Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 175
Filtrar
1.
Cancer Gene Ther ; 17(10): 700-7, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20559333

RESUMO

Vascular endothelial-specific cadherin (VE-cadherin) is an endothelial cell-specific adhesion molecule, localized at cell-cell contact sites. It is involved in physiological and pathological angiogenesis. In this study, we showed that in vitro a soluble N-terminal fragment of VE-cadherin (EC1-3) corresponding to cadherin 1-3 ectodomains inhibited vascular endothelial growth factor-stimulated endothelial cell proliferation and capillary tube structure formation in the matrigel model. In vivo, EC1-3 was tested in a murine colon cancer model. EC1-3-expressing colon cancer C51 cells were subcutaneously grafted into nude mice, and tumor growth and angiogenesis were evaluated. At day 33, the mean volume of the tumors developed was reduced (510±104 versus 990±120 mm(3) for control). Similarly, injection of EC1-3 virus-producing cells into established C51 tumors resulted in an inhibition by 33% of tumor growth. Immunohistological staining of vessels on tumor sections showed a significantly reduced intratumoral angiogenesis. Furthermore, EC1-3 did not induce vessel injury in the lung, liver, spleen, heart and brain in the mice. These results suggest that the soluble N-terminal fragment of VE-cadherin EC1-3 could exert an antitumoral effect by targeting tumor angiogenesis, which included blocking endothelial cell proliferation and capillary tube formation with no obvious toxicity on normal organs.


Assuntos
Adenocarcinoma/metabolismo , Inibidores da Angiogênese/farmacologia , Antígenos CD/farmacologia , Caderinas/farmacologia , Neoplasias do Colo/metabolismo , Células Endoteliais/metabolismo , Inibidores da Angiogênese/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Endotélio Vascular/metabolismo , Humanos , Camundongos , Camundongos Nus , Transdução Genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
2.
Gene Ther ; 16(12): 1405-15, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19641530

RESUMO

Adenovirus (Ad)-mediated delivery of anti-angiogenic molecules into tumors constitutes an appealing approach for growth inhibition. However, lack of expression on tumors of Ad receptors leads to weak tumor transduction. Therefore, to provide Ad with a new entry pathway into tumors, an NGR peptide was inserted into either fiber (AdFNGR) or hexon (AdHNGR) capsid proteins. This strategy provided Ad with a very efficient entry pathway in both endothelial cells and tumor cells, with the highest efficacy observed for AdHNGR. Using pharmacological, biochemical and genetic approaches, AdHNGR and AdFNGR were shown to bind not only to CD13 receptor, but also to alphavbeta3 integrins. Both vectors were efficient tools to deliver angiostatin K1-5 cDNA into endothelial cells, thus leading to a dramatic inhibition of their proliferation and increased cell death. Although AdHNGR and Adwt were found to display similar gene transduction efficacy in Lewis lung carcinoma (LLC), pseudotyping AdHNGR with an Ad3-fiber unmasked the ability of NGR-peptide to target these tumors. As a result, delivery of angiostatin K1-5 cDNA into highly aggressive tumors translated into a stronger inhibition of their growth. Altogether, our results suggest that NGR-bearing Ad are valuable tools to realize the potential of this anti-angiogenic approach to anti-tumor therapy.


Assuntos
Adenoviridae/genética , Angiostatinas/uso terapêutico , Técnicas de Transferência de Genes , Neoplasias/terapia , Oligopeptídeos , Animais , Antígenos CD13 , Capsídeo , Carcinoma Pulmonar de Lewis , Linhagem Celular Tumoral , Receptor gp130 de Citocina , Células Endoteliais , Terapia Genética/métodos , Vetores Genéticos , Humanos , Camundongos , Transdução Genética
3.
Carcinogenesis ; 29(11): 2236-42, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18753414

RESUMO

Plasminogen activator inhibitor-1 is known to play a paradoxical positive role in tumor angiogenesis, but its contribution to metastatic spread remains unclear. We studied the impact of plasminogen activator inhibitor (PAI)-1 deficiency in a transgenic mouse model of ocular tumors originating from retinal epithelial cells and leading to brain metastasis (TRP-1/SV40 Tag mice). PAI-1 deficiency did not affect primary tumor growth or vascularization, but was associated with a smaller number of brain metastases. Brain metastases were found to be differentially distributed between the two genotypes. PAI-1-deficient mice displayed mostly secondary foci expanding from local optic nerve infiltration, whereas wild-type animals displayed more disseminated nodules in the scissura and meningeal spaces. SuperArray GEarray analyses aimed at detecting molecules potentially compensating for PAI-1 deficiency demonstrated an increase in fibroblast growth factor-1 (FGF-1) gene expression in primary tumors, which was confirmed by reverse transcription-polymerase chain reaction and western blotting. Our data provide the first evidence of a key role for PAI-1 in a spontaneous model of metastasis and suggest that angiogenic factors, such as FGF-1, may be important for primary tumor growth and may compensate for the absence of PAI-1. They identify PAI-1 and FGF-1 as important targets for combined antitumor strategies.


