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Virus-like particles (VLPs) of human papillomavirus (HPV) are used as a vaccine against HPV-induced cancer, and recently we have shown that these VLPs are able to activate natural killer (NK) cells. Since NK cells collaborate with dendritic cells (DCs) to induce an immune response against viral infections and tumors, we studied the impact of this crosstalk in the context of HPV vaccination. NK cells in the presence of HPV-VLPs enhanced DC-maturation as shown by an upregulation of CD86 and HLA-DR and an increased production of IL-12p70, but not of the immunosuppressive cytokine IL-10. This activation was bidirectional. Indeed, in the presence of HPV-VLPs, DCs further activated NK cells by inducing the upregulation of cell surface activation markers (CD69 and HLA-DR). The function of NK cells was also improved as shown by an increase in IFN-γ secretion and cytotoxic activity against an HPV(+) cell line. This crosstalk between NK cells and DCs needed CD40 interaction and IL-12p70 secretion, whereas NKG2D was not implicated. Our results provide insight into how VLPs interact with innate immune cells and how NK cells and DCs play a role in the immune response induced by this vaccine agent.
Assuntos
Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Células Matadoras Naturais/imunologia , Regulação para Cima/imunologia , Neoplasias do Colo do Útero/prevenção & controle , Vacinas de Partículas Semelhantes a Vírus/imunologia , Antígeno B7-2/imunologia , Células Dendríticas/patologia , Feminino , Antígenos HLA-DR/imunologia , Humanos , Interferon gama/imunologia , Interleucina-12/imunologia , Células Matadoras Naturais/patologia , Ativação Linfocitária , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Neoplasias do Colo do Útero/imunologia , Neoplasias do Colo do Útero/patologiaRESUMO
Human papillomavirus (HPV) infection, particularly type 16, is causally associated with cancer of the uterine cervix, which mainly develops at the squamocolumnar (SC) junction. The progression of cervical HPV infections into (pre)neoplastic lesions suggests that viral antigens are not adequately recognized by innate immunity or presented to the adaptive immune system. Members of the defensin family have recently been found to inhibit viral and bacterial pathogens, to stimulate the migration of immune cells and to play a role in anticancer responses. In the present study, we focused on the poorly characterized human α-defensin 5 (HD-5) and its possible role in these processes. We showed that HD-5 was able to prevent HPV virion entry into cervical keratinocytes and to influence adaptive immunity. Indeed, this peptide specifically induced the chemoattraction and proliferation of both activated T lymphocytes and immature dendritic cells in a CCR2/CCR6-dependent manner and stimulated the infiltration of these professional antigen-presenting cells in a (pre)neoplastic epithelium transplanted in vivo in immunodeficient mice. No chemotactic effect was observed with plasmacytoid dendritic cells, macrophages or natural killer cells. Proliferative and angiogenic effects of HD-5 were also assessed in vitro and in vivo. However there was a striking regional disparity in expression of HD-5, being prominent in ectocervical, vaginal and vulvar neoplasia, while absent, or nearly so, in the cervical SC junction. Taken together, these results suggest one possible explanation for why the SC junction is uniquely vulnerable to both high-risk HPV infection (via reduced HD-5 expression and viral entry) and progression of neoplasia (via altered cell-mediated immune responses and altered microenvironment).
