RESUMO
Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents. It is characterized by highly complex karyotypes with structural and numerical chromosomal alterations. The observed OS-specific characteristics in localization and frequencies of chromosomal breakages strongly implicate a specific set of responsible driver genes or a specific mechanism of fragility induction. In this study, a comprehensive assessment of somatic copy number alterations (SCNAs) was performed in 160 OS samples using whole-genome CytoScan High Density arrays (Affymetrix, Santa Clara, CA). Genes or regions frequently targeted by SCNAs were identified. Breakage analysis revealed OS specific unstable regions in which well-known OS tumor suppressor genes, including TP53, RB1, WWOX, DLG2 and LSAMP are located. Certain genomic features, such as transposable elements and non-B DNA-forming motifs were found to be significantly enriched in the vicinity of chromosomal breakage sites. A complex breakage pattern-chromothripsis-has been suggested as a widespread phenomenon in OS. It was further demonstrated that hyperploidy and in particular chromothripsis were strongly correlated with OS patient clinical outcome. The revealed OS-specific fragility pattern provides novel clues for understanding the biology of OS.
Assuntos
Neoplasias Ósseas/genética , Quebra Cromossômica , Variações do Número de Cópias de DNA , Osteossarcoma/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Cromotripsia , Feminino , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
BACKGROUND: The human genome contains multiple LTR elements including human endogenous retroviruses (HERVs) that together account for approximately 8-9% of the genomic DNA. At least 40 different HERV groups have been assigned to three major HERV classes on the basis of their homologies to exogenous retroviruses. Although most HERVs are silenced by a variety of genetic and epigenetic mechanisms, they may be reactivated by environmental stimuli such as exogenous viruses and thus may contribute to pathogenic conditions. The objective of this study was to perform an in-depth analysis of the influence of HIV-1 infection on HERV activity in different cell types. RESULTS: A retrovirus-specific microarray that covers major HERV groups from all three classes was used to analyze HERV transcription patterns in three persistently HIV-1 infected cell lines of different cellular origins and in their uninfected counterparts. All three persistently infected cell lines showed increased transcription of multiple class I and II HERV groups. Up-regulated transcription of five HERV taxa (HERV-E, HERV-T, HERV-K (HML-10) and two ERV9 subgroups) was confirmed by quantitative reverse transcriptase PCR analysis and could be reversed by knock-down of HIV-1 expression with HIV-1-specific siRNAs. Cells infected de novo by HIV-1 showed stronger transcriptional up-regulation of the HERV-K (HML-2) group than persistently infected cells of the same origin. Analysis of transcripts from individual members of this group revealed up-regulation of predominantly two proviral loci (ERVK-7 and ERVK-15) on chromosomes 1q22 and 7q34 in persistently infected KE37.1 cells, as well as in de novo HIV-1 infected LC5 cells, while only one single HML-2 locus (ERV-K6) on chromosome 7p22.1 was activated in persistently infected LC5 cells. CONCLUSIONS: Our results demonstrate that HIV-1 can alter HERV transcription patterns of infected cells and indicate a correlation between activation of HERV elements and the level of HIV-1 production. Moreover, our results suggest that the effects of HIV-1 on HERV activity may be far more extensive and complex than anticipated from initial studies with clinical material.
Assuntos
Retrovirus Endógenos/fisiologia , Infecções por HIV/virologia , HIV-1/crescimento & desenvolvimento , Transcrição Gênica , Ativação Viral , Linhagem Celular , Retrovirus Endógenos/genética , Perfilação da Expressão Gênica , Humanos , Análise em MicrossériesRESUMO
BACKGROUND: Emerging evidence suggests that human cytomegalovirus (HCMV) is highly prevalent in tumours of different origin. This virus is implied to have oncogenic and oncomodulatory functions, through its ability to control host gene expression. Human endogenous retroviruses (HERV) are also frequently active in tumours of different origin, and are supposed to contribute as cofactors to cancer development. Due to the high prevalence of HCMV in several different tumours, and its ability to control host cell gene expression, we sought to define whether HCMV may affect HERV transcription. FINDINGS: Infection of 3 established cancer cell lines, 2 primary glioblastoma cells, endothelial cells from 3 donors and monocytes from 4 donors with HCMV (strains VR 1814 or TB40/F) induced reverse transcriptase (RT) activity in all cells tested, but the response varied between donors. Both, gammaretrovirus-related class I elements HERV-T, HERV-W, HERV-F and ERV-9, and betaretrovirus-related class II elements HML-2 - 4 and HML-7 - 8, as well as spuma-virus related class III elements of the HERV-L group were up-regulated in response to HCMV infection in GliNS1 cells. Up-regulation of HERV activity was more pronounced in cells harbouring active HCMV infection, but was also induced by UV-inactivated virus. The effect was only slightly affected by ganciclovir treatment and was not controlled by the IE72 or IE86 HCMV genes. CONCLUSIONS: Within this brief report we show that HCMV infection induces HERV transcriptional activity in different cell types.
