RESUMO
BACKGROUND: Sexual and reproductive health of women is a major public health problem in Nepal. Screening of cervico-vaginal clinical syndromes could potentially provide insights to the prevalence of sexually transmitted infections (STIs), which is not known. OBJECTIVE: To investigate the prevalence and factors associated with cervico-vaginal clinical syndromes in the socio-behavioral, medical, and public health context of Nepal. METHODS: Married women attending a clinical health camp held by the Nepal Fertility Care Centerin Khokana of Lalitpur district were recruited to the study. Seventy-three participants completed face-to-face questionnaires on basic socio-demographic, behavioral and reproductive health factors and underwent pelvic screening including clinical diagnosis of cervicitis and vaginitis. An univariate analysis was performed to determine if any of the self-reported variables were associated with abnormal pelvic examination (cervicitis and/or vaginitis). RESULTS: Vaginitis was diagnosed in three (4.4%) participants, while cervicitis was detected in 16 (23.5%) women. None of the participants reported any high risk sexual behavior. However, 28% of the participants reported having had STI diagnosis in the past and was associated (P<0.008) with abnormal pelvic results. Additionally, women with lower education were associated (p<0.02) with abnormal pelvic results. CONCLUSIONS: The high occurrence of cervicitis in our exploratory could indicate the high prevalence of STIs. However, while there could potentially be an unknown epidemic of STIs related to the clinical syndromes, point of care testing practice might help to understand the true prevalence of STIs in Nepali women and also reduce the health burden and consequences of over treatment based on the current symptomatic diagnosis.
Assuntos
Infecções Sexualmente Transmissíveis/epidemiologia , Cervicite Uterina/epidemiologia , Vaginite/epidemiologia , Adulto , Comportamento Contraceptivo , Feminino , Doenças dos Genitais Femininos/epidemiologia , Comportamentos Relacionados com a Saúde , Humanos , Pessoa de Meia-Idade , Nepal/epidemiologia , Prevalência , Saúde Reprodutiva , Fatores de Risco , Comportamento Sexual , Fatores Socioeconômicos , Síndrome , Saúde da MulherRESUMO
We have demonstrated previously that oncogenic human papillomaviruses (HPVs) induce basal cell tetrasomy in low-grade squamous intraepithelial lesions of the cervix. To identify HPV genes and growth conditions involved in this process, we analyzed: (a) organotypic raft cultures of primary human keratinocytes transfected with whole HPV-18 genomes; and (b) organotypic raft cultures acutely infected with recombinant retroviruses expressing the HPV-18 E6, E7, or E6/E7 genes from the differentiation-dependent HPV-18 enhancer-promoter. Cultures were examined for HPV DNA by in situ hybridization and for karyotype by interphase cytogenetics. Tetrasomy occurred in the suprabasal strata of raft cultures expressing E7 and E6/E7 but not in those expressing E6 alone or in a control culture. These data indicate that suprabasal tetrasomy occurs in association with expression of the E7 gene alone. Basal cell tetrasomy was additionally observed in the raft culture transfected with whole HPV-18 genomes, consistent with observations in low-grade squamous intraepithelial lesions. The distribution of tetrasomic cells in these raft cultures may reflect the involvement of additional viral genes or possibly differences in the pattern of viral oncogene and host gene expression.