Assuntos
Colo do Útero/metabolismo , Infecções por Papillomavirus/imunologia , Lesões Pré-Cancerosas/imunologia , Neoplasias Uterinas/virologia , alfa-Defensinas/biossíntese , Animais , Western Blotting , Células Cultivadas , Colo do Útero/imunologia , Progressão da Doença , Ensaio de Imunoadsorção Enzimática , Feminino , Xenoenxertos , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/virologia , Reação em Cadeia da Polimerase em Tempo Real , Neoplasias Uterinas/imunologia , Displasia do Colo do Útero/imunologia , Displasia do Colo do Útero/virologiaRESUMO
Tracheobronchial squamous metaplasia is common in smokers, and is associated with both airway obstruction in chronic obstructive pulmonary disease (COPD) and increased risk of lung cancer. Although this reversible epithelial replacement is almost always observed in association with chronic inflammation, the role of inflammatory mediators in the pathogenesis of squamous metaplasia remains unclear. In the present study, we investigated the implication of cigarette smoke-mediated proinflammatory cytokine up-regulation in the development and treatment of tracheobronchial epithelial hyperplasia and squamous metaplasia. Using immunohistological techniques, we showed a higher epithelial expression of TNF-α, IL-1ß, and IL-6, as well as an activation of NF-κB and activator protein-1/mitogen-activated protein kinase signaling pathways in the respiratory tract of smoking patients, compared with the normal ciliated epithelium of nonsmoking patients. In addition, we demonstrated that these signaling pathways strongly influence the proliferation and differentiation state of in vitro-generated normal human airway epithelial basal cells. Finally, we exposed mice to cigarette smoke for 16 weeks, and demonstrated that anti-TNF-α (etanercept), anti-IL-1ß (anakinra), and/or anti-IL-6R (tocilizumab) therapies significantly reduced epithelial hyperplasia and the development of squamous metaplasia. These data highlight the importance of soluble inflammatory mediators in the pathogenesis of tracheobronchial squamous metaplasia. Therefore, the administration of proinflammatory cytokine antagonists may have clinical applications in the management of patients with COPD.
Assuntos
Brônquios/patologia , Citocinas/metabolismo , Fumaça/efeitos adversos , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Células Cultivadas , Citocinas/antagonistas & inibidores , Etanercepte , Humanos , Hiperplasia , Imunoglobulina G/farmacologia , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/patologia , Proteína Antagonista do Receptor de Interleucina 1/farmacologia , Interleucina-1beta/antagonistas & inibidores , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Sistema de Sinalização das MAP Quinases , Masculino , Metaplasia , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Neutrófilos/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Receptores de Interleucina-6/antagonistas & inibidores , Receptores de Interleucina-6/metabolismo , Receptores do Fator de Necrose Tumoral , Fumar/efeitos adversos , Fator de Transcrição AP-1/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Human papillomavirus (HPV) infections account for more than 50% of infection-linked cancers in women worldwide. The immune system controls, at least partially, viral infection and around 90% of HPV-infected women clear the virus within two years. However, it remains unclear which immune cells are implicated in this process and no study has evaluated the direct interaction between HPVs and NK cells, a key player in host resistance to viruses and tumors. We demonstrated an NK-cell infiltration in HPV-associated preneoplastic cervical lesions. Since HPVs cannot grow in vitro, virus-like particles (VLPs) were used as a model for studying the NK-cell response against the virus. Interestingly, NK cells displayed higher cytotoxic activity and cytokine production (TNF-α and IFN-γ) in the presence of HPV-VLPs. Using flow cytometry and microscopy, we observed that NK-cell stimulation was linked to rapid VLP entry into these cells by macropinocytosis. Using CD16(+) and CD16(-) NK-cell lines and a CD16-blocking antibody, we demonstrated that CD16 is necessary for HPV-VLP internalization, as well as for degranulation and cytokine production. Thus, we show for the first time that NK cells interact with HPVs and can participate in the immune response against HPV-induced lesions.
Assuntos
Carcinoma de Células Escamosas/virologia , Citotoxicidade Imunológica , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/virologia , Papillomaviridae/imunologia , Receptores de IgG/imunologia , Neoplasias do Colo do Útero/virologia , Western Blotting , Carcinoma de Células Escamosas/imunologia , Carcinoma de Células Escamosas/metabolismo , Separação Celular , Citocinas/metabolismo , Feminino , Humanos , Imunoprecipitação , Células Matadoras Naturais/metabolismo , Microscopia Confocal , Infecções por Papillomavirus/imunologia , Lesões Pré-Cancerosas/imunologia , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/virologia , Receptores de IgG/biossíntese , Neoplasias do Colo do Útero/imunologia , Neoplasias do Colo do Útero/metabolismo , Internalização do Vírus , Displasia do Colo do Útero/imunologia , Displasia do Colo do Útero/metabolismo , Displasia do Colo do Útero/virologiaRESUMO
Human papillomavirus (HPV) infection, particularly type 16, is causally associated with cancer of the uterine cervix. The progression of cervical lesions suggests that viral antigens are not adequately presented to the immune system. The aim of this study was to determine whether HPV16 viral particles can influence the trafficking of human DC/Langerhans cells (LC), either by direct interactions with DC or following incubation with human normal keratinocytes that are in close contact with LC in the squamous epithelium. We first demonstrated that HPV16 L1 major capsid protein, when self-assembled into virus-like particles (VLP), is able to induce in DC an over-expression of CXC receptor 4 (CXCR4) via the activation of the NF-κB signaling pathway and to enhance DC motility in the presence of CXCL12, suggesting an ability to migrate towards lymph nodes. We also showed that conditioned media of HPV16 VLP-treated keratinocytes induce a lower LC migration than those from untreated keratinocytes and that prostaglandin E2 (PGE(2)), detected in HPV16 VLP-treated keratinocyte supernatants, may reduce LC recruitment into the squamous epithelium. Taken together, our data demonstrate that HPV16 VLP may differentially regulate the immune protective response according to their target cells.
