Deletion of eIF2ß lysine stretches creates a dominant negative that affects the translation and proliferation in human cell line: A tool for arresting the cell growth.

BACKGROUND: Eukaryote initiation factor 2 subunit ß (eIF2ß) plays a crucial role in regulation protein synthesis, which mediates the interaction of eIF2 with mRNA. eIF2ß contains evolutionarily conserved polylysine stretches in amino-terminal region and a zinc finger motif in the carboxy-terminus. METHODS: The gene eIF2ß was cloned under tetracycline transcription control and the polylysine stretches were deleted by site-directed mutagenesis (eIF2ßΔ3K). The plasmid was transfected into HEK 293 TetR cells. These cells were analyzed for their proliferative and translation capacities as well as cell death rate. Experiments were performed using gene reporter assays, western blotting, flow cytometry, cell sorting, cell proliferation assays and confocal immunofluorescence. RESULTS: eIF2ßΔ3K affected negatively the protein synthesis, cell proliferation and cell survival causing G2 cell cycle arrest and increased cell death, acting in a negative dominant manner against the native protein. Polylysine stretches are also essential for eIF2ß translocated from the cytoplasm to the nucleus, accumulating in the nucleolus and eIF2ßΔ3K did not make this translocation. DISCUSSION: eIF2ß is involved in the protein synthesis process and should act in nuclear processes as well. eIF2ßΔ3K reduces cell proliferation and causes cell death. Since translation control is essential for normal cell function and survival, the development of drugs or molecules that inhibit translation has become of great interest in the scenario of proliferative disorders. In conclusion, our results suggest the dominant negative eIF2ßΔ3K as a therapeutic strategy for the treatment of proliferative disorders and that eIF2ß polylysine stretch domains are promising targets for this.

Mitochondria and redox homoeostasis as chemotherapeutic targets of Araucaria angustifolia (Bert.) O. Kuntze in human larynx HEp-2 cancer cells.

Natural products are among one of the most promising fields in finding new molecular targets in cancer therapy. Laryngeal carcinoma is one of the most common cancers affecting the head and neck regions, and is associated with high morbidity rate if left untreated. The aim of this study was to examine the antiproliferative effect of Araucaria angustifolia on laryngeal carcinoma HEp-2 cells. The results showed that A. angustifolia extract (AAE) induced a significant cytotoxicity in HEp-2 cells compared to the non-tumor human epithelial (HEK-293) cells, indicating a selective activity of AAE for the cancer cells. A. angustifolia extract was able to increase oxidative damage to lipids and proteins, and the production of nitric oxide, along with the depletion of enzymatic antioxidant defenses (superoxide dismutase and catalase) in the tumor cell line. Moreover, AAE was able to induce DNA damage, nuclear fragmentation and chromatin condensation. A significant increase in the Apoptosis Inducing Factor (AIF), Bax, poly-(ADP-ribose) polymerase (PARP) and caspase-3 cleavage expression were also found. These effects could be related to the ability of AAE to increase the production of reactive oxygen species through inhibition of the mitochondrial electron transport chain complex I activity and ATP production by the tumor cells. The phytochemical analysis of A. angustifolia, performed using High Resolution Mass Spectrometry (HRMS) in MS and MS/MS mode, showed the presence of dodecanoic and hexadecanoic acids, and phenolic compounds, which may be associated with the chemotherapeutic effect observed in this study.

Betacellulin overexpression in mesenchymal stem cells induces insulin secretion in vitro and ameliorates streptozotocin-induced hyperglycemia in rats.