Assuntos
Aberrações Cromossômicas , Proteínas de Ligação a DNA , Queratinócitos/virologia , Proteínas Oncogênicas Virais/genética , Papillomaviridae/genética , Células do Tecido Conjuntivo , Replicação do DNA , DNA Viral/biossíntese , DNA Viral/genética , Expressão Gênica , Humanos , Hibridização In Situ , Queratinócitos/fisiologia , Queratinócitos/ultraestrutura , Proteínas Oncogênicas Virais/biossíntese , TransfecçãoRESUMO
The human papillomavirus (HPV) E7 protein promotes S-phase reentry in a fraction of postmitotic, differentiated keratinocytes. Here we report that these cells contain an inherent mechanism that opposes E7-induced DNA replication. In organotypic raft cultures of primary human keratinocytes, neither cyclin E nor p21cip1 is detectable in situ. However, E7-transduced differentiated cells not in S phase accumulate abundant cyclin E and p21cip1. We show that normally p21cip1 protein is rapidly degraded by proteasomes. In the presence of E7 or E6/E7, p21cip1, cyclin E, and cyclin E2 proteins were all up-regulated. The accumulation of p21cip1 protein is a posttranscriptional event, and ectopic cyclin E expression was sufficient to trigger it. In constract, cdk2 and p27kip1 were abundant in normal differentiated cells and were not significantly affected by E7. Cyclin E, cdk2, and p21cip1 or p27kip1 formed complexes, and relatively little kinase activity was found associated with cyclin E or cdk2. In patient papillomas and E7 raft cultures, all p27kip1-positive cells were negative for bromodeoxyuridine (BrdU) incorporation, but only some also contained cyclin E and p21cip1. In contrast, all cyclin E-positive cells also contained p27kip1. When the expression of p21cip1 was reduced by rottlerin, a PKC delta inhibitor, p27kip1- and BrdU-positive cells remained unchanged. These observations show that high levels of endogenous p27kip1 can prevent E7-induced S-phase reentry. This inhibition then leads to the stabilization of cyclin E and p21cip1. Since efficient initiation of viral DNA replication requires cyclin E and cdk2, its inhibition accounts for heterogeneous viral activities in productively infected lesions.
Assuntos
Acetilcisteína/análogos & derivados , Ciclina E/fisiologia , Ciclinas/metabolismo , Proteínas de Ligação a DNA , Queratinócitos/metabolismo , Proteínas Oncogênicas Virais/fisiologia , Acetofenonas/farmacologia , Acetilcisteína/farmacologia , Animais , Benzopiranos/farmacologia , Ciclina E/análise , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/análise , Ciclinas/química , Cisteína Endopeptidases/fisiologia , Humanos , Recém-Nascido , Complexos Multienzimáticos/fisiologia , Complexo de Endopeptidases do Proteassoma , Proteína Quinase C/fisiologia , CoelhosRESUMO
Epidermodysplasia-verruciformis-associated human papilloma virus DNA has been detected in skin cancers, in premalignant and benign skin lesions, and in plucked hairs from immunocompetent and immunosuppressed patients. The role of epidermodysplasia-verruciformis-associated human papilloma virus in the pathogenesis of nonmelanoma skin cancer is still enigmatic. In organotypic cultures we investigated the effects of retroviral transduction of the E6 and E7 genes of epidermodysplasia-verruciformis-associated human papilloma virus types 5, 12, 15, 17, 20, and 38 on the growth and differentiation of human keratinocytes. Differentiation was disturbed to different degrees as revealed by histology and by the expression patterns of differentiation markers keratin 10 and small proline rich protein 2. Conversely, proliferating cell nuclear antigen was induced in some of the suprabasal, differentiated cells to varying extent. No unscheduled DNA synthesis was detected in these cells, however, as probed by 5'-bromo-2'-deoxyuridine incorporation. Most intriguingly, when the E6 and E7 genes of epidermodysplasia-verruciformis-associated human papilloma virus types 15 and 17 were transduced, a broadening layer of basal cells and an accelerated differentiation were observed. In addition, "papilla-like structures" comprising basal-like keratinocytes arose from the basal layer into the differentiated layers. These cells did not express the differentiation markers keratin 10 and small proline rich protein 2, but did actively replicate DNA. These observations warrant further research by using this system to elucidate the replication strategy of epidermodysplasia-verruciformis-associated human papilloma virus types in keratinocytes and to shed light on the role of these human papilloma virus types in the pathogenesis of skin cancer.
