RESUMO
In recent years, the contribution of viruses to cutaneous oncogenesis has steadily gained recognition. The archetype is human herpesvirus 8, which is well established as the causative agent in Kaposi sarcoma. Other viruses believed to play a role in nonmelanoma skin cancer include human papillomavirus and the recently described Merkel cell polyomavirus. We review the mechanisms by which these three viruses interact with the host cell, ultraviolet radiation and immunosuppression to result in carcinogenesis.
Assuntos
Neoplasias Cutâneas/virologia , Infecções Tumorais por Vírus/complicações , Carcinoma de Célula de Merkel/virologia , Carcinoma de Células Escamosas/virologia , Transformação Celular Neoplásica , Transformação Celular Viral , Previsões , Herpesvirus Humano 8 , Humanos , Tolerância Imunológica/fisiologia , Terapia de Imunossupressão/efeitos adversos , Infecções por Papillomavirus/complicações , Infecções por Polyomavirus/complicações , Sarcoma de Kaposi/virologia , Raios Ultravioleta/efeitos adversosRESUMO
Globally approximately 15% of all malignant tumors are caused by viruses and even a higher percentage is observed in organ transplant recipients (OTR). Here, nonmelanoma skin cancer (NMSC) is the most frequent malignancy, which correlates with cutaneous human papilloma virus (HPV) infection. In the present review, we reflect on some recent general concepts how tumor viruses can either act as direct or indirect carcinogens in the multistep process of carcinogenesis. Immunosuppressive drugs in OTR, which reduce the risk of organ rejection, could be critical in increasing the activation of persisting viral infections, thereby enhancing the probability to develop skin tumors. We discuss virus-induced transformation with special emphasis on the function of HPV as an indirect and HHV-8 as direct carcinogen in the development of NMSC and Kaposi sarcoma (KS), respectively. Moreover, we describe a rodent model system useful to examine future strategies in preventing skin tumor formation in immunosuppressed OTR.
Assuntos
Transplante de Órgãos/efeitos adversos , Neoplasias Cutâneas/etiologia , Infecções Tumorais por Vírus/etiologia , Animais , Carcinógenos , Modelos Animais de Doenças , Rejeição de Enxerto , Herpesvirus Humano 8/metabolismo , Humanos , Imunossupressores/efeitos adversos , Imunossupressores/uso terapêutico , Papillomaviridae/metabolismo , Sarcoma de Kaposi/complicações , Sarcoma de Kaposi/etiologia , Sarcoma de Kaposi/virologia , Neoplasias Cutâneas/complicações , Neoplasias Cutâneas/virologia , Infecções Tumorais por Vírus/complicaçõesRESUMO
Histone deacetylase (HDAC) inhibitors sodium butyrate and trichostatin A arrest human papillomavirus (HPV)-positive carcinoma cells in G1 to S transition of the cell cycle, which is paralleled by an up-regulation of the cyclin-dependent kinase inhibitors (CKIs) p21CIP1 and p27KIP1 as well as the complete loss of cdk2 activity. Although HPV expression was hitherto thought to be required to maintain a proliferative phenotype of these cells, cdk2 suppression is achieved even in the presence of ongoing viral transcription. While CKIs normally cannot exert their cdk2-inhibitory function in the presence of the viral oncoprotein E7, co-immunoprecipitation experiments revealed that E7 binding is prevented. Increase of p27KIP1 correlates with down-regulation of p45SKP2, a component of the ubiquitin-protein ligase SCF(SKP2) controlling the half-life of regulatory proteins during the cell cycle. HDAC inhibition also triggered an E7-dependent degradation of pRb, while the levels of E2F remained unaffected. The presence of free intracellular E2F and the concomitant up-regulation of CKIs during G1 arrest results in a 'conflicting growth situation', which finally renders the cells to undergo apoptosis. These data provide novel molecular insights into how the transforming potential of HPV can be bypassed and open new therapeutical perspectives for the treatment of cervical cancer.