Betacellulin (BTC), a ligand of the epidermal growth factor receptor, has been shown to promote growth and differentiation of pancreatic ß-cells and to improve glucose metabolism in experimental diabetic rodent models. Mesenchymal stem cells (MSCs) have been already proved to be multipotent. Recent work has attributed to rat and human MSCs the potential to differentiate into insulin-secreting cells. Our goal was to transfect rat MSCs with a plasmid containing BTC cDNA to guide MSC differentiation into insulin-producing cells. Prior to induction of cell MSC transfection, MSCs were characterized by flow cytometry and the ability to in vitro differentiate into mesoderm cell types was evaluated. After rat MSC characterization, these cells were electroporated with a plasmid containing BTC cDNA. Transfected cells were cultivated in Dulbecco's modified Eagle medium high glucose (H-DMEM) with 10 mM nicotinamide. Then, the capability of MSC-BTC to produce insulin in vitro and in vivo was evaluated. It was possible to demonstrate by radioimmunoassay analysis that 10(4) MSC-BTC cells produced up to 0.4 ng/mL of insulin, whereas MSCs transfected with the empty vector (negative control) produced no detectable insulin levels. Moreover, MSC-BTC were positive for insulin in immunohistochemistry assay. In parallel, the expression of pancreatic marker genes was demonstrated by molecular analysis of MSC-BTC. Further, when MSC-BTC were transplanted to streptozotocin diabetic rats, BTC-transfected cells ameliorated hyperglycemia from over 500 to about 200 mg/dL at 35 days post-cell transplantation. In this way, our results clearly demonstrate that BTC overabundance enhances glucose-induced insulin secretion in MSCs in vitro as well as in vivo.

Experiência da adoção do I e II Consensos Brasileiros de Fator Antinuclear por Imunofluorescência Indireta em Células HEp-2 em um hospital universitário / Experience of an university hospital on the implementation of I and II Brazilian Consensuses for Standardization of ANA in HEp-2 Cells

OBJETIVO: Analisar a prevalência, padrões e títulos do Fator Antinuclear (FAN) por imunofluorescência indireta (IFI) em células HEp-2 em um hospital universitário após a adoção do I e II Consensos Nacional para Padronização dos Laudos de FAN em Células HEp-2. MÉTODO: Foi realizado um estudo transversal, em que foram revisados os laudos de FAN por IFI originários de solicitações encaminhadas ao Serviço de Patologia Clínica do Hospital de Clínicas de Porto Alegre (SPC/HCPA) entre 2002 e 2005. RESULTADOS: Foram analisados 12.095 testes de FAN no período entre 2002 e 2005. As solicitações com resultado reagente foram de 2.577 (21,30 por cento), com média anual de 644±233). Houve um aumento significativo na proporção de resultados reagentes posterior à adoção dos Consensos (p < 0,001). A Reumatologia foi a especialidade que mais solicitou exames por paciente atendido, mas houve um declínio nesse número nos anos posteriores à adoção do Consenso, ocorrido em 2004 (p < 0,001). O padrão de imunofluorescência de FAN mais frequentemente encontrado foi o padrão nuclear pontilhado fino, presente em 52,3 por cento dos resultados reagentes (453/866), e os títulos mais encontrados foram 1/80 e 1/160 (27,8 por cento e 29,4 por cento, respectivamente). CONCLUSÃO: Após a adoção do Consenso Nacional de Padronização de Laudos de FAN, observou-se um aumento de exames com resultados reagentes, na sua maioria, com títulos baixos e padrão nuclear pontilhado fino. Na Reumatologia, observou-se uma diminuição no número de solicitações desse exame. As potenciais causas para essas observações são discutidas, mas seu real impacto sobre a situação clínica do paciente e seu tratamento merece ser mais bem estudado.