Assuntos
Epidermodisplasia Verruciforme/patologia , Epidermodisplasia Verruciforme/virologia , Queratinócitos/citologia , Queratinócitos/virologia , Proteínas Oncogênicas Virais/genética , Antimetabólitos/farmacocinética , Bromodesoxiuridina/farmacocinética , Diferenciação Celular , Divisão Celular/fisiologia , Células Epidérmicas , Regulação Viral da Expressão Gênica , Humanos , Hibridização In Situ , Técnicas de Cultura de Órgãos , Antígeno Nuclear de Célula em Proliferação/genética , RNA Viral/análise , Transdução GenéticaRESUMO
Papillomaviruses are ubiquitous pathogens of humans and other vertebrates. Productive infections lead to hyperproliferative lesions in squamous epithelia from diverse anatomic sites, both cutaneous and mucosal. The 7,900 bp double-stranded, circular DNA genome replicates as extrachromosomal plasmids in the nuclei of infected cells. The productive phase of the HPV infection takes place in differentiated, post-mitotic squamous keratinocytes. However, viral DNA replication requires the host cells to supply much of the replication machinery and substrates. Consequently, these viruses usurp the cellular control mechanisms via protein interactions and provide an excellent model system to investigate cellular processes. This paper summarize our investigations and insight into the virus-host interactions observed in productively infected patient lesions, in a model organotypic culture system of primary human keratinocytes transduced with viral genes, and in a cell-free viral DNA replication system with purified viral and host protein.
Assuntos
Replicação do DNA/fisiologia , DNA Viral/biossíntese , Papillomaviridae/fisiologia , Humanos , Papillomaviridae/crescimento & desenvolvimentoRESUMO
Infection by the oncogenic human papillomavirus (HPV) types 16 and type 18 can progress to cancers. Two well studied cervical carcinoma cell lines, SiHa and CaSki, contain two to four copies, or several hundred copies of integrated HPV-16, respectively. To define the chromosomal loci from which HPV mRNAs are transcribed in these cells, we have simultaneously visualized chromosomal DNA territories, HPV DNA or nascent HPV RNA sequences by using a highly sensitive in situ hybridization (T-FISH) technique employing deposition of fluorescent tyramides. We found that, in SiHa cells, nascent HPV RNAs co-localized with both integrated HPV copies on chromosome 13. Surprisingly, in CaSki cells, nascent HPV RNA only co-localized with one minor HPV DNA-positive locus on chromosome 14. The DNA signal intensity of this locus was consistent with a single to a few HPV intergrants. The tyramide methodologies described here provide an in-depth molecular cytological analyses applicable to research and diagnosis.
Assuntos
Amidas/química , DNA Viral/genética , Hibridização in Situ Fluorescente/métodos , Papillomaviridae/genética , Transcrição Gênica , RNA Viral/genéticaRESUMO
To investigate the prevalence and the natural history of human papillomavirus infections, we monitored HPV DNA shedding as a consequence of immunosuppression, with the expectation that latent viral infections would reactivate and become detectable. The study populations consisted of women who were in end-stage renal failure, those who ultimately received kidney transplantations, and those who had HIV/AIDS with various degrees of immune depression at entry. For each woman, cervico-vaginal lavage to sample viral shedding from the lower genital tract was performed at approximately six month intervals, and the cohorts have been followed since 1996. Nested polymerase chain reaction amplification of papillomavirus DNA using novel pairs of primers was followed by diagnostic restriction endonuclease cleavage or by DNA sequencing. This strategy is particularly capable of identifying single and multiple infections and determining the genotypes of any viruses present. Of the 225 women in the HIV cohort, 177 (79%) were HPV-positive and 111 (49%) shed from two up to eight different HPV types over the course of the survey. Thirty-five different mucosotropic HPV types, virtually all that have ever been described worldwide, were isolated from these 225 women, and nine additional new (provisional) types were discovered. As is always the case, HPV-6 was very common. However, all the other frequently detected HPV types (45, 52, 53, 54, 58, 74) were more prevalent than the types typically reported forthe general population (HPV-11, 16, 18, 31, 33, 35). Notably, the 14 members of the A3 phylogenetic subgroup (HPV-61, 62, 72, 81, 83, 84, and all the new types) were by far the most frequently observed viral types in the AIDS cohort. The HPV prevalence in the cohorts of kidney transplantation candidates and recipients was only slightly lower than that in the AIDS cohort. We conclude that HPV infections are extraordinarily common and are normally held in a sub-clinical state by functional immune systems, but can be reactivated by immunosuppressive conditions. The question of how so many distinct types persist in the human population and can be repeatedly isolated from specimens collected around the world raises complex issues concerning the nature of viral transmission, reproduction, shedding, and mutational drift. These molecular epidemiological observations signal the likelihood that HPV is part of the commensal microflora of human epithelia. Their prevalence elicits a caution that latent HPV DNA may be present in primary human epithelial tissues.