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Proteínas do Capsídeo/imunologia , Movimento Celular/imunologia , Papillomavirus Humano 16/imunologia , Células de Langerhans/imunologia , Proteínas Oncogênicas Virais/imunologia , Infecções por Papillomavirus/imunologia , Vacinas contra Papillomavirus/imunologia , Dinoprostona/imunologia , Regulação da Expressão Gênica/imunologia , Queratinócitos/imunologia , Queratinócitos/virologia , Linfonodos/imunologia , Receptores CXCR4/imunologiaRESUMO
TP63 is a p53-related gene that contains two alternative promoters, which give rise to transcripts that encode proteins with (TAp63) or without (DeltaNp63) an amino-transactivating domain. Whereas the expression of p63 is required for proper development of epithelial structures, the role of p63 in tumorigenesis remains unclear. Here, we investigated the role of Snail and Slug transcription factors, known to promote epithelial-to-mesenchymal transitions during development and cancer, in the regulation of p63 isoforms in human squamous cell carcinoma (SCC). In the present study, we observed that the expressions of DeltaN and TAp63 isoforms were, respectively, down- and up-regulated by both Snail and Slug. However, the induction of TAp63 was not directly caused by these two transcription factors but resulted from the loss of DeltaNp63, which acts as dominant-negative inhibitor of TAp63. In SCC cell lines and cancer tissues, high expression of Snail and Slug was also significantly associated with altered p63 expression. Finally, we showed that DeltaNp63 silencing reduced cell-cell adhesion and increased the migratory properties of cancer cells. These data suggest that the disruption of p63 expression induced by Snail and Slug plays a crucial role in tumor progression. Therefore, p63 and its regulating factors could constitute novel prognosis markers in patients with SCC and attractive targets for the therapeutic modulation of neoplastic cell invasiveness.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Cutâneas/metabolismo , Transativadores/química , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/química , Idoso , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Isoformas de Proteínas , Fatores de Transcrição da Família SnailRESUMO
BACKGROUND: The epidermal growth factor receptor (EGFR), a member of the ErbB family of receptors, is a transmembrane tyrosine kinase (TK) activated by the binding of extracellular ligands of the EGF-family and involved in triggering the MAPK signaling pathway, which leads to cell proliferation. Mutations in the EGFR tyrosine kinase domain are frequent in non-small-cell lung cancer (NSCLC). However, to date, only very few, mainly non-European, studies have reported rare EGFR mutations in colorectal cancer (CRC). METHODS: We screened 236 clinical tumor samples from European patients with advanced CRC by direct DNA sequencing to detect potential, as yet unknown mutations, in the EGFR gene exons 18 to 21, mainly covering the EGFR TK catalytic domain. RESULTS: EGFR sequences showed somatic missense mutations in exons 18 and 20 at a frequency of 2.1% and 0.4% respectively. Somatic SNPs were also found in exons 20 and 21 at a frequency of about 3.1% and 0.4% respectively. Of these mutations, four have not yet been described elsewhere. CONCLUSIONS: These mutation frequencies are higher than in a similarly sized population characterized by Barber and colleagues, but still too low to account for a major role played by the EGFR gene in CRC.
