RESUMO
Ewing sarcoma is an aggressive neoplasm of pediatric and adolescent patients. Immunohistochemistry (IHC) can be used to support the morphologic diagnosis of Ewing sarcoma family of tumors (ESFT) in a convincing clinical/radiological context. Although neither NKX2.2 nor CD99 alone are entirely specific, when combined, the diagnostic specificity is high. The aim of the present study was to investigate the IHC expression of NKX2.2, ETV4 and BCOR in a large series of genetically confirmed ESFT. The results for CD99 and CAV-1 immunoreactivity, and the histological and fusion gene subtypes were retrieved from our previous study. NKX2.2 demonstrated moderate or strong nuclear positivity in 91.2% of the tumors. The staining intensity was heterogeneous. Many of the ESFT with negative NKX2.2 immunoreactivity were in bone. Strong/moderate ETV4 nuclear expression was detected in two small round cell tumors, both were negative for NKX2.2. No relationships could be found between expression of NKX2.2 and the histological subgroups or ESFT gene fusion subtypes. BCOR was negative in all ESFT. In conclusion, NKX2.2, ETV4 and BCOR IHC may be helpful in daily practice for distinguishing ESFT from CIC or BCOR-associated sarcomas, especially in hospitals without access to molecular assays. In addition, the combination of strong CD99 membranous positivity and nuclear NKX2.2 positivity seems to be very reliable for ESFT diagnosis in an appropriate clinicoradiological setting. So far no antibody is entirely specific for ESFT diagnosis, and the IHC or molecular results in round cell tumors of bone may be strongly influenced by decalcification processes.
Assuntos
Proteínas E1A de Adenovirus/análise , Biomarcadores Tumorais/análise , Neoplasias Ósseas/diagnóstico , Proteínas de Homeodomínio/análise , Proteínas Proto-Oncogênicas/análise , Proteínas Repressoras/análise , Sarcoma de Ewing/diagnóstico , Fatores de Transcrição/análise , Proteína Homeobox Nkx-2.2 , Humanos , Imuno-Histoquímica , Proteínas Nucleares , Proteínas Proto-Oncogênicas c-ets , Proteínas de Peixe-ZebraRESUMO
AIMS: A recently characterized group of undifferentiated small round cell sarcomas harbours fusions of the genes CIC and DUX4. Studies report a distinctive gene expression profile for these sarcomas, including expression of E26 transformation-specific (ETS) family proto-oncogenic transcription factors ETV1, ETV4 and ETV5. To test the utility of an ancillary diagnostic technique for these tumours, we evaluated chromogenic RNA in-situ hybridization assays for ETV1, ETV4 and ETV5 as diagnostic adjuncts for this emerging group of highly malignant sarcomas. METHODS AND RESULTS: We tested six confirmed CIC-DUX4 sarcomas and 105 lesions in the differential, including 48 Ewing sarcomas for expression of ETV1, ETV4 and ETV5, scoring expression utilizing a previously validated scale. ETV1 and ETV4 were positive in five of six cases, while ETV5 was positive in six of six. No Ewing sarcoma or other sarcoma tested showed coexpression of these transcripts, while one ETV1/ETV4/ETV5 triple positive previously unclassified round cell sarcoma was identified as harbouring a CIC rearrangement by break-apart fluorescence in-situ hybridization (FISH). CONCLUSION: We identified overexpression of ETV1, ETV4 and ETV5 transcripts in situ in CIC-DUX4 sarcomas using a robust assay in routine archival sections. One previously unclassified round cell sarcoma showed ETV1/4/5 positivity, and was proved to harbour a CIC rearrangement by break-apart FISH. The sensitivity and specificity observed with our in-situ hybridization assay implies potential utility as an ancillary diagnostic technique, particularly when faced with limited biopsy samples.