Assuntos
Modelos Biológicos , Papillomaviridae/fisiologia , Latência Viral , Estudos de Coortes , Feminino , Infecções por HIV/complicações , Humanos , Transplante de Rim , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/virologia , Esfregaço VaginalRESUMO
The human papillomavirus type 18 E7 protein subverts the pRb/E2F pathway to promote S-phase reentry by postmitotic, differentiated primary human keratinocytes in support of viral DNA amplification. We prepared a panel of HPV-18 E7 mutations in pRb binding or in casein kinase II (CKII) phosphorylation. Our results showed that the ability of E7 binding to pRb correlated with the activation of DNA polymerase alpha or cyclin E to various extents in differentiated keratinocytes of organotypic cultures but was insufficient to induce the proliferating cell nuclear antigen. Proteins mutated in the CKII recognition sequence or in one or both serine substrates (S32 and S34) bound pRb in vitro, but only those with negative charges at these two residues induced proliferating cell nuclear antigen effectively. Nevertheless, unscheduled cellular DNA synthesis occurred very inefficiently relative to the wild-type E7, if at all. Thus, both pRb binding and CKII phosphorylation of E7 are critical for activating cellular genes essential for S-phase entry.
Assuntos
Proteínas de Ligação a DNA , Queratinócitos/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Fase S , Motivos de Aminoácidos , Sequência de Aminoácidos , Caseína Quinase II , Linhagem Celular , Células Cultivadas , Ciclina E/metabolismo , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/metabolismo , DNA Polimerase I/metabolismo , Replicação do DNA , Humanos , Recém-Nascido , Queratinócitos/virologia , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas Virais/química , Proteínas Oncogênicas Virais/genética , Fosforilação , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteína p107 Retinoblastoma-Like , Alinhamento de Sequência , Ativação TranscricionalRESUMO
The E2 protein of papillomaviruses is a site-specific DNA binding nuclear protein. It functions as the primary replication origin recognition protein and assists in the assembly of the preinitiation complex. It also helps regulate transcription from the native viral promoter. The E2 protein consists of an amino-terminal (N) trans-acting domain, a central hinge (H) domain, and a carboxyl-terminal (C) protein dimerization and DNA binding domain. The hinge is highly divergent among papillomaviruses, and little is known about its functions. We fused the enhanced green fluorescent protein (GFP) with the full-length human papillomavirus type 11 (HPV-11) E2 protein and showed that the resultant fusion, called gfpE2, maintained transcription and replication functions of the wild-type protein and formed similar subnuclear foci. Using a series of GFP fusion proteins, we showed that the hinge conferred strong nuclear localization, whereas the N or C domain was present in both cytoplasm and nucleus. Biochemical fractionation demonstrated that the N domain and hinge, but not the C domain, independently associated with the nuclear matrix. Mutational analyses showed that a cluster of basic amino acid residues, which is conserved among many mucosotropic papillomaviruses, was required for efficient nuclear localization and nuclear matrix association. This mutation no longer repressed the HPV-11 upstream regulatory region-controlled reporter expression. However, a very small fraction of this mutant colocalized with E1 in the nucleus, perhaps by a piggyback mechanism, and was able to support transient replication. We propose that the hinge is critical for the diverse regulatory functions of the HPV-11 E2 protein during mRNA transcription and viral DNA replication.