Assuntos
Betaretrovirus/genética , Citomegalovirus/crescimento & desenvolvimento , Gammaretrovirus/genética , Transcrição Gênica , Betaretrovirus/enzimologia , Células Cultivadas , Gammaretrovirus/enzimologia , Regulação Viral da Expressão Gênica , Humanos , DNA Polimerase Dirigida por RNA/metabolismo , Regulação para CimaRESUMO
The human genome comprises approximately 8-9â% of human endogenous retroviruses (HERVs) that are transcribed with tissue specificity. However, relatively few organs have been examined in detail for individual differences in HERV transcription pattern, nor have tissue-to-cell culture comparisons been frequently performed. Using an HERV-specific DNA microarray, a core HERV transcription profile was established for the human kidney comparing 10 tissue samples. This core represents HERV groups expressed uniformly or nearly so in non-tumour kidney tissue. The profiles obtained from non-tumour tissues were compared to 10 renal tumour tissues (renal cell carcinoma, RCC) derived from the same individuals and additionally, to 22 RCC cell lines. No RCC cell line or tumour-specific differences were observed, suggesting that HERV transcription is not altered in RCC. However, when comparing tissue transcription to cell line transcription, there were consistent differences. The differences were irrespective of cancer state and included cell lines derived from non-tumour kidney tissue, suggesting that a specific alteration of HERV transcription occurs when establishing cell lines. In contrast to previous publications, all known HERV-derived tumour antigens, including those identified in RCC, were expressed both in multiple RCC cell lines and several non-tumour tissue-derived cell lines, a result that contrasts with findings from patient samples. The results establish the core kidney transcription pattern of HERVs and reveal differences between cell culture lines and tissue samples.
Assuntos
Retrovirus Endógenos/patogenicidade , Perfilação da Expressão Gênica , Rim/virologia , Transcrição Gênica , Carcinoma de Células Renais/virologia , Linhagem Celular , Humanos , Neoplasias Renais/virologia , Análise em MicrossériesRESUMO
Prion diseases are neurodegenerative diseases associated with the accumulation of a pathogenic isoform of the host-encoded prion protein. The cellular responses to prion infection are not well defined. By performing microarray analysis on cultured neuronal cells infected with prion strain 22L, in the group of up-regulated genes we observed predominantly genes of the cholesterol pathway. Increased transcript levels of at least nine enzymes involved in cholesterol synthesis, including the gene for the rate-limiting hydroxymethylglutaryl-CoA reductase, were detected. Up-regulation of cholesterogenic genes was attributable to a prion-dependent increase in the amount and activity of the sterol regulatory element-binding protein Srebp2, resulting in elevated levels of total and free cellular cholesterol. The up-regulation of cholesterol biosynthesis appeared to be a characteristic response of neurons to prion challenge, as cholesterogenic transcripts were also elevated in persistently infected GT-1 cells and prion-exposed primary hippocampal neurons but not in microglial cells and primary astrocytes. These results convincingly demonstrate that prion propagation not only depends on the availability of cholesterol but that neuronal cells themselves respond to prions with specific up-regulation of cholesterol biosynthesis.
