RESUMO
Transmembrane protein 16A (TMEM16A) localizes at plasma membrane and controls chloride influx in various type of cells. We here showed an intracellular localization pattern of TMEM16A molecules. In myoblasts, TMEM16A was primarily localized to the cytosolic compartment and partially co-localized with intracellular organelles. The global deletion of TMEM16A led to severe skeletal muscle developmental defect. In vitro observation showed that the proliferation of Tmem16a-/- myoblasts was significantly promoted along with activated ERK1/2 and Cyclin D expression; the myogenic differentiation was impaired accompanied by the enhanced caspase 12/3 activation, implying enhanced endoplasmic reticulum (ER) stress. Interestingly, the bradykinin-induced Ca2+ release from ER calcium store was significantly enhanced after TMEM16A deletion. This suggested a suppressing role of intracellular TMEM16A in ER calcium release whereby regulating the flux of chloride ion across the ER membrane. Our findings reveal a unique location pattern of TMEM16A in undifferentiated myoblasts and its role in myogenesis.
RESUMO
BACKGROUND: Previous studies showed that alpha-1,2-fucosyltransferase (HT), decay accelerating factor (DAF), and CD59 have an inhibitory effect on the immunological rejection of xenogenic transplantation. METHODS: To investigate their possible synergistic effects in suppression of heterogeneic transplantation, we produced transgenic mouse lines expressing human HT, DAF, and/or CD59 by the standard pronuclear injection approach. PCR and Southern blot were used to identify the transgenic founder lines. Flow cytometry confirmed the high-level expression of HT, DAF, or CD59 in the transgenic mice. RESULTS: The deposition of IgM, C3c, or C9 in the cardiac vascular endothelial cells of the HT, HT/CD59, and/or DAF multiple positive transgenic mice was markedly decreased. The survival time and function of the hearts of the co-transgenic mice were significantly longer and higher than that of the single HT-positive transgenic mice (P < 0.05). CONCLUSION: The mice co-expressing HT/DAF or HT/CD59 could resist the hyperacute rejection better than those expressing HT alone. It is feasible to use HT and C-reactive proteins co-transgenic tissues to resist hyperacute rejection and xenograft rejection.
Assuntos
Anticorpos Heterófilos/metabolismo , Antígenos CD55/metabolismo , Antígenos CD59/metabolismo , Fucosiltransferases/metabolismo , Transplante de Coração/imunologia , Transplante Heterólogo/imunologia , Animais , Antígenos CD55/genética , Antígenos CD59/genética , Complemento C3c/metabolismo , Complemento C9/metabolismo , Endotélio Vascular/imunologia , Fucosiltransferases/genética , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Humanos , Imunoglobulina M/metabolismo , Camundongos , Camundongos Transgênicos , Miocárdio/imunologiaRESUMO
OBJECTIVE: To evaluate the anticancer effects of exogenous human WT-PTEN overexpression on bladder transitional carcinoma cell line EJ. METHODS: The plasmid containing WT-PTEN or mutant PTEN was separately transfected into bladder transitional carcinoma cell line EJ, and the protein expression of PTEN in the EJ cells was detected by Western blot. Cell morphological changes were observed under the inverted microscope and transmission electron microscope. MTT test was used to assess the effect of PTEN on proliferation and anticancer effects for mitomycin and theraubicin. The change of bcl-2 expression in the cells was measured by Western blot. The empty plasmid was used as control. RESULTS: Western blot analysis showed that EJ cells expressed high level of PTEN protein after transfection with WT-PTEN or mutant PTEN plasmid. Abnormal morphological changes of the cells were observed in WT-PTEN transfected groups. The growth of EJ cells treated with WT-PTEN was significantly inhibited by 40.1% and anticancer effects were enhanced by mitomycin and theraubicin, but the cells transfected with mutant PTEN plasmid did not show such similar biological behavior. CONCLUSION: WT-PTEN gene transfection can suppress the in vitro growth and induce apoptosis of bladder transitional carcinoma cell line EJ cells. Mutant PTEN does not show similar biological behavior. Overexpression of WT-PTEN inhibits cancer cell proliferation by down-regulating bcl-2 expression in the cells.