Assuntos
Neoplasias Encefálicas/prevenção & controle , Neoplasias Encefálicas/secundário , Neoplasias Oculares/patologia , Inibidor 1 de Ativador de Plasminogênio/fisiologia , Animais , Western Blotting , Neoplasias Encefálicas/genética , Neoplasias Oculares/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , Inibidor 1 de Ativador de Plasminogênio/genética , Epitélio Pigmentado da Retina/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa
4.
Gene Ther ; 15(21): 1436-45, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18548116

RESUMO

Given as a prophylactic treatment, a single muscle electrogene transfer of plasmid coding canstatin fused to human serum albumin (CanHSA), slowed down the development of two xenografted human carcinomas from mammary (MDA-MB-231) and prostate origin (PC-3) in nude mice and delayed lung metastatic spreading of B16F10 melanoma cells in syngenic mice. No effect was observed with unfused canstatin. The long lasting circulating blood level of CanHSA (20 ng ml(-1)) resulted in a profound disorganization of the tumor blood vessel network. However, when used as a curative treatment, on well-established tumors, CanHSA electrogenetherapy was ineffective in reducing tumor growth. As radiation is known to increase the alpha v beta3 and alpha v beta5 integrins, which are canstatin receptors, to extend the use of CanHSA electrogenetherapy, as a curative treatment, we explored the combination of CanHSA and ionizing radiation. We demonstrated a better efficacy (P=0.01) of the bitherapy over irradiation alone, as a result of strong vessel disorganization and dramatic increase of tumor cells apoptosis. This extremely simple virus free curative protocol could open the door to potential clinical applications, especially for prostate cancer that often develops radioresistance.


Assuntos
Neoplasias da Mama/terapia , Colágeno Tipo IV/genética , Terapia Genética/métodos , Fragmentos de Peptídeos/genética , Neoplasias da Próstata/terapia , Proteínas Recombinantes de Fusão/uso terapêutico , Animais , Apoptose , Neoplasias da Mama/radioterapia , Linhagem Celular Tumoral , Colágeno Tipo IV/sangue , Terapia Combinada , Eletroporação , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Músculo Esquelético/metabolismo , Fragmentos de Peptídeos/sangue , Neoplasias da Próstata/radioterapia , Dosagem Radioterapêutica , Proteínas Recombinantes de Fusão/genética , Albumina Sérica/genética , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Gene Ther ; 14(2): 108-17, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16943854

RESUMO

Tumor growth is dependent both on endothelial and tumor cells. The aim of this study was to investigate dynamically whether changes in tumor vasculature implicate tumor tissue degeneration during antiangiogenic therapies. In order to quantify intra-tumor vascularization and necrosis, we have used ultrasound technology. This study has identified essential parameters needed to quantify specifically and sensitively the number of microvessels and the extent of necrosis in xenografted human carcinomas during natural tumor evolution, using contrast-enhanced high-frequency ultrasonography with (HFCDUS) or without (HFUS) color Doppler. We showed that quantification of intra-tumor microvessels between HFCDUS and immunohistochemistry is correlated using an anti-CD31 antibody. Furthermore, quantification of tumor necrosis with HFUS was confirmed by histological examination of hematoxylin-eosin-saffranin-stained sections over the observation period. Subsequently, for the assessment of novel angiogenic inhibitors, HFCDUS and HFUS were used to elucidate the underlying dynamics linking vessel inhibition and tumor eradication. We describe a novel application for HFCDUS/HFUS that constitutes an effective, convenient, and non-invasive method for clinical assessment of angiogenic inhibitors. In conclusion, we showed that tumor cells abruptly became necrotic following an antivascular therapy, whereas untreated tumors were protected from degeneration by a significant blood supply.


Assuntos
Inibidores da Angiogênese/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/tratamento farmacológico , Terapia Genética/métodos , Transdução Genética/métodos , Adenoviridae/genética , Inibidores da Angiogênese/genética , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais Humanizados , Bevacizumab , Neoplasias da Mama/diagnóstico por imagem , Linhagem Celular Tumoral , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Humanos , Camundongos , Camundongos Nus , Necrose , Neovascularização Patológica , Distribuição Aleatória , Ultrassonografia Doppler em Cores , Ultrassonografia de Intervenção , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Cancer Gene Ther ; 14(3): 251-61, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17082795