Assuntos
Apoptose/efeitos dos fármacos , Quinases relacionadas a CDC2 e CDC28 , Transformação Celular Neoplásica , Proteínas de Ligação a DNA , Inibidores Enzimáticos/farmacologia , Inibidores de Histona Desacetilases , Proteínas Musculares , Proteínas Oncogênicas Virais/fisiologia , Neoplasias do Colo do Útero/tratamento farmacológico , Ciclo Celular/efeitos dos fármacos , Quinase 2 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Quinases Ciclina-Dependentes/antagonistas & inibidores , Ciclinas/análise , Ciclinas/fisiologia , Feminino , Células HeLa , Humanos , Proteínas dos Microfilamentos/fisiologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteína do Retinoblastoma/metabolismo , Neoplasias do Colo do Útero/virologiaRESUMO
AP-1 represents a transcription factor, which plays a pivotal role in initiating and maintaining the expression of human papillomavirus (HPV) oncoproteins E6 and E7 during HPV-linked carcinogenesis of the uterine cervix. AP-1 stands as a synonym for different proteins such as c-Jun, JunB, JunD, c-Fos, FosB as well as the Fos-related antigens Fra-1 and Fra-2, which can either homo- or heterodimerize to build up a functional transcription complex. AP-1 is mainly considered as a positive regulator, which binds to cognate DNA sequences within the viral upstream regulatory region. By using non-tumorigenic HeLa-fibroblast hybrids ('444'), their tumorigenic segregants ('CGL3') as well as HPV 18 positive HeLa cells as a experimental model system, evidence is provided that AP-1 composition differs considerably between these cell lines. In nuclear extracts obtained from non-tumorigenic cells, Jun-family members (in the order c-Jun>JunD>JunB) were mainly heterodimerized with Fra-1, a protein, known to be involved in the abrogation of AP-1 activity under certain experimental conditions. In contrast, Fra-1 concentration is low in extracts from tumorigenic cells. Conversely, c-Fos, the canonical dimerization partner of Jun proteins is expressed in substantial quantity in HeLa- and 'CGL3' cells, but it is completely absent in AP-1 complexes from non-tumorigenic '444' cells. Ectopical expression of c-fos under a heterologous promoter in '444'-cells induces tumorigenicity and a change of the Jun/Fra-1 ratio towards a constellation initially detected in 'CGL3'-and HeLa cells. Furthermore, conversion to tumorigenicity is accompanied with a resistance against TNF-alpha, a cytokine, capable to selectively suppress HPV 18 transcription in formerly non-malignant cells. These data propose a novel role for AP-1 as an essential component of an inter- and intracellular surveillance mechanism negatively controlling HPV transcription in non-tumorigenic cells.
Assuntos
Regulação Viral da Expressão Gênica/efeitos dos fármacos , Papillomaviridae/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Fator de Transcrição AP-1/metabolismo , Transcrição Gênica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Western Blotting , Divisão Celular , Eletroforese em Gel de Poliacrilamida , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Vetores Genéticos , Células HeLa , Humanos , Células Híbridas , Invasividade Neoplásica , Proteínas Proto-Oncogênicas c-fos/genética , Fator de Transcrição AP-1/genética , TransfecçãoRESUMO
The expression of the monocyte-chemoattractant-protein-1 (MCP-1) is closely linked with a non-tumorigenic phenotype in somatic cell hybrids made between the human papillomavirus type 18 (HPV 18) positive cervical carcinoma cell line HeLa and normal human fibroblasts. In contrast, MCP-1 transcription is absent in tumorigenic segregants derived from the same hybrids or in parental HeLa cells. Selectivity of MCP-1 transcription, which is regulated at the level of initiation of transcription, is mainly based on differences in the location and extension of DNAse I-hypersensitive regions (DHSR) at both ends of the gene. While TNF-alpha only moderately increases the sensitivity of pre-existing 5'-DHSRs, a 3'-end DHSR became strongly induced exclusively in non-malignant hybrids. DNA sequencing showed that the 3'-DHSR coincides with an additional AP-1 site located approximately 600 bp downstream of the polyadenylation site. Analyses of AP-1 composition revealed that MCP-1 is only expressed in those cells where jun-family members were mainly heterodimerized with the fos-related protein fra-1. In contrast, in tumorigenic cells the 1: 1 ratio between jun and fra-1 is disturbed and the MCP-1 gene is no longer expressed. Hence, alterations in the heterodimerization pattern of AP-1 and its selective accessibility to opened chromatin may represent a novel regulatory pathway in the regulation of chemokines in malignant and non-malignant HPV-positive cells.
