RESUMO
BACKGROUND: Cutaneous squamous cell carcinoma (cSCC) is one of the most common malignancies in fair-skinned populations worldwide and its incidence is increasing. Despite previous observations of multiple genetic abnormalities in cSCC, the oncogenic process remains elusive. The purpose of this study was to elucidate key molecular events associated with progression from premalignant actinic keratoses (AKs) to invasive cSCC by transcriptome profiling. METHODS: We combined laser capture microdissection with the Affymetrix HGU133 Plus 2.0 microarrays to profile 30 cSCC and 10 AKs. RESULTS: We identified a core set of 196 genes that are differentially expressed between AK and cSCC, and are enriched for processes including epidermal differentiation, cell migration, cell-cycle regulation and metabolism. Gene set enrichment analysis highlighted a key role for the mitogen activated protein kinase (MAPK) pathway in cSCC compared with AK. Furthermore, the histological subtype of the tumour was shown to influence the expression profile. CONCLUSION: These data indicate that the MAPK pathway may be pivotal to the transition from AK to cSCC, thus representing a potential target for cSCC prevention. In addition, transcriptome differences identified between cSCC subtypes have important implications for future development of targeted therapies for this malignancy.
Assuntos
Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Ceratose Actínica/genética , Ceratose Actínica/patologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas/irrigação sanguínea , Adesão Celular/genética , Ciclo Celular/genética , Diferenciação Celular/genética , Movimento Celular/genética , Proliferação de Células , Progressão da Doença , Epiderme/patologia , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Quinases Ativadas por Mitógeno/genética , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Neoplasias Cutâneas/irrigação sanguínea , TranscriptomaRESUMO
The incidence of skin cancer is increasing worldwide and cutaneous squamous cell carcinomas (SCCs) are associated with considerable morbidity and mortality, particularly in immunosuppressed individuals ('carcinomatous catastrophy'). Yet, molecular mechanisms are still insufficiently understood. Besides ultraviolet (UV)-indicative mutations, chromosomal aberrations are prominent. As telomeres are essential in preserving chromosome integrity, and telomere erosion as well as aberrant spatial telomere distribution contribute to genomic instability, we first established telomere length profiles across the whole tissue and identified normal skin (10/30) harboring discrete epidermal sites (stem cell territories) of evenly short telomeres. Precancerous actinic keratoses (AKs) (17) and SCCs (27) expressed two telomere phenotypes: (i) tissue-wide evenly short to intermediate and (ii) longer and tissue-wide heterogeneous telomere lengths, suggesting two modes of initiation, with one likely to originate in the epidermal stem cells. Although tumor histotype, location, patient gender or age failed to distinguish the two SCC telomere phenotypes, as did telomerase activity, we found a trend for a higher degree of aberrant p53 and cyclin D1 expression with long/heterogeneous telomeres. In addition, we established an association for the short/homogeneous telomeres with a simpler and the heterogeneous telomeres with a more complex karyotype correlating also with distinct chromosomal changes. SCCs (13) from renal transplant recipients displayed the same telomere dichotomy, suggesting that both telomere subtypes contribute to 'carcinomatous catastrophy' under immunosuppression by selecting for a common set (3, 9p and 17q) and subtype-specific aberrations (e.g., 6p gain, 13q loss). As a second mechanism of telomere-dependent genomic instability, we investigated changes in telomere distribution with its most severe form of telomeric aggregates (TAs). We identified a telomere length-independent but progression-dependent increase in cells with small telomere associations in AKs (17/17) and additional TAs in SCCs (24/32), basal cell carcinomas (30/31) and malignant melanomas (15/15), and provide evidence for a reactive oxygen species-dependent mechanism in this UV-induced telomere organization-dependent genomic instability.