Assuntos
Colesterol/biossíntese , Expressão Gênica , Neurônios/metabolismo , Doenças Priônicas/genética , Príons/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Animais , Linhagem Celular Tumoral , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , Doenças Priônicas/metabolismo , Príons/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Regulação para CimaRESUMO
The human genome contains more than half a million human endogenous retrovirus (HERV) long terminal repeats (LTRs) that can be regarded as mobile regulatory modules. Many of these HERV LTRs have been recruited during evolution as transcriptional control elements for cellular gene expression. We have cloned LTR sequences from two HERV families, HERV-H and HERV-L, differing widely in their activity and tissue specificity into a murine leukemia virus (MLV)-based promoter conversion vector (ProCon). Various human cell lines were infected with the HERV-MLV hybrid vectors, and cell type-specific expression of the reporter gene was compared with the promoter specificity of the corresponding HERV LTRs in transient-transfection assays. Transcription start site analysis of HERV-MLV hybrid vectors revealed preferential use of the HERV promoter initiation site. Our data show that HERV LTRs function in the context of retroviral vectors in certain cell types and have the potential to be useful as cell type-specific promoters in vector construction.
Assuntos
Retrovirus Endógenos/genética , Engenharia Genética/métodos , Vetores Genéticos , Vírus da Leucemia Murina/genética , Regiões Promotoras Genéticas , Sequências Repetidas Terminais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Expressão Gênica , Perfilação da Expressão Gênica , Genes Reporter , Humanos , Dados de Sequência Molecular , Recombinação Genética , Sítio de Iniciação de Transcrição , TransfecçãoRESUMO
Human endogenous retroviruses (HERVs) account for up to 9% of the human genome and include more than 800 elements related to betaretroviruses. While mouse mammary tumor virus (MMTV) is the accepted etiological agent of mammary tumors in mice, the role of retroviral elements in human breast cancer remains elusive. Here, we performed a comprehensive microarray-based analysis of overall retroviral transcriptional activities in 46 mammary gland tissue specimens representing pairs of nonmalignant and tumor samples from 23 patients. An analysis of nonmalignant tissue samples revealed a distinct, mammary gland-specific HERV expression profile that consists of 18 constitutively active HERV taxa. For corresponding tumor samples, a general trend toward lower levels of HERV transcription was observed, suggesting common regulatory mechanisms. In various subsets of patients, however, increased transcript levels of single class I HERV families (HERV-T, HERV-E, and HERV-F) and several class II families, including HML-6, were detected. An analysis of transcribed HML-6 sequences revealed either the activation of some or the increased activity of several proviral loci. No evidence for MMTV or human MMTV-like virus transcripts was found, indicating that transcriptionally active, MMTV analogous, exogenous viruses were not present in the breast cancer samples analyzed.
Assuntos
Neoplasias da Mama/virologia , Retrovirus Endógenos/genética , Transcrição Gênica , Sequência de Bases , Betaretrovirus/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: A significant proportion of the human genome is comprised of human endogenous retroviruses (HERVs). HERV transcripts are found in every human tissue. Expression of proviruses of the HERV-K(HML-2) family has been associated with development of human tumors, in particular germ cell tumors (GCT). Very little is known about transcriptional activity of individual HML-2 loci in human tissues, though. RESULTS: By employing private nucleotide differences between loci, we assigned approximately 1500 HML-2 cDNAs to individual HML-2 loci, identifying, in total, 23 transcriptionally active HML-2 proviruses. Several loci are active in various human tissue types. Transcription levels of some HML-2 loci appear higher than those of other loci. Several HML-2 Rec-encoding loci are expressed in GCT and non-GCT tissues. A provirus on chromosome 22q11.21 appears strongly upregulated in pathologic GCT tissues and may explain high HML-2 Gag protein levels in GCTs. Presence of Gag and Env antibodies in GCT patients is not correlated with activation of individual loci. HML-2 proviruses previously reported capable of forming an infectious HML-2 variant are transcriptionally active in germ cell tissue. Our study furthermore shows that Expressed Sequence Tag (EST) data are insufficient to describe transcriptional activity of HML-2 and other HERV loci in tissues of interest. CONCLUSION: Our, to date, largest-scale study reveals in greater detail expression patterns of individual HML-2 loci in human tissues of clinical interest. Moreover, large-scale, specialized studies are indicated to better comprehend transcriptional activity and regulation of HERVs. We thus emphasize the need for a specialized HERV Transcriptome Project.
