent membrane protein 1 (LMP1) oncogene downregulates DNMT1 and that DNMT3A binds to the viral promoter Wp. Genome-wide promoter arrays performed with these cells showed that EBV-associated methylation changes in cellular genes were not randomly distributed across the genome but clustered at chromosomal locations, consistent with an instructive pattern of methylation, and were in part determined by promoter CpG content. Both DNMT3B and DNMT1 were downregulated and DNMT3A was upregulated in HL cell lines, recapitulating the pattern of expression observed following EBV infection of GC B cells. We also found, by using gene expression profiling, that genes differentially expressed following EBV infection of GC B cells were significantly enriched for those reported to be differentially expressed in HL. These observations suggest that EBV-infected GC B cells are a useful model for studying virus-associated changes contributing to the pathogenesis of HL.
Assuntos
Linfócitos B/imunologia , Epigênese Genética , Regulação da Expressão Gênica , Centro Germinativo/imunologia , Herpesvirus Humano 4/patogenicidade , Doença de Hodgkin/virologia , Transcrição Gênica , Linfócitos B/virologia , Células Cultivadas , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/biossíntese , Metilação de DNA , DNA Metiltransferase 3A , Perfilação da Expressão Gênica , Centro Germinativo/virologia , Doença de Hodgkin/imunologia , Doença de Hodgkin/patologia , Humanos , Proteínas da Matriz Viral/metabolismo , DNA Metiltransferase 3BRESUMO
An important pathogenic event in Epstein-Barr virus (EBV)-associated lymphomas is the suppression of virus replication, which would otherwise lead to cell death. Because virus replication in B cells is intimately linked to their differentiation toward plasma cells, we asked whether the physiologic signals that drive normal B-cell differentiation are absent in EBV-transformed cells. We focused on BLIMP1α, a transcription factor that is required for plasma cell differentiation and that is inactivated in diffuse large B-cell lymphomas. We show that BLIMP1α expression is down-regulated after EBV infection of primary germinal center B cells and that the EBV oncogene, latent membrane protein-1 (LMP-1), is alone capable of inducing this down-regulation in these cells. Furthermore, the down-regulation of BLIMP1α by LMP-1 was accompanied by a partial disruption of the BLIMP1α transcriptional program, including the aberrant induction of MYC, the repression of which is required for terminal differentiation. Finally, we show that the ectopic expression of BLIMP1α in EBV-transformed cells can induce the viral lytic cycle. Our results suggest that LMP-1 expression in progenitor germinal center B cells could contribute to the pathogenesis of EBV-associated lymphomas by down-regulating BLIMP1α, in turn preventing plasma cell differentiation and induction of the viral lytic cycle.
Assuntos
Linfócitos B/virologia , Diferenciação Celular , Herpesvirus Humano 4/fisiologia , Linfoma de Células B/etiologia , Plasmócitos/patologia , Proteínas Repressoras/metabolismo , Proteínas da Matriz Viral/metabolismo , Linfócitos B/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Transformação Celular Viral , Células Cultivadas , Criança , Infecções por Vírus Epstein-Barr/complicações , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/virologia , Perfilação da Expressão Gênica , Centro Germinativo , Humanos , Técnicas Imunoenzimáticas , Linfoma de Células B/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Tonsila Palatina/citologia , Tonsila Palatina/metabolismo , Plasmócitos/metabolismo , Fator 1 de Ligação ao Domínio I Regulador Positivo , RNA Mensageiro/genética , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas da Matriz Viral/genética , Latência Viral , Replicação ViralRESUMO
BACKGROUND: It has been suggested that in women who test positive for high-risk human papillomavirus (HPV) types, viral load can distinguish women who are at increased risk of cervical neoplasia from those who are not. METHODS: Quantitative PCR (qPCR) was used to measure HPV copy number in serial samples taken from 60 and 58 young women previously found to have incident cervical HPV16 or HPV18 infections, respectively, using GP5+/GP6+ primers; women provided at least three samples for qPCR testing, at least one of which was positive. RESULTS: A 10-fold increase in HPV16 or HPV18 copy number was associated with a modestly increased risk of acquiring a cytologic abnormality [HPV16: hazards ratio, 1.76 (95% confidence interval, 1.38-2.25); HPV18: hazards ratio, 1.59 (95% confidence interval, 1.25-2.03)]. However, in most women, copy number increased during follow-up, before decreasing again. In women with a HPV16 infection, the median copy number per 1,000 cells was 7.7 in their first qPCR HPV-positive sample, 1,237 in the sample yielding the maximum copy number, and 7.8 in their last qPCR HPV-positive sample; corresponding copy numbers for women with HPV18 infection were 2.3, 87, and 2.4. Maximum HPV16 and HPV18 copy number did not differ significantly between women who acquired an incident cervical cytologic abnormality and those who did not. CONCLUSION: Whereas large relative increases in copy number are associated with an increased risk of abnormality, a single measurement of viral load made at an indeterminate point during the natural history of HPV infection does not reliably predict the risk of acquiring cervical neoplasia. Therefore, a single measure of HPV viral load cannot be considered a clinically useful biomarker.
