Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 9 de 9
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Acta Diabetol ; 49(2): 125-30, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20309589

RESUMO

Diabetes is often associated with atherothrombosis. It is unknown whether high glucose can modulate the expression of tissue factor (TF) and thrombomodulin (TM) in human aortic endothelial cells (HAECs). HAECs were treated with a lower-degree high glucose condition (LG, 11.2 mM) for 8 days and a higher-degree high glucose condition (HG, 30 mM) for 4-6 h. Methoxyphenyl tetrazolium inner salt assay, real-time polymerase chain reaction, western blot, and TF activity assay were performed. In HAECs, both LG and HG conditions were nontoxic. LG caused a 74 ± 20% decrease (P = 0.273) and HG caused a 57 ± 5% decrease in TF mRNA expression (P = 0.001). LG caused a 53 ± 13% decrease (P = 0.036) and HG caused a 75 ± 10% decrease in TF protein expression (P = 0.096). TF activity was not significantly changed by LG (127 ± 13%, P = 0.40) or HG treatments (120 ± 42%, P = 0.70). In contrast, LG caused a 153 ± 16% increase (P = 0.03) and HG caused a 211 ± 20% increase in TM mRNA expression (P = 0.005). LG caused a 131 ± 31% increase (P = 0.35) and HG caused a 140 ± 9% increase in TM protein expression (P = 0.006). Different high glucose conditions do not provide the sufficient stress required to induce TF expression in HAECs. In contrast, high glucose conditions can induce TM expression in HAECs.


Assuntos
Aorta/citologia , Regulação da Expressão Gênica , Glucose/metabolismo , Trombomodulina/genética , Tromboplastina/genética , Aorta/metabolismo , Linhagem Celular , Células Endoteliais/metabolismo , Humanos , Trombomodulina/metabolismo , Tromboplastina/metabolismo
2.
J Clin Neurosci ; 18(8): 1048-54, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21715171

RESUMO

Previous clinical trials of dendritic cell (DC)-based immunotherapy in patients with glioblastoma multiforme (GBM) have reported induction of systemic immune responses and prolonged survival. From 2003 to 2005, we performed a clinical trial in which patients with malignant glioma underwent surgery for maximal cytoreduction followed by a 6-month 10-injection course of autologous DC-tumor vaccine therapy, each injection containing 1-6×10(7) DC. Of the 17 treated patients (16 with World Health Organization grade IV and one with grade III glioma), eight (47.1%) had an initial transient elevation in aspartate aminotransferase (AST)/alanine aminotransferase (ALT). Vaccination caused some tumor shrinkage and increased concentration of tumor-infiltrating CD8(+) lymphocytes. Median survival and 5-year survival were 525 days and 18.8%, respectively, for 16 patients with grade IV glioma (381 days and 12.5% for eight newly diagnosed; 966 days and 25% for eight relapsed patients) compared to 380 days and 0% for 63 historical control patients. We concluded that autologous DC-tumor immunotherapy benefits patients with malignant glioma but may cause transient but reversible elevation of serum AST/ALT levels.


Assuntos
Neoplasias Encefálicas/terapia , Células Dendríticas/imunologia , Glioma/terapia , Imunoterapia Adotiva/métodos , Adolescente , Adulto , Idoso , Antígenos CD/metabolismo , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/mortalidade , Vacinas Anticâncer , Feminino , Seguimentos , Proteína Glial Fibrilar Ácida/metabolismo , Glioma/imunologia , Glioma/mortalidade , Humanos , Imunoterapia Adotiva/efeitos adversos , Estimativa de Kaplan-Meier , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Período Pós-Operatório , Fatores de Tempo , Resultado do Tratamento , Adulto Jovem
3.
PLoS One ; 6(6): e18643, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21694768