Assuntos
Núcleo Celular/metabolismo , Sinais de Localização Nuclear , Matriz Nuclear/metabolismo , Papillomaviridae/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Animais , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Regulação Viral da Expressão Gênica , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Papillomaviridae/genética , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Frações Subcelulares , Transcrição Gênica , Transfecção , Células Tumorais Cultivadas , Proteínas Virais/genéticaRESUMO
Human papillomaviral (HPV) origin-containing plasmids replicate efficiently in human 293 cells or cell extracts in the presence of HPV origin-recognition protein E2 and replication initiation protein E1, whereas cervical carcinoma-derived, HPV-18-positive HeLa cells or cell extracts support HPV DNA replication poorly. We recently showed that HPV-11 E1 interacts with cyclin/cyclin-dependent kinase (cdk) complexes through an RXL motif and is a substrate for these kinases. E1 mutations in this motif or in candidate cdk phosphorylation sites are impaired in replication, suggesting a role for cdks in HPV replication. We now demonstrate that one limiting activity in HeLa cells is cyclin E/CDK2. Purified cyclin E/CDK2 or cyclin E/CDK3 complex, but not other cdks, partially complemented HeLa cell extracts. Cyclin E/CDK2 expression vectors also enhanced transient HPV replication in HeLa cells. HeLa cell-derived HPV-18 E1 protein is truncated at the carboxyl terminus but can associate with cyclin E/CDK2. This truncated E1 was replication-incompetent and inhibited cell-free HPV replication. These results indicate that HeLa cells are phenotypically limiting in cyclin E/CDK2 for efficient HPV replication, most likely due to sequestration by the endogenous, defective HPV-18 E1 protein. Further analyses of the regulation of HPV E1 and HPV replication by cyclin E may shed light on the roles of cyclin E/CDK2 in cellular DNA replication.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Ciclina E/metabolismo , Papillomaviridae/metabolismo , Replicação Viral , Extratos Celulares , Quinase 2 Dependente de Ciclina , Quinases Ciclina-Dependentes , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Humanos , Proteínas Oncogênicas Virais/metabolismo , Plasmídeos , Proteínas Serina-Treonina Quinases , Origem de Replicação , Vírus 40 dos Símios/genética , Transfecção , Proteínas Virais/metabolismoRESUMO
The function of several known oncogenes is restricted to specific host cells in vitro, suggesting that new genes may be identified by using alternate hosts. RK3E cells exhibit characteristics of epithelia and are susceptible to transformation by the G protein RAS and the zinc finger protein GLI. Expression cloning identified the major transforming activities in squamous cell carcinoma cell lines as c-MYC and the zinc finger protein gut-enriched Kruppel-like factor (GKLF)/epithelial zinc finger. In oral squamous epithelium, GKLF expression was detected in the upper, differentiating cell layers. In dysplastic epithelium, expression was prominently increased and was detected diffusely throughout the entire epithelium, indicating that GKLF is misexpressed in the basal compartment early during tumor progression. The results demonstrate transformation of epithelioid cells to be a sensitive and specific assay for oncogenes activated during tumorigenesis in vivo, and identify GKLF as an oncogene that may function as a regulator of proliferation or differentiation in epithelia.
Assuntos
Proteínas E1A de Adenovirus/genética , Proteínas de Ligação a DNA , Vetores Genéticos , Vírus da Leucemia Murina de Moloney , Proteínas Proto-Oncogênicas c-myc/genética , Fatores de Transcrição/genética , Dedos de Zinco , Animais , Sequência de Bases , Northern Blotting , Linhagem Celular , Transformação Celular Viral , Clonagem Molecular , DNA Complementar , Células Epiteliais , Dosagem de Genes , Expressão Gênica , Biblioteca Gênica , Humanos , Rim/citologia , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like , Dados de Sequência Molecular , Proteínas Oncogênicas/genética , Oncogenes , Ratos , Transativadores , Células Tumorais Cultivadas , Proteína GLI1 em Dedos de ZincoRESUMO
Human papillomavirus (HPV) gene expression in squamous epithelia is differentiation dependent in benign patient lesions and in organotypic raft cultures of primary human keratinocytes (PHKs). Using the lacZ reporter in raft cultures, we previously showed that this transcriptional regulation of the HPV type 11 (HPV-11) enhancer-promoter located in the upstream regulatory region (URR) appears to have resulted from coordination between the transcription transactivators AP1, Oct1, and Sp1 in differentiated upper strata and the repressor C/EBP in proliferating basal cells. We report here that trichostatin A, a specific inhibitor of histone deacetylase, dramatically stimulated reporter gene activity from the wild-type HPV-11 URR or the C/EBP mutation in PHKs grown in undifferentiated submerged cultures. In epithelial raft cultures, up-regulation occurred predominantly in basal and parabasal strata; this effect was promoter specific, as expression of the lacZ reporter gene driven by the murine leukemia virus long terminal repeat (LTR), the keratin 14 promoter, or the involucrin promoter was not altered, nor was expression of endogenous keratin 10 and profilaggrin affected. However, the responses were not cell type or species specific, as identical results were observed for both HPV-11 URR-lacZ and LTR-lacZ in murine retrovirus producer cell lines of fibroblast origin.