Assuntos
Retrovirus Endógenos/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Provírus/genética , Sequência de Bases , Encéfalo/citologia , Encéfalo/metabolismo , Encéfalo/patologia , Mama/citologia , Mama/metabolismo , Mama/patologia , Linhagem Celular Tumoral , DNA Complementar/genética , Retrovirus Endógenos/metabolismo , Etiquetas de Sequências Expressas , Regulação Neoplásica da Expressão Gênica , Genoma Humano , Humanos , Neoplasias Embrionárias de Células Germinativas/genética , Provírus/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
Using a versatile and highly sensitive retroviral microarray, we have investigated particle preparations from three different human packaging cell lines harboring retroviral vector systems based on human immunodeficiency virus (HIV) and murine leukemia virus (MLV). 293Rev/Gag/Pol(i) cells inducibly express high titers of HIV-derived particles for packaging of HIV vectors. The Phoenix-GP and the Anjou 65 cell lines constitutively express MLV vector particles. We compared the transcription profiles of human endogenous retroviruses (HERVs) in all cell lines with the HERV sequences present in the particles. In addition, the influence of the transfected vector plasmid on the copackaging of HERVs was investigated. All particle preparations showed a defined pattern of endogenous retroviral sequences that differed from the cellular HERV expression pattern. HERV transcripts were observed in the particle preparations independent of whether a vector construct was coexpressed or not. Furthermore, our results suggest that particle preparations are frequently contaminated by cellular vesicles (exosomes) containing cellular RNAs including HERV transcripts.
Assuntos
Retrovirus Endógenos/genética , Vetores Genéticos , HIV-1/genética , Vírus da Leucemia Murina/genética , Animais , Linhagem Celular , Retrovirus Endógenos/isolamento & purificação , Perfilação da Expressão Gênica , Terapia Genética , HIV-1/fisiologia , Humanos , Vírus da Leucemia Murina/fisiologia , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Transfecção , Montagem de VírusRESUMO
BACKGROUND: The human genome comprises numerous human endogenous retroviruses (HERVs) that formed millions of years ago in ancestral species. A number of loci of the HERV-K(HML-2) family are evolutionarily much younger. A recent study suggested an infectious HERV-K(HML-2) variant in humans and other primates. Isolating such a variant from human individuals would be a significant finding for human biology. RESULTS: When investigating expression patterns of specific HML-2 proviruses we encountered HERV-K(HML-2) cDNA sequences without proviral homologues in the human genome, named HERV-KX, that could very well support recently suggested infectious HML-2 variants. However, detailed sequence analysis, using the software RECCO, suggested that HERV-KX sequences were produced by recombination, possibly arising ex vivo, between transcripts from different HML-2 proviral loci. CONCLUSION: As RT-PCR probably will be instrumental for isolating an infectious HERV-K(HML-2) variant, generation of "new" HERV-K(HML-2) sequences by ex vivo recombination seems inevitable. Further complicated by an unknown amount of allelic sequence variation in HERV-K(HML-2) proviruses, newly identified HERV-K(HML-2) variants should be interpreted very cautiously.
Assuntos
Retrovirus Endógenos/genética , Expressão Gênica , Genoma Humano , RNA Viral/biossíntese , Recombinação Genética , Sequência de Bases , DNA Complementar/química , DNA Complementar/genética , DNA Complementar/isolamento & purificação , DNA Viral/química , DNA Viral/genética , DNA Viral/isolamento & purificação , Humanos , Dados de Sequência Molecular , Filogenia , Provírus/genética , RNA Viral/genética , Análise de Sequência de DNA , Homologia de Sequência , SoftwareRESUMO
The overall impact of prion disease on gene expression is not well characterized. We have carried out a large-scale expression analysis of specific cell types commonly employed in studies of prion disease. Neuroblastoma cells (N2a) and hypothalamic neuronal cells (GT1) can be persistently infected with mouse-adapted scrapie prions, the latter demonstrating cytopathologic effects associated with prion neuropathology. Exploiting a mouse DNA microarray containing approximately 21,000 spotted cDNAs, we have identified several hundred differentially expressed sequences in the two cell lines when infected with prion strain RML. ScN2a and ScGT1 cells demonstrate unique changes in RNA profiles and both differ from the reported changes in human microglia and prion-infected brain studies albeit with some overlap. In addition, several of the identified changes are shared in common with other neurodegenerative diseases such as Alzheimer's disease. The results illustrate that prion infection differs in effect depending on cell type, which could be exploited for diagnostic or therapeutic intervention.