Assuntos
Quimiocina CCL2/genética , Cromatina/fisiologia , Regulação da Expressão Gênica , Papillomaviridae/genética , Fator de Transcrição AP-1/metabolismo , Sequência de Bases , Linhagem Celular , Núcleo Celular/metabolismo , Mapeamento Cromossômico , Desoxirribonuclease I/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Dados de Sequência Molecular , RNA Mensageiro , Análise de Sequência de DNA , Fator de Transcrição AP-1/genética , Transcrição Gênica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
The Notch1 signaling pathway contributes to tumorigenesis by influencing differentiation, proliferation and apoptosis. Here, we demonstrate that inhibition of the Notch1 signaling pathway sensitizes glioblastoma cell lines and glioblastoma initiating cells to apoptosis induced by the death ligand TRAIL. This sensitization occurs through transcriptional upregulation of the death receptor 5 (DR5, TRAIL-R2). The increase in DR5 expression is abrogated by concomitant repression of the transcription factor Sp1, which directly binds to the DR5 promoter in the absence of Notch1 as revealed by chromatin immunoprecipitation. Consistent with these findings, Notch1 inhibition resulted in increased DR5 promoter activity, which was impaired by mutation of one out of two Sp1-binding sites within the proximal DR5 promoter. Moreover, we demonstrate that JNK signaling contributes to the regulation of DR5 expression by Notch1. Taken together, our results identify Notch1 as key driver for TRAIL resistance and suggest Notch1 as a promising target for anti-glioblastoma therapy.
Assuntos
Antineoplásicos/farmacologia , Receptor Notch1/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Fator de Transcrição Sp1/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Apoptose/efeitos dos fármacos , Caspase 8/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Receptor Notch1/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Transdução de Sinais , Regulação para CimaRESUMO
PURPOSE: Inhibitors of histone deacetylase, such as sodium butyrate, block proliferation of cervical carcinoma cells by inhibiting the G1 to S transition of the cell cycle. The derivative phenylbutyrate (PB), characterized by its higher pharmacological half-life, and its metabolite phenylacetate (PA) were tested for their growth-inhibitory function on cervical cancer cells differing in their HPV type, copy number, and integration sites. METHODS AND RESULTS: Using flow cytometric and Western blot analyses, we show that a 24-h incubation period with PB, but not with PA, was already sufficient to cause a dose-dependent growth arrest by increasing the G1 fraction with a concomitant drop in the S-phase. Consistent with the cell cycle block, only PB, but not PA, induced the cyclin-dependent kinase inhibitors p21(CIP1) and p27(KIP1). The inhibitory effect was not the result of a non-specific cytotoxic effect of PB, since cessation of cellular growth was already completely reversible 5 h after drug removal. CONCLUSIONS: Due to its broad growth inhibitory properties on different cervical carcinoma cells in vitro, and its low toxic profile demonstrated in preceding clinical studies, PB may serve as an effective drug in handling pre-cancerous lesions and cervical cancer in patients.