RESUMO

Different antiangiogenic and antimetastatic recombinant adenoviruses were tested in a transgenic mouse model of metastatic ocular cancer (TRP1/SV40 Tag transgenic mice), which is a highly aggressive tumor, developed from the pigmented epithelium of the retina. These vectors, encoding amino-terminal fragments of urokinase plasminogen activator (ATF), angiostatin Kringles (K1-3), endostatin (ES) and canstatin (Can) coupled to human serum albumin (HSA) were injected to assess their metastatic and antiangiogenic activities in our model. Compared to AdCO1 control group, AdATF-HSA did not significantly reduce metastatic growth. In contrast, mice treated with AdK1-3-HSA, AdES-HSA and AdCan-HSA displayed significantly smaller metastases (1.19+/-1.19, 0.87+/-1.5, 0.43+/-0.56 vs controls 4.04+/-5.12 mm3). Moreover, a stronger inhibition of metastatic growth was obtained with AdCan-HSA than with AdK1-3-HSA (P=0.04). Median survival was improved by 4 weeks. A close correlation was observed between the effects of these viruses on metastatic growth and their capacity to inhibit tumor angiogenesis. Our study indicates that systemic antiangiogenic factors production by recombinant adenoviruses, particularly Can, might represent an effective way of delaying metastatic growth via inhibition of angiogenesis.


Assuntos
Inibidores da Angiogênese/genética , Neoplasias Encefálicas/terapia , Neoplasias Oculares/terapia , Técnicas de Transferência de Genes , Terapia Genética , Vetores Genéticos/genética , Albumina Sérica/genética , Fatores Ativadores da Transcrição/genética , Adenoviridae/genética , Inibidores da Angiogênese/uso terapêutico , Angiostatinas/genética , Animais , Proteínas Sanguíneas/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/secundário , Colágeno Tipo IV/genética , Endostatinas/genética , Neoplasias Oculares/genética , Neoplasias Oculares/patologia , Humanos , Camundongos , Camundongos Nus , Camundongos Transgênicos , Neovascularização Patológica , Fragmentos de Peptídeos/genética , Taxa de Sobrevida , Células Tumorais Cultivadas , Ativador de Plasminogênio Tipo Uroquinase/genética
7.
Gene Ther ; 14(6): 533-44, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17109009

RESUMO

The immunogenicity of recombinant adenoviruses (Ad) constitutes a major concern for their use in gene therapy. Antibody- and cell-mediated immune responses triggered by adenoviral vectors hamper long-term transgene expression and efficient viral readministration. We previously reported that interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha play an essential role in both the acute phase and antibody response against Ad, respectively. As TNF-alpha controls the immune response and the development of the immune system, we examined here the consequence of blockade of TNF-alpha activity through Ad-mediated gene delivery of a dimeric mouse TNFR1-IgG fusion protein on transgene expression from a second Ad. Ad encoding TNFR1-IgG (AdTNFR1-Ig) was injected intravenously along with Ad encoding beta-galactosidase or alpha1-antitrypsin transgene in wild-type (IL-6(+/+)) but also in IL-6-deficient mice (IL-6(-/-)) to analyze how TNF-alpha and IL-6 diminish liver gene transfer efficacy. Blockade of TNF-alpha leads to increased transgene expression in both wild-type and IL-6(-/-) mice due to a reduced inflammatory response and to diminished recruitment of macrophages and NK cells towards the liver. Antibody responses against adenoviral particles and expressed transgenes were only delayed in AdTNFR1-Ig-treated wild-type mice, but were markedly reduced in AdTNFR1-Ig-treated IL-6(-/-) mice. Finally, treatment of mice with etanercept, a clinically approved anti-TNF-alpha drug, confirmed the importance of controlling proinflammatory cytokines during gene therapy by adenoviral vectors.


Assuntos
Infecções por Adenoviridae/imunologia , Adenoviridae/genética , Terapia Genética/métodos , Interleucina-6/imunologia , Transdução Genética/métodos , Fator de Necrose Tumoral alfa/imunologia , Adenoviridae/imunologia , Animais , Anticorpos Antivirais/sangue , Autoanticorpos/sangue , DNA Viral/genética , Etanercepte , Expressão Gênica , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Hepatite/imunologia , Humanos , Imunoglobulina G/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , RNA Mensageiro/análise , Receptores do Fator de Necrose Tumoral/uso terapêutico , Receptores Tipo I de Fatores de Necrose Tumoral/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transgenes , Fator de Necrose Tumoral alfa/antagonistas & inibidores
8.
Gene Ther ; 13(3): 235-44, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16208422

RESUMO

The BRCA1 (breast cancer 1) breast cancer susceptibility gene is recognized as responsible for most familial breast and ovarian cancers and is suggested to be a tissue-specific tumor suppressor gene. In this report, we investigated the tissue specificity of tumor inhibitory activities induced by a recombinant adenovirus coding for wild-type BRCA1 (wtAdBRCA1). We demonstrated a pronounced in vitro antiproliferative effect on H1299 lung and HT29 colon cells upon infection with AdBRCA1. We describe a prolonged G1 cell cycle arrest associated with a decrease in the hyperphosphorylated form of Rb, suggesting that the Rb/E2F pathway is implicated in BRCA1-induced cell growth arrest. We also observed a significant antitumor effect in these pre-established subcutaneous tumors after in situ delivery of AdBRCA1, although these two tumors do not express wt p53, and also estrogen alpha and beta, progesterone and androgen receptors. Moreover, BRCA1 can induce a strong prolonged cell cycle arrest and apoptotic cell death but no significant antiangiogenic effect in H1299 tumors. Finally, our data indicate that intratumor administration of wtAdBRCA1 significantly inhibits growth of lung and colon steroid hormone-independent tumors.