Assuntos
Neurônios/metabolismo , Príons/metabolismo , RNA/metabolismo , Animais , Linhagem Celular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Hipotálamo/metabolismo , Camundongos , Cadeias Leves de Miosina , Canal de Sódio Disparado por Voltagem NAV1.2 , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Príons/genética , Proteínas/genética , Proteínas/metabolismo , Canais de Sódio/genética , Canais de Sódio/metabolismoRESUMO
A DNA chip-based assay is described for parallel detection and identification of a wide variety of human and mammalian exogenous and endogenous retroviruses. The assay combines multiplex polymerase chain reaction (PCR) using fluorochrome-modified primer mixtures and chip hybridization. The microarray is composed of retrovirus-specific synthetic oligonucleotides as capture probes deposited on glass slides. The retrovirus chip can be used to assess the occurrence of reverse transcriptase (RT)-related transcripts in biological samples of human and mammalian origin. For example, distinct expression profiles of human endogenous retroviruses (HERV) were established reproducibly in human white blood cells, mammary gland and other human tissues. In particles released by human cells, packaging of specific HERV transcripts could be observed. Monitoring of human exogenous retroviruses (HIV, HTLV) and detection of putative cross-species transmissions (MLV, PERV) in human samples was efficient and reliable. The DNA chip should be an excellent tool for the detection of most relevant retroviruses and offers insights into differential retroviral activities and replication strategies. Furthermore, it could improve significantly the safety of gene therapy, tissue engineering, xenotransplantation and production of therapeutic polypeptides in cell culture.
Assuntos
Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA Viral/análise , Retroviridae/fisiologia , Animais , Técnicas de Cultura de Células , Primers do DNA , Retrovirus Endógenos/genética , Retrovirus Endógenos/fisiologia , Perfilação da Expressão Gênica , Humanos , Leucócitos Mononucleares/virologia , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase , Retroviridae/classificação , Retroviridae/genética , Retroviridae/isolamento & purificação , Transplante HeterólogoRESUMO
The pathomechanism of mycosis fungoides (MF), the most common type of primary cutaneous T-cell lymphomas (CTCLs) and a malignancy of non-recirculating, skin-resident T-cells, is unknown albeit underlying viral infections have been sought for. Human endogenous retroviruses (HERVs) are ancient retroviral sequences in the human genome and their transcription is often deregulated in cancers. We explored the transcriptional activity of HERV sequences in a total of 34 samples comprising MF and psoriasis skin lesions, as well as corresponding non-malignant skin using a retrovirus-specific microarray and quantitative RT-PCR. To identify active HERV-W loci, we cloned the HERV-W specific RT-PCR products, sequenced the cDNA clones and assigned the sequences to HERV-W loci. Finally, we used immunohistochemistry on MF patient and non-malignant inflammatory skin samples to confirm specific HERV-encoded protein expression. Firstly, a distinct, skin-specific transcription profile consisting of five constitutively active HERV groups was established. Although individual variability was common, HERV-W showed significantly increased transcription in MF lesions compared to clinically intact skin from the same patient. Predominantly transcribed HERV-W loci were found to be located in chromosomes 6q21 and 7q21.2, chromosomal regions typically altered in CTCL. Surprisingly, we also found the expression of 7q21.2/ERVWE1-encoded Syncytin-1 (Env) protein in MF biopsies and expression of Syncytin-1 was seen in malignant lymphocytes, especially in the epidermotropic ones, in 15 of 30 cases studied. Most importantly, no Syncytin-1 expression was detected in inflammatory dermatosis (Lichen ruber planus) with skin-homing, non-malignant T lymphocytes. The expression of ERVWE1 mRNA was further confirmed in 3/7 MF lesions analyzed. Our observations strengthen the association between activated HERVs and cancer. The study offers a new perspective into the pathogenesis of CTCL since we demonstrate that differences in HERV-W transcription levels between lesional MF and non-malignant skin are significant, and that ERVWE1-encoded Syncytin-1 is expressed in MF lymphoma cells.