Assuntos
Carcinoma de Células Escamosas/classificação , Carcinoma de Células Escamosas/genética , Neoplasias Cutâneas/classificação , Neoplasias Cutâneas/genética , Telômero/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas/enzimologia , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Criança , Ciclina D1/metabolismo , Progressão da Doença , Instabilidade Genômica/efeitos da radiação , Humanos , Masculino , Melanoma/enzimologia , Melanoma/genética , Melanoma/patologia , Pessoa de Meia-Idade , Neoplasias Cutâneas/enzimologia , Neoplasias Cutâneas/patologia , Telomerase/metabolismo , Telômero/efeitos da radiação , Proteína Supressora de Tumor p53/metabolismo , Raios Ultravioleta , Adulto JovemRESUMO
BACKGROUND: Novel prognostic biomarkers and therapeutic strategies are urgently required for malignant melanoma. Ecto-5-prime-nucleotidase (NT5E; CD73) overexpression has been reported in several human cancers. The mechanism(s) underlying deregulated expression and the clinical consequences of changes in expression are not known. METHODS: We used RT-PCR, qPCR, methylation-specific PCR and pyrosequencing to analyse expression and regulation of NT5E in malignant melanoma cell lines and primary and metastatic melanomas. RESULTS: NT5E is subject to epigenetic regulation in melanoma. NT5E mRNA is downregulated by methylation-dependent transcriptional silencing in the melanoma cell lines SKMel2, SKMel23, WM35, Mel501, Mel505 and C81-61 and expression is reactivated by azacytidine. In contrast, the CpG island is unmethylated and the gene expressed in cultured normal melanocytes. In clinical cases of melanoma, methylation in the NT5E CpG island occurs in both primary and metastatic melanomas and correlates with transcriptional downregulation of NT5E mRNA. Relapse with metastatic disease, particularly to the visceral sites and brain, is more common in primary melanomas lacking NT5E methylation. Primary melanomas with methylation in NT5E show limited metastatic potential or more commonly metastasise predominantly to nodal sites rather than viscera and brain (P=0.01). CONCLUSION: Deregulation of NT5E expression in melanoma occurs via epigenetic changes in the NT5E CpG island. Confirmation of our results in larger clinical series would support the candidacy of NT5E as a clinical biomarker in melanoma, which could be applied in both primary and relapsed disease. Inhibition of NT5E may have therapeutic potential in melanoma, particularly in patients with more aggressive disease metastatic to viscera or the brain.
Assuntos
5'-Nucleotidase/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/secundário , Epigênese Genética/genética , Melanoma/genética , Melanoma/patologia , 5'-Nucleotidase/metabolismo , Linhagem Celular Tumoral , Ilhas de CpG/genética , Metilação de DNA/genética , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Inativação Gênica , Humanos , Especificidade de Órgãos , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Gênica/genéticaRESUMO
AIMS: The association between squamous cell carcinoma of the head and neck (HNSCC) and infection with human papilloma viruses (HPV) has created considerable interest. Rates of primary oropharyngeal cancers have shown increasing incidence and declining age at presentation over the last decade, believed to relate to infection with oncogenic or high-risk subtypes of HPV (HR-HPV). HR-HPV-associated tumours have reportedly improved outcomes when compared with HPV-negative cancers. Within the UK, rates of HR-HPV in HNSCC have not yet been reported. MATERIALS AND METHODS: We analysed consecutive retrospective cases of oropharyngeal cancer presenting between 2004 and 2007. RESULTS: Thirty-seven per cent of 83 oropharyngeal tumours stained positively for p16(INK4A), a marker of HPV infection (73% tonsillar cancers being p16 (INK4A) positive, 30% tongue and 43% floor of mouth tumours). HPV16 DNA was demonstrated in 75% p16 (INK4A) cases. Despite being more advanced with higher T-stage and nodal burden at presentation, HR-HPV-associated HNSCC showed significantly improved rates of disease-free and overall survival, in particular with improved rates of response to radical radiotherapy. CONCLUSION: HPV16 infection seems to be a clinically significant cause of oropharyngeal HNSCC in the UK and the collection of national data should be supported.