Assuntos
Proteínas E1A de Adenovirus/biossíntese , Biomarcadores Tumorais/análise , Proteínas de Ligação a DNA/biossíntese , Hibridização In Situ/métodos , Proteínas Proto-Oncogênicas/biossíntese , Sarcoma de Células Pequenas/diagnóstico , Fatores de Transcrição/biossíntese , Proteínas E1A de Adenovirus/análise , Adulto , Proteínas de Ligação a DNA/análise , Feminino , Humanos , Masculino , Proteínas de Fusão Oncogênica/análise , Proteínas de Fusão Oncogênica/genética , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas c-ets , RNA/análise , Estudos Retrospectivos , Sarcoma de Células Pequenas/genética , Sensibilidade e Especificidade , Fatores de Transcrição/análiseRESUMO
Subsets of primitive round-cell sarcomas remain difficult to diagnose and classify. Among these is a rare round-cell sarcoma that harbors a CIC gene rearrangement known as CIC-rearranged undifferentiated round-cell sarcoma, which is most commonly fused to the DUX4 gene. Owing to its aggressive clinical behavior and potential therapeutic implications, accurate identification of this novel soft tissue sarcoma is necessary. Definitive diagnosis requires molecular confirmation, but only a few centers are as yet able to perform this test. Several studies have shown that PEA3 subfamily genes, notably ETV4 (belonging to the family of ETS transcription factors), are upregulated in CIC-rearranged undifferentiated round-cell sarcomas. We performed a detailed immunohistochemical analysis to investigate ETV4 expression in CIC-rearranged undifferentiated round-cell sarcomas and their potential mimics (especially Ewing sarcomas). The study cohort included 17 cases of CIC-rearranged undifferentiated round-cell sarcomas, and 110 tumors that morphologically mimic CIC-rearranged undifferentiated round-cell sarcomas: 43 Ewing sarcomas, 25 alveolar rhabdomyosarcomas, 20 poorly differentiated round-cell synovial sarcomas, 10 desmoplastic round-cell tumors, 5 BCOR-CCNB3 sarcomas, 5 lymphoblastic lymphomas, and 2 rhabdoid tumors. All CIC-rearranged undifferentiated round-cell sarcomas (on core needle biopsies and open biopsies) were ETV4-positive with a strong diffuse nuclear pattern. Among the other 110 tumors, only six cases (four Ewing sarcomas, one alveolar rhabdomyosarcoma, and one desmoplastic round-cell tumor) showed focal (<5% of tumor cells) and very weak nuclear expression of ETV4; all other tumors were completely negative for ETV4. We conclude that systematic immunohistochemical analysis of ETV4 makes it possible to diagnose undifferentiated round-cell sarcomas (with no molecular markers for sarcoma-associated translocation) such as CIC-rearranged undifferentiated round-cell sarcoma.
Assuntos
Proteínas E1A de Adenovirus/biossíntese , Biomarcadores Tumorais/análise , Proteínas Proto-Oncogênicas/biossíntese , Sarcoma de Células Pequenas/diagnóstico , Proteínas E1A de Adenovirus/análise , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Diagnóstico Diferencial , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas c-ets , Proteínas Repressoras/genética , Tumor Rabdoide/diagnóstico , Rabdomiossarcoma Alveolar/diagnóstico , Sarcoma de Ewing/diagnóstico , Sarcoma de Células Pequenas/genética , Sarcoma de Células Pequenas/patologia , Sarcoma Sinovial/diagnóstico , Adulto JovemRESUMO
A distinct subset of round cell sarcomas harbors capicua transcriptional repressor (CIC) rearrangement. Diagnosing these sarcomas can be difficult owing to their resemblance to Ewing sarcoma and other 'small round blue cell tumors'; molecular techniques are generally required. Recent gene expression studies of CIC-rearranged sarcomas identified the upregulation of ETV4. We assessed the sensitivity and specificity of ETV4 and WT1 immunohistochemistry for CIC-rearranged sarcoma. We evaluated whole-tissue sections from 40 CIC-rearranged sarcomas, 40 Ewing sarcomas, 4 BCOR-CCNB3 sarcomas, 6 unclassified round cell sarcomas, and 150 histologic mimics. Moderate-to-strong nuclear immunoreactivity for ETV4 in at least 50% of cells was observed in 36 (90%) CIC-rearranged sarcomas and 10 (5%) other tumors, including 5 unclassified round cell sarcomas, 2 Wilms tumors, and 1 each desmoplastic small round cell tumor, melanoma, and small cell carcinoma. Thirty-eight (95%) CIC-rearranged sarcomas showed nuclear staining for WT1, and 34 (85%) were positive for both ETV4 and WT1. Of 182 other tumors evaluated, 34 (19%) showed nuclear WT1 positivity, including all Wilms tumors and desmoplastic small round cell tumors, 5 unclassified round cell sarcomas, and a subset of lymphoblastic lymphomas, rhabdomyosarcomas, mesenchymal chondrosarcomas, carcinomas, and melanomas. In summary, diffuse moderate-to-strong ETV4 expression is present in most CIC-rearranged sarcomas and unclassified round cell sarcomas. More limited expression is seen in small subsets of various other round cell neoplasms. Nuclear WT1 expression is also present in most CIC-rearranged sarcomas and unclassified round cell sarcomas, along with Wilms tumors and desmoplastic small round cell tumors, and subsets of various histologic mimics. The sensitivity and specificity of diffuse ETV4 expression for CIC-rearranged sarcomas are 90% and 95%, respectively, whereas the sensitivity and specificity of WT1 are 95% and 81%, respectively. Diffuse ETV4 along with at least focal WT1 expression is helpful to distinguish CIC-rearranged sarcoma from Ewing sarcoma and other histologic mimics.
