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1.
Oncotarget ; 7(49): 81208-81222, 2016 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-27783991

RESUMO

Advanced extremity melanoma and sarcoma present a significant therapeutic challenge, requiring multimodality therapy to treat or even palliate disease. These aggressive tumours are relatively chemo-resistant, therefore new treatment approaches are urgently required. We have previously reported on the efficacy of oncolytic virotherapy (OV) delivered by isolated limb perfusion. In this report, we have improved therapeutic outcomes by combining OV with radiotherapy. In vitro, the combination of oncolytic vaccinia virus (GLV-1h68) and radiotherapy demonstrated synergistic cytotoxicity. This effect was not due to increased viral replication, but mediated through induction of intrinsic apoptosis. GLV-1h68 therapy downregulated the anti-apoptotic BCL-2 proteins (MCL-1 and BCL-XL) and the downstream inhibitors of apoptosis, resulting in cleavage of effector caspases 3 and 7. In an in vivo ILP model, the combination of OV and radiotherapy significantly delayed tumour growth and prolonged survival compared to single agent therapy. These data suggest that the virally-mediated down-regulation of anti-apoptotic proteins may increase the sensitivity of tumour cells to the cytotoxic effects of ionizing radiation. Oncolytic virotherapy represents an exciting candidate for clinical development when delivered by ILP. Its ability to overcome anti-apoptotic signals within tumour cells points the way to further development in combination with conventional anti-cancer therapies.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/efeitos da radiação , Fibrossarcoma/terapia , Terapia Viral Oncolítica , Vírus Oncolíticos/patogenicidade , Vaccinia virus/patogenicidade , Animais , Proteínas Reguladoras de Apoptose/genética , Caspase 3/metabolismo , Caspase 7/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta à Radiação , Fibrossarcoma/metabolismo , Fibrossarcoma/patologia , Fibrossarcoma/virologia , Regulação Neoplásica da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Masculino , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Radioterapia Adjuvante , Ratos Endogâmicos BN , Transdução de Sinais/efeitos da radiação , Fatores de Tempo , Proteína bcl-X/metabolismo
2.
Arch Virol ; 161(12): 3473-3481, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27654667

RESUMO

In our previous study, six subgroup J strains of avian leukosis virus (ALV-J)-associated acutely transforming viruses carrying different lengths of the v-fps oncogene, designated as Fu-J and Fu-J1-5, were isolated and characterized from fibrosarcomas in ALV-J-infected chickens. In the present study, the oncogenic potential of Fu-J and Fu-J1-5 was investigated using a reverse genetics technique. Six replication-defective viruses, named rFu-J and rFu-J1-5, were rescued with the replication-competent rescued ALV-J strain rSDAU1005 as a helper virus by co-transfection of chicken embryo fibroblast monolayers with infectious clone plasmids. Experimental bird studies were performed, demonstrating that only the rescued rFu-J virus carrying the complete v-fps oncogene with rSDAU1005 as the helper virus could induce acute fibrosarcoma after inoculation in specific-pathogen-free (SPF) chickens. These results provide direct evidence that the replication-defective acutely transforming Fu-J virus, with the complete v-fps oncogene, was associated with acute fibrosarcoma in chickens infected with ALV-J in the field, as reported previously.


Assuntos
Vírus da Leucose Aviária/genética , Vírus da Leucose Aviária/isolamento & purificação , Fibrossarcoma/veterinária , Proteínas Oncogênicas/genética , Doenças das Aves Domésticas/patologia , Doenças das Aves Domésticas/virologia , Experimentação Animal , Animais , Vírus da Leucose Aviária/patogenicidade , Testes de Carcinogenicidade , Galinhas , Fibrossarcoma/virologia , Vírus Auxiliares , Organismos Livres de Patógenos Específicos , Replicação Viral
3.
Avian Pathol ; 45(2): 202-7, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27100152

RESUMO

To understand the cytogenetic characteristics of acute fibrosarcoma in chickens infected with the subgroup J avian leukosis virus associated with the v-src oncogene, we performed a karyotype analysis of fibrosarcoma cell cultures. Twenty-nine of 50 qualified cell culture spreads demonstrated polyploidy of some macrochromosomes, 21 of which were trisomic for chromosome 7, and others were trisomic for chromosomes 3, 4, 5 (sex chromosome w), and 10. In addition, one of them was trisomic for both chromosome 7 and the sex chromosome 5 (w). In contrast, no aneuploidy was found for 10 macrochromosomes of 12 spreads of normal chicken embryo fibroblast cells, although aneuploidy for some microchromosomes was demonstrated in five of the 12 spreads. The cytogenetic mosaicism or polymorphism of the aneuploidy in the acute fibrosarcoma described in this study suggests that the analysed cells are polyclonal.


Assuntos
Vírus da Leucose Aviária/genética , Leucose Aviária/virologia , Galinhas/virologia , Aberrações Cromossômicas , Fibrossarcoma/veterinária , Genes src/genética , Doença Aguda , Animais , Leucose Aviária/genética , Vírus da Leucose Aviária/isolamento & purificação , Embrião de Galinha , Feminino , Fibrossarcoma/genética , Fibrossarcoma/virologia , Cariótipo , Cariotipagem/veterinária , Polimorfismo Genético
4.
Virus Genes ; 52(3): 365-71, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27108997

RESUMO

Transduction of oncogenes by ALVs and generation of acute transforming viruses is common in natural viral infections. In order to understand the molecular basis for the rapid oncogenicity of Fu-J, an acutely transforming avian leukosis virus isolated from fibrosarcomas in crossbreed broilers infected with subgroup J avian leukosis virus (ALV-J) in China, complete genomic structure of Fu-J virus was determined by PCR amplification and compared with those of Fu-J1, Fu-J2, Fu-J3, Fu-J4, and Fu-J5 reported previously. The results showed that the genome of Fu-J was defective, with parts of gag gene replaced by the complete v-fps oncogene and encoded a 137 kDa Gag-fps fusion protein. Sequence analysis revealed that Fu-J and Fu-J1 to Fu-J5 were related quasi-species variants carrying different lengths of v-fps oncogenes generated from recombination between helper virus and c-fps gene. Comparison of virus carrying v-fps oncogene also gave us a glimpse of the molecular characterization and evolution process of the acutely transforming ALV.


Assuntos
Vírus da Leucose Aviária/genética , Leucose Aviária/virologia , Proteínas de Fusão gag-onc/genética , Proteínas Oncogênicas/genética , Vírus Oncogênicos/genética , Doenças das Aves Domésticas/virologia , Proteínas Tirosina Quinases/genética , Animais , Vírus da Leucose Aviária/isolamento & purificação , Vírus da Leucose Aviária/patogenicidade , Vírus do Sarcoma Aviário/genética , Sequência de Bases , Embrião de Galinha , Galinhas/virologia , DNA Viral , Fibrossarcoma/virologia , Produtos do Gene gag/genética , Genes Virais , Vírus Auxiliares/genética , Retroviridae/genética , Replicação Viral
5.
Mol Med Rep ; 12(5): 6517-26, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26352782

RESUMO

Heat shock factor 1 (Hsf1) serves an important role in regulating the proliferation of human tumor cell lines in vitro and tissue specific tumorigenesis in certain mouse models. However, its role in viral­oncogenesis remains to be fully elucidated. In the current study, the role of Hsf1 in fibroblastoma derived from simian virus 40/T antigen (SV40/TAG)­transformed mouse embryonic fibroblast (MEF) cell lines was investigated. Knockout of Hsf1 inhibited MEF cell proliferation in vitro and fibroblastoma growth and metastasis to the lungs in vivo in nude mice. Knockout of Hsf1 increased the protein expression levels of p53 and phosphorylated retinoblastoma protein (pRb), however reduced the expression of heat shock protein 25 (Hsp25) in addition to the expression of the angiogenesis markers vascular endothelial growth factor, cluster of differentiation 34 and factor VIII related antigen. Furthermore, immunoprecipitation indicated that knockout of Hsf1 inhibited the association between SV40/TAG and p53 or pRb. These data suggest that Hsf1 is involved in the regulation of SV40/TAG­derived fibroblastoma growth and metastasis by modulating the association between SV40/TAG and tumor suppressor p53 and pRb. The current study provides further evidence that Hsf1 may be a novel therapeutic target in the treatment of cancer.


Assuntos
Antígenos Virais de Tumores/genética , Proteínas de Ligação a DNA/genética , Fibroblastos/virologia , Fibrossarcoma/genética , Neoplasias Pulmonares/genética , Vírus 40 dos Símios/genética , Neoplasias Cutâneas/genética , Fatores de Transcrição/genética , Animais , Antígenos CD34/genética , Antígenos CD34/metabolismo , Antígenos Virais de Tumores/metabolismo , Linhagem Celular Transformada , Proliferação de Células , Proteínas de Ligação a DNA/deficiência , Embrião de Mamíferos , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrossarcoma/metabolismo , Fibrossarcoma/secundário , Fibrossarcoma/virologia , Regulação Neoplásica da Expressão Gênica , Técnicas de Inativação de Genes , Fatores de Transcrição de Choque Térmico , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/virologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Chaperonas Moleculares , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Transplante de Neoplasias , Fosforilação , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais , Vírus 40 dos Símios/metabolismo , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/virologia , Fatores de Transcrição/deficiência , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator de von Willebrand/genética , Fator de von Willebrand/metabolismo
6.
Poult Sci ; 94(4): 668-72, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25713393

RESUMO

The diagnosis of avian leukosis virus subgroup J (ALV-J) infection in Chinese Partridge Shank chickens was confirmed by necropsy, histopathological examinations, antibody tests, viral isolation, immunofluorescence assays, and sequence analysis. Myelocytoma, myeloma, and fibrosarcoma were simultaneously found in Partridge Shank flock with ALV-J infection. Sequence analysis of the env genes of ALV-J demonstrated that both gp85 and gp37 were highly homologous among the three strains from local chickens of those among ALV-J strains isolated from white meat-type chickens. The phylogenetic trees indicated that the three strains isolated in this study were closely related to reference strains isolated in so-called Chinese yellow chickens and some strains isolated from white meat-type chickens, both from the USA and China. The observed ALV-J infection was the first report on Partridge Shank chickens, and myelocytoma, myeloma, and fibrosarcoma were found at the same time in this batch of local chickens.


Assuntos
Vírus da Leucose Aviária/genética , Leucose Aviária/diagnóstico , Galinhas , Doenças das Aves Domésticas/diagnóstico , Animais , Leucose Aviária/epidemiologia , Leucose Aviária/virologia , Vírus da Leucose Aviária/metabolismo , China/epidemiologia , Fibrossarcoma/epidemiologia , Fibrossarcoma/veterinária , Fibrossarcoma/virologia , Incidência , Dados de Sequência Molecular , Mieloma Múltiplo/epidemiologia , Mieloma Múltiplo/veterinária , Mieloma Múltiplo/virologia , Filogenia , Doenças das Aves Domésticas/epidemiologia , Doenças das Aves Domésticas/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Análise de Sequência de DNA/veterinária , Especificidade da Espécie
7.
Wei Sheng Wu Xue Bao ; 53(3): 299-305, 2013 Mar 04.
Artigo em Chinês | MEDLINE | ID: mdl-23678577

RESUMO

OBJECTIVE: To prepare anti-fps mono-specific serum, and detect the fps antigen in tumors induced by acute transforming avian leukosis/sarcoma virus containing v-fps oncogene. METHODS: Two part of v-fps gene was amplified by RT-PCR using the Fu-J viral RNA as the template. Mono-specific serum was prepared by immuning Kunming white mouse with both two recombinant infusion proteins expressed by the prokaryotic expression system. Indirect immunofluorescent assay was used to detect fps antigen in tumor tissue suspension cells and CEF infected by sarcoma supernatant. Immunohistochemical method was used to detect fps antigen in tumor tissue. RESULTS: The mouse mono-specific serum was specific as it had no cross reaction with classical ALV-J strains. The result reveals that the tumor tissue suspension cells, the CEF infected by sarcoma supernatant, and the slice immunohistochemistry of the sarcoma showed positive results. CONCLUSION: The anti-fps mono-specific serum was prepared, and the detection method was established, which laid the foundation for the study of viral biological characteristics and mechanism of tumourgenesis of acute transforming avian leukosis/sarcoma virus containing v-fps oncogene.


Assuntos
Vírus da Leucose Aviária/imunologia , Vírus do Sarcoma Aviário/imunologia , Galinhas , Fibrossarcoma/imunologia , Doenças das Aves Domésticas/imunologia , Proteínas Proto-Oncogênicas c-fes/imunologia , Animais , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Leucose Aviária/imunologia , Leucose Aviária/virologia , Transformação Celular Neoplásica , Fibrossarcoma/virologia , Camundongos , Doenças das Aves Domésticas/virologia , Proteínas Proto-Oncogênicas c-fes/genética , RNA Viral/genética , Sarcoma Aviário/imunologia , Sarcoma Aviário/virologia , Organismos Livres de Patógenos Específicos
8.
Acta Virol ; 57(1): 69-74, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23530826

RESUMO

This study investigated the anti-neoplastic potential of avian reovirus σC (sigma C) protein on Rous sarcoma virus-induced fibrosarcoma in chicken. The recombinant vector expressing σC protein was injected intra-tumorally into specific pathogen free chicken with fibro-sarcoma at the dose 100µg per bird, while control birds were mock-treated with 100µg of empty vector per bird. Recombinant σC protein induced apoptosis in tumors of treated birds resulting in progressive tumor regression, while similar changes were absent in tumors of mock-treated controls. The σC protein-induced apoptosis in tumors was quantified by flow cytometry and the mean level of apoptosis up to 66% was observed in treated tumors, whereas any significant level of apoptosis was absent in mock-treated controls.


Assuntos
Antineoplásicos/administração & dosagem , Proteínas do Capsídeo/administração & dosagem , Galinhas , Orthoreovirus Aviário/genética , Vírus do Sarcoma de Rous/fisiologia , Sarcoma Aviário/terapia , Animais , Apoptose , Proteínas do Capsídeo/genética , Embrião de Galinha , Feminino , Fibrossarcoma/terapia , Fibrossarcoma/virologia , Terapia Genética , Vetores Genéticos , Humanos , Orthoreovirus Aviário/metabolismo , Proteínas Recombinantes , Sarcoma Aviário/virologia , Organismos Livres de Patógenos Específicos
9.
Hum Gene Ther ; 23(2): 231-7, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21981728

RESUMO

Concerns surrounding the oncogenic potential of recombinant gammaretroviral vectors has spurred a great deal of interest in vector integration site (VIS) preferences. Although gammaretroviral vectors exhibit a modest preference for integration near transcription start sites (TSS) of active genes, such associations only account for about a third of all VIS. Previous studies suggested a correlation between gammaretroviral VIS and DNase hypersensitive sites (DHS), which mark chromatin regions associated with cis-regulatory elements. In order to study this issue directly, we assessed the correlation between 167 validated gammaretroviral VIS and a deep genome-wide map of DHS, both determined in the same cell line (the human fibrosarcoma HT1080). The DHS map was developed by sequencing individual DNase I cleavage sites using massively parallel sequencing technologies. These studies revealed an overwhelming preference for integrations associated with DHS, with a median distance of only 238 bp between individual VIS and the nearest DHS for the experimental dataset, compared to 3 kb for a random dataset and 577 to 1457 bp for two unrelated cell lines (p<0.001). Indeed, nearly 84% of all VIS were found to