Assuntos
Carcinoma de Células Escamosas/virologia , Neoplasias de Cabeça e Pescoço/virologia , Infecções por Papillomavirus/complicações , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/radioterapia , DNA Viral/análise , Intervalo Livre de Doença , Feminino , Neoplasias de Cabeça e Pescoço/mortalidade , Neoplasias de Cabeça e Pescoço/radioterapia , Papillomavirus Humano 16 , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Infecções por Papillomavirus/epidemiologia , Reação em Cadeia da Polimerase , Reino UnidoRESUMO
Identifying therapeutic targets for cancer treatment relies on consistent changes within particular types or sub-types of malignancy. The ability to define either consistent changes or sub-types of malignancy is often masked by tumor heterogeneity. To elucidate therapeutic targets in cutaneous squamous cell carcinoma (cSCC), the most frequent skin neoplasm with malignant potential, we have developed an integrated approach to gene expression profiling beginning with primary keratinocytes in culture. Candidate drivers of cSCC development were derived by first defining a set of in vitro cancer genes and then comparing their expression in a range of clinical data sets containing normal skin, cSCC and the benign hyper-proliferative condition psoriasis. A small interfering RNA (siRNA) screen of the resulting 21 upregulated genes has yielded targets capable of reducing xenograft tumor volume in vivo. Small-molecule inhibitors for one target, Polo-like kinase-1 (PLK1), are already in clinical trials for other malignancies, and our data show efficacy in cSCC. Another target, C20orf20, is identified as being overexpressed in cSCC, and siRNA-mediated knockdown induces apoptosis in vitro and reduces tumor growth in vivo. Thus, our approach has shown established and uncharacterized drivers of tumorigenesis with potent efficacy as therapeutic targets for the treatment of cSCC.
Assuntos
Carcinoma de Células Escamosas/genética , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Neoplasias Cutâneas/genética , Apoptose , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/terapia , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Histona Acetiltransferases , Humanos , Queratinócitos/metabolismo , Terapia de Alvo Molecular , Proteínas Nucleares , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA Interferente Pequeno , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/terapia , Células Tumorais Cultivadas , Quinase 1 Polo-LikeRESUMO
BACKGROUND: The incidence of human papillomavirus-associated vulval neoplasia is increasing worldwide; yet the associated genetic changes remain poorly understood. METHODS: We have used single-nucleotide polymorphism microarray analysis to perform the first high-resolution investigation of genome-wide allelic imbalance in vulval neoplasia. Our sample series comprised 21 high-grade vulval intraepithelial neoplasia and 6 vulval squamous cell carcinomas, with paired non-lesional samples used to adjust for normal copy number variation. RESULTS: Overall the most common recurrent aberrations were gains at 1p and 20, with the most frequent deletions observed at 2q, 3p and 10. Copy-neutral loss of heterozygosity at 6p was a recurrent event in vulval intraepithelial neoplasia. The pattern of genetic alterations differed from the characteristic changes we previously identified in cutaneous squamous cell carcinomas. Vulval neoplasia samples did not exhibit gain at 5p, a frequent recurrent aberration in a series of cervical tumours analysed elsewhere using an identical protocol. CONCLUSION: This series of 27 vulval samples comprises the largest systematic genome-wide analysis of vulval neoplasia performed to date. Despite shared papillomavirus status and regional proximity, our data suggest that the frequency of certain genetic alterations may differ in vulval and cervical tumours.