Assuntos
Proteínas E1A de Adenovirus/biossíntese , Biomarcadores Tumorais/análise , Proteínas Proto-Oncogênicas/biossíntese , Sarcoma de Ewing/diagnóstico , Sarcoma de Células Pequenas/diagnóstico , Neoplasias de Tecidos Moles/diagnóstico , Proteínas WT1/biossíntese , Proteínas E1A de Adenovirus/análise , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Ósseas/diagnóstico , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Criança , Pré-Escolar , Diagnóstico Diferencial , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas c-ets , Proteínas Repressoras/genética , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Sarcoma de Células Pequenas/genética , Sarcoma de Células Pequenas/metabolismo , Neoplasias de Tecidos Moles/genética , Neoplasias de Tecidos Moles/metabolismo , Proteínas WT1/análise , Adulto JovemRESUMO
Transcriptional activity of the retinoic acid receptor (RAR) is regulated by diverse binding partners, including classical corepressors and coactivators, in response to its ligand retinoic acid (RA). Recently, we identified a novel corepressor of RAR called the retinoic acid resistance factor (RaRF) (manuscript submitted). Here, we report how adenovirus E1A stimulates RAR activity by associating with RaRF. Based on immunoprecipitation (IP) assays, E1A interacts with RaRF through the conserved region 2 (CR2), which is also responsible for pRb binding. The first coiled-coil domain of RaRF was sufficient for this interaction. An in vitro glutathione-S-transferase (GST) pull-down assay was used to confirm the direct interaction between E1A and RaRF. Further fluorescence microscopy indicated that E1A and RaRF were located in the nucleoplasm and nucleolus, respectively. However, RaRF overexpression promoted nucleolar translocation of E1A from the nucleoplasm. Both the RA-dependent interaction of RAR with RaRF and RAR translocation to the nucleolus were disrupted by E1A. RaRF-mediated RAR repression was impaired by wild-type E1A, but not by the RaRF binding-defective E1A mutant. Taken together, our data suggest that E1A is sequestered to the nucleolus by RaRF through a specific interaction, thereby leaving RAR in the nucleoplasm for transcriptional activation.
Assuntos
Infecções por Adenoviridae/metabolismo , Adenoviridae/fisiologia , Proteínas E1A de Adenovirus/metabolismo , Nucléolo Celular/virologia , Interações Hospedeiro-Patógeno , Receptores do Ácido Retinoico/metabolismo , Proteínas Repressoras/metabolismo , Adenoviridae/química , Adenoviridae/metabolismo , Infecções por Adenoviridae/virologia , Proteínas E1A de Adenovirus/análise , Linhagem Celular , Nucléolo Celular/metabolismo , Humanos , Mapas de Interação de Proteínas , Estrutura Terciária de Proteína , Receptores do Ácido Retinoico/análise , Proteínas Repressoras/análise , Tretinoína/metabolismoRESUMO
PURPOSE: CD40 ligand (CD40L, CD154) plays a central role in immunoregulation and also directly modulates epithelial cell growth and differentiation. We previously showed that the CD40 receptor is commonly expressed in primary breast cancer tissues. In this proof-of-principle study, we examined the breast cancer growth-regulatory activities of an oncolytic adenoviral construct carrying the CD40L transgene (AdEHCD40L). EXPERIMENTAL DESIGN: In vitro and in vivo evaluations were carried out on AdEHCD40L to validate selective viral replication and CD40L transgene activity in hypoxia inducing factor-1alpha and estrogen receptor-expressing human breast cancer cells. RESULTS: AdEHCD40L inhibited the in vitro growth of CD40+ human breast cancer lines (T-47D, MDA-MB-231, and BT-20) by up to 80% at a low multiplicity of infection of 1. Incorporation of the CD40L transgene reduced the effective dose needed to achieve 50% growth inhibition (ED50) by approximately 10-fold. In contrast, viral and transgene expression of AdEHCD40L, as well its cytotoxicity, was markedly attenuated in nonmalignant cells. Intratumoral injections with AdEHCD40L reduced preexisting MDA-MB-231 xenograft growth in severe combined immunodeficient mice by >99% and was significantly more effective (P<0.003) than parental virus AdEH (69%) or the recombinant CD40L protein (49%). This enhanced antitumor activity correlated with cell cycle blockade and increased apoptosis in AdEHCD40L-infected tumor cells. CONCLUSIONS: These novel findings, together with the previously known immune-activating features of CD40L, support the potential applicability of AdEHCD40L for experimental treatment of human breast cancer.
Assuntos
Adenoviridae/genética , Neoplasias da Mama/terapia , Ligante de CD40/genética , Terapia Genética/métodos , Transgenes , Proteínas E1A de Adenovirus/análise , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos SCID , Fenótipo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The recent identification of fusion genes involving ETS family members in human prostate adenocarcinoma has confirmed the hypothesis that recurrent specific aberrations such as fusion genes may be as frequent in epithelial tumors as they are in leukemias and sarcomas. However, reciprocal translocations with fusion genes are often not detectable in carcinomas by conventional karyotyping because of additional complex chromosomal abnormalities. We retrospectively analyzed a large series of formalin-fixed, paraffin-embedded samples including 55 prostate carcinomas and 11 benign prostate tumors. We identified the fusion gene TMPRSS2-ERG by reverse-transcriptase polymerase chain reaction (RT-PCR) in 40/55 carcinomas (72%). Our study demonstrates that the detection of ETS fusion gene by RT-PCR is feasible on formalin-fixed and paraffin-embedded samples. No significant association between the presence of the fusion gene and any clinical feature, such as preoperative serum prostate-specific antigen (PSA) level (PSA>20 or PSA< or =20), pTNM stage including capsule invasion, seminal vesicle invasion, and lymph nodes metastases, or recurrence was observed in our series.