RESUMO

Alternative splicing involves differential exon selection of a gene transcript to generate mRNA and protein isoforms with structural and functional diversity. Abnormal alternative splicing has been shown to be associated with malignant phenotypes of cancer cells, such as chemo-resistance and invasive activity. Screening small molecules and drugs for modulating RNA splicing in human hepatocellular carcinoma cell line Huh-7, we discovered that amiloride, distinct from four pH-affecting amiloride analogues, could "normalize" the splicing of BCL-X, HIPK3 and RON/MISTR1 transcripts. Our proteomic analyses of amiloride-treated cells detected hypo-phosphorylation of splicing factor SF2/ASF, and decreased levels of SRp20 and two un-identified SR proteins. We further observed decreased phosphorylation of AKT, ERK1/2 and PP1, and increased phosphorylation of p38 and JNK, suggesting that amiloride treatment down-regulates kinases and up-regulates phosphatases in the signal pathways known to affect splicing factor protein phosphorylation. These amiloride effects of "normalized" oncogenic RNA splicing and splicing factor hypo-phosphorylation were both abrogated by pre-treatment with a PP1 inhibitor. Global exon array of amiloride-treated Huh-7 cells detected splicing pattern changes involving 584 exons in 551 gene transcripts, many of which encode proteins playing key roles in ion transport, cellular matrix formation, cytoskeleton remodeling, and genome maintenance. Cellular functional analyses revealed subsequent invasion and migration defects, cell cycle disruption, cytokinesis impairment, and lethal DNA degradation in amiloride-treated Huh-7 cells. Other human solid tumor and leukemic cells, but not a few normal cells, showed similar amiloride-altered RNA splicing with devitalized consequence. This study thus provides mechanistic underpinnings for exploiting small molecule modulation of RNA splicing for cancer therapeutics.


Assuntos
Processamento Alternativo/efeitos dos fármacos , Amilorida/farmacologia , Neoplasias/genética , Neoplasias/patologia , RNA Neoplásico/genética , Processamento Alternativo/genética , Amilorida/química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Citocinese/efeitos dos fármacos , Citoesqueleto/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Éxons/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Neoplásicos/genética , Genoma Humano/genética , Humanos , Mitose/efeitos dos fármacos , Invasividade Neoplásica , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Fosforilação/efeitos dos fármacos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Fosfatase 1/metabolismo , Proteômica , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Processamento de Serina-Arginina
4.
J Nucl Med ; 47(5): 877-84, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16644759

RESUMO

UNLABELLED: Noninvasive imaging in lung metastatic tumor models is used infrequently because of technical limitations in detecting metastases. We have previously used 2'-fluoro-2'-deoxy-5-iodo-1-beta-d-arabinofuranosyluracil labeled with (131)I ((131)I-FIAU) and demonstrated the applicability of noninvasive imaging for monitoring cancer gene therapy in an experimental animal model of herpes simplex virus type 1 thymidine kinase (HSV1-tk)-expressing tumor xenografts. We have now used the same animal model to effectively and noninvasively monitor the location, magnitude, and duration of therapeutic gene expression over time for the lung metastases model. METHODS: To improve the detectability of lung metastases, an experimental blood-borne lung metastases model in mice was established using intravenously administered HSV1-tk-expressing NG4TL4 fibrosarcoma cells (NG4TL4-TK) and simulated the clinical application of HSV1-tk plus ganciclovir (GCV) prodrug activation gene therapy. The efficacy of noninvasively monitoring the sites of development of lung metastatic lesions and their GCV-induced regression were assessed by SPECT with (131)I-FIAU. RESULTS: The results of this study showed that the lung metastases model of NG4TL4-TK cells could be successfully detected as early as 24 h after intravenous injection of tumor cells radiolabeled with (131)I-FIAU and also subsequently detected by extended monitoring with the intravenous injection of (131)I-FIAU on day 10. In mice treated with GCV, gamma-camera imaging demonstrated a significant growth inhibition of NG4TL4-TK cells of primary tumors and lung metastases on day 7 after initiating treatment. CONCLUSION: We conclude that this in vivo imaging approach will be useful for future studies of the lung metastases model and for the assessment of novel anticancer and antimetastatic therapies.