Assuntos
Neoplasias Colorretais/genética , Receptores ErbB/genética , Mutação de Sentido Incorreto/genética , População Branca/genética , Sequência de Bases , Análise Mutacional de DNA , Éxons , Frequência do Gene , Humanos , Polimorfismo de Nucleotídeo Único , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/genéticaRESUMO
Although communication skills training programs have been recommended to reduce physicians' burnout, few studies have investigated their efficacy. This study assessed the impact of two training programs on cancer physicians' burnout. Especially, it identified some variables leading to burnout in order to develop effective interventions. Burnout was assessed with the Maslach Burnout Inventory. No statistically significant impact of training programs on burnout was observed. The amount of clinical workload and the overuse of some facilitative communication skills were associated with cancer physicians' burnout. The content of such programs must be redefined to reduce burnout.
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Esgotamento Profissional/prevenção & controle , Comunicação , Capacitação em Serviço/organização & administração , Oncologia , Feminino , Humanos , Masculino , Estresse PsicológicoRESUMO
Although human papillomavirus (HPV) DNA is detected in the majority of squamous intraepithelial lesions (SIL) and carcinoma (SCC) of the uterine cervix, the persistence or progression of cervical lesions suggest that viral antigens are not adequately presented to the immune system. This hypothesis is reinforced by the observation that most SIL show quantitative and functional alterations of Langerhans cells (LC). The aim of this study was to determine whether prostaglandins (PG) may affect LC density in the cervical (pre)neoplastic epithelium. We first demonstrated that the epithelial expression of PGE(2) enzymatic pathways, including cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase 1 (mPGES-1), is higher in SIL and SCC compared to the normal exocervical epithelium and inversely correlated to the density of CD1a-positive LC. By using cell migration assays, we next showed that the motility of immature dendritic cells (DC) and DC partially differentiated in vitro in the presence of PGE(2) are differentially affected by PGE(2). Immature DC had a lower ability to migrate in the presence of PGE(2) compared to DC generated in vitro in the presence of PGE(2). Finally, we showed that PGE(2) induced a cytokine production profile and phenotypical features of tolerogenic DC, suggesting that the altered expression of PGE(2) enzymatic pathways may promote the cervical carcinogenesis by favouring (pre)cancer immunotolerance.
Assuntos
Dinoprostona/biossíntese , Tolerância Imunológica/imunologia , Células de Langerhans/imunologia , Lesões Pré-Cancerosas/enzimologia , Displasia do Colo do Útero/enzimologia , Neoplasias do Colo do Útero/enzimologia , Antígenos Virais/imunologia , Western Blotting , Diferenciação Celular/imunologia , Movimento Celular/imunologia , Ensaio de Imunoadsorção Enzimática , Epitélio/imunologia , Feminino , Humanos , Imuno-Histoquímica , Células de Langerhans/citologia , Papillomaviridae/imunologia , Lesões Pré-Cancerosas/imunologia , Lesões Pré-Cancerosas/virologia , Transdução de Sinais/fisiologia , Neoplasias do Colo do Útero/imunologia , Neoplasias do Colo do Útero/virologia , Displasia do Colo do Útero/imunologia , Displasia do Colo do Útero/virologiaRESUMO
Human papillomavirus (HPV) infection, particularly type 16, is causally associated with cancer of the uterine cervix. The persistence or progression of cervical lesions suggests that viral antigens are not adequately presented to the immune system. This hypothesis is reinforced by the observation that most squamous intraepithelial lesions (SILs) show quantitative and functional alterations of Langerhans cells (LC). The infiltration of immature LC in the squamous epithelium is mainly controlled by Macrophage Inflammatory Protein 3alpha/CCL20. After having shown that CCL20 production is altered in HPV-transformed keratinocytes (KC), the possible role of HPV16 E6 and E7 viral oncoproteins in the reduced CCL20 levels observed in SILs was investigated by silencing HPV16 E6 and E7 oncogenes by RNA interference (siRNA). This treatment not only increased CCL20 secretion but also resulted in the modulation of NF-kappaB p50, p52 and p65 precursor localization. Moreover, silencing of E6 and E7 oncogenes in HPV16-transformed KC induced a significantly higher migratory capacity of LC in a Boyden chamber assay and in an in vitro formed (pre)neoplastic epithelium reminiscent of high-grade SILs. Anti-CCL20 neutralizing antibody experiments showed that the increased migration of LC is due to the re-expression of CCL20 in E6 and E7 siRNA transfected KC. These data suggest that HPV16 E6/E7-induced down-regulation of CCL20 observed during the cervical carcinogenesis may contribute to a diminished capacity of the immune system to control HPV infection.