Assuntos
Adenocarcinoma/genética , Adenocarcinoma/patologia , Inclusão em Parafina , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-ets/análise , Serina Endopeptidases/análise , Proteínas E1A de Adenovirus/análise , Proteínas E1A de Adenovirus/genética , Idoso , Idoso de 80 Anos ou mais , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/genética , Estudos de Viabilidade , Fixadores/farmacologia , Formaldeído/farmacologia , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas de Fusão Oncogênica/análise , Proteínas de Fusão Oncogênica/genética , Inclusão em Parafina/métodos , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-ets/genética , Estudos Retrospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Serina Endopeptidases/genética , Fatores de Transcrição/análise , Fatores de Transcrição/genéticaRESUMO
OBJECTIVE: This study is to investigate the inhibitory effect of E1A gene on the cell proliferation of HeLa cells and its mechanism related to apoptosis. METHODS: MTT assay and soft agar colony formation assay were employed to justify the inhibition activity of E1A on the proliferation of HeLa cells transfected with E1A gene. Western Blot, RT-PCR and Real-time quantitative RT-PCR were used to detect the gene expression of E1A, HER-2/Neu and Caspase-3 in HeLa cells, respectively. The Caspase-3 activity was monitored by ApoAlert Caspase-3 Assay. The redistribution of cell cycles and apoptosis of HeLa cells regulated by E1A expression were evaluated by flow cytometry. RESULTS: E1A expression significantly inhibits the cell proliferation and anchorage-independent cell growth of HeLa, with the respective highest inhibition rate of 40.7% and 43.4% (P < 0.01). HER-2/Neu expression in HeLa was significantly down-regulated by E1A, while the protein expression and activity of Caspase-3 was up-regulated by E1A expression. Flow cytometry revealed that E1A transfection in HeLa increased the cell number at G1 stage and simultaneously decreased the cell number at S stage. E1A transfection induced 8.71% of HeLa cells at apoptosis status. CONCLUSIONS: E1A significantly inhibits the cell proliferation of HeLa by the apoptosis induction through HER-2/Neu/Caspase-3 pathway. These results encourage us to continue an in-vivo study and preclinical development of LPD-E1A as a novel gene therapeutic agent for human cervical cancer.
Assuntos
Proteínas E1A de Adenovirus/genética , Apoptose , Caspase 3/fisiologia , Terapia Genética , Receptor ErbB-2/fisiologia , Neoplasias do Colo do Útero/terapia , Proteínas E1A de Adenovirus/análise , Proliferação de Células , Ativação Enzimática , Feminino , Células HeLa , Humanos , RNA Mensageiro/análiseRESUMO
Gene expression patterns of testicular seminoma were analysed applying oligonucleotide microarrays in 40 specimens of different tumour stages (pT1, pT2, pT3) and in normal testes. Transcripts of maternally expressed 3 transcripts were expressed in seminoma without correlation with delta-like 1 homologue expression indicating an impaired imprinting status in seminoma. Interestingly, the transcripts of bromodomain-containing 2 and nuclear autoantigenic sperm protein associated with spermatogenesis were significantly upregulated in progressing tumour stages. Transcription factors TEA domain family member 4 and ETS variant gene 4 (ETV4), weakly expressed in normal testis, were strongly augmented during tumourigenesis. For ETV4 expression, a significant correlation with the increased expression of matrix metalloproteinase 2 and a disintegrin and metalloproteinase domain 15 was determined. The ETV4 protein was localised to nuclei of spermatogonia and revealed an intense staining in seminoma cells. Taken together, we characterised additional transcription factors and spermatogenesis-associated genes involved in the progression of seminoma.