Assuntos
Antineoplásicos/farmacologia , Carcinoma/tratamento farmacológico , Papillomaviridae/classificação , Fenilbutiratos/farmacologia , Lesões Pré-Cancerosas/tratamento farmacológico , Neoplasias do Colo do Útero/tratamento farmacológico , Western Blotting , Carcinoma/virologia , Feminino , Citometria de Fluxo , Humanos , Infecções por Papillomavirus/complicações , Lesões Pré-Cancerosas/virologia , Infecções Tumorais por Vírus/complicações , Neoplasias do Colo do Útero/virologiaRESUMO
BACKGROUND: Immune response modifiers including imiquimod can be topically applied for the treatment of both genital warts and benign and malignant skin tumours (e.g. actinic keratosis). In an initial pilot study, we examined the response of spontaneously papillomavirus caused skin lesions (e.g. papillomas, keratoacanthomas) vs. chemically induced skin tumours of Mastomys coucha to imiquimod. METHODS: Fourteen spontaneously and 16 chemically [initiation with 7,12-dimethylbenzanthracene (DMBA) followed by repeated 12-O-Tetradecanoylphorbol-13-acetate (TPA) applications] induced skin tumours were treated two to three times per week with 5% imiquimod or placebo. RESULTS: Notably, significant higher regression or growth arrest rates of imiquimod treated animals were observed in chemically vs. spontaneously induced skin tumours [9/14 (64%) vs. 2/11 (18%), P < 0.05]. Regression or growth arrest of both skin tumours from placebo treated animals were similar (1/2 vs. 1/3). Tumour growth of nonresponders was lowest in imiquimod treated animals compared to the placebo or untreated group. CONCLUSIONS: Imiquimod was able to reduce skin tumour growth particularly in chemically induced lesions of Mastomys coucha. The different clearance rates are most likely due to lower differentiation status of the DMBA/TPA-induced tumours, allowing a better uptake of imiquimod than spontaneously induced papillomas or keratoacanthomas, known to be highly keratinized.
Assuntos
Aminoquinolinas/uso terapêutico , Antineoplásicos/uso terapêutico , Neoplasias Cutâneas/tratamento farmacológico , 9,10-Dimetil-1,2-benzantraceno , Animais , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos/métodos , Imiquimode , Murinae , Papiloma/tratamento farmacológico , Papiloma/patologia , Papiloma/virologia , Infecções por Papillomavirus/complicações , Neoplasias Cutâneas/induzido quimicamente , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/virologia , Acetato de Tetradecanoilforbol , Resultado do TratamentoRESUMO
Sedimentation analysis of isolated episomal bovine papillomavirus type 1 (BPV-1) nucleoprotein complexes in sucrose gradients and subsequent separation of the purified DNA in chloroquine gels revealed different classes of molecules, varying in their degree of superhelicity. Since torsionally stressed DNA favors the adoption of secondary structures, we employed the single-strand-specific S1 nuclease to detect such structural alterations in both naked DNA and native chromatin. Direct examination of nuclease digestion products in chloroquine gels showed that neither the naked DNA nor the BPV-1 nucleoprotein complexes in isolated nuclei were cleaved randomly by the enzyme. Instead, there was a strict dependence on nuclease susceptibility and the degree of supercoiling, strongly suggesting that the structural features detected by S1 nuclease are due to the occurrence of torsionally stressed viral chromatin. Mapping analysis using the indirect end-labeling method demonstrated an S1-nuclease cleavage site adjacent to 20 homopurine residues known to be hypersensitive to S1 attack. Furthermore, direct methylation experiments with viral chromatin in isolated nuclei indicated that only circular, covalently closed nucleoprotein complexes served as substrate, whereas linearized BPV-1 chromatin was not susceptible to exogenously added Hhal methylase. This observation raises the possibility that the modulation of topology in nucleosomally organized DNA might also play a role in eukaryotic DNA methylation.
Assuntos
Papillomavirus Bovino 1/genética , Cromatina/química , DNA Viral/química , Conformação de Ácido Nucleico , Nucleoproteínas/genética , Animais , Linhagem Celular , Núcleo Celular/química , Transformação Celular Viral , Centrifugação com Gradiente de Concentração , Metilação , Camundongos , Modelos Moleculares , Proteínas Virais/genéticaRESUMO
The association of topoisomerase II enzymes with papovavirus-chromatin has been described recently. We used cells infected with simian virus 40 (SV40) to investigate the in vivo effect of the topoisomerase II inhibitors nalidixic acid and novobiocin on the viral chromatin template. Blocking of topoisomerases inhibits DNA and RNA synthesis. The block with the inhibitors resulted in a conversion of a 95- to 75-svedberg chromatin to a 180- to 150-svedberg component accompanied with a variation in the topological linking number of the DNA. The condensed chromatin fractions (180-150 S) from inhibited cells resemble encapsidation intermediates and their DNA has a linking number as the DNA extracted from purified virions. Moreover, the virion DNA has a higher superhelical density compared to intracellular chromatin isolated from untreated cells and argues for a supercoiling activity in the cell.