Assuntos
Alphapapillomavirus/fisiologia , Carcinoma in Situ/genética , Carcinoma de Células Escamosas/genética , Perfilação da Expressão Gênica/métodos , Neoplasias Vulvares/genética , Carcinoma in Situ/etiologia , Carcinoma in Situ/virologia , Carcinoma de Células Escamosas/etiologia , Carcinoma de Células Escamosas/virologia , Aberrações Cromossômicas , DNA Viral/análise , Feminino , Regulação Neoplásica da Expressão Gênica , Genômica/métodos , Papillomavirus Humano 16/fisiologia , Humanos , Perda de Heterozigosidade , Análise de Sequência com Séries de Oligonucleotídeos , Polimorfismo de Nucleotídeo Único , Neoplasias do Colo do Útero/genética , Neoplasias Vulvares/etiologia , Neoplasias Vulvares/virologia , Displasia do Colo do Útero/genéticaRESUMO
BACKGROUND: Human papillomaviruses (HPVs) of the beta genus (beta-PV), especially HPV5 and HPV36, are proposed to play a pathogenic role in psoriasis, but many previous studies have failed to control for potential confounders, including treatment. OBJECTIVES: To re-examine the relationship between beta-PV and psoriasis addressing limitations present in previous studies and analyse intra-patient concordance for carriage of HPV. METHODS: Plucked eyebrow hairs and forearm skin scrapes were collected from 20 newly diagnosed, previously untreated adult patients with psoriasis and 23 normal controls. A combination of type-specific and degenerate polymerase chain reaction methods was used to achieve comprehensive HPV DNA detection. RESULTS: The prevalence of HPV in hair and skin from psoriasis patients was higher than in controls (83.3% vs. 46.7%, respectively, P < 0.03 corrected for age and clustering). HPV5 or HPV36 were not over-represented. The profile of diverse beta-PV types was comparable in the two groups. Intra-patient concordance for HPV DNA at separate sites was high (P < 0.00001). CONCLUSIONS: Our data do not support a specific causal role for HPV5 or HPV36 in psoriasis, but suggest that psoriatic skin may be more permissive for viral presence than normal skin. High intra-patient concordance for specific HPV types at separate sites, together with the ubiquity of HPV DNA in normal human skin, suggests that an individual becomes colonized with a particular beta-PV profile presumably to the exclusion of other types. To what extent this HPV profile is then causal in the subsequent development of hyperproliferative skin disease is unknown.
Assuntos
Betapapillomavirus , Portador Sadio/virologia , Doenças do Cabelo/virologia , Infecções por Papillomavirus/complicações , Psoríase/virologia , Neoplasias Cutâneas/virologia , Adulto , Idoso , Estudos de Casos e Controles , DNA Viral/genética , Sobrancelhas/metabolismo , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Pele/metabolismoRESUMO
AIM: To investigate the role of human papillomavirus (HPV) in the development of bladder transitional cell carcinoma (TCC). METHODS: Seventy eight paraffin wax embedded TCC samples were tested for the presence of HPV by two methods. First, immunohistochemistry was carried out using a polyclonal antibody capable of detecting the capsid protein of all known papillomaviruses. The second method was a consensus GP5+/6+ primer mediated polymerase chain reaction (PCR) technique, with the products analysed by both agarose gel electrophoresis and an enzyme immunoassay using type specific oligonucleotide probes for 10 different mucosal genotypes. To exclude false negative results because of the poor quality of DNA extracted from paraffin wax embedded samples, the series was extended to include 20 further blocks for which the corresponding snap frozen unfixed tissue was available. RESULTS: The two methods produced contrasting results, with 47 of the 78 samples positive for HPV antigen and none positive for HPV DNA. HPV DNA was not detected in the 20 additional paraffin wax embedded TCCs or in the 20 paired unfixed samples. In contrast, HPV DNA was amplified by PCR from all six of the paraffin wax embedded cervical carcinoma and anogenital wart control samples. CONCLUSION: The disparity between the two sets of results is probably caused by false positives resulting from the non-specificity of the polyclonal antibody used for immunohistochemistry. These results suggest that HPV is unlikely to play an aetiological role in the development of bladder TCC.