Assuntos
Inibidores Enzimáticos/farmacologia , Inibidores de Histona Desacetilases , Ácidos Hidroxâmicos/farmacologia , Queratinócitos/virologia , Papillomaviridae/genética , Regiões Promotoras Genéticas , Proteínas Estimuladoras de Ligação a CCAAT , Diferenciação Celular , Células Cultivadas , Proteínas de Ligação a DNA/fisiologia , Histona Desacetilases/fisiologia , Humanos , Proteínas Nucleares/fisiologia , Papillomaviridae/efeitos dos fármacos , Sequências Repetidas Terminais , Regulação para Cima , Proteínas do Envelope Viral/genéticaRESUMO
Human papillomaviruses (HPVs) such as types 6 and 11 can establish lifelong infections in airway epithelial cells in patients, and long-term infection can lead to pulmonary involvement and death. The mechanisms underlying this persistence depend on both the transcriptional activity of the viral enhancers and promoters and the ability of this virus to maintain its double-stranded circular DNA genome in infected tissues. We investigated the transcription and replication properties of HPV sequence elements and protein products in a human airway cell line. We showed that incorporation of the upstream regulatory region and cotransfection with expression vectors of two virus-encoded proteins, E1 and E2, conferred approximately 5,000-fold stimulation of reporter gene expression. Transient plasmid replication in transfected human airway cells and lungs of FVB/N-C57BL/6 mice was demonstrated by a modified transient replication assay. These results have important implications for viral pathogenesis in airway cells and the potential of HPV-based replicons for gene transfer into airway epithelium.
Assuntos
Papillomaviridae/fisiologia , Plasmídeos , Traqueia/metabolismo , Transcrição Gênica/fisiologia , Animais , Sequência de Bases , Primers do DNA , Técnicas de Transferência de Genes , Genes Reporter , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Traqueia/citologia , Traqueia/virologia , Regulação para CimaRESUMO
Productive infections by human papillomaviruses (HPVs) occur only in differentiated keratinocytes in squamous epithelia in which the HPV E7 protein reactivates the host DNA replication machinery to support viral DNA replication. In a fraction of the differentiated keratinocytes, E7 also posttranscriptionally induces p21Cip1, which is distributed in a mutually exclusive manner with unscheduled cellular DNA synthesis. In this study, double immunofluorescence labeling unexpectedly revealed that E7 caused a concordant accumulation of both cyclin E and p21Cip1 to high levels in patient papillomas and in organotypic cultures of primary human keratinocytes. The induction of cyclin E is mutually exclusive with unscheduled cellular DNA synthesis or abundant viral DNA. These novel virus-host interactions in differentiated keratinocytes are in contrast to previous observations made in submerged proliferating cultures, in which HPV E7 induces cyclin E and overcomes p21Cip1 inhibition of S-phase entry. We propose that an appropriately timed induction of cyclin E/cyclin-dependent kinase 2 by HPV E7 in postmitotic cells enables S-phase reentry and HPV DNA amplification, whereas prematurely induced cyclin E stabilizes p21Cip1 protein, which then inhibits cyclin E/cyclin-dependent kinase 2. Consequently, cyclin E and p21Cip1 both fail to turn over, and DNA synthesis does not occur.