Assuntos
Ganciclovir/uso terapêutico , Terapia Genética/métodos , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/diagnóstico , Animais , Técnicas de Transferência de Genes , Genes Reporter , Herpesvirus Humano 1/genética , Processamento de Imagem Assistida por Computador/métodos , Camundongos , Metástase Neoplásica , Cintilografia , Timidina Quinase/genética , Distribuição Tecidual
5.
J Biomed Mater Res A ; 76(3): 512-20, 2006 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-16278875

RESUMO

Chondrocytes are useful as a cell culture system for studying arthritic degeneration in tissue engineered cartilage. However, primary chondrocytes have short in vitro lifespan and rapid shift of collagen phenotype. In this study, we used a high dosage of retroviral vector LXSN16E6E7 to transduce human primary chondrocytes and obtained an actively proliferating cell line, designated hPi, which expresses HPV-16 E6/E7 mRNA in early passages. Parental primary chondrocytes cease to grow after five passages, whereas hPi could be propagated beyond 100 passages without requiring additional cell elements in defined medium. After 48 passages, hPi can also give many profiles similar to those of parental primary chondrocyte, including type II collagen in mRNA and protein level, aggrecan in mRNA level, lacunae in type I collagen matrices, and morphology with GAG-specific Alcian blue staining. hPi has shown neoplastic transformation, as examined by NOD-SCID mice tumorigenicity assays for 3 months. Our results indicated that human primary chondrocytes could be immortalized by transduction with HPV-16 E6/E7, preserving stable cartilage-specific differentiation markers. The established chondrocyte cell line could provide a novel model to engineer cartilage in vitro and in vivo for cartilage repair research and clinical application.


Assuntos
Cartilagem/crescimento & desenvolvimento , Condrócitos/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Proteínas Repressoras/metabolismo , Engenharia Tecidual , Animais , Cartilagem/citologia , Linhagem Celular Transformada , Transformação Celular Viral/genética , Condrócitos/citologia , Condrócitos/transplante , Expressão Gênica/genética , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus , Proteínas Repressoras/genética , Engenharia Tecidual/métodos
6.
Clin Cancer Res ; 11(21): 7749-56, 2005 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-16278396

RESUMO

The aim of this study was to assess the efficacy human mesenchymal stem cells (hMSC) for targeting microscopic tumors and suicide gene or cytokine gene therapy. Immunodeficient mice were transplanted s.c. with human colon cancer cells of HT-29 Inv2 or CCS line, and 3 to 4 days later, i.v. with "tracer" hMSCs expressing herpes simplex virus type 1 thymidine kinase (HSV1-TK) and enhanced green fluorescent protein (EGFP) reporter genes. Subsequently, these tumors were examined for specificity and magnitude of HSV1-TK(+), EGFP(+) stem cell engraftment and proliferation in tumor stroma by in vivo positron emission tomography (PET) with (18)F-labeled 9-(4-fluoro-3-hydroxymethylbutyl)-guanine ([(18)F]-FHBG). In vivo PET images of tumors growing for 4 weeks showed the presence of HSV1-TK(+) tumor stroma with an average of 0.36 +/- 0.24% ID/g [(18)F]-FHBG accumulation. In vivo imaging results were validated by in situ correlative histochemical, immunofluorescent, and cytometric analyses, which revealed EGFP expression in vWF(+) and CD31(+) endothelial cells of capillaries and larger blood vessels, in germinal layer of dermis and hair follicles proximal to the s.c. tumor site. These differentiated HSV1-TK(+), GFP(+) endothelial cells had limited proliferative capacity and a short life span of <2 weeks in tumor fragments transplanted into secondary hosts. We conclude that hMSCs can target microscopic tumors, subsequently proliferate and differentiate, and contribute to formation of a significant portion of tumor stroma. PET imaging should facilitate clinical translation of stem cell-based anticancer gene therapeutic approaches by providing the means for in vivo noninvasive whole-body monitoring of trafficking, tumor targeting, and proliferation of HSV1-tk-expressing "tracer" hMSCs in tumor stroma.