Assuntos
Adenoviridae/genética , Neoplasias do Colo/terapia , Genes BRCA1 , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Neoplasias Pulmonares/terapia , Animais , Apoptose , Ciclo Celular , Linhagem Celular Tumoral , Feminino , Fase G1 , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Predisposição Genética para Doença , Vetores Genéticos/genética , Células HT29 , Humanos , Injeções Intralesionais , Masculino , Camundongos , Transplante de Neoplasias , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo
9.
Gene Ther ; 12(10): 831-42, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15789064

RESUMO

Since kringle (K)5, not present in the angiostatin molecule, was shown to be a key functional domain possessing potent antiangiogenic activity, we have evaluated a new plasminogen-derived fragment, consisting of the N-terminal part of human plasminogen, that included the complete secondary structure of K1-5 (aa 1-566). In contrast to other fragments described to date, K1-5 includes cysteine residues at positions 543, 555 and 560 allowing the formation of the three disulfide bonds lying within K5. Vascular endothelial cell proliferation and migration assays revealed that a replication-defective adenovirus (AdK1-5(1-566)), expressing K1-5 (aa 1-566), was dose dependently more potent that AdK1-3(1-354), an adenovirus that expresses only the first three kringles. In contrast to AdK1-3(1-354), a single intratumoral injection of AdK1-5(1-566) into MDA-MB-231 breast human carcinoma tumors was followed by a total regression of 40% of the tumor and by significant arrest of tumor growth (90%), which was correlated with a drastic decrease of functional neovascularization into the tumors. Furthermore, systemic delivery of AdK1-5(1-566) in mice inhibited the lung invasion of melanoma B16-F10 cells by 87%. Our findings provide evidence that the full kringles of plasminogen (aa 1-566) may be much more potent than K1-3 (aa 1-354), for the suppression of angiogenesis, tumor growth and metastatic dissemination.


Assuntos
Adenoviridae/genética , Neoplasias da Mama/terapia , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Kringles/genética , Plasminogênio/genética , Animais , Neoplasias da Mama/patologia , Feminino , Humanos , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/terapia , Melanoma/patologia , Melanoma/terapia , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neovascularização Patológica , Distribuição Aleatória , Transplante Heterólogo
10.
Gene Ther ; 11(21): 1599-605, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15334112

RESUMO

We describe here a clonal approach for efficient and robust construction of recombinant adenoviral genomes that holds certain advantages over existing approaches. Transgenes of interest are cloned into a small, conditionally replicating plasmid containing the left end of a recombinant adenoviral genome, encompassing pIX coding regions. Transformation of this plasmid into recombination-competent Escherichia coli bearing a plasmid containing the right end of a recombinant adenoviral genome, commencing from pIX coding regions, yields a stable co-integrated plasmid encoding a full adenoviral genome, by virtue of shared homology in pIX coding regions contained in both plasmids. The recombination process yielding the full adenoviral plasmid requires only one step, and always results in the formation of only the desired recombinant adenoviral genome. Thus, no screening is required to identify the correct plasmid encoding the desired recombinant adenoviral genome. In addition, the plasmid encoding the right-hand side of the adenoviral genome is itself incapable of producing contaminating adenovirus. We have successfully employed this approach to generate over 200 recombinant adenoviruses, obtaining only the desired recombinant adenoviral species each time. The process is amenable to medium-to-high-throughput parallel construction of adenoviral genomes, and as such should aid efforts aimed towards high-throughput functional annotation of therapeutic gene targets, which aim to leverage the benefits of adenoviruses as gene delivery and expression vectors.


Assuntos
Adenoviridae/genética , Engenharia Genética , Vetores Genéticos/genética , Genoma Viral , Recombinação Genética , Animais , Clonagem Molecular , Escherichia coli/genética , Luciferases de Vaga-Lume/genética , Plasmídeos , Transgenes
11.
Gene Ther ; 10(25): 2098-103, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14595383

RESUMO

Diabetic retinopathy and retinopathy of prematurity are among the leading causes of vision impairment throughout the world. Both diseases are characterized by pathological angiogenesis, which severely impairs vision. Extracellular proteinases play important roles in endothelial cell migration during angiogenesis. Amino-terminal fragment (ATF) is an angiostatic molecule that targets the uPA/uPAR system and inhibits endothelial cell migration. The angiostatic effect of ATF has been demonstrated in models of cancer, but has never been assessed in pathological retinal neovascularization. Endostatin also has angiostatic effects on tumor growth and retinal neovascularization. We used an adenoviral vector carrying the murine ATF (AdATFHSA) or endostatin gene coupled to human serum albumin (HSA) (AdEndoHSA) to increase the half-life of the therapeutic protein in the circulation. We induced retinopathy by exposing 7-day-old mice to high levels of oxygen. They were intravitreally injected with the vectors. Local injection of AdATFHSA or AdEndoHSA reduced retinal neovascularization by 78.1 and 79.2%, respectively. Thus, the adenovirus-mediated delivery of ATFHSA or EndoHSA reduces retinal neovascularization in a mouse model of hypoxia-induced neovascularization.