Assuntos
Cromatina/análise , Replicação do DNA/efeitos dos fármacos , DNA Super-Helicoidal/análise , Vírus 40 dos Símios/genética , Inibidores da Topoisomerase II , Transcrição Gênica/efeitos dos fármacos , Animais , Centrifugação com Gradiente de Concentração , Microscopia Eletrônica , Ácido Nalidíxico/farmacologia , Novobiocina/farmacologia , Vírus 40 dos Símios/efeitos dos fármacosRESUMO
The investigation of papillomavirus chromatin has been hampered by the unavailability of a tissue culture system for vegetative growth of these viruses. We have used, therefore, bovine papillomavirus type 1-transformed hamster embryo fibroblasts containing 200 to 250 episomal genome equivalents per cell as a source of viral chromatin. The selectively isolated chromatin was shown to be slightly larger (80S) than the mature simian virus 40 chromatin, which was cosedimented in a sucrose density gradient. Both Fo I and Fo II were present in the bovine papillomavirus type 1 chromatin. A fast-sedimenting fraction, whose structure is still unknown, also contained oligomeric bovine papillomavirus type 1 DNA. By in situ DNase digestion of isolated nuclei and subsequent cleavage of the bovine papillomavirus type 1 DNA with various restriction endonucleases, a major DNase-hypersensitive region was detected in the chromatin. This region, comprising approximately 320 base pairs, is located between the relative physical map positions 0.88 and 0.92.
Assuntos
Papillomavirus Bovino 1/genética , Cromatina/análise , Endodesoxirribonucleases/farmacologia , Genes Virais , Papillomaviridae/genética , Plasmídeos , Animais , Linhagem Celular , Transformação Celular Viral , Cromatina/isolamento & purificação , Cricetinae , Enzimas de Restrição do DNA , Desoxirribonuclease IRESUMO
The origin of replication of bovine papilloma virus type 1 (BPV-1) has been determined by isolating replicative intermediates (RI) of BPV-transformed hamster embryo fibroblasts (HEF-BPV). These RI were treated with single cut restriction enzymes to determine the start-position (origin) of the extending replication eyes using electron microscopic techniques. 'Cairns'-type RI molecules were shown to contain one replication eye in monomeric as well as in dimeric molecules. The position of this eye was localized at 6940 +/- 5% bp in the physical map. In a second set of experiments BPV-1 DNA fragments cloned in pBR322 were tested for transient episomal replication. Transfected cells were harvested after increasing periods of time and screened for replication with isoschizomeric restriction enzymes to differentiate between input and replicated DNA. The part of the BPV genome harboring the replication origin spans the BPV ClaI-C restriction fragment corresponding to the non-coding region of the BPV genome and coincides with the DNase I-hypersensitive control region in the chromatin, isolated from transformed cells.
Assuntos
Papillomavirus Bovino 1/genética , Transformação Celular Neoplásica , Replicação do DNA , DNA Viral/genética , Papillomaviridae/genética , Animais , Sequência de Bases , Linhagem Celular , Enzimas de Restrição do DNA , DNA Viral/isolamento & purificação , Microscopia Eletrônica , Plasmídeos , TransfecçãoRESUMO
The transcription of human papillomavirus type 18 (HPV 18) is selectively suppressed in non-tumorigenic HeLa x fibroblast or HeLa x keratinocyte cell hybrids by 5-azacytidine. In contrast, viral gene expression is not influenced by 5-azacytidine in both tumorigenic hybrid segregants and in the parental HeLa cells. The suppression mechanism seems to operate at the level of initiation of transcription since nuclear run-on experiments show the absence of elongated nascent viral RNA, whereas the transcription of cellular reference genes remains unaffected. Down-regulation of HPV 18 mRNA correlates directly with cessation of cellular growth and can be abolished using the protein synthesis inhibitor cycloheximide. Furthermore human keratinocytes immortalized by HPV 16 but still retaining the non-tumorigenic phenotype reveal the same inhibitory effect on viral transcription after treatment with 5-azacytidine. These results support a model of a postulated intracellular control mechanism, directed against papillomavirus transcription, which can be induced by 5-azacytidine and appears to correlate with the presence of specific chromosomes in non-tumorigenic cells.