Assuntos
Ciclina E/metabolismo , Ciclinas/metabolismo , Queratinócitos/metabolismo , Inibidores da Síntese de Ácido Nucleico/metabolismo , Proteínas Oncogênicas Virais/farmacologia , Gânglios da Base/metabolismo , Bromodesoxiuridina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21 , Replicação do DNA , Epitélio/anatomia & histologia , Epitélio/metabolismo , Feminino , Imunofluorescência , Humanos , Imuno-Histoquímica , Hibridização In Situ , Recém-Nascido , Neoplasias Laríngeas/imunologia , Masculino , Papiloma/imunologia , Papillomaviridae , Proteínas E7 de Papillomavirus , Pênis/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Retroviridae/metabolismo , Neoplasias Vaginais/metabolismo , Replicação ViralRESUMO
In benign squamous lesions and in organotypic epithelial cultures, the human papillomavirus (HPV) E6 and E7 genes are transcriptionally up-regulated in differentiated, spinous keratinocytes. We previously identified sequence elements in the enhancer-promoter regions of HPV types 18 and 11 important for this promoter regulation by using the bacterial LacZ reporter gene in stratified raft cultures of primary human keratinocytes (PHKs) or in submerged, proliferating cultures acutely transduced with recombinant retroviruses. Notably, mutations in the promoter-proximal Sp1, Oct1, and AP1 sites each significantly reduce reporter activity in differentiated cells, indicating that the bound factors are transcription transactivators. In the present study, we performed further mutagenesis on distal motifs in the HPV-11 regulatory region in PHKs in submerged and raft cultures. Mutations in an AP2-like site, three individual NF-1 sites, or five NF-1 sites collectively reduced promoter activity slightly in differentiated cells. A mutation in a putative glucocorticoid response element had no discernable effect in the presence or the absence of dexamethasone. However, mutations in a C/EBP binding site, especially the distal site, strikingly up-regulated reporter gene expression, particularly in basal and lower spinous cells, implicating bound protein as a transcription repressor. Collectively, these results demonstrate that the overall differentiation-dependent papillomaviral gene expression observed in vivo and in vitro involves promoter repression in the lower strata and activation in the upper, differentiated strata.
Assuntos
Regulação Viral da Expressão Gênica , Queratinócitos/virologia , Papillomaviridae/genética , Regiões Promotoras Genéticas , Sítios de Ligação , Proteínas Estimuladoras de Ligação a CCAAT , Diferenciação Celular , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Genes Reporter , Células HeLa , Humanos , Mutagênese , Fatores de Transcrição NFI , Proteínas Nucleares/metabolismo , Fator de Transcrição AP-2 , Fatores de Transcrição/metabolismo , Regulação para Cima , beta-Galactosidase/genéticaRESUMO
Productive infection by human papillomaviruses (HPVs) occurs only in differentiated squamous epithelial cells in papillomas, condylomata, and low grade intraepithelial neoplasias. Host DNA replication is reactivated in a fraction of terminally differentiated keratinocytes in benign human lesions and in organotypic raft cultures of primary human keratinocytes (PHKs) transduced with retroviruses expressing HPV-18 E7 oncogene from its native upstream regulatory region (URR). Thus the natural function of E7 protein, which inactivates pRB family proteins, is to induce host genes essential to support viral DNA replication in post-mitotic cells. Using this raft culture model system, we show that HPV-18 URR-E7 induces the universal cyclin-dependent kinase inhibitor p21cip1 protein in a fraction of differentiated PHKs. Induction is mediated by posttranscriptional mechanisms independent of p53. Double immunofluorescence studies demonstrate that, in raft cultures and in laryngeal papillomas, p21cip1 induction and reactivated host DNA synthesis take place in a mutually exclusive manner in PCNA-positive, differentiated keratinocytes. We suggest that p21cip1 induction effectively blocks unscheduled DNA synthesis reactivated by E7. These results begin to explain the inverse relationship between p21cip1 induction and HPV activities previously observed in a spectrum of benign lesions regardless of HPV types present.