Assuntos
Carcinoma de Células de Transição/virologia , Infecções por Papillomavirus/complicações , Neoplasias da Bexiga Urinária/virologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antivirais/análise , Especificidade de Anticorpos/imunologia , Antígenos Virais/análise , Proteínas do Capsídeo/análise , Carcinoma de Células de Transição/imunologia , DNA Viral/análise , Humanos , Imuno-Histoquímica/métodos , Pessoa de Meia-Idade , Papillomaviridae/imunologia , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/imunologia , Reação em Cadeia da Polimerase/métodos , Neoplasias da Bexiga Urinária/imunologiaRESUMO
A unique series of epidermal cell lines representing different stages of malignant transformation were spontaneously derived from a single adult immunosuppressed individual. Four keratinocyte lines (PM1-4) established from forehead skin are here compared with 4 squamous cell carcinoma (SCC) lines (MET1-4) derived respectively from a primary cutaneous tumour, two local recurrences and a distant metastasis of invasive SCC. Despite altered growth properties, the PM lines retained many features of normal keratinocytes including keratin phenotype, differentiation capacity and non-tumorigenicity in athymic mice. In contrast, from early passage, the MET lines displayed markedly reduced growth requirements, abnormal differentiation, aberrant K18 expression and tumorigenicity in athymic mice. The abnormal keratin profile of individual MET lines closely reflected the keratin phenotype of the tumour of origin. Although unusual HPV types were identified in the original tissue, there was no evidence of persistent virus within any cell line and it appears that HPV is not critical for maintenance of the immortal phenotype. The PM lines were distinctly different from invasive SCC lines and are likely to be useful for studies of mutations important early in neoplastic progression. The SCC series represent primary, recurrent and metastatic carcinoma. Availability of such a series from the same individual will facilitate genetic analysis of the malignant process.
Assuntos
Transformação Celular Neoplásica , Epiderme/patologia , Queratinócitos/patologia , Estadiamento de Neoplasias , Adaptação Fisiológica , Adulto , Animais , Testes de Carcinogenicidade , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/secundário , Carcinoma de Células Escamosas/virologia , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Linhagem Celular , Face , Humanos , Queratinócitos/metabolismo , Queratinócitos/fisiologia , Queratinócitos/virologia , Queratinas/metabolismo , Masculino , Camundongos , Camundongos Nus , Transplante de Neoplasias , Papillomaviridae/isolamento & purificação , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/virologiaRESUMO
An aetiological role has been proposed for human papillomavirus (HPV) in skin carcinogenesis within the immunosuppressed patient population. To examine this possibility, we have focused on an HPV type that, to date, has been identified only in the cutaneous lesions of renal transplant recipients despite a high degree of sequence homology with other HPVs commonly found in warts in the general population. We report that the non-coding region of this virus, HPV type 77, contains a consensus binding site for the tumour suppressor protein p53, and we show by gel-retardation analysis that this sequence does indeed bind p53. Furthermore, using reporter gene assays, we demonstrate that HPV77 promoter activity is stimulated by UV radiation and that this response is mediated through the p53 binding site. This is the first report of a p53-dependent positive response element within a viral genome. Our results suggest a possible novel mechanism by which specific types of HPV might act as cofactors with UV radiation in cutaneous transformation.
Assuntos
Sequência Consenso , Papillomaviridae/genética , Regiões Promotoras Genéticas , Neoplasias Cutâneas/virologia , Proteína Supressora de Tumor p53/metabolismo , Adulto , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Células Cultivadas , Cocarcinogênese , DNA Viral , Regulação Viral da Expressão Gênica , Genes Reporter , Humanos , Dados de Sequência Molecular , Neoplasias Induzidas por Radiação/virologia , Homologia de Sequência do Ácido Nucleico , Transcrição Gênica , Proteína Supressora de Tumor p53/química , Raios UltravioletaRESUMO
Gene expression of human papillomaviruses (HPV) is tightly controlled by cellular factors and by the virally encoded E2 protein through binding to distinct sites within the regulatory noncoding region. While for the high-risk genital papillomaviruses a single promoter drives the expression of all early genes, a second promoter present in the E6 open reading frame of the low-risk HPV type 6 (HPV6) would allow an independent regulation of E6 and E7 oncogene expression. In this report, we provide the first evidence that E2 regulates both early promoters of HPV6 separately and we show that promoter usage as well as E2 regulation is cell type dependent. Among the different epithelial cell lines tested, only RTS3b cells allowed an expression pattern similar to that observed in naturally infected benign condylomas. While the E6 promoter was repressed by E2 to 50% of its basal activity, the E7 promoter was simultaneously stimulated up to