Assuntos
Proliferação de Células , Mutação , PTEN Fosfo-Hidrolase/fisiologia , Antibióticos Antineoplásicos/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Carcinoma de Células de Transição/genética , Carcinoma de Células de Transição/metabolismo , Carcinoma de Células de Transição/patologia , Linhagem Celular Tumoral , Doxorrubicina/análogos & derivados , Doxorrubicina/farmacologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Microscopia Eletrônica de Transmissão , Mitomicina/farmacologia , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Plasmídeos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/fisiologia , Transfecção , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologiaRESUMO
OBJECTIVE: To investigate the expression of prostate cancer antigen-1 (PCA-1) in different prostate tissues and analyze its correlation with the clinical parameters of prostate cancer (PCa). METHODS: The expression of PCA-1 mRNA was detected by RT-PCR in the samples from 45 cases of PCa with various clinico-pathologic characteristics, 30 cases of high-grade prostatic intraepithelial neoplasia (HG-PIN), 43 cases of BPH and 39 cases of other carcinoma tissues. The correlation of PCA-1 mRNA expression with the clinical parameters of PCa was statistically analyzed and the PCA-1 expression was examined in different samples by immunohistochemistry. RESULTS: The positive expression rate of PCA-1 mRNA was 88.9% and 60.0% and that of PCA-1 protein was 84.4% and 50.0% in the patients with PCa and HG-PIN, respectively. PCA-1 mRNA and PCA-1 proteins were not expressed in the BPH and other carcinoma tissues. The expression of PCA-1 mRNA was unrelated with the clinical parameters of PCa (P > 0.05). CONCLUSION: It is suggested that PCA-1 is a PCa-specific gene and its expression is unrelated to the clinical parameters of PCa. It might serve as a specific biomarker for the early diagnosis of PCa.
Assuntos
Regulação Neoplásica da Expressão Gênica , Antígeno Prostático Específico/genética , Neoplasias da Próstata/patologia , Idoso , Idoso de 80 Anos ou mais , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Antígeno Prostático Específico/biossíntese , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
To investigate the effect of small-interfering RNA (siRNA) targeted against Ki-67, which is an attractive molecular target for cancer therapy, on inhibiting Ki-67 expression and cell proliferation in human renal carcinoma cells (HRCCs), siRNAs were used to inhibit the expression of Ki-67 in HRCCs. Ki-67 mRNA levels were detected by RT-PCR and in situ hybridization analysis. Ki-67 protein levels were detected by Western blot and immunocytochemistry analysis. TUNEL assay was used to measure the apoptosis of carcinoma cells. Results of RT-PCR and in situ hybridization demonstrated reduction of Ki-67 mRNA expression in Ki-67 siRNAs treated 786-0 cells. Similar reduction in Ki-67 protein measured by Western blot and immunocytochemistry was observed in cells transfected with Ki-67 siRNA. Ki-67-siRNA treatment of HRCCs resulted in specific inhibition of proliferation and increased apoptotic cell death. From these findings we conclude that inhibition of Ki-67 expression by siRNA may be a reasonable approach in renal cancer therapy.
Assuntos
Apoptose/fisiologia , Carcinoma/metabolismo , Inativação Gênica/fisiologia , Antígeno Ki-67/metabolismo , Neoplasias Renais/metabolismo , RNA Interferente Pequeno/metabolismo , Apoptose/efeitos dos fármacos , Carcinoma/tratamento farmacológico , Carcinoma/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inativação Gênica/efeitos dos fármacos , Humanos , Hibridização In Situ , Marcação In Situ das Extremidades Cortadas , Antígeno Ki-67/genética , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
AIM: To investigate the effect of androgen on the proliferation, differentiation and regression of canine prostatic stromal cells in vivo and human stromal cells in vitro. METHODS: Twenty-two dogs, including 15 normal prostate dogs and 7 prostatic hyperplasia dogs, had their serum concentration of testosterone and estrodiol determined by radioimmunoassay before and after castration. The expression of androgen receptor (AR) and estrogen receptor (ER) in the prostate were analysed by immunohistochemistry and semi-quantitative RT-PCR before and after castration. Light microscopy, transmission electron microscopy and TUNEL assay were carried out successively before and after castration to evaluate the prostatic histomorphology. In vitro serum-free cell cultures from human prostatic stroma were established and exposed to dihydrotestosterone (DHT). The proliferation of the cell culture was detected by MTT assay. The expression of TGFbgr, bFGF, AR, and smooth muscle cell (SMC) specific