Assuntos
Azacitidina/farmacologia , Papillomaviridae/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Cicloeximida/farmacologia , DNA Viral/metabolismo , Humanos , Células Híbridas , Metilação , Papillomaviridae/genética , Papillomaviridae/metabolismoRESUMO
Episomal nucleoprotein complexes of bovine papillomavirus type 1 (BPV-1) in transformed cells were exposed to DNase I treatment to localize hypersensitive regions. Such regions, which are indicative for gene expression, were found within the noncoding part of the genome, coinciding with the origin of replication and the 5' ends of most of the early mRNAs. However, there were also regions of hypersensitivity within the structural genes. These intragenic perturbations of the chromatin structure coincide with regulatory sequences at the DNA level. One of these regions maps in close proximity to a Z-DNA antibody-binding site which is located near the putative BPV-1 enhancer sequence.
Assuntos
Papillomavirus Bovino 1/genética , Cromatina/análise , Genes Virais , Papillomaviridae/genética , Animais , Anticorpos Monoclonais , Linhagem Celular , Replicação do DNA , DNA Viral/biossíntese , DNA Viral/genética , DNA Viral/imunologia , Desoxirribonuclease I/farmacologia , Elementos Facilitadores Genéticos , Genes , Genes Reguladores , Camundongos , RNA Mensageiro/genética , RNA Viral/genéticaRESUMO
Integration of human papillomaviruses (HPVs) into the host genome is considered to be an early and important event in HPV-linked cervical carcinogenesis. Consequently, the viral DNA potentially becomes a target for cellular control mechanisms normally acting on the corresponding integration site. Besides resulting position effects, host-specific DNA methylation may play a functional role in HPV gene regulation. To elucidate the influence of such a kind of epigenetic modification on viral transcription, in vitro methylation studies on HPV-18 upstream regulatory region (URR)-controlled reporter plasmids were carried out. Selective methylation of the viral URR results in a down-regulation of the transcriptional activity, which can be attributed to nonrandom distribution of methyl-acceptor sites clustered within the constitutive enhancer region. In vivo competition experiments show that suppression is not directly mediated by steric hindrance of methyl residues with transcription factors, but rather is due to the association with methyl-CpG DNA-binding proteins. Using a restriction enzyme accessibility assay on both the DNA and chromatin levels, it could be demonstrated that, in vivo, extensively methylated viral DNA is nucleosomally organized, characteristic of transcriptionally inactive chromatin. These data suggest that DNA methylation is an important regulatory pathway in the modulation of HPV expression and as a consequence the proliferation rate of virus-infected cells.
Assuntos
DNA Viral/metabolismo , Regulação Viral da Expressão Gênica , Papillomaviridae/genética , Ligação Competitiva , Células Cultivadas , Cromatina/ultraestrutura , Clonagem Molecular , Proteínas de Ligação a DNA/metabolismo , Humanos , Metilação , Nucleossomos/metabolismo , Transcrição Gênica , Transfecção , Células Tumorais CultivadasRESUMO
Mapping analysis of the nucleosomal organization of integrated human papillomavirus type 18 (HPV18) DNA in HeLa cells reveals a very prominent nuclease-hypersensitive site within the viral noncoding regulatory region that harbors transcriptional control sequences and coincides with most of the 5' ends of the cytoplasmic early mRNAs. Moreover, it is shown that the conserved coamplified 5' cellular flank, common to all HPV18 copies in HeLa cells and located close to the virus-cell integration site, also contains several distinct hypersensitive sites, accessible not only to DNase I but also to restriction enzymes. Nuclear run-on analysis in isolated HeLa nuclei demonstrates the occurrence of nascent transcripts covering the cellular flank (the late and the viral noncoding regulatory region), indicating that a cellular promoter, marked by the hypersensitive sites, cooperates with the viral control region in generating the HPV18 transcripts. Cycloheximide treatment of HeLa cells results in a reduction of the cytoplasmic steady-state level of the 3.5-kb mRNA corresponding to the viral E6, E7, and parts of the E1 open reading frames (ORFs), whereas the expression of the 1.6-kb transcript corresponding only to the E6 and E7 ORFs is not influenced. Nuclear run-on analysis carried out after the cycloheximide chase reveals that the distribution of nascent transcripts spanning the viral E6, E7, and parts of the E1 region is substantially decreased. In contrast to this finding, an even, pronounced increase of the elongation rate of those transcripts, which cover the cellular flank, the late and the viral noncoding regulatory region was noted indicating a different involvement of regulatory factors in the activity of both promoters.