fivefold. Activation of the E7 promoter was mediated predominantly by the binding of E2 to the most promoter-distal E2 binding site. Repression of the E6 promoter depended on the presence of two intact promoter-proximal binding sites. Mutation of both of these repressor binding sites reversed the effect of E2 on the E6 promoter from repression to activation. In contrast, in HT3 cells we observed an E2-mediated activation of the E6 promoter in the context of the wild-type noncoding region. This indicated that repression of the E6 promoter by binding of E2 to both promoter-proximal binding sites did not function in the cellular environment provided by HT3 cells. These data suggest that the separate regulation of the E6 and E7 promoters of HPV6 is mediated through successive occupation of binding sites with different affinities for E2 depending on the intracellular concentration of E2 and on the cellular environment provided by the infected cell.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação Viral da Expressão Gênica , Proteínas Oncogênicas Virais/genética , Papillomaviridae/genética , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Proteínas Virais/metabolismo , Animais , Sítios de Ligação , Células COS , Linhagem Celular , Humanos , Mutação , Ativação Transcricional , Células Tumorais CultivadasRESUMO
Epidermodysplasia verruciformis (EV) is a rare inherited condition in which there is widespread infection with human papillomavirus (HPV). Patients have a high risk of developing squamous cell carcinoma and Bowen's disease on sun-exposed sites. We describe a Jamaican man with the typical clinical and histopathological features of EV.HPV 8, 24 and a subtype of HPV 38, along with a novel HPV sequence most closely related to HPV 9 have been detected in his skin lesions. Although skin tumours are rare in black patients with EV and he has lived in a temperate climate most of his life, several of the lesions showed bowenoid atypia and he is at risk of developing invasive cutaneous malignancies.
Assuntos
Epidermodisplasia Verruciforme/virologia , Papillomaviridae/classificação , Infecções por Papillomavirus/complicações , Infecções Tumorais por Vírus/complicações , Epidermodisplasia Verruciforme/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Papillomaviridae/isolamento & purificação , Lesões Pré-Cancerosas/virologia , Neoplasias Cutâneas/virologiaRESUMO
Hybridization sites of an rDNA probe coding for the 5.8S, 18S and 26S genes were detected on the chromosomes of sugarcane and a related genus, Erianthus, using fluorescence in situ hybridization. One unpaired and five paired hybridization sites were detected in a Saccharum spp. hybrid. A first introgression hybrid (I1) between Saccharum officinarum and Saccharum spontaneum had seven pairs of hybridization sites. A clone of Erianthus arundinaceus showed six hybridization sites in somatic tissue.
Assuntos
Cromossomos/genética , DNA Ribossômico/genética , Hibridização in Situ Fluorescente , Região Organizadora do Nucléolo/genética , Plantas/genética , Austrália , Sondas de DNA , Corantes Fluorescentes , Indóis , Meiose , MetáfaseRESUMO
Twenty-three wheat/alien addition or substitution lines were screened using restriction fragment length polymorphisms for the presence or absence of 4/5 and 4/7 reciprocal translocations in the alien chromosomes. Such translocations have previously been identified in wheat and rye. Group 4 and group 5 Aegilops umbellulata, Triticum urartu, and Thinopyrum bessarabicum chromosomes were found to carry 4/5 translocations. Evidence for a 4/7 translocation was also found in Secale montanum. The presence of the 4/5 translocations in T. urartu indicates that the translocation predates the polyploidization of wheat. The implications of these results are discussed.
RESUMO
Cultured wart keratinocytes have previously been described as having a limited proclivity to maintain episomal human papillomavirus (HPV) DNA. To investigate the nature of episome loss, and to determine keratinocyte-specific factors involved in it, we have examined a large series of anogenital and oral wart keratinocyte cultures, tracing episomal copy number with culture passage. We report that a higher proportion of oral wart keratinocytes maintain episomal HPV DNA to first passage (70% compared with 37% of anogenital wart cultures) when screened by slot blot hybridization. Furthermore, oral wart keratinocytes maintain episomal HPV copy through a greater number of passages (60% positive at passage 2 compared with 2% of anogenital wart cultures) with this technique. When anogenital cultures were examined at first passage for HPV infection by PCR with Southern blot hybridization of the product, a further 34% were found to be HPV-positive. To determine the mechanism of loss of episomal DNA from these cultures we examined the relative HPV copy number in cells which adhered to the culture vessel following passage and in those which did not adhere. Those which remained floating contained episomal HPV at high copy number whereas those which adhered were negative by slot blotting. The adherent cells, however, remained positive by PCR at subsequent passages until senescence. We conclude that a subpopulation of HPV-positive keratinocytes may be maintained in culture through serial passage until senescence.