proteins (myosin and/or smoothelin) were detected using immunohistochemistry and RT-PCR. The differentiation from fibroblasts to smooth muscle cells was deduced by measuring the expression of SMC specific proteins. RESULTS: Before castration, the serum concentrations of testosterone and estrodiol were not statistically different between normal and hyperplasia groups. Following castration, the serum concentration of testosterone decreased rapidly in 2 days, and the concentration of estrodiol had no significant change compared with the pre-castration data. In the prostate, AR was presented in both the epithelial and stromal cells and the AR mRNA level was higher in hyperplasia than in normal prostate tissues (P<0.05). While ER predominantly existed in the prostate stromal cells and the ER mRNA had no difference between the hyperplasia and the normal group. Within the early phase of castration (Assuntos
Próstata/fisiologia
, Hiperplasia Prostática/fisiopatologia
, Células Estromais/fisiologia
, Testosterona/sangue
, Animais
, Biomarcadores
, Diferenciação Celular/efeitos dos fármacos
, Diferenciação Celular/fisiologia
, Divisão Celular/efeitos dos fármacos
, Divisão Celular/fisiologia
, Células Cultivadas
, Di-Hidrotestosterona/farmacologia
, Cães
, Estradiol/sangue
, Fator 2 de Crescimento de Fibroblastos/genética
, Fator 2 de Crescimento de Fibroblastos/farmacologia
, Expressão Gênica
, Humanos
, Masculino
, Músculo Liso/citologia
, Músculo Liso/fisiologia
, Orquiectomia
, Próstata/citologia
, RNA Mensageiro/análise
, Receptores Androgênicos/genética
, Receptores de Estrogênio/genética
, Células Estromais/citologia
, Fator de Crescimento Transformador beta/genética
, Fator de Crescimento Transformador beta/farmacologia
RESUMO
OBJECTIVE: To investigate the inhibitory effects of antisense TGF beta1 on proliferation of human bladder transitional cell carcinoma in vitro and in vivo. METHODS: Human bladder carcinoma cell line EJ was transfected with pRevT beta-AS, a replication defective retroviral vector carried antisense TGF beta1 fragment. The growth of the transfected cells was observed in vitro and in vivo. TGF beta1 mRNA expression and protein expression were detected by RT-PCR and ELISA. The proliferative activity was evaluated by immunohistochemistry method. The ultrastructure of cells was observed by image analysis system and electron microscopy. Cell cycle was determined by flow cytometry. RESULTS: The expression of TGF beta1 mRNA and protein in EJ cells was inhibited by pRevT beta-AS, G(1) to S transition was restrained in cell cycle and cell proliferation decreased in vitro. The tumorigenesis and growth of EJ cells and DNA heteroploidy were reduced by antisense TGF beta1 in vivo. CONCLUSION: TGF beta1 plays a role in vitro proliferation and in vivo growth of bladder transitional cell carcinoma.
Assuntos
RNA Antissenso/uso terapêutico , Fator de Crescimento Transformador beta/antagonistas & inibidores , Neoplasias da Bexiga Urinária/tratamento farmacológico , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos SCID , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta1 , Neoplasias da Bexiga Urinária/patologiaRESUMO
OBJECTIVE: To investigate the specific induction of cytotoxic lymphocyte (CTL) by tumor-derived heat shock protein 90-peptide complexes (HSP90-PC). METHODS: Heat shock protein 90-peptide complex (HSP90-PC) was isolated and purified by liquid chromatography after precipitation by 50% - 70% (NH4) 2SO4 saturation from 10 specimens of renal carcinoma resected from 10 patients aged 40 - 60 during operation. The component containing HSP90-PC was filtered and sterilized. The molecular weight and the identity of the purified HSP90-PC were confirmed by SDS-PAGE and Western blotting. 10 - 15 ml peripheral blood was extracted from these patients. T cells were amplified. Flow cytometry was used to detect the phenotype of dendritic cells (DCs). The DCs in the experimental group were cultured for 5 days and then HSP90-PC and tumor necrosis factor (TNF)-alpha was added into the culture. The HSP90-PC pulsed DCs were collected and co-cultured with auto-T cells for 72 hours. Flow cytometry was used to detect the content of CD8(+) T cells. The DC of the control group were mixed directly with auto-T cells and the content of CD8(+) T cells was examined by flow cytometry. RESULTS: The proliferation of T cells co-cultured with the HSP90-PC pulsed DCs was significantly remarkable and the content of CD8(+) CTLs was significantly more in comparison with the control DC (P < 0.01). CONCLUSION: HSP90-PC prepared from tumor tissues has strong immunogenicity and the DC sensitized thereby effectively induces the proliferation of CTL. Application of HSP90-PC provides a new approach in tumor immunotherapy.