Assuntos
Retrovirus Endógenos/genética , Produtos do Gene env/genética , Linfoma Cutâneo de Células T/genética , Proteínas da Gravidez/genética , Análise por Conglomerados , Feminino , Perfilação da Expressão Gênica , Produtos do Gene env/metabolismo , Loci Gênicos , Humanos , Líquen Plano/genética , Líquen Plano/metabolismo , Líquen Plano/patologia , Linfoma Cutâneo de Células T/patologia , Masculino , Micose Fungoide/genética , Proteínas da Gravidez/metabolismo , Psoríase/genética , RNA Mensageiro/genética , Pele/metabolismo , Pele/patologia , Transcrição GênicaRESUMO
Ku, a cellular complex required for human cell survival and involved in double strand break DNA repair and multiple other cellular processes, may modulate retroviral multiplication, although the precise mechanism through which it acts is still controversial. Recently, Ku was identified as a possible anti-human immunodeficiency virus type 1 (HIV-1) target in human cells, in two global approaches. Here we investigated the role of Ku on the HIV-1 replication cycle by analyzing the expression level of a panel of non-replicative lentiviral vectors expressing the green fluorescent protein in human colorectal carcinoma HCT 116 cells, stably or transiently depleted of Ku. We found that in this cellular model the depletion of Ku did not affect the efficiency of (pre-)integrative steps but decreased the early HIV-1 expression by acting at the transcriptional level. This negative effect was specific of the HIV-1 promoter, required the obligatory step of viral DNA integration and was reversed by transient depletion of p53. We also provided evidence on a direct binding of Ku to HIV-1 LTR in transduced cells. Ku not only promotes the early transcription from the HIV-1 promoter, but also limits the constitution of viral latency. Moreover, in the presence of a normal level of Ku, HIV-1 expression was gradually lost over time, likely due to the counter-selection of HIV-1-expressing cells. On the contrary, the reactivation of transgene expression from HIV-1 by means of trichostatin A- or tumor necrosis factor α-administration was enhanced under condition of Ku haplodepletion, suggesting a phenomenon of provirus latency. These observations plead in favor of the hypothesis that Ku has an impact on HIV-1 expression and latency at early- and mid-time after integration.
Assuntos
DNA Helicases/metabolismo , HIV-1/fisiologia , DNA Helicases/genética , Células HCT116 , Humanos , Autoantígeno Ku , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Integração Viral/fisiologia , Latência Viral/fisiologiaRESUMO
Human endogenous retroviruses (HERVs) have been associated with various neurological and neuropsychiatric disorders. Transcripts and proteins of at least three HERV groups, HERV-W, ERV9 and HERV-K(HML-2) have been detected repeatedly in brain samples or cerebrospinal fluid of patients with schizophrenia suggesting that alterations in HERV activity may play a role in etiopathogenesis. Current therapies otherwise include neuroleptics and/or antidepressants that may induce epigenetic alterations and thus influence HERV expression. To investigate the effects of these drugs on HERV transcriptional activity, HERV expression profiles of a broad range of human brain cell lines treated with valproic acid (VPA), haloperidol, risperidone, and clozapine were analyzed using a retrovirus-specific microarray and qRT-PCR. Investigation of 52 HERV subgroups revealed upregulation of several class I and class II HERV elements by VPA in a dose-dependent manner. The strongest effect was observed on HERV-W and ERV9 groups in the human glioblastoma cell lines SK-N-SH and SK-N-MC, respectively. The transcript level of HERV-K(HML-2) elements was not influenced. Transcription of HERV-W, ERV9 and HERV-K(HML-2) taxa was further quantified in postmortem brain samples of patients with schizophrenia, bipolar disorders and a healthy control group with regard to their medication. Patients with schizophrenia showed a significantly higher HERV-W transcription associated with VPA treatment. However in case of ERV9, enhanced transcript levels could not be explained solely by VPA treatment, since a slight increase was also found in untreated patients compared to healthy controls. HERV-K(HML-2) elements appeared to be upregulated in some patients with bipolar disorders independent from medication. In conclusion, these results suggest that antipsychotic medication may contribute to increased expression of distinct HERV taxa in patients with neuropsychiatric diseases.