Assuntos
Quimiocina CCL20/fisiologia , Inativação Gênica , Células de Langerhans/imunologia , Proteínas Oncogênicas Virais , Proteínas Repressoras , Neoplasias do Colo do Útero/fisiopatologia , Western Blotting , Linhagem Celular Tumoral , Movimento Celular , Quimiocina CCL20/genética , Regulação para Baixo , Feminino , Citometria de Fluxo , Humanos , Células de Langerhans/metabolismo , Células de Langerhans/virologia , Proteínas Oncogênicas Virais/genética , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/metabolismo , Infecções por Papillomavirus/virologia , Proteínas Repressoras/genética , Transdução de Sinais , Neoplasias do Colo do Útero/imunologiaRESUMO
Epithelial metaplasia (EpM) is an acquired tissue abnormality resulting from the transformation of epithelium into another tissue with a different structure and function. This adaptative process is associated with an increased frequency of (pre)cancerous lesions. We propose that EpM is involved in cancer development by altering the expression of adhesion molecules important for cell-mediated antitumor immunity. Langerhans cells (LCs) are intraepithelial dendritic cells that initiate immune responses against viral or tumor antigens on both skin and mucosal surfaces. In the present study, we showed by immunohistology that the density of CD1a(+) LCs is reduced in EpM of the uterine cervix compared with native squamous epithelium and that the low number of LCs observed in EpM correlates with the down-regulation of cell-surface E-cadherin. We also demonstrated that transforming growth factor-beta1 is not only overexpressed in metaplastic tissues but also reduces E-cadherin expression in keratinocytes in vitro by inducing the promoter activity of Slug and Snail transcription factors. Finally, we showed that in vitro-generated LCs adhere poorly to keratinocytes transfected with either Slug or Snail DNA. These data suggest that transforming growth factor-beta1 indirectly reduces antigen-presenting cell density in EpM by affecting E-cadherin expression, which might explain the increased susceptibility of abnormal tissue differentiation to the development of cancer by the establishment of local immunodeficiency responsible for EpM tumorigenesis.
Assuntos
Caderinas/metabolismo , Transformação Celular Neoplásica , Colo do Útero/patologia , Células de Langerhans/citologia , Fatores de Transcrição/biossíntese , Fator de Crescimento Transformador beta1/fisiologia , Adulto , Antígenos CD , Antígenos CD1/metabolismo , Linhagem Celular , Colo do Útero/metabolismo , Metilação de DNA , Epitélio/patologia , Feminino , Inativação Gênica , Humanos , Queratinócitos/metabolismo , Células de Langerhans/metabolismo , Células de Langerhans/fisiologia , Metaplasia/metabolismo , Metaplasia/patologia , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta1/farmacologia , Regulação para CimaRESUMO
Adipocytes are part of hematopoietic microenvironment, even though up to now in humans, their role in hematopoiesis is still questioned. We have previously shown that accumulation of fat cells in femoral bone marrow (BM) coincides with increased expression of neuropilin-1 (NP-1), while it is weakly expressed in hematopoietic iliac crest BM. Starting from this observation, we postulated that adipocytes might exert a negative effect on hematopoiesis mediated through NP-1. To test this hypothesis, we set up BM adipocytes differentiated into fibroblast-like fat cells (FLFC), which share the major characteristics of primitive unilocular fat cells, as an experimental model. As expected, FLFCs constitutively produced macrophage colony stimulating factor and induced CD34(+) differentiation into macrophages independently of cell-to-cell contact. By contrast, granulopoiesis was hampered by cell-to-cell contact but could be restored in transwell culture conditions, together with granulocyte colony stimulating factor production. Both functions were also recovered when FLFCs cultured in contact with CD34(+) cells were treated with an antibody neutralizing NP-1, which proved its critical implication in contact inhibition. An inflammatory cytokine such as interleukin-1 beta or dexamethasone modulates FLFC properties to restore granulopoiesis. Our data provide the first evidence that primary adipocytes exert regulatory functions during hematopoiesis that might be implicated in some pathological processes. Disclosure of potential conflicts of interest is found at the end of this article.