Assuntos
Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/virologia , Neoplasias do Colo do Útero/virologia , Carga Viral , Adolescente , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , DNA Viral/sangue , Feminino , Dosagem de Genes , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/isolamento & purificação , Papillomavirus Humano 18/genética , Papillomavirus Humano 18/isolamento & purificação , Humanos , Estudos Longitudinais , Reação em Cadeia da Polimerase , Fatores de Risco , Neoplasias do Colo do Útero/sangue , Adulto JovemRESUMO
Repeated measurements of smoking, cervical human papillomavirus (HPV) status and sexual behaviour were used to measure the risk of high-grade cervical intraepithelial neoplasia (CIN) in relation to changes in smoking and cervical HPV status, and to explore the impact of smoking on the acquisition and duration of incident cervical HPV infection. Included in this longitudinal analysis are 1485 women aged 15-19 years: 1075 were HPV-negative and cytologically normal at recruitment; 410 were HPV-positive, cytologically abnormal or both, at this time. Women re-attended every 6 months, when samples were taken for cytological and virological examination. Current smoking intensity was associated with an increased risk of high-grade CIN, after controlling for cervical HPV status (compared to non-smokers, hazards ratio (HR) for 10 or more cigarettes per day=2.21, 95% confidence interval (CI) 1.19-4.12, p-trend=0.008). In women who were HPV-negative and cytologically normal at recruitment, current smoking was not significantly associated with the risk of acquiring a cervical HPV infection, after controlling for life-time number of partners and age of oldest partner (HR=1.13, 95% CI 0.90-1.41); nor did it prolong the length of time during which HPV could be detected (HR=1.03, 95% CI 0.78-1.34). Current smoking intensity is an independent risk factor for high-grade CIN in young women, after controlling for cervical HPV infection.
Assuntos
Infecções por Papillomavirus/etiologia , Fumar/efeitos adversos , Displasia do Colo do Útero/etiologia , Neoplasias do Colo do Útero/etiologia , Adolescente , Inglaterra/epidemiologia , Feminino , Humanos , Incidência , Estudos Longitudinais , Infecções por Papillomavirus/epidemiologia , Fatores de Risco , Parceiros Sexuais , Neoplasias do Colo do Útero/epidemiologia , Adulto Jovem , Displasia do Colo do Útero/epidemiologiaRESUMO
Integration of high-risk human papillomavirus (HPV) types into the host-cell genome disrupts the HPV regulatory E2 protein, resulting in a loss of negative feedback control of viral oncogene expression; this disruption has been considered a critical event in the pathogenesis of cervical neoplasia, and a potential biomarker of progressive disease. However, using serial samples taken from a cohort of young women who were recruited soon after they first had sexual intercourse, we show that disruption of the E2 gene is a common and early event in the natural history of incident cervical HPV infections. The E2 gene was significantly more likely to be disrupted in women who tested positive for HPV18 in their baseline sample than in those who tested positive for HPV16 [26% versus 58%; relative risk, 2.26; 95% confidence interval (CI), 1.38-3.71; chi(2), 9.23; 1 degree of freedom (df); P = 0.002]. Among women with an intact E2 gene in their baseline sample, the median time to first detection of E2 disruption was also shorter for those who tested positive for HPV18 than HPV16 (5.7 versus 10.9 months; hazards ratio, 1.93; 95% CI, 0.84-4.44; chi(2), 2.49; 1 df; P = 0.11). This tendency for HPV18 to integrate early, coupled with the substantial reduction in viral load in HPV18-positive samples in which E2 is disrupted, may explain why HPV18-associated disease is often reported to be characterized by minor cytologic changes, which underestimate the severity of the underlying histologic abnormality.