Assuntos
Condiloma Acuminado/virologia , DNA Viral/análise , Queratinócitos/virologia , Papillomaviridae/crescimento & desenvolvimento , Southern Blotting , Células Cultivadas , Humanos , Queratinócitos/citologia , Hibridização de Ácido Nucleico , Papillomaviridae/isolamento & purificação , Reação em Cadeia da Polimerase , Inoculações SeriadasRESUMO
Genomic in-situ hybridization (GISH) was used to determine the amount of wheat-rye chromosome pairing in wheat (Triticum aestivum) x rye (Secale cereale) hybrids having chromosome 5B present, absent, or replaced by an extra dose of chromosome 5D. The levels of overall chromosome pairing were similar to those reported earlier but the levels of wheat-rye pairing were higher than earlier determinations using C-banding. Significant differences in chromosome pairing were found between the three genotypes studied. Both of the chromosome-5B-deficient hybrid genotypes showed much higher pairing than the euploid wheat hybrid. However, the 5B-deficient hybrid carrying an extra chromosome 5D had significantly less wheat-rye pairing than the simple 5B-deficient genotype, indicating the presence of a suppressing factor on chromosome 5D. Non-homologous/non-homoeologous chromosome pairing was observed in all three hybrid genotypes. The value of GISH for assessing the level of wheat-alien chromosome pairing in wheat/alien hybrids and the effectiveness of wheat genotypes that affect homoeologous chromosome pairing is demonstrated.
RESUMO
Renal allograft recipients are at greatly increased risk of developing squamous cell carcinomas. As these frequently arise adjacent to areas of multiple viral warts, we have investigated a possible role for human papillomavirus in malignant transformation within this population. We established, as primary cultures, keratinocytes from 24 lesions of varying degrees of squamous atypia from 9 patients. Ten of 14 cultures screened for the presence of episomal human papillomavirus DNA were positive using a mixed probe for cutaneous human papillomaviruses, although episomal copy was universally lost with continued passage. Three cultures, 2 of which were derived from malignant tissue and 1 from a benign lesion, were positive when screened with a probe for potentially oncogenic human papillomavirus DNAs 5 or 8. Both positive cultures of malignant origin exhibited extended lifespan and have been briefly characterized by morphology and growth requirements.
Assuntos
Transformação Celular Neoplásica , Transplante de Rim/patologia , Papillomaviridae/patogenicidade , Infecções por Papillomavirus/patologia , Complicações Pós-Operatórias/patologia , Neoplasias Cutâneas/patologia , Pele/patologia , Infecções Tumorais por Vírus/patologia , Biópsia , Southern Blotting , Divisão Celular , Células Cultivadas , Técnicas de Cultura/métodos , DNA Viral/análise , Seguimentos , Humanos , Cariotipagem , Queratinócitos/citologia , Queratinócitos/microbiologia , Queratinócitos/patologia , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/microbiologia , Neoplasias Cutâneas/microbiologia , Fatores de Tempo , Transplante Homólogo/patologia , Infecções Tumorais por Vírus/microbiologiaRESUMO
Ten random amplified polymorphic DNA (RAPD) markers specific to chromosome 5E(b) of Thinopyrum bessarabicum were detected. Genomic in situ hybridization and standard cytological observations revealed that six of the markers are located on the 5E(b) short arm and four are located on the 5E(b) long arm. These RAPD markers have been used to confirm the identity of putative 5E(b) (5A) and 5E(b) (5D) substitution individuals. The potential of RAPDs for the detection of wheat/alien recombinants is discussed.