Assuntos
Adipócitos/fisiologia , Células da Medula Óssea/citologia , Fator Estimulador de Colônias de Granulócitos/antagonistas & inibidores , Neuropilina-1/fisiologia , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Animais , Antígenos CD/fisiologia , Antígenos CD34/fisiologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/fisiologia , Proteínas de Ligação ao Cálcio , Diferenciação Celular , Primers do DNA , Regulação da Expressão Gênica , Fator Estimulador de Colônias de Granulócitos/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Macrófagos/citologia , Macrófagos/fisiologia , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
CONTEXT: Since 2000, problem-based learning (PBL) seminars have been introduced into the curriculum of medical studies at the University of Liège. We aimed to carry out a cross-sectional investigation of the maturational increase in biomedical reasoning capacity in comparison with factual knowledge retention throughout the curriculum. METHODS: We administered a factual knowledge test (i.e. a true/false test with ascertainment degree) and a biomedical reasoning test (i.e. an adapted script concordance test [SCT]) to 104 students (Years 3-6) and a reference panel. The selected topic was endocrinology. RESULTS: On the SCT, the students obtained higher scores in Years 5 and 6 than in Years 3 and 4. In Year 3, the scores obtained on SCT questions in a new context indicated transfer of reasoning skills. On the true/false test, the scores of Year 3 students were significantly higher than those of students in the other three year groups. A positive correlation between SCT scores and true/false test scores was observed only for students in Years 3 and 4. In each group, the ascertainment degree scores were higher for correct than for incorrect responses and the difference was calculated as an index of self-estimation of core knowledge. This index was found to be positively correlated to SCT scores in the four year groups studied. CONCLUSIONS: Biomedical reasoning skills are evidenced early in a curriculum involving PBL and further increase during training. This is accompanied by a decrease in factual knowledge retention. The self-estimation of core knowledge appears to be related to reasoning capacity, which suggests there is a link between the two processes.
Assuntos
Currículo , Educação Médica/métodos , Aprendizagem Baseada em Problemas/métodos , Bélgica , Competência Clínica , Avaliação Educacional/métodos , Endocrinologia/educação , Feminino , Humanos , Masculino , Resolução de ProblemasRESUMO
Human papillomavirus (HPV) infection, particularly type 16, is causally associated with cancer of the uterine cervix. The persistence or progression of cervical lesions suggests that viral antigens are not adequately presented to the immune system. This hypothesis is reinforced by the observation that most squamous intra-epithelial lesions show quantitative and functional alterations of Langerhans cells (LCs). Moreover, E-cadherin-dependent adhesion of LC to keratinocytes (KCs) is defective in cervical HPV16-associated (pre)neoplastic lesions. The possible role of viral oncoprotein E7 in the reduced levels of cell surface E-cadherin was investigated by silencing HPV16 E7 by RNA interference (siRNA). This treatment induced an increased cell surface E-cadherin expression in HPV16-positive KC and a significant adhesion of LC to these squamous cells. The E-cadherin re-expression following HPV16 E7 silencing was associated with increased detection levels of retinoblastoma protein and the activating protein (AP)-2alpha transcription factor. These data suggest that HPV16 E7-induced alterations of LC/KC adhesion may play a role in the defective immune response during cervical carcinogenesis.