Assuntos
Proteínas de Ligação a DNA/genética , Papillomavirus Humano 16/genética , Papillomavirus Humano 18/genética , Proteínas Oncogênicas Virais/genética , Infecções por Papillomavirus/virologia , Doenças do Colo do Útero/virologia , Adolescente , Estudos de Coortes , DNA Viral/genética , Feminino , Humanos , Estudos Longitudinais , Carga ViralRESUMO
A micro-array analysis using biopsies from patients with EBV-positive undifferentiated nasopharyngeal carcinoma (NPC) and from cancer-free controls revealed down-regulation of tumour suppressor genes (TSG) not previously associated with this disease; one such gene was the ataxia telangiectasia mutated (ATM) gene. Q-PCR confirmed down-regulation of ATM mRNA and ATM protein expression in tumour cells was weak or absent in almost all cases. In NPC cell lines, however, ATM was down-regulated only in the EBV-positive line, C666.1, and in none of five EBV-negative lines. In vitro infection of EBV-negative NPC cell lines with a recombinant EBV was followed by the down-regulation of ATM mRNA and protein, and only EBV-positive cells showed a defective DNA damage response following gamma-irradiation. Our data suggest that loss of ATM function could be an important step in the pathogenesis of NPC, and may have implications for the treatment of this disease.
Assuntos
Adenocarcinoma/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Regulação Viral da Expressão Gênica , Neoplasias Nasofaríngeas/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Supressoras de Tumor/genética , Adenocarcinoma/virologia , Proteínas Mutadas de Ataxia Telangiectasia , Estudos de Casos e Controles , Proteínas de Ciclo Celular/análise , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/análise , Perfilação da Expressão Gênica/métodos , Herpesvirus Humano 4 , Humanos , Imuno-Histoquímica , Neoplasias Nasofaríngeas/virologia , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Serina-Treonina Quinases/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Supressoras de Tumor/análise , Infecções Tumorais por Vírus/complicações , Infecções Tumorais por Vírus/genéticaRESUMO
We have evaluated a neutralizing antibody assay which uses human papillomavirus (HPV) type 16 (HPV-16) and HPV-18 pseudovirions carrying a secretory alkaline phosphatase reporter gene and which can potentially measure functionally relevant HPV type-specific neutralizing antibodies. The reproducibility of the assay was excellent; for HPV-16, the intra- and interassay kappa values were 0.95 and 0.90, respectively; and for HPV-18, the corresponding values were 0.90 and 0.90. This assay was used to describe the kinetics of the neutralizing antibody response in a cohort of 42 young women who were recruited soon after first intercourse and who first tested positive for HPV-16 DNA or HPV-18 DNA, or both, during follow-up. Most women seroconverted following the first detection of type-specific HPV DNA and remained seropositive until the end of follow-up. Our findings are broadly consistent with those of two other cohort studies which have measured the serological response following an incident infection by using the technically simpler virus-like-particle-based enzyme-linked immunosorbent assay.
Assuntos
Anticorpos Antivirais/sangue , Papillomavirus Humano 16/isolamento & purificação , Papillomavirus Humano 18/isolamento & purificação , Infecções por Papillomavirus/diagnóstico , Virossomos , Adolescente , Adulto , Estudos de Coortes , Feminino , Papillomavirus Humano 16/imunologia , Papillomavirus Humano 18/imunologia , Humanos , Testes de Neutralização , Infecções por Papillomavirus/virologia , Reprodutibilidade dos TestesRESUMO
In approximately 50% of patients with Hodgkin's lymphoma (HL), the Epstein-Barr virus (EBV), an oncogenic herpesvirus, is present in tumor cells. After microarray profiling of both HL tumors and cell lines, we found that EBV infection increased the expression of the chemokine CCL20 in both primary Hodgkin and Reed-Sternberg cells and Hodgkin and Reed-Sternberg cell-derived cell lines. Additionally, this up-regulation could be mediated by the EBV nuclear antigen 1 protein. The higher levels of CCL20 in the supernatants of EBV-infected HL cell lines increased the migration of CD4(+) lymphocytes that expressed FOXP3, a marker of regulatory T cells (Tregs), which are specialized CD4(+) T cells that inhibit effector CD4(+) and CD8(+) T cells. In HL, an increased number of Tregs is associated with the loss of EBV-specific immunity. Our results identify a mechanism by which EBV can recruit Tregs to the microenvironment of HL by inducing the expression of CCL20 and, by doing so, prevent immune responses against the virus-infected tumor population. Further investigation of how EBV recruits and modifies Tregs will contribute not only to our understanding of the pathogenesis of virus-associated tumors but also to the development of therapeutic strategies designed to manipulate Treg activity.