Assuntos
Antipsicóticos/farmacologia , Encéfalo/patologia , Encéfalo/virologia , Retrovirus Endógenos/efeitos dos fármacos , Retrovirus Endógenos/genética , Transcrição Gênica/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Linhagem Celular Tumoral , Humanos , Mudanças Depois da Morte , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ácido Valproico/farmacologiaRESUMO
Human endogenous retroviruses (HERV) and related elements account for more than 8% of the human genome and significantly contribute to the human transcriptome by long terminal repeat (LTR) promoter activity. In this context, HERVs are thought to intervene in the expression of adjacent genes by providing regulatory sequences (cis-effect) or via noncoding RNA including natural antisense transcripts. To address the potential impact of HERV activity in urothelial carcinoma, we comparatively analyzed the HERV transcription profiles in paired samples of non-malignant urothelium and urothelial carcinoma derived from 13 patients with bladder cancer by means of a retrovirus-specific microarray (RetroArray). We established a characteristic HERV signature consisting of six ubiquitously active HERV subgroups (E4-1, HERV-Rb, ERV9, HERV-K-T47D, NMWV3, HERV-KC4). The transcription pattern is largely identical in human urothelial carcinoma, non-malignant urothelial tissue, four tumor-derived cell lines and in a non-malignant urothelial cell line (UROtsa). Quantitative reverse transcriptase PCR (qRT-PCR) of HERV-E4-1, HERV-K(HML-6) and HERV-T(S71-TK1) revealed a bias to lower HERV activity in carcinoma samples compared to non-malignant tissue. Determination of active HERV-E4-1 loci by cloning and sequencing revealed six HERV-E4-1 proviral loci that are differentially regulated in urothelial carcinoma cells and normal tissue. Two full-length HERV-E4-1 proviruses, HERV-Ec1 and HERV-Ec6, are located in antisense orientation in introns of the genes PLA2G4A and RNGTT, respectively. PLA2G4A encodes a cytosolic phospholipase A2 (cPLA2) that is dysregulated in many human tumors. PLA2G4A and HERV-Ec1 displayed reciprocal transcript levels in 7 of 11 urothelial carcinoma patients. Moreover, reciprocal shifts were observed after treatment of UROtsa cells with HERV-Ec1 and PLA2G4A-directed siRNAs or 5-aza-2'-deoxycytidine (aza-dC) pointing to an antagonistic regulation of PLA2G4A and HERV-Ec1 transcription in human urothelial cells. We suggest that transcription of HERV-Ec1 contributes to fine tuning of cPLA2 expression, thereby facilitating tumorigenesis.
Assuntos
Carcinoma/virologia , Retrovirus Endógenos/genética , Fosfolipases A2 do Grupo IV/genética , Transcrição Gênica , Neoplasias Urológicas/virologia , Carcinoma/genética , Carcinoma/patologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/metabolismo , Neoplasias Urológicas/genética , Neoplasias Urológicas/patologia , Urotélio/metabolismo , Urotélio/virologiaRESUMO
Human endogenous retroviruses (HERVs) represent approximately 8% of our genome. HERVs influence cellular gene expression and contribute to normal physiological processes such as cellular differentiation and morphogenesis. HERVs have also been associated with certain pathological conditions, including cancer and neurodegenerative diseases. As HTLV-1 causes adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and has been shown to modulate host gene expression mainly through the expression of the powerful Tax transactivator, herein we were interested in looking at the potential modulation capacity of HTLV-1 Tax on HERV expression. In order to evaluate the promoter activity of different HERV LTRs, pHERV-LTR-luc constructs were co-transfected in Jurkat T-cells with a Tax expression vector. Tax expression potently increased the LTR activity of HERV-W8 and HERV-H (MC16). In parallel, Jurkat cells were also stimulated with different T-cell-activating agents and HERV LTRs were observed to respond to different combination of Forskolin, bpV[pic] a protein tyrosine phosphatase inhibitor, and PMA. Transfection of expression vectors for different Tax mutants in Jurkat cells showed that several transcription factors including CREB appeared to be important for HERV-W8 LTR activation. Deletion mutants were derived from the HERV-W8 LTR and the region from -137 to -123 was found to be important for LTR response following Tax expression in Jurkat cells, while a different region was shown to be required in cells treated with activators. Our results thus demonstrated that HTLV-1 Tax activates several HERV LTRs. This raises the possibility that upregulated HERV expression could be involved in diseases associated with HTLV-1 infection.