Assuntos
Caderinas/biossíntese , Carcinoma de Células Escamosas/virologia , Transformação Celular Viral/genética , Papillomavirus Humano 16/genética , Queratinócitos/virologia , Proteínas Oncogênicas Virais/genética , Neoplasias do Colo do Útero/virologia , Antígenos CD1/biossíntese , Caderinas/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/imunologia , Carcinoma de Células Escamosas/patologia , Adesão Celular/genética , Linhagem Celular Tumoral , Transformação Celular Viral/imunologia , Feminino , Inativação Gênica , Humanos , Queratinócitos/imunologia , Queratinócitos/metabolismo , Queratinócitos/patologia , Células de Langerhans/imunologia , Células de Langerhans/metabolismo , Células de Langerhans/patologia , Células de Langerhans/virologia , Proteínas Oncogênicas Virais/biossíntese , Proteínas E7 de Papillomavirus , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/imunologia , Infecções por Papillomavirus/metabolismo , Infecções por Papillomavirus/patologia , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Fator de Transcrição AP-2/biossíntese , Fator de Transcrição AP-2/genética , Transfecção , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/imunologia , Neoplasias do Colo do Útero/patologiaRESUMO
INTRODUCTION: Overexpression of the ERBB2 oncogene is observed in about 20% of human breast tumors and is the consequence of increased transcription rates frequently associated with gene amplification. Several studies have shown a link between activator protein 2 (AP-2) transcription factors and ERBB2 gene expression in breast cancer cell lines. Moreover, the Yin Yang 1 (YY1) transcription factor has been shown to stimulate AP-2 transcriptional activity on the ERBB2 promoter in vitro. In this report, we examined the relationships between ERBB2, AP-2alpha, and YY1 both in breast cancer tissue specimens and in a mammary cancer cell line. METHODS: ERBB2, AP-2alpha, and YY1 protein levels were analyzed by immunohistochemistry in a panel of 55 primary breast tumors. ERBB2 gene amplification status was determined by fluorescent in situ hybridization. Correlations were evaluated by a chi2 test at a p value of less than 0.05. The functional role of AP-2alpha and YY1 on ERBB2 gene expression was analyzed by small interfering RNA (siRNA) transfection in the BT-474 mammary cancer cell line followed by real-time reverse transcription-polymerase chain reaction and Western blotting. RESULTS: We observed a statistically significant correlation between ERBB2 and AP-2alpha levels in the tumors (p < 0.01). Moreover, associations were found between ERBB2 protein level and the combined high expression of AP-2alpha and YY1 (p < 0.02) as well as between the expression of AP-2alpha and YY1 (p < 0.001). Furthermore, the levels of both AP-2alpha and YY1 proteins were inversely correlated to ERBB2 gene amplification status in the tumors (p < 0.01). Transfection of siRNAs targeting AP-2alpha and AP-2gamma mRNAs in the BT-474 breast cancer cell line repressed the expression of the endogenous ERBB2 gene at both the mRNA and protein levels. Moreover, the additional transfection of an siRNA directed against the YY1 transcript further reduced the ERBB2 protein level, suggesting that AP-2 and YY1 transcription factors cooperate to stimulate the transcription of the ERBB2 gene. CONCLUSION: This study highlights the role of both AP-2alpha and YY1 transcription factors in ERBB2 oncogene overexpression in breast tumors. Our results also suggest that high ERBB2 expression may result either from gene amplification or from increased transcription factor levels.
Assuntos
Neoplasias da Mama/metabolismo , Genes erbB-2/genética , Fator de Transcrição AP-2/biossíntese , Fator de Transcrição YY1/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Feminino , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Fator de Transcrição AP-2/genética , Fator de Transcrição YY1/genéticaRESUMO
The current study attempts to characterize the eosinophilia associated with T-cell lymphomas and to investigate its possible relationship with the secretion of eosinophil-stimulating factors by lymphoma cells and/or intra-tumoral surrounding cells. Paraffin-embedded specimens from 50 patients diagnosed with peripheral T-cell lymphomas, either unspecified (PTCL-U, n=30) or angioimmunoblastic (AITL, n=20) were morphologically assessed for intra-tumoral eosinophilia and analyzed by immunohistochemistry using specific antibodies directed against TARC, IL-5, RANTES, and eotaxin. The AITL and PTCL-U cases contained a mean of 147+/-41 and 102+/-37 eosinophils per 10 high power fields, respectively. Thirty-two of 47 cases (68%) showed IL-5-positive lymphoma cells while 15/50 (30%) tumors showed variable staining for TARC in scattered non-lymphoid cells with dendritic morphology. TARC and IL-5-positive cases possessed significantly more eosinophils. Our data indicate that IL-5 and TARC expression highly correlate with eosinophilia in T-cell lymphomas, suggesting that these chemokines are involved in the recruitment of eosinophils into the tumors.