Assuntos
Cromatina , DNA Viral/genética , Papillomaviridae/genética , Sequências Reguladoras de Ácido Nucleico , Transcrição Gênica , Southern Blotting , Cicloeximida/farmacologia , Sondas de DNA , DNA Viral/efeitos dos fármacos , Desoxirribonuclease I , Genes Virais , Células HeLa , Humanos , RNA Nuclear/biossíntese , Mapeamento por Restrição , Transcrição Gênica/efeitos dos fármacosRESUMO
'Universal fuser' clones of a human papillomavirus type 16 positive cervical carcinoma cell line (SiHa) were established to study the effect of a non-tumorigenic fusion partner on the regulation of a stably integrated chloramphenicol acetyltransferase (CAT) gene controlled by the HPV18 upstream regulatory region under non-selective conditions. The CAT expressing cells were fused with both non-tumorigenic, spontaneously immortalized human keratinocytes (HaCaT) and non-modified SiHa cells. The resulting hybrids were characterized by restriction enzyme fragment length polymorphism analysis and flow cytometry. While the non-selectable, HPV18-driven indicator gene is constitutively expressed in SiHa cells, the CAT activity is extinguished in SiHa x HaCaT cells, but still present in SiHa x SiHa hybrids. Examination of the cytokeratin expression pattern reveals that the keratinocyte phenotype seems not only to be dominant in terms of the extinction of the HPV18 regulatory region but also by the conservation of most of the differentiation markers of the non-tumorigenic fusion partner. Cycloheximide treatment and intracellular competition experiments using the transient COS7 fusion-amplification technique are accompanied by the reactivation of the marker gene in previously CAT- SiHa x HaCaT hybrids. These data strongly suggest that trans-acting negative regulatory factors derived from the non-malignant human keratinocytes are responsible for the extinction phenomenon.
Assuntos
Carcinoma/genética , Regulação Viral da Expressão Gênica , Queratinócitos/fisiologia , Papillomaviridae/genética , Neoplasias do Colo do Útero/genética , Animais , Northern Blotting , Carcinoma/patologia , Fusão Celular , Chlorocebus aethiops , Eletroforese em Gel Bidimensional , Feminino , Humanos , Técnicas In Vitro , Queratinas/química , Queratinas/metabolismo , Polimorfismo de Fragmento de Restrição , RNA Mensageiro/genética , Sequências Reguladoras de Ácido Nucleico , Proteínas Repressoras/fisiologia , Transcrição Gênica , Transfecção , Neoplasias do Colo do Útero/patologiaRESUMO
Considering the involvement of a redox-regulatory pathway in the expression of human papillomaviruses (HPVs), HPV type 16 (HPV-16)-immortalized human keratinocytes were treated with the antioxidant pyrrolidine-dithiocarbamate (PDTC). PDTC induces elevated binding of the transcription factor AP-1 to its cognate recognition site within the viral regulatory region. Despite of increased AP-1 binding, normally indispensable for efficient HPV-16 transcription, viral gene expression was selectively suppressed at the level of initiation of transcription. Electrophoretic mobility supershift assays showed that the composition of the AP-1 complex, predominantly consisting of Jun homodimers in untreated cells, was altered. Irrespective of enhanced c-fos expression, c-jun was phosphorylated and became primarily heterodimerized with fra-1, which was also induced after PDTC incubation. Additionally, there was also an increased complex formation between c-jun and junB. Because both fra-1 and junB overexpression negatively interferes with c-jun/c-fos trans-activation of AP-1-responsive genes, our results suggest that the observed block in viral transcription is mainly the consequence of an antioxidant-induced reconstitution of the AP-1 transcription complex. Since expression of the c-jun/c-fos gene family is tightly regulated during cellular differentiation, defined reorganization of a central viral transcription factor may represent a novel mechanism controlling the transcription of pathogenic HPVs during keratinocyte differentiation and in the progression to cervical cancer.
