RESUMEN
OBJECTIVE: To investigate the effect of the down-regulated expression of pituitary tumor-transforming gene 1 (PTTG1) on the senescence of human castration-resistant prostate cancer LNCaP-AI cells. METHODS: Human castration-resistant prostate cancer LNCaP-AI cells were induced in vitro and transfected with siRNA targeting PTTG1 (the siRNA-PTTG1 group), the reagent lip3000 only (the mock group) or siRNA negative control vector (the NC group). All the cells were cultured in fetal bovine serum (FBS) or charcoal-stripped bovine serum (CSS) and counted with the cell counting chamber. The senescence characteristics of the transfected LNCaP-AI cells were examined by senescence-associated ß-galactosidase (SA-ß-Gal) staining, and the expressions of the senescence-related ß-galactosidase-1-like proteins (Glb1), the cyclin-dependent kinase inhibitors p-21CIP1 and p-27Kip1, and the chromatin-regulating heterochromatin protein 1γ (HP1γ) were detected by Western blot. RESULTS: The expression of PTTG1 in the human prostate cancer LNCaP-AI cells was significantly reduced in the siRNA-PTTG1 group compared with those in the mock and NC groups (0.21 ± 0.01 vs 0.56 ± 0.02 and 0.61 ± 0.02, P < 0.05). Culture with FBS markedly increased while that with CSS decreased the number of LNCaP-AI cells transfected with siRNA, but both FBS and CSS enhanced the proliferation of the LNCaP-AI cells in the mock and NC groups. SA-ß-Gal staining revealed that reducing the expression of PTTG1 induced a remarkably higher positive rate of the LNCaP-AI cells in the siRNA-PTTG1 than in the mock and NC groups (ï¼»63.5 ± 2.35ï¼½% vs ï¼»11.3 ± 1.24ï¼½% and ï¼»12.4 ± 1.15ï¼½%, P < 0.05). The siRNA-PTTG1 group, in comparison with the mock and NC groups, showed a significantly down-regulated expression of PTTG1 (0.21 ± 0.01 vs 0.56 ± 0.02 and 0.61 ± 0.02, P < 0.05), but up-regulated expressions of p-21CIP1 (0.32 ± 0.03 vs 0.20 ± 0.02 and 0.21 ± 0.03, P < 0.05), p-27Kip1 (0.38 ± 0.02 vs 0.20 ± 0.03 and 0.22 ± 0.01, P < 0.05), Glb1 (0.24 ± 0.01 vs 0.13 ± 0.01 and 0.15 ± 0.01, P < 0.05), and HP1γ (0.41 ± 0.01 vs 0.26 ± 0.01 and 0.27 ± 0.02, P < 0.05) in the LNCaP-AI cells. CONCLUSIONS: Down-regulated expression of PTTG1 induces senescence of human castration-resistant prostate cancer LNCaP-AI cells.
Asunto(s)
Neoplasias de la Próstata Resistentes a la Castración/genética , Securina/genética , Línea Celular Tumoral , Proliferación Celular , Humanos , Masculino , Neoplasias de la Próstata Resistentes a la Castración/patología , ARN Interferente Pequeño , beta-Galactosidasa/genéticaRESUMEN
OBJECTIVE: To develop a specific SARS virus-targeted antibody preparation for emergent prophylaxis and treatment of SARS virus infection. METHODS: By using phage display technology, we constructed a naive antibody library from convalescent SARS patient lymphocytes. To obtain the neutralizing antibody to SARS virus surface proteins, the library panning procedure was performed on purified SARS virions and the specific Fab antibody clones were enriched by four rounds of repeated panning procedure and screened by highthroughput selection. The selected Fab antibodies expressed in the periplasma of E. coli were soluble and further purified and tested for their binding properties and antiviral function to SARS virus. The functional Fab antibodies were converted to full human IgG antibodies with recombinant baculovirus/insect cell systems and their neutralizing activities were further determined. RESULTS: After four rounds of the panning, a number of SARS-CoV virus-targeted human recombinant Fab antibodies were isolated from the SARS patient antibody library. Most of these were identified to recognize both natural and recombinant SARS spike (S) proteins, two Fab antibodies were specific for the virus membrane (M) protein, only one bound to SARS-CoV nucleocapsid protein. The SARS-CoV S and M protein-targeted Fab or IgG antibodies showed significant neutralizing activities in cytopathic effect (CPE) inhibition neutralization test, these antibodies were able to completely neutralize the SARS virus and protect the Vero cells from CPE after virus infection. However, the N protein-targeted Fab or IgG antibodies failed to neutralize the virus. In addition, the SARS N protein-targeted human Fab antibody reacted with the denatured N proteins, whereas none of the S and M protein specific neutralizing antibodies did. These results suggested that the S and M protein-specific neutralizing antibodies could recognize conformational epitopes which might be involved in the binding of virions to cellular receptors and the fusion activity of the virus. CONCLUSION: The SARS-CoV spike protein and membrane proteins are able to elicite efficient neutralizing antibodies in SARS patients. The neutralizing antibodies we generated in this study may be more promising candidates for prophylaxis and treatment of SARS infection.