Assuntos
Proteínas de Choque Térmico HSP90/farmacologia , Neoplasias Renais/química , Linfócitos T Citotóxicos/citologia , Adulto , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/imunologia , Feminino , Proteínas de Choque Térmico HSP90/isolamento & purificação , Humanos , Imunoterapia , Neoplasias Renais/imunologia , Neoplasias Renais/patologia , Masculino , Pessoa de Meia-Idade , Linfócitos T Citotóxicos/imunologiaRESUMO
OBJECTIVE: To construct a recombinant human CD59 gene containing intercellular adhesion molecule-2 promoter for high level endothelial-specific expression in xenotransplantation. METHODS: ICAM-2 promotor fragment and CD59-intron 1 fragment were produced by PCR from the human blood genome, and then clone these fragments into a pcDNA3-CD59 eukaryotic expression vector which was followed by digestion with the specific restricted endonuclease (for example: EcoRI, Hind III). The ICAM-2 promoter and CD59-intron 1 fragments were identified by PCR, and sequencing. The recombinant was then transfected into pig aorta endothelial cells with Lipofection, and the expression was measured by flow cytometer. RESULTS: Products of the sequences measured were in accord with the frames of the gene bank. The expression of the protein of this recombinant was positive. CONCLUSION: The CD59 recombinant gene is constructed successfully, providing a basis for transgenic research.
Assuntos
Antígenos CD/genética , Antígenos CD59/genética , Moléculas de Adesão Celular/genética , Endotélio Vascular/metabolismo , Regiões Promotoras Genéticas , Animais , Antígenos CD59/biossíntese , Clonagem Molecular , Endotélio Vascular/citologia , Células Eucarióticas/metabolismo , Regulação da Expressão Gênica , Humanos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Suínos , Transfecção , Transplante HeterólogoRESUMO
Short hairpin RNAs (shRNAs) transcribed by RNA polymerase III promoters can trigger sequence-selective gene silencing in mammalian cells. By virtue of their excellent function in knocking down expression of cancer-associated genes, shRNAs could be used as new therapeutic agents for cancer. As overexpression of Ki67 in renal cancer has been correlated to a more aggressive tumor phenotype, inhibition of Ki67 protein expression by means of shRNAs seems to be a promising approach for the therapy of renal cancer. In this study, we constructed an expression plasmid encoding shRNAs against the Ki67 gene, named pSilencerKi67, and transfected it into human renal carcinoma cells. The pSilencerKi67 was shown to significantly knock down the expression of the Ki67 gene in human renal carcinoma cells, resulting in inhibiting proliferation and inducing apoptotic cell death that can be maintained for at least 6 d. These findings offer the promise of using vector-based shRNAs against Ki67 in renal cancer gene therapy.
Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Antígeno Ki-67/fisiologia , RNA Interferente Pequeno/farmacologia , Linhagem Celular Tumoral , Vetores Genéticos/uso terapêutico , Humanos , Marcação In Situ das Extremidades Cortadas , Neoplasias Renais , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
Telomerase is an attractive molecular target for cancer therapy because it is present in most malignant cells but is undetectable in most normal somatic cells. Human telomerase consists of two subunits, an RNA component (hTR) and a human telomerase reverse transcriptase component (hTERT). Small interfering RNA (siRNA), one kind of RNA interferences, has been demonstrated to be an effective method to inhibit target gene expression in human cells. We investigated the effects of siRNA targeting at both hTR and hTERT mRNA on the inhibition of telomerase activity in human renal carcinoma cells (HRCCs). The proliferation and apoptosis of HRCCs were examined. The treatment of HRCCs using hTR and hTERT siRNAs resulted in significant decrease of hTR mRNA, hTERT mRNA and hTERT protein. The siRNA can also inhibit the telomerase activity and the proliferation of HRCCs. Moreover, they can induce apoptotic cell death in a dose-dependent manner. From these findings, we propose that the inhibition of telomerase activity using siRNA targeting hTR and hTERT might be a rational approach in renal cancer therapy.