Assuntos
Antioxidantes/farmacologia , Papillomaviridae/genética , Pirrolidinas/farmacologia , Tiocarbamatos/farmacologia , Fator de Transcrição AP-1/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/genética , Regulação para Baixo , Genes p53 , Células HeLa , Humanos , Queratinócitos/citologia , Fator de Transcrição AP-1/genética , Células Tumorais Cultivadas , Regulação para CimaRESUMO
The transcription factor AP-1 represents a central key element in the expression of human pathogenic papillomaviruses (HPV). We here propose a novel role for AP-1 as an essential component of an intracellular surveillance mechanism negatively controlling the proliferation of HPV-positive cells under in vivo conditions. The dissection of AP-1 composition in cervical-carcinoma cells revealed an inverse relationship between the Fos-related antigen Fra-1 and the tumorigenic phenotype. Cervical-carcinoma cell lines were either negative or expressed only low amounts of Fra-1 (jointly with c-Fos) within their AP-1 complexes. Somatic-cell hybridization technique was used to fuse different HPV-positive malignant cell lines. This resulted either in tumorigenic hybrids or in cells in which the malignant phenotype of the parental fusion partners was completely suppressed. The monitoring of AP-1 composition in electrophoretic mobility super-shift assays showed that the amount of Fra-1 was substantially increased within the AP-1 complex of non-malignant cells. In contrast, Fra-1 was even diminished in malignant hybrids, while c-Fos remained expressed. This correlation suggests that the concentration of Fra-1 within the AP-1 transcription complex might be an important marker for predicting the in vivo growth properties of HPV-positive cells.
Assuntos
Papillomaviridae/isolamento & purificação , Fator de Transcrição AP-1/análise , Neoplasias do Colo do Útero/etiologia , Feminino , Teste de Complementação Genética , Humanos , Proteínas Proto-Oncogênicas c-fos/análise , Células Tumorais Cultivadas , Neoplasias do Colo do Útero/virologiaRESUMO
To investigate the effect of tumor-associated macrophages on the in vivo growth properties of cervical carcinoma cells, tumorigenic human papilloma virus (HPV) 18-positive HeLa cells were transfected with an expression vector harboring the cDNA for the macrophage chemoattractant protein-1 JE (MCP-1). Although the endogenous gene is present and not structurally rearranged, its expression seems to be negatively affected by a still unknown mechanism. Inoculation of JE (MCP-1)-negative HeLa cells into nude mice led to rapidly growing tumors, where macrophage infiltration into the inner tumor mass was not detectable immunohistochemically. The activity that attracted mononuclear cells under both in vitro and in vivo condition was reconstituted in HeLa cells after transfection with the JE (MCP-1) expression vector. Heterotransplantation of those cells into immunocompromised animals resulted in significant growth retardation that was accompanied by a strong infiltration of macrophages. On the other hand, in vivo selection of nonmalignant hybrids made between wild-type HeLa cells and normal human fibroblasts in nude mice resulted in tumorigenic segregants 4 mo after inoculation into the animals. Monitoring JE (MCP-1) expression directly within those nodules, we found that transcription was either absent or only weakly detectable. Recultivation of JE (MCP-1)-positive tissue grafts under in vitro conditions revealed that the gene was only marginally inducible by tumor necrosis factor-alpha, a cytokine that normally induces a very strong activation of transcription in nontumorigenic cells. These findings suggest that functional JE (MCP-1) expression and in turn activated macrophages may play a pivotal role in controlling the proliferation rate of HPV-positive cells in vivo.