Assuntos
Mesoderma/citologia , Neoplasias/patologia , Tomografia por Emissão de Pósitrons/métodos , Células-Tronco/citologia , Animais , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Proliferação de Células , Citocinas/metabolismo , Citometria de Fluxo , Genes Reporter , Terapia Genética/métodos , Vetores Genéticos , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imuno-Histoquímica , Lentivirus/genética , Camundongos , Transplante de Neoplasias , Neoplasias/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/biossíntese , Reação em Cadeia da Polimerase , Biossíntese de Proteínas , Fatores de Tempo
7.
J Interferon Cytokine Res ; 24(4): 231-43, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15144569

RESUMO

Various human colon cancer cell lines tested in vitro differed significantly in susceptibility to growth inhibition of recombinant human interferon-beta (rHuIFN-beta). Two p53-mutant lines, COH and CC-M2, derived from high-grade colon adenocarcinoma, showed signs of apoptosis after treatment with 250 IU/ml of HuIFN- beta in the culture medium. The similarly p53-mutated HT-29 line from a grade I adenocarcinoma showed no apoptosis, however, and only cell cycle G1/G0 or S phase retardation with 1000 IU/ml HuIFN-beta. After HuIFN-beta exposure, COH and CC-M2 cells showed increased levels of Fas and FasL proteins, alteration of mitochondrial membrane potential, and activation of caspase-9, caspase-8, and caspase-3 in a time-dependent manner. Treatment of COH and CC-M2 cells with anti-FasL antibodies or rFas/Fc fusion protein, however, could not prevent the apoptosis induced by HuIFN-beta. In contrast, cell-permeable specific inhibitors of the three caspases could inhibit the DNA fragmentation and cell death but not the mitochondrial membrane potential changes. Treatment with mitochondria-stabilizing reagents could significantly abrogate the apoptosis and caspase activation induced by HuIFN-beta. These results suggest that in COH and CC-M2 colon cancer cell lines, HuIFN-beta induces apoptosis mainly through mitochondrial membrane alteration and subsequent activation of the caspase cascade pathway, but not by the Fas/FasL interaction or the p53-dependent apoptotic mechanism.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Neoplasias do Colo/enzimologia , Neoplasias do Colo/patologia , Interferon Tipo I/farmacologia , Mitocôndrias/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2 , Receptores do Fator de Necrose Tumoral , Adenocarcinoma/enzimologia , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Antineoplásicos/toxicidade , Inibidores de Caspase , Linhagem Celular Tumoral , Neoplasias do Colo/metabolismo , Citocromos c/metabolismo , Ativação Enzimática/efeitos dos fármacos , Proteína Ligante Fas , Genes p53/genética , Humanos , Interferon Tipo I/toxicidade , Glicoproteínas de Membrana/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Mutação/genética , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Recombinantes , Proteína X Associada a bcl-2 , Receptor fas
8.
Int J Cancer ; 110(3): 313-9, 2004 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-15095294

RESUMO

hMSCs derived from bone marrow are useful as a species-specific cell culture system for studying cell lineage differentiation and tissue remodeling. However, hMSCs usually have a short in vitro life span due to replicative senescence. We therefore used a high dose of retroviral vector LXSN-16E6E7 to transduce hMSCs of an aging donor and obtained an actively proliferating cell line, designated KP-hMSCs, which expressed HPV16 E6/E7 mRNA. Whereas parental hMSCs ceased to grow after 30 PDs, KP-hMSCs could be propagated beyond 100 PDs. With culture procedures to avoid selection pressure and crowded cell growth, KP-hMSCs showed no signs of neoplastic transformation as examined by soft-agar anchorage-independent growth and NOD-SCID mouse tumorigenicity assays. KP-hMSCs gave similar cytofluorimetric profiles of 31 CD markers to those of the parental primary hMSCs, except with some morphologic changes and expansion of an originally very minor CD34(dim)CD38(+)CD50+ cell population. Upon exposure to specific stimulating conditions in vitro, KP-hMSCs could respond and differentiate along the mesenchymal (bone, fat and cartilage) and nonmesenchymal (neuron) cell lineages. Our results indicated that hMSCs could be immortalized by transduction with HPV16 E6/E7, maintained without neoplastic transformation by careful culture procedures and thus useful for stem cell research and clinical application.