Assuntos
Retrovirus Endógenos/genética , Produtos do Gene tax/metabolismo , Repetição Terminal Longa de HIV , Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Ativação Linfocitária , Linfócitos T/imunologia , Ativação Transcricional , Linhagem Celular , Retrovirus Endógenos/metabolismo , Regulação Viral da Expressão Gênica , Produtos do Gene tax/genética , Infecções por HTLV-I/imunologia , Vírus Linfotrópico T Tipo 1 Humano/genética , HumanosRESUMO
OBJECTIVE: In human astrocytes, restriction of HIV replication involves inhibition of HIV Rev activity. We previously identified a Rev-interacting human protein fragment (16.4.1) that can reduce Rev activity. The 16.4.1 sequence is contained in a group of highly similar host cell proteins, which we call the Risp family. Here we investigate whether the Risp family is connected to HIV replication in astrocytes. METHODS: Cell/tissue lysates were analyzed for Risp expression by western blot with various anti-Risp antibodies. The interaction of astrocytic Risp members with Rev was investigated by affinity chromatography. Astrocytes were transfected with expression plasmids containing cDNAs encoding full-length Risp or the isolated 16.4.1 region for Risp overexpression or with siRNAs designed for Risp knock-down. Rev activity was investigated with a Rev-reporter assay. RNA levels were quantified by real-time RT-PCR, HIV Gag levels by p24ELISA. RESULTS: Expression of the Risp family was demonstrated in human brain tissues and astrocytes. Astrocytes were shown to produce Risp family members that interact with Rev. Production of HIV Gag proteins and Rev-dependent RNAs in persistently infected astrocytes increased upon Risp knock-down and decreased upon Risp overexpression. Risp knock-down increased Rev activity and raised proportions of Rev proteins in the nucleus of astrocytes. CONCLUSION: Our results link the Risp family to restriction of HIV production and inhibition of Rev activity in astrocytes. We conclude that the Risp family represents a novel family of host factors that can control HIV replication and may be important for the containment of HIV infection in brain reservoirs.
Assuntos
Astrócitos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Infecções por HIV/metabolismo , HIV-1/fisiologia , Replicação Viral/fisiologia , Produtos do Gene rev do Vírus da Imunodeficiência Humana/metabolismo , Astrócitos/virologia , Western Blotting , Células Cultivadas , Proteínas de Ligação a DNA/genética , Infecções por HIV/genética , Infecções por HIV/virologia , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Produtos do Gene rev do Vírus da Imunodeficiência Humana/genéticaRESUMO
Human endogenous retroviruses (HERVs) are a major component of the human genome and an active part of the transcriptome. Some HERVs play vital biological roles, while others potentially contribute to diseases. Many HERVs are relatively new in the primate genome, having entered or expanded after the lineages leading to the platyrrhines (New World monkeys) and catarrhines (Old World monkeys and apes) separated. Most HERVs are active in at least some tissues, though tissue specificity is common for most elements. We analyzed multiple tissues from several Old World monkeys using retroviral pol-based DNA microarrays and quantitative PCR methods to determine their ERV expression profiles. The results demonstrate that while many ERVs are active in nonhuman primates, overall the tissue expression specificity is unique to each species. Most striking is that while the majority of HERVs analyzed in this study are expressed in human brain, almost none are expressed in Old World monkey brains or are only weakly expressed.
Assuntos
Cercopithecidae/virologia , Retrovirus Endógenos/genética , Perfilação da Expressão Gênica , Regulação Viral da Expressão Gênica , Animais , Feminino , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Transcrição Gênica/genéticaRESUMO
Human endogenous retroviruses (HERVs) arose in antiquity from stable integration into the human genome. The mechanism for activation of HERVs has not been fully elucidated. Toxoplasmosis, caused by Toxoplasma gondii, is a medically important parasitic infection with worldwide distribution. To search for a tentative link between toxoplasmosis and HERV activation, HERV expression profiles of human neuroepithelial SK-N-MC cells infected with T. gondii were analyzed. Increased transcriptional activity of class I, II, and III HERV elements was observed in infected cells, suggesting that T. gondii can influence the transcription of HERVs in neuronal cells.