Assuntos
Proteínas E1A de Adenovirus/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Proto-Oncogênicas/genética , Seminoma/genética , Neoplasias Testiculares/genética , Proteínas E1A de Adenovirus/análise , Núcleo Celular/química , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Masculino , Metaloproteinase 2 da Matriz/genética , Estadiamento de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas c-ets , Seminoma/metabolismo , Testículo/metabolismo , Regulação para CimaRESUMO
Solid tumors contain normoxic and hypoxic regions depending on the distance from the capillary. Normal cells may also be exposed to hypoxia under certain physiological conditions. Tumor hypoxia has been shown to associate strongly with tumor propagation and malignant progression. Hypoxia-inducible factor (HIF)-1alpha is stable under hypoxia and induces transcription of target genes by binding to the hypoxia-response element (HRE). Here we investigated the oncolytic effects of a novel adenovirus mutant with a deleted E1B55 gene (Ad.Delta55.HRE), in which the expression of E1A, which is essential for adenoviral replication, is regulated under the control of an HRE-expression system. Ad.Delta55.HRE expressed E1A under normoxia and more E1A under hypoxia and exhibited oncolytic effects on various cultured tumor cells, but its cytotoxic effect is relatively attenuated in normal fibroblast cells under normoxic and hypoxic conditions. Ad.Delta55.HRE lysed Huh-7 hepatoma cells stably expressing HIF-1alpha more effectively compared to parental cells. Ad.Delta55.HRE treatment exhibited significant antitumor activity in PC-3 prostate- and MDA-MB-435 breast tumor-bearing nude mice in which HIF-1alpha protein was immunohistochemically detected. The E1A and hexon proteins of adenovirus were immunostained in MDA-MB-435 xenografts after Ad.Delta55.HRE treatment, suggestive of viral replication. Our results suggest that Ad.Delta55.HRE may be useful for the treatment of solid tumors.
Assuntos
Adenoviridae/genética , Proteínas E1A de Adenovirus/genética , Terapia Genética/métodos , Vetores Genéticos/genética , Neoplasias/terapia , Proteínas E1A de Adenovirus/análise , Proteínas E1B de Adenovirus/genética , Proteínas E1B de Adenovirus/metabolismo , Animais , Proteínas do Capsídeo/análise , Hipóxia Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Deleção de Genes , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia , Camundongos , Transplante de Neoplasias , Regiões Promotoras Genéticas/genética , Elementos de Resposta/genética , Fatores de Transcrição/análise , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Replicação Viral/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Replication competent adenoviruses with various E1 modifications designed to restrict their replication to tumor cells are being evaluated as oncolytic agents in clinical trials. In mouse models, we observed that such oncolytic adenoviruses showed greater hepatotoxicity than E1-deleted adenovirus vectors following intravenous administration. Additional studies in congenic BALB/c, nude, and beige/Scid mice demonstrated dose-dependent hepatotoxicity and indicated that beige/Scid was the most sensitive strain. Comparison of E1-containing viruses showed that hepatotoxicity correlated with expression of wild-type E1a in the liver. Pharmacokinetic analysis showed rapid increases in viral DNA levels in the liver with a virus containing wild-type E1a. This was correlated with rapid induction of TNF-alpha to high levels and with rapid elevation of serum ALT. Hepatotoxicity was significantly reduced for an adenovirus with deletions in the region E1a (dl01/07) or a virus lacking E1a. The results suggest a mechanism for hepatotoxicity involving virus-induced production of local TNF-alpha release and E1a-mediated sensitization of hepatocyte killing.
Assuntos
Adenoviridae/genética , Proteínas E1A de Adenovirus/metabolismo , Vetores Genéticos/farmacocinética , Hepatite/virologia , Fígado/virologia , Fator de Necrose Tumoral alfa/biossíntese , Proteínas E1A de Adenovirus/análise , Proteínas E1A de Adenovirus/genética , Alanina Transaminase/sangue , Animais , DNA Viral/análise , DNA Viral/biossíntese , Feminino , Vetores Genéticos/genética , Vetores Genéticos/fisiologia , Hepatite/metabolismo , Hepatite/patologia , Hospedeiro Imunocomprometido , Injeções Intravenosas , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Camundongos SCID , Modelos Animais , Mutação , Especificidade da Espécie , Fatores de Tempo , Fator de Necrose Tumoral alfa/análise , Vírus/genéticaRESUMO
Adenoviral E1A is an indispensable protein for virus-host interaction. To provide a suitable environment for viral replication, E1A physically interacts with multiple cellular proteins to reprogram gene expression and other processes of the host cells. Proteins targeted by E1A include the pRb family of pocket proteins, p300/CBP, cyclin/Cdk, the carboxyl terminal binding protein (CtBP), transcriptional regulator YY1, and the recently identified RACK1 and SWI/SNF complex. Reprogramming activity of E1A and the host cell response to this reprogramming lead to transformation, growth arrest or apoptosis. Based on the ability of E1A to override the fundamental controls of host cells, E1A has been being utilized to make continuous contributions not only to a better understanding of the molecular mechanisms underlying the regulation of transcription, cell division, apoptosis and tumorigenesis but also to new therapeutics such as gene therapy.
Assuntos
Proteínas E1A de Adenovirus , Proteínas E1A de Adenovirus/análise , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/metabolismo , Proteínas E1A de Adenovirus/farmacologia , Apoptose , Divisão Celular , Transformação Celular Viral , Cromatina/metabolismo , Previsões , Inativação Gênica , Terapia Genética , Humanos , Modelos Biológicos , Neoplasias/terapia , Fatores de Transcrição/metabolismo , Ativação TranscricionalRESUMO
This study examines the hypothesis that the cigarette smoke-induced inflammatory process is amplified in severe emphysema and explores the association of this response with latent adenoviral infection. Lung tissue from patients with similar smoking histories and either no (n = 7), mild (n = 7), or severe emphysema (n = 7) was obtained by lung resection. Numbers of polymorphonuclear cells (PMN), macrophages, B cells, CD4, CD8 lymphocytes, and eosinophils present in tissue and airspaces and of epithelial cells expressing adenoviral E1A protein were determined using quantitative techniques. Severe emphysema was associated with an absolute increase in the total number of inflammatory cells in the lung tissue and airspaces. The computed tomography (CT) determined extent of lung destruction was related to the number of cells/m(2) surface area by R(2) values that ranged from 0.858 (CD8 cells) to 0.483 (B cells) in the tissue and 0.630 (CD4 cells) to 0.198 (B cells) in the airspaces. These changes were associated with a 5- to 40-fold increase in the number of alveolar epithelial cells expressing adenoviral E1A protein in mild and severe disease, respectively. We conclude that cigarette smoke-induced lung inflammation is amplified in severe emphysema and that latent expression of the adenoviral E1A protein expressed by alveolar epithelial cells influenced this amplification process.
Assuntos
Infecções por Adenoviridae/complicações , Proteínas E1A de Adenovirus/biossíntese , Enfisema/imunologia , Inflamação/fisiopatologia , Fumar/efeitos adversos , Proteínas E1A de Adenovirus/análise , Idoso , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Enfisema/fisiopatologia , Enfisema/virologia , Feminino , Humanos , Inflamação/virologia , Macrófagos , Masculino , Pessoa de Meia-Idade , Tomografia Computadorizada por Raios XRESUMO
A selective cyclooxygenase-2 (COX-2) inhibitor, NS-398, was shown to produce an anti-proliferative and pro-apoptotic effect on different types of cell lines. We describe the presence of COX-1 and COX-2 pathways in the human osteosarcoma 1547 cell line, as well as the conflicting effects of NS-398 (10, 50 and 100 microM) on programmed cell death, PGE2 release and COX-2 expression in 1547 cells cultured under apoptotic conditions. We demonstrate a link between the effects of 10 and 100 microM NS-398 on cell apoptosis, PGE2 release, and expression of COX-2 in 1547 cells undergoing apoptosis. At 10 microM, NS-398 acted as a selective COX-2 inhibitor moderately increasing apoptosis without any effect on COX-2 expression. In contrast, at 100 microM, NS-398 induced a cell cycle slowing or arrest, strongly enhanced COX-2 expression which was associated with a high PGE2 release and a marked decrease in apoptosis. This latter property of NS-398 at 100 microM in 1547 human osteosarcoma cells is novel compared to the described NS-398 pro-apoptotic effect on other cell lines.
Assuntos
Apoptose/efeitos dos fármacos , Neoplasias Ósseas/enzimologia , Inibidores de Ciclo-Oxigenase/farmacologia , Isoenzimas/biossíntese , Nitrobenzenos/farmacologia , Osteossarcoma/enzimologia , Prostaglandina-Endoperóxido Sintases/biossíntese , Sulfonamidas/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacos , Proteínas E1A de Adenovirus/análise , Ácido Araquidônico/metabolismo , Western Blotting , Neoplasias Ósseas/patologia , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Primers do DNA/química , Dinoprostona/análise , Relação Dose-Resposta a Droga , Humanos , Indóis , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Proteínas de Membrana , Osteossarcoma/patologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína X Associada a bcl-2RESUMO
The presence of replication-competent adenoviruses (RCAs) in batches of replication-defective adenovirus (Ad) vectors is a major problem for the application of these vectors in gene therapy. RCAs are generated by recombination between sequences in the Ad vector and homologous Ad sequences in the helper cells, resulting in the acquisition by the vector of early region 1. To prevent the formation of RCAs, we have developed helper cell lines, which we named PER, and matched Ad vectors that do not have sequence overlap. PER cells contain the Ad serotype 5 (Ad5) E1A- and E1B-encoding sequences (Ad5 nucleotides 459-3510) under the control of the human phosphoglycerate kinase (PGK) promoter. We demonstrate that PER cells synthesize high levels of the Ad5 E1A and E1B proteins. The yields from PER cells of E1-deleted Ads are similar to those obtained from earlier helper cells, such as 911 and 293 cells. Propagation of matched Ad vectors, which lack Ad5 nucleotides 459-3510, in one of the PER clones, PER.C6, does not result in the generation of RCAs, in contrast to propagation in 293 cells. We conclude that the combination of PER.C6 cells and nonoverlapping E1-deleted adenoviral vectors eliminates the problem of RCA generation by homologous recombination, and allows cost-effective production of safe, clinical-grade batches of recombinant Ad vectors.