OBJECTIVE: To evaluate the prevalence of patterns and titers of antinuclear antibodies (ANA) detected by indirect immunofluorescence (IIF) technique on HEp-2 cells in a university hospital following the introduction of I and II Brazilian Consensuses for Standardization of ANA in HEp-2 Cells. METHODS:A transversal study was performed between 2002 and 2005 during which all ANA orders to Serviço de Hospital de Clínicas de Porto Alegre (SPC/HCPA) and cognate results were reviewed. RESULTS:12.095 tests of ANA were revised. The number of positive results during this period was 2.577 (21.30 percent), annual mean 644 (SD: 233). A marked increase in the number of positive results was observed following the introduction of the Consensuses (p < 0.001). Rheumatology was the medical specialty which requested the highest number of ANA testing per patient although a significant decrease of these numbers was observed after the introduction of the Consensus in 2004 (p < 0,001). Nuclear fine speckled immunofluorescence labeling was the most frequently ANA pattern observed, 52.3 percent (453/866), and low ANA titers (1/80 and 1/160) more commonly detected (27.8 percent and 29.4 percent, respectively). CONCLUSION: Following the introduction of the Brazilian Consensus for standardization of ANA in HEp-2 cells an increased number of positive results was observed, mostly in low titers and with nuclear fine speckled immunofluorescence pattern. Moreover, there were decreasing numbers of ANA orders by rheumatologists in the same period. Potential causes for these observations are discussed but the real impact in the clinical condition of the patient and therapy deserves to be better studied.

Padrões de imunofluorescência do fator antinuclear (FAN) em células HEp-2 de soros reagentes para anti-SSA/Ro / Antinuclear antibodies (ANA) immunofluorescent patterns in HEp-2 cells on samples positive for anti-SSA/Ro

OBJETIVO: avaliar os padrões de imunofluorescência do fator antinuclear (FAN) em soros reagentes para anticorpos anti-SSA/Ro e sua associação clínica. MÉTODO: foi realizado um estudo transversal retrospectivo, no qual foram revisadas as solicitações de anticorpos antiantígenos nucleares extraíveis (anti-ENA) encaminhadas ao SPC/HCPA no período de dois anos. Das solicitações com resultado positivo para anti-ENA identificou-se qual ou quais auto-anticorpos estavam envolvidos (anti-SSA/Ro, anti-SSB/La, anti-RNP, anti-Sm, anti-Scl-70), bem como os padrões de imunofluorescência do FAN e os quadros clínicos dos pacientes anti-SSA/Ro positivo. As técnicas usadas para detecção e identificação foram FAN por imunofluorescência indireta (IFI) em células HEp-2 e anti-ENA por hemaglutinação. RESULTADOS: das 392 solicitações analisadas 90 eram anti-ENA positivo. Houve um predomínio do sexo feminino (94 por cento) (86/91) e a idade média foi de 42 anos. O anti-SSA/Ro foi o mais freqüente (67,8 por cento) (61/90), sendo que todas as amostras anti-SSA/Ro positivas eram positivas para o FAN. O padrão de imunofluorescência nuclear pontilhado fino foi o predominante (68,9 por cento) (42/61) nos pacientes com anti-SSA/Ro positivo, e o quadro clínico mais encontrado foi de lúpus eritematoso sistêmico, em 50,8 por cento (31/61) dos pacientes. CONCLUSÃO: o teste de FAN por IFI utilizando células HEp-2 é um bom método de triagem para detecção de auto-anticorpos anti-SSA/Ro, apresentando maior associação com o padrão nuclear pontilhado fino. Diferente do que tem sido descrito na literatura, não encontramos nenhuma amostra de pacientes com anti-SSA/Ro que tenham apresentado FAN falso-negativo na IFI. Pelo menos na nossa experiência, esses dados questionam o custo-efetividade da solicitação de rotina desse exame em pacientes FAN negativo pelo teste de IFI.