Assuntos
Ciclinas/biossíntese , Proteínas de Ligação a DNA , DNA/biossíntese , Queratinócitos/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Papillomaviridae/metabolismo , Transcrição Gênica , Antígenos de Neoplasias , Bromodesoxiuridina , Diferenciação Celular , Fracionamento Celular , Criança , Inibidor de Quinase Dependente de Ciclina p21 , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Neoplasias Laríngeas , Proteínas Oncogênicas Virais/genética , Papiloma , Antígeno Nuclear de Célula em Proliferação/análise , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismoRESUMO
Human papillomavirus replication initiator, the E1 helicase, binds weakly to the origin of DNA replication. Purified human chaperone proteins Hsp70 and Hsp40 (HDJ-1 and HDJ-2) independently and additively enhanced E1 binding to the origin. The interaction between E1 and Hsp70 was transient and required ATP hydrolysis, whereas Hsp40 bound to E1 directly and remained in the complex. A peptide of 20 residues spanning the HPD loop and helix II of the J domain of YDJ-1 also stimulated E1 binding to the origin, alone or in combination with Hsp70 or Hsp40. A mutated peptide (H34Q) had a reduced activity, while an adjacent or an overlapping peptide had no effect. Neither Hsp70 nor the J peptide altered the E1/DNA ratio in the complex. Electron microscopy showed that E1 mainly bound to DNA as a hexamer. In the presence of Hsp40, E1 primarily bound to DNA as a dihexamer. Preincubation of chaperones with viral E1 and template shortened the lag time and increased replication in a cell-free system. Since two helicases are essential for bidirectional replication of human papillomavirus DNA, these results demonstrate that, as in prokaryotes, chaperones play an important role in the assembly of preinitiation complexes on the origin.
Assuntos
Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/metabolismo , Origem de Replicação , Proteínas Virais/metabolismo , Proteínas de Transporte/metabolismo , Sistema Livre de Células , DNA Viral/biossíntese , Proteínas de Choque Térmico HSP40 , Humanos , Conformação de Ácido Nucleico , PapillomaviridaeRESUMO
We have established cell-free replication for the human papillomavirus type 18 (HPV-18) origin of replication (ori)-containing DNA by using purified HPV-18 E1 and E2 gene products expressed as fusion proteins in Escherichia coli. The transcription factor YY1 has been shown to regulate RNA transcription by binding to a sequence overlapping the putative E1 protein binding site in the HPV-18 ori. We show that exogenously added YY1 fusion protein inhibited HPV-18 ori replication. Cotransfection of YY1 expression vectors also inhibited transient replication in 293 cells. However, inhibition did not appear to be mediated by binding to its cognate site in the ori as YY1 also inhibited the replication of the HPV-11 ori, which does not have a known or suspected YY1 binding site. Moreover, inhibition was not alleviated by the inclusion of YY1 binding oligonucleotides in the replication reaction mixtures. Rather, we demonstrated a direct interaction between purified fusion E2 protein and fusion YY1 protein by the pull-down assay and a partial restoration of replication activity by an elevated E2 protein concentration. These results suggest that YY1 can inhibit HPV ori replication by interfering with E2 protein functions.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Papillomaviridae/fisiologia , Fatores de Transcrição/fisiologia , Replicação Viral/fisiologia , Sistema Livre de Células , Fatores de Ligação de DNA Eritroide Específicos , Regulação Viral da Expressão Gênica , Humanos , Plasmídeos , Transfecção , Fator de Transcrição YY1RESUMO
Infections of the genital and oral epithelia by human papillomaviruses cause condylomata, papillomas, and squamous intraepithelial neoplasms, some of which can progress to invasive cancers. We describe an induction of p21cip1/WAF1/sdi1 protein in a fraction of the spinous cells in benign lesions and in cervical intraepithelial neoplasia grades I and II. The induction appears to be post-transcriptional and independent of p53. p21cip1 antigen-positive cells were sporadic in cervical intraepithelial neoplasia III and rare and focal in carcinomas. In contrast, p21cip1 protein was below or at the threshold of detection in the differentiated cells of normal squamous epithelia from different body sites despite an up-regulation of p21cip1 RNA. In cervical intraepithelial neoplasias from patients who were also positive for the human immunodeficiency virus, there was an additional increase in p21cip1 RNA in the upper spinous cells without concomitant p21cip1 protein induction. A consistent inverse relationship was observed between the p21cip1 protein induction and abundant human papillomavirus DNA and RNAs. We propose that p21cip1 protein induction is a novel host response that inhibits viral DNA replication and thus prevents elevated viral transcription. This hypothesis can partly account for the heterogeneity and the differentiation-dependent viral activities commonly observed in benign human papillomavirus lesions.