Assuntos
Quimiocina CCL17/metabolismo , Eosinofilia/metabolismo , Interleucina-5/metabolismo , Linfoma de Células T Periférico/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Quimiocina CCL11/metabolismo , Quimiocina CCL5/metabolismo , Eosinofilia/patologia , Eosinófilos/patologia , Feminino , Humanos , Linfadenopatia Imunoblástica/metabolismo , Linfadenopatia Imunoblástica/patologia , Técnicas Imunoenzimáticas , Linfoma de Células T Periférico/patologia , Masculino , Pessoa de Meia-Idade , PrognósticoRESUMO
BACKGROUND: Follicular lymphoma, the neoplastic counterpart of germinal center B cells, typically recapitulates a follicular architecture. Several observations point to the crucial role of the cellular microenvironment in the development and/or progression of follicular lymphoma cells in vivo. The aim of our study was to characterize the spontaneous apoptosis of follicular lymphoma cells in vitro, and the modulation of this apoptosis by follicular dendritic cells. DESIGN AND METHODS: We used a cell line derived from follicular dendritic cells to model the functional interactions of these cells and lymphoma cells in co-culture. Follicular lymphoma cells were isolated from tissue biopsies. Apoptosis was quantified by flow cytometry and apoptotic pathways were investigated by western blotting. RESULTS: The spontaneous apoptosis of follicular lymphoma cells in vitro involves the activation of caspases-3 and -8 but not of caspase-9, occurs despite persistent high levels of BCL-2 and MCL-1, and is associated with down-regulation of c-FLIP(L). Spontaneous apoptosis of follicular lymphoma cells is partially prevented by co-culture with the follicular dendritic cells, which prevents activation of caspase-8, caspase-3 and induces an upregulation of c-FLIP(L). Using neutralizing antibodies, we demonstrated that interactions involving CD54 (ICAM-1), CD106 (VCAM-1) and CD40 are implicated in this biological process. CONCLUSIONS: Follicular dendritic cells constitute a useful tool to study the functional interactions between follicular lymphoma cells and follicular dendritic cells in vitro. Understanding the molecular mechanisms involved in these protective interactions may lead to the identification of therapeutic agents that might suppress the survival and growth of follicular lymphoma cells.
Assuntos
Linfócitos B/patologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Caspase 3/metabolismo , Caspase 8/metabolismo , Células Dendríticas Foliculares/patologia , Linfoma Folicular/patologia , Idoso , Idoso de 80 Anos ou mais , Apoptose , Linfócitos B/enzimologia , Biópsia , Linhagem Celular Tumoral , Células Dendríticas Foliculares/enzimologia , Ativação Enzimática , Feminino , Citometria de Fluxo , Humanos , Linfoma Folicular/enzimologia , Linfoma Folicular/epidemiologia , Linfoma Folicular/genética , Masculino , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Estados Unidos/epidemiologiaRESUMO
BACKGROUND: The 21-kDa Vaccinia virus VH1-related (VHR) dual-specific protein phosphatase (encoded by the DUSP3 gene) plays a critical role in cell cycle progression and is itself regulated during the cell cycle. We have previously demonstrated using RNA interference that cells lacking VHR arrest in the G1 and G2 phases of the cell cycle and show signs of beginning of cell senescence. METHODS: In this report, we evaluated successfully the expression levels of VHR protein in 62 hysterectomy or conization specimens showing the various (pre) neoplastic cervical epithelial lesions and 35 additional cases of hysterectomy performed for non-cervical pathologies, from patients under 50 years of age. We used a tissue microarray and IHC technique to evaluate the expression of the VHR phosphatase. Immunofluorescence staining under confocal microscopy, Western blotting and RT-PCR methods were used to investigate the localization and expression levels of VHR. RESULTS: We report that VHR is upregulated in (pre) neoplastic lesions (squamous intraepithelial lesions; SILs) of the uterine cervix mainly in high grade SIL (H-SIL) compared to normal exocervix. In the invasive cancer, VHR is also highly expressed with nuclear localization in the majority of cells compared to normal tissue where VHR is always in the cytoplasm. We also report that this phosphatase is highly expressed in several cervix cancer cell lines such as HeLa, SiHa, CaSki, C33 and HT3 compared to primary keratinocytes. The immunofluorescence technique under confocal microscopy shows that VHR has a cytoplasmic localization in primary keratinocytes, while it localizes in both cytoplasm and nucleus of the cancer cell lines investigated. We report that the up-regulation of this phosphatase is mainly due to its post-translational stabilization in the cancer cell lines compared to primary keratinocytes rather than increases in the transcription of DUSP3 locus. CONCLUSION: These results together suggest that VHR can be considered as a new marker for cancer progression in cervix carcinoma and potential new target for anticancer therapy.