Assuntos
Adenovírus Humanos/genética , Inibidores da Angiogênese/genética , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Fragmentos de Peptídeos/genética , Neovascularização Retiniana/terapia , Inibidores da Angiogênese/metabolismo , Animais , Endostatinas/genética , Endostatinas/metabolismo , Expressão Gênica , Vetores Genéticos/genética , Humanos , Injeções , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Fragmentos de Peptídeos/metabolismo , Albumina Sérica/genética , Albumina Sérica/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Corpo Vítreo
12.
Mol Ther ; 7(2): 174-84, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12597905

RESUMO

We previously reported that intratumoral injection of AdK3, a recombinant adenovirus encoding human angiostatin kringle (K) 1 to 3, inhibits tumor vascularization and tumor growth. To reduce the serum clearance of this factor, we constructed an adenovirus (AdK3-HSA) that carries a chimeric gene encoding a fusion protein between angiostatin K1-3 and human serum albumin (HSA). This conjugate inhibited endothelial cell proliferation as efficiently as K1-3. K3-HSA serum concentrations in immunodeficient mice systemically injected with AdK3-HSA were dramatically higher than in AdK3-injected mice. Furthermore, the growth of MDA-MB-231 tumors grafted into nude mice that had been injected intravenously with AdK3-HSA was inhibited by 79% (versus 17% with AdK3). In TRP-1/SV40 Tag transgenic mice, which spontaneously develop eye tumors with brain metastases, intravenous injections of AdK3-HSA in newborn mice blocked metastatic dissemination efficiently and significantly, and prolonged survival by 3 weeks. After 2 months, only 46% of AdK3-HSA-treated animals developed micrometastases, whereas 94% of the AdCO1-injected group displayed numerous macrometastases. Nevertheless, ocular tumor growth was not modified because of impaired diffusion of the conjugate in the eye compartment. Our results show that HSA genetic coupling is an efficient way to increase the pharmacokinetics of circulating angiogenic inhibitors and thus their antitumoral activity.


Assuntos
Adenoviridae/genética , Fragmentos de Peptídeos/genética , Plasminogênio/genética , Albumina Sérica/genética , Angiostatinas , Animais , Western Blotting , Divisão Celular , Linhagem Celular , Endotélio/citologia , Ensaio de Imunoadsorção Enzimática , Neoplasias Oculares/genética , Deleção de Genes , Humanos , Imuno-Histoquímica , Kringles , Camundongos , Camundongos Nus , Camundongos Transgênicos , Modelos Genéticos , Metástase Neoplásica , Transplante de Neoplasias , Fragmentos de Peptídeos/metabolismo , Plasminogênio/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo , Células Tumorais Cultivadas
13.
Gene Ther ; 10(2): 153-62, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12571644

RESUMO

The development of genetically modified adenoviral vectors capable of specifically transducing a given cell population requires the addition and functional presentation of particular tropism determinants within the virus capsid, together with the abrogation of the molecular determinants that dictate their natural tropism in vivo. The human adenovirus serotype 5 (Ad5) first attaches to the cell surface following high-affinity binding of the C-terminal knob of the fiber capsid protein to the coxsackie and adenovirus receptor (CAR). Here we have assessed whether genetic shortening of the fiber shaft (virus BS1), or replacing the Ad5 fiber shaft and knob with their Ad3 counterparts (virus DB6), could cripple this interaction in vitro and in vivo. A 10-fold decrease in the binding of the modified capsids to soluble CAR was evidenced, which correlated with a similar reduction of their ability to transduce CAR-positive cells in vitro. The ability of BS1 to interact with cellular integrins was also impaired, suggesting that the penton base and the short-shafted fiber when embedded in the capsid preclude each other from efficiently interacting with their cognate cell surface receptors (CAR and integrins respectively). BS1 and DB6 intravenous injections in mice further supported a profound impairment of the ability of the capsid-modified viruses to transduce the liver as demonstrated by a 10-fold reduction of intracellular viral DNA and transgene expression. Interestingly enough, the host humoral response was also specifically weakened in BS1- and DB6-inoculated animals. Taken together, these observations indicate that (i) fiber shortening and (ii) pseudo-typing of Ad5-based vectors with the shaft and knob from non-CAR-binding serotypes constitute two promising strategies to successfully attenuate their native tropism in vitro and most importantly in vivo.


Assuntos
Proteínas do Capsídeo/genética , Proteínas do Olho , Marcação de Genes/métodos , Terapia Genética/métodos , Vetores Genéticos/genética , Lipoproteínas , Fígado/metabolismo , Proteínas do Tecido Nervoso , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular , DNA/análise , Expressão Gênica , Engenharia Genética , Hipocalcina , Camundongos , Camundongos Endogâmicos C57BL , Recoverina , Transdução Genética/métodos
14.
Gene Ther ; 9(3): 192-200, 2002 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11859422

RESUMO

Plasmin is essential for metalloproteases activation, endothelial cell migration and degradation of the extracellular matrix. The process is common to neoangiogenesis pannus formation and cartilage degradation within arthritic joints. Since 80% of synovial cells express urokinase plasminogen activator receptor (uPAR), we investigated the inhibition of plasmin activation in a collagen-induced arthritis (CIA) mice model, by expressing a uPA/uPAR antagonist molecule (ATF) fused to human serum albumin (HSA) to extend its serum half-life. Overexpression was obtained with an adenoviral vector expressing the chimeric murine ATF-HSA. We showed that the genetic coupling did not significantly reduce the ability of the ATF moiety to interact with its receptor uPAR. The chimeric protein was detectable in the sera of injected mice 7 days following Ad-mATF-HSA injection, then decreased in parallel with the anti-HSA titer increase. Systemic Ad-mATF-HSA injection performed on day 25 following CIA induction decreased the incidence of arthritis and the severity of the disease. Moreover, synovial angiogenesis in arthritic paws was decreased after Ad-mATF-HSA gene transfer, as assessed by smooth muscle actin immunostaining. The preventive effect observed on arthritis was related to the decrease in angiogenesis, rather than inhibition of extracellular matrix degradation.


Assuntos
Adenoviridae/genética , Artrite Experimental/terapia , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Neovascularização Patológica , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Animais , Anticorpos/sangue , Artrite Experimental/patologia , Meia-Vida , Injeções Intravenosas , Masculino , Camundongos , Camundongos Endogâmicos DBA , Proteínas Recombinantes de Fusão/genética , Albumina Sérica/genética , Albumina Sérica/imunologia , Células Tumorais Cultivadas , Ativador de Plasminogênio Tipo Uroquinase/genética
15.
Cancer Gene Ther ; 8(10): 759-70, 2001 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11687899

RESUMO

The loss of BRCA1 function appears as an essential step in breast and ovarian epithelial cells oncogenesis and is consistent with the concept that BRCA1 acts as a tumor suppressor gene. However, the mechanism underlying this activity is not understood. In 1996, a retroviral vector was used for BRCA1 delivery to demonstrate that the transfer of BRCA1 inhibits breast and ovarian cancer cell growth. Since this early observation, the tumor growth inhibitory activity of BRCA1 in vivo has not been further documented. Here we re-address this issue and report experiments designed to evaluate the potential of adenovirus-mediated BRCA1 delivery to suppress the growth of cells with various status of endogenous BRCA1 in comparison with p53 and p21. Delivery of wild-type BRCA1 by an adenovirus vector in breast and ovarian tumor cells, decreases in vitro proliferation and tumorigenicity. Similarly, in vivo administration of BRCA1 provokes tumor growth retardation or regression comparable to that obtained with p53 or p21. The antitumor effect of BRCA1 is not observed upon transfer of a mutant lacking the 542 C-terminal residues. The p53- or p21-mediated antiproliferative activities are likely to bear on their capacity to induce apoptosis and/or interfere with cell cycle checkpoint. By contrast, the data presented here show that neither of these mechanisms can account for the BRCA1-mediated antitumor activity and suggest the activation of an alternative route.


Assuntos
Proteína BRCA1/genética , Ciclinas/genética , Genes Supressores de Tumor/fisiologia , Neoplasias Mamárias Animais/terapia , Neoplasias Ovarianas/terapia , Proteína Supressora de Tumor p53/genética , Adenoviridae/genética , Animais , Apoptose , Proteína BRCA1/metabolismo , Ciclo Celular , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/metabolismo , Feminino , Terapia Genética/métodos , Humanos , Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/patologia , Camundongos , Camundongos Nus , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Deleção de Sequência , Ativação Transcricional , Transfecção , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismo
16.
J Gene Med ; 3(3): 228-39, 2001.
Artigo em Inglês | MEDLINE | ID: mdl-11437328

RESUMO

BACKGROUND: Melanomas of the uveal tract are the most common intraocular malignancies in adults, with an incidence of six cases per million adults per year. Enucleation, which may enhance the dissemination of tumour cells into the systemic circulation, is still required for eyes with large tumours. Gene therapy is proposed as a new therapeutic approach for uveal melanoma management. METHODS: The potential of adenovirus-mediated gene transfer to normal eyes of two laboratory Beagles and in an iris tumour of a Great Dane were evaluated. Replication-defective adenoviral vectors (Adbetagal) were used to assess the feasibility, efficiency and safety of direct adenoviral delivery to the anterior chamber of normal eyes and to an iris tumour. The expression of angiostatin into the aqueous humour following an adenoviral-mediated delivery of human angiostatin (AdK3) was also investigated. RESULTS: The ciliary body was the area preferentially transduced after adenoviral injection into the anterior chamber. It was also demonstrated that a direct intratumoral injection of a recombinant adenovirus efficiently transduces a canine uveal melanoma. Western blot analysis performed on the aqueous humour revealed that the expression of the angiostatin recombinant protein in the aqueous humour correlated with the dose of AdK3 administered. Lymphocyte infiltrates at the site of AdK3 injection indicated induction of a strong cellular immune response, and humoral immune responses developed in all three dogs. CONCLUSIONS: The present study involving adenovirus-mediated gene transfer to dog eyes provides an essential basis for gene therapy treatment of uveal melanoma-bearing patients.


Assuntos
Adenoviridae/genética , Técnicas de Transferência de Genes , Terapia Genética/métodos , Vetores Genéticos/genética , Neoplasias Uveais/terapia , Adenoviridae/imunologia , Angiostatinas , Animais , Câmara Anterior/virologia , Formação de Anticorpos , Humor Aquoso/metabolismo , Modelos Animais de Doenças , Cães , Estudos de Viabilidade , Terapia Genética/tendências , Vetores Genéticos/imunologia , Humanos , Fragmentos de Peptídeos/genética , Plasminogênio/genética , Neoplasias Uveais/genética , Neoplasias Uveais/patologia
17.
Cancer Res ; 61(8): 3450-7, 2001 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-11309307

RESUMO

The tissue inhibitor of metalloproteinases-2 (TIMP-2) has at least two independent functions, i.e., regulation of matrix metalloproteinases and growth promoting activity. We investigated the effects of TIMP-2 overexpression, induced by retroviral mediated gene transfer, on the in vivo development of mammary tumors in syngeneic mice inoculated with EF43.fgf-4 cells. The EF43.fgf-4 cells established by stably infecting the normal mouse mammary EF43 cells with a retroviral expression vector for the fgf-4 oncogene, are highly tumorigenic and overproduce vascular endothelial growth factor (VEGF). Despite a promotion of the in vitro growth rate of EF43.fgf-4 cells overexpressing timp-2, the in vivo tumor growth was delayed. At day 17 post-cell injection, the volume of tumor derived from TIMP-2-overexpressing cells was reduced by 80% as compared with that obtained with control cells. Overexpression of TIMP-2 was associated with a down-regulation of VEGF expression in vitro and in vivo, a reduction of vessel size, density, and blood supply in the induced tumors. In addition, TIMP-2 completely inhibited the angiogenic activity of EF43.fgf-4 cell-conditioned medium in vitro using a rat aortic ring model. Our findings suggest that overexpression of TIMP-2 delays growth and angiogenesis of mammary carcinoma in vivo and that down-regulation of VEGF expression may play an important role in this TIMP-2-mediated antitumoral and antiangiogenic effects. Finally the in vivo delivery of TIMP-2, as assessed by i.v. injection of recombinant adenoviruses vectors, significantly reduced the growth of the EF43.fgf-4-induced tumors. This effect of TIMP-2 was shown to be equally comparable with that of angiostatin, a known potent inhibitor of angiogenesis.


Assuntos
Fatores de Crescimento Endotelial/biossíntese , Linfocinas/biossíntese , Neoplasias Mamárias Experimentais/irrigação sanguínea , Neoplasias Mamárias Experimentais/patologia , Neovascularização Patológica/patologia , Inibidor Tecidual de Metaloproteinase-2/fisiologia , Adenoviridae/genética , Angiostatinas , Animais , Divisão Celular , Regulação para Baixo , Fatores de Crescimento Endotelial/genética , Feminino , Fatores de Crescimento de Fibroblastos/genética , Técnicas de Transferência de Genes , Linfocinas/genética , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/fisiologia , Plasminogênio/genética , Plasminogênio/fisiologia , Ratos , Inibidor Tecidual de Metaloproteinase-2/biossíntese , Inibidor Tecidual de Metaloproteinase-2/genética , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular
18.
Hum Gene Ther ; 12(5): 515-26, 2001 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-11268284

RESUMO

TIMP-2 is a natural matrix metalloproteinase (MMP) inhibitor that prevents the degradation of extracellular matrix proteins. It abolishes the hydrolytic activity of all activated members of the metalloproteinase family and in particular that of MT1-MMP, MMP-2, and MMP-9, which are selective for type IV collagenolysis. Since MMPs have been implicated in both cancer progression and angiogenesis, we generated a recombinant adenovirus to deliver human TIMP-2 (AdTIMP-2) and evaluated its anticancer efficiency in three murine models. Our results demonstrated that overexpression in vitro of TIMP-2 inhibited the invasion of both tumor and endothelial cells without affecting cell proliferation. Its in vivo efficiency has been evaluated in murine lung cancer LLC, and colon cancer C51 in syngeneic mice as well as in human breast cancer MDA-MB231 in athymic mice. Preinfection of tumor cells by AdTIMP-2 resulted in an inhibition of tumor establishment in more than 50% of mice in LLC and C51 models and in 100% mice in the MDA-MB231 model. A single local injection of AdTIMP-2 into preestablished tumors of these three types significantly reduced tumor growth rates by 60--80% and tumor-associated angiogenesis index by 25--75%. Lung metastasis of LLC tumor was inhibited by >90%. In addition, AdTIMP-2-treated mice showed a significantly prolonged survival in all the cancer models tested. These data demonstrate the potential of adenovirus-mediated TIMP-2 therapy in cancer treatment.


Assuntos
Adenoviridae/genética , Terapia Genética/métodos , Inibidor Tecidual de Metaloproteinase-2/genética , Animais , Apoptose , Western Blotting , Colágeno/metabolismo , Meios de Cultivo Condicionados/farmacologia , DNA Complementar/metabolismo , Combinação de Medicamentos , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imuno-Histoquímica , Laminina/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Metástase Neoplásica , Neovascularização Patológica , Proteoglicanas/metabolismo , Fatores de Tempo , Células Tumorais Cultivadas
19.
Cancer Immunol Immunother ; 49(11): 593-602, 2001 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11225990

RESUMO

The genes of the MAGE-A family code for antigens that are strictly tumor-specific and are shared by many human tumors. Melanoma patients have been immunized against these antigens and some tumor regressions have been observed. However, no unequivocal evidence of cytolytic T cell responses has been obtained by analyzing the blood lymphocytes of these patients. Hence it was considered worthwhile to examine in mouse systems whether or not immunization against antigens derived from the mouse Mage homologs can produce cytolytic T cell responses. We have identified an antigenic peptide encoded by mouse gene Mage-a2, and here we show that immunization of DBA/2 mice with a recombinant adenovirus containing either just the sequence encoding this peptide or a large part of the Mage-a2 coding sequence produces strong cytolytic T cell responses. The Mage-a2 system should prove useful for the comparison of vaccination modalities that could be applied to human patients in therapeutic vaccination trials with MAGE antigens.


Assuntos
Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Linfócitos T Citotóxicos/imunologia , Adenoviridae/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Testes Imunológicos de Citotoxicidade , Feminino , Antígenos H-2/imunologia , Humanos , Imunização , Mastocitose/imunologia , Camundongos , Camundongos Endogâmicos DBA , Dados de Sequência Molecular , Peptídeos/imunologia , Transfecção , Células Tumorais Cultivadas
20.
Cardiovasc Res ; 49(2): 288-97, 2001 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-11164839

RESUMO

OBJECTIVE: We examined the functional consequences of expressing adult rabbit fast skeletal sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA1a) in isolated adult rat ventricular myocytes. METHODS: Myocytes were infected with a recombinant adenovirus harboring SERCA1a. Then 2 days after myocyte infection, protein expression was estimated using Western blot and SDS-PAGE analysis. We also measured the ATP-dependent oxalate-facilitated Ca(2+) uptake of myocyte homogenates and monitored Ca(2+) transient in myocytes loaded with the Ca(2+) dye, indo-1. RESULTS: SERCA1a gene expression resulted in a 36% increase in the total SERCA protein level in infected myocytes compared to controls (P<0.01), while SERCA2 and phospholamban levels did not change. This increase was associated with a 42% rise in SR Ca(2+) uptake (P<0.01), while tau (the time constant of Ca(2+) transient decay), and the time to peak fell by 32% (P<0.01) and 38% (P<0.001), respectively. Increasing the frequency of stimulation from 0.2 to 2 Hz decreased tau in both cell types (P<0.01). However, the decrease was much smaller in infected (P<0.01) than in uninfected cells (P<0.001). Isoproterenol (1 microM) further decreased tau in infected myocytes by 23% (P<0.05). In these cells, the diastolic [Ca(2+)](i) decreased by 50% (P<0.05) while the systolic [Ca(2+)](i) increased by 19% (P<0.05). No difference was found in the speed of SR Ca(2+) reloading after caffeine washout between the two cell types. CONCLUSION: Adenovirus-mediated SERCA1a gene transfer to adult rat ventricular myocytes enhances SR Ca(2+) handling to a degree similar to that observed following physiological stimulation.


Assuntos
Adenoviridae/genética , ATPases Transportadoras de Cálcio/genética , Cálcio/metabolismo , Vetores Genéticos/administração & dosagem , Miocárdio/metabolismo , Retículo Sarcoplasmático/enzimologia , Análise de Variância , Animais , Western Blotting , ATPases Transportadoras de Cálcio/análise , ATPases Transportadoras de Cálcio/metabolismo , Células Cultivadas , Técnicas de Transferência de Genes , Masculino , Microscopia Confocal , Miocárdio/enzimologia , Ratos , Ratos Wistar , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...