OBJECTIVE: to evaluate the pattern at immunofluorescence of the antinuclear antibodies (ANA) detected by the indirect immunofluorescence (IIF) technique in positive samples for anti-SSA/Ro autoantibody and the clinical associations. METHODS: a retrospective transversal study was performed in a period of two years where the all the solicitations of testing for the presence of anti-extractable nuclear antigen (anti-ENA) antibodies delivered to the SPC/HCPA were analyzed. We selected the positive samples and identified which autoantibodies were involved (anti-SSA/RO, anti-SSB/La, anti-RNP, anti-Sm and anti-Scl70) as well as the immunofluorescence patterns by ANA testing and the clinical associations found in the patients presenting anti-SSA/Ro positive serum. IIF was used for ANA using HEp-2 cells and hemagglutination for anti-ENA antibodies detection. RESULTS: 90 out of the 392 solicitations analyzed were anti-ENA positive, with a predominance of women (86/91 - 94 percent) and the mean age was 42 years old. The most frequent autoantibody was anti-SSA/Ro (61/90 - 67.8 percent) and all samples that were anti-SSA/Ro positive were also ANA positive. Speckled nuclear immunofluorescence was the most frequent ANA pattern (42/61 - 68.9 percent) among the anti-SSA/Ro positive samples and systemic lupus erythematosus was the most common clinical diagnosis (31/61 - 50.8 percent). CONCLUSION: ANA testing by IIF using HEp-2 cells proved to be a good screening test for the detection of anti-SSA/Ro antibodies, that showed a strong positive association to the speckled nuclear IIF pattern. As opposed to what has been described in the literature, there was no ANA negative among the anti-SSA/Ro positive samples. At least in our experience, these data question the cost-effectiveness of performing routine screening for anti-SSA/Ro antibodies in ANA negative samples by IIF testing.

Caracterização de sistemas de anti-corpos-auto-antígenos associados ao padrão de imunofluorescência de pontos citoplasmáticos isolados / Caracterization of autoantibody-autoantigen systems associated with cytoplasm discrete speckled immunofluorescence pattern

Objetivo - Identificação e caracterização de auto-anticorpos e auto-antígenos associados ao padrão de imunofluorescéncia indireta (IFI) de Pontos Citoplasmáticos Isolados (PU). Materiais e métodos - Foram selecionados soros que apresentavam o padrão de PCI à IFI. Esses soros foram analisados por IFI dupla em microscopia confocal, imunomicroscopia eletrônica, immunoblotting, imunoprecipitação de antígenos protéicos sintetizados in vitro, HPTLC de lipídeos seguida de imunocoloração e análise de espectrometria de massas para antígenos lipídicos. Os dados clínicos foram obtidos por revisão de prontuários e entrevistas com os médicos assistentes. Resultados Dois sistemas de auto-antigeno-auto-anticorpo associados com o padrão de IFI de pontos citoplasmáticos isolados (PCI) foram identificados e denominados de padrão PCI-I e PCI-II. O padrão PCI-I foi caracterizado por 3 a 20 pontos brilhantes bem delimitados e homogeneamente distribuídos pelo citoplasma. O padrão PCI-11 foi caracterizado por apresentar mais de 30 pontos por todo o citoplasma, com predominância perinuclear, grande heterogeneidade de tamanho e tendência a aglomeração. De acordo com esta classificação foram obtidos 27 soros PCI-I e 6 soros PCI-II. Por IFI dupla em microscopia confocal e imuno-microscopia eletrônica os soros PCI-11 mostraram co-localização com os anticorpos monoclonais anti-LAMP-2 (proteína-2 associada à membrana do lisossomo) e anti-EEA1 (antígeno-1 do endossomo primário) e com receptor de transferrina, marcadores da via endocíticaexocítica. Os domínios citoplasmáticos reconhecidos pelos auto-anticorpos PCI-11 parecem ser delimitados por membrana. Por immunoblotting soros PCI-11 reconheceram uma banda com mobilidade relativa de 180kDa e imunoprecipitaram a proteína EEA1. Em contraste, os anticorpos PCI-I não apresentaram co-localização com anti-EEA1 e anti-LAMP-2, porém apresentaram co-localização parcial com o anticorpo monoclonal anti-GW182, que reconhece uma proteína de 182 kDa (GW182), presente em aglomerados citoplasmáticos de mRNA. Os soros PCI-I não demonstraram qualquer reatividade ao immunoblotting com diversos extratos celulares e não imunoprecipitaram as proteínas GW182 e EEA1 sintetizadas in vitro...