Assuntos
Adenovírus Humanos/genética , Proteínas do Capsídeo , Linhagem Celular Transformada/virologia , Vetores Genéticos/genética , Vírus Auxiliares/genética , Replicação Viral/genética , Proteínas E1A de Adenovirus/análise , Proteínas E1A de Adenovirus/genética , Proteínas E1B de Adenovirus/análise , Proteínas E1B de Adenovirus/genética , Adenovírus Humanos/fisiologia , Capsídeo/biossíntese , Linhagem Celular Transformada/citologia , Temperatura Alta , Humanos , Retina/citologia , Vírion , Cultura de VírusRESUMO
A model of adenovirus 5 (Ad5) infection was developed in guinea pigs to begin to study its role in the pathogenesis of peripheral lung inflammation. Forty animals were inoculated intranasally with 10(7.0) pfu of Ad5/animal, and 15 animals inoculated with sterile culture media served as controls. Viral titres were 10(4.4), 10(6.1), 10(5.2), and 10(2.9) pfu/animal, on days 1, 3, 4, and 7 after infection, respectively. In situ hybridization to viral DNA and immunocytochemistry for Ad5 E1A protein localized the virus to airway and alveolar epithelial cells. Histologic examination showed an extensive inflammatory cell infiltration around the airways, with epithelial necrosis and an alveolar exudate that caused localized alveolar collapse in the infected areas. Immunocytochemistry identified the cells in the infiltrate as cytotoxic T cells. Although all animals 20 and 47 days after infection had seroconverted to Ad5, virus was not detected in these groups either by viral plaque assay or in situ hybridization. Ad5 E1A DNA was detected by polymerase chain reaction in five of six animals 20 days after infection and in five of five animals 47 days after infection. In these same animals, E1A protein was detected 20 days after infection in two and 47 days after infection in one while persistent bronchiolitis was observed in four and three animals 20 and 47 days after infection, respectively. These results demonstrate that the guinea pig provides a useful model to study the role of Ad5 infection in chronic airway inflammation.
Assuntos
Infecções por Adenovirus Humanos , Modelos Animais de Doenças , Cobaias , Pulmão/virologia , Pneumonia Viral , Proteínas E1A de Adenovirus/análise , Infecções por Adenovirus Humanos/imunologia , Infecções por Adenovirus Humanos/patologia , Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/imunologia , Adenovírus Humanos/isolamento & purificação , Animais , Anticorpos Antivirais/sangue , Sequência de Bases , Bronquiolite/imunologia , Bronquiolite/virologia , DNA Viral/análise , Epitélio/imunologia , Epitélio/patologia , Feminino , Humanos , Pulmão/imunologia , Dados de Sequência Molecular , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Alvéolos Pulmonares/imunologia , Alvéolos Pulmonares/patologia , Linfócitos T Citotóxicos/imunologiaRESUMO
The reasons for the dissimilar oncogenicities of human adenoviruses and human papillomaviruses (HPV) in humans are unknown but may relate to differences in the capacities of the E1A and E7 proteins to target cells for rejection by the host natural killer (NK) cell response. As one test of this hypothesis, we compared the abilities of E1A- and E7-expressing human fibroblastic or keratinocyte-derived human cells to be selectively killed by either unstimulated or interferon (IFN)-activated NK cells. Cells expressing the E1A oncoprotein were selectively killed by unstimulated NK cells, while the same parental cells but expressing the HPV type 16 (HPV-16) or HPV-18 E7 oncoprotein were resistant to NK cell lysis. The ability of IFN-activated NK cells to selectively kill virally transformed cells depends on IFN's ability to induce resistance to NK cell lysis in normal (i.e., non-viral oncogene-expressing) but not virally transformed cells. E1A blocked IFN's induction of cytolytic resistance, resulting in the selective lysis of adenovirus-transformed cells by IFN-activated NK cells. The extent of IFN-induced NK cell killing of E1A-expressing cells was proportional to the level of E1A expression and correlated with the ability of E1A to block IFN-stimulated gene expression in target cells. In contrast, E7 blocked neither IFN-stimulated gene expression nor IFN's induction of cytolytic resistance, thereby precluding the selective lysis of HPV-transformed cells by IFN-activated NK cells. In conclusion, E1A expression marks cells for destruction by the host NK cell response, whereas the E7 oncoprotein lacks this activity.
Assuntos
Adenovírus Humanos/patogenicidade , Transformação Celular Neoplásica , Citotoxicidade Imunológica , Células Matadoras Naturais/imunologia , Papillomaviridae/patogenicidade , Proteínas E1A de Adenovirus/análise , Proteínas E1A de Adenovirus/biossíntese , Adenovírus Humanos/imunologia , Linhagem Celular , Citotoxicidade Imunológica/efeitos dos fármacos , Fibroblastos , Expressão Gênica , Células HeLa , Humanos , Interferon-alfa/farmacologia , Queratinócitos , Proteínas Oncogênicas Virais/análise , Proteínas Oncogênicas Virais/biossíntese , Papillomaviridae/imunologia , Proteínas E7 de Papillomavirus , Células Tumorais CultivadasRESUMO
The adenoviral E1A proteins possess the ability to associate with the DNA binding domains of a number of transcription factors, and in this manner promiscuously to activate a wide variety of genes. The present study was designed to determine whether this protein is expressed in human lungs where nonlytic adenoviral infection has been demonstrated. Lung tissue from 12 patients harboring trace amounts of viral DNA were examined along with A549 cells infected with adenovirus 5 and uninfected Graham 293 (G293) cells as controls. Immunohistochemical staining was used to identify E1A proteins. The control studies examined both types of cultured cells either grown on coverslips, as cryosections of cells embedded in blocks, and or as formalin-fixed, paraffin-embedded sections. E1A proteins were detected in all three control preparations in both types of cells and were detected in the nucleus of adenovirus 5-infected A549 cells 4 h postinfection. Generally, all preparations of infected A549 cells showed greater of staining than the corresponding preparation of G293 cells. Formalin-fixed, paraffin-embedded cells gave the best morphology. Immunolabeling for adenovirus E1A proteins was also demonstrated in six of 12 paraffin-embedded lung samples. We conclude that adenovirus E1A proteins are expressed in human lung tissue and speculate that they may contribute to the pathogenesis of chronic obstructive pulmonary disease by amplifying the airways inflammation associated with cigarette smoking.
Assuntos
Proteínas E1A de Adenovirus/análise , Pulmão/metabolismo , Adenoviridae/metabolismo , Adenoviridae/patogenicidade , Linhagem Celular Transformada , Transformação Celular Viral , Humanos , Imuno-Histoquímica , Pulmão/virologiaRESUMO
The localization and distribution of three adenoviral proteins, hexon, E1A, and 55-kDa E1B, in 16 cases of fatal adenovirus bronchopneumonia in infants and children, are described. The proteins were immunohistochemically demonstrated in paraffin sections using monoclonal antibodies followed by the avidin-biotin-peroxidase method. The hexon antigen was present in inclusion-bearing bronchial, bronchiolar, and alveolar cells, mainly in the so-called rosette cells, as well as in necrotic debris in necrotizing areas. E1A antigen was also recognized in cells with nuclear inclusions where the reaction decorated the inclusion, nuclear chromatin, and cytoplasm but distributed mainly in alveolar cells and to a lesser extent in bronchial and bronchiolar cells. The 55-kDa E1B protein was extensively present in "activated," reactive-appearing, nuclei of bronchial, bronchiolar, and alveolar epithelial cells and in the cytoplasm of rare cells having nuclear inclusions. These activated nuclei did not stain for the other two antigens. "Smudge" cells reacted poorly or not at all with any of the antibodies. The reactivity found produced a sort of complementary pattern between the hexon-positive, inclusion-containing cells and the 55-kDa E1B-positive, inclusion-noncontaining cells. The relationships of present findings and virologic data are discussed.
Assuntos
Infecções por Adenoviridae/virologia , Proteínas E1A de Adenovirus/análise , Proteínas E1B de Adenovirus/análise , Broncopneumonia/virologia , Proteínas do Capsídeo , Capsídeo/análise , Infecções por Adenoviridae/imunologia , Infecções por Adenoviridae/mortalidade , Broncopneumonia/imunologia , Broncopneumonia/mortalidade , Pré-Escolar , Humanos , Corpos de Inclusão Viral/química , Lactente , Recém-NascidoRESUMO
The follicular form of bronchiectasis originally described by Whitwell (1) has been associated with adenoviral infection. The present study compares resected lungs from 16 patients with follicular bronchiectasis (in 10 of whom a nonviral etiology was identified) with those from eight patients with a nonfollicular histologic pattern. DNA isolated from sections of 45 paraffin-embedded lung samples was subjected to the polymerase chain reaction (PCR) using primers for the E1A region of the adenovirus genome and the human HLA DQ alpha gene. In situ hybridization (ISH) was performed on separate sections cut from the same blocks using a probe for the entire adenovirus 5 genome. E1A was demonstrated in six of eight patients (75%) with non-follicular bronchiectasis (non-FB) and in four of 16 (25%) with follicular bronchiectasis (FB) (p < 0.03, Fisher's exact test). The optical density ratio for the E1A product of PCR (ratio of E1A product from specimen to that from 1 pg adenovirus DNA) was significantly lower in FB than in non-FB (0.057 +/- 0.054 versus 0.365 +/- 0.223 [mean +/- SEM], p < 0.05). Moreover, the duration of symptoms of bronchiectasis in patients without E1A in bronchial specimens was significantly shorter than that of patients with positive E1A PCR products (3.09 +/- 1.44 versus 14.41 +/- 3.33 yr; p < 0.05). By ISH, adenovirus was demonstrated in three patients with FB and in two with non-FB (17.2 and 18.8% of tissue blocks, respectively; NS).(ABSTRACT TRUNCATED AT 250 WORDS)