Assuntos
Transformação Celular Neoplásica , Terapia Genética/métodos , Mesoderma/citologia , Proteínas Oncogênicas Virais/genética , Proteínas Repressoras , Células-Tronco/citologia , ADP-Ribosil Ciclase/biossíntese , ADP-Ribosil Ciclase 1 , Animais , Antígenos CD/biossíntese , Antígenos CD34/biossíntese , Moléculas de Adesão Celular , Diferenciação Celular , Divisão Celular , Linhagem Celular , Linhagem da Célula , Feminino , Citometria de Fluxo , Humanos , Glicoproteínas de Membrana , Camundongos , Camundongos SCID , Microscopia de Fluorescência , Pessoa de Meia-Idade , Proteínas E7 de Papillomavirus , Fenótipo , RNA Mensageiro/metabolismo , Retroviridae/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Fatores de Tempo
9.
Eur J Nucl Med Mol Imaging ; 31(1): 99-109, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14513292

RESUMO

An experimental cancer gene therapy model was employed to develop a non-invasive imaging procedure using radiolabelled 2'-fluoro-2'-deoxy-5-iodo-1-beta- d-arabinofuranosyluracil (FIAU) as an enzyme substrate for monitoring retroviral vector-mediated herpes simplex virus type 1 thymidine kinase gene ( HSV1-tk) transgene expression. Iodine-131 labelled FIAU was prepared by a no-carrier-added (n.c.a.) synthesis process and lyophilised to give "hot kits". The labelling yield was over 95%, with a radiochemical purity of more than 98%. The stability of [(131)I]FIAU in the form of lyophilised powder (the hot kit) was much better than that in the normal saline solution. The shelf life of the final [(131)I]FIAU hot kit product is as long as 4 weeks. Cellular uptake of [(131)I]FIAU after different periods of storage was investigated in vitro with HSV1-tk-retroviral vector transduced NG4TL4-STK and parental non-transduced NG4TL4 murine sarcoma cell lines over an 8-h incubation period. The NG4TL4-STK cells accumulated more radioactivity than NG4TL4 cells in all conditions, and accumulation increased with time up to 8 h. The kinetic profile of the cellular uptake of n.c.a. [(131)I]FIAU formulated from the lyophilised hot kit or from the stock solution was qualitatively similar. For animal model cancer gene therapy studies, FVB/N mice were inoculated subcutaneously with the HSV1-tk(+) and tk(-) sarcoma cells into the flank to produce tumours. Biodistribution studies showed that tumour/blood ratios were 2, 3.5, 8.2 and 386.8 at 1, 4, 8 and 24 h post injection, respectively, for the HSV1-tk(+) tumours, and 0.5, 0.5, 0.7 and 5.4, respectively, for the HSV1-tk(-) tumours. Radiotracer clearance from blood was completed in 24 h and was bi-exponential. A significant difference in radioactivity accumulation was revealed among the HSV1-tk(+) tumours, the tk(-) tumours and other tissues. At 24 h p.i., higher activity retention was observed in HSV1-tk(+) tumours (9.67%+/-3.89%ID/g) than in HSV1-tk(-) tumours (0.48%+/-0.19%ID/g). After seven consecutive daily treatments with the prodrug ganciclovir, planar gamma camera imaging showed HSV1-tk(+) tumour regression at day 4, and complete tumour regression at day 7. These results clearly demonstrate that the simplified n.c.a. synthesis process developed in this study is reliable and that the [(131)I]FIAU product is useful for in vivo monitoring of HSV1-tk gene transfer, expression and gene therapy.


Assuntos
Arabinofuranosiluracila/análogos & derivados , Arabinofuranosiluracila/farmacocinética , Terapia Genética/métodos , Sarcoma/diagnóstico por imagem , Sarcoma/metabolismo , Timidina Quinase/metabolismo , Proteínas Virais/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Ganciclovir/administração & dosagem , Ganciclovir/uso terapêutico , Radioisótopos do Iodo/farmacocinética , Marcação por Isótopo/métodos , Taxa de Depuração Metabólica , Camundongos , Camundongos Endogâmicos , Especificidade de Órgãos , Cintilografia , Compostos Radiofarmacêuticos/farmacocinética , Sarcoma/genética , Sarcoma/terapia , Timidina Quinase/genética , Timidina Quinase/uso terapêutico , Distribuição Tecidual , Proteínas Virais/genética , Proteínas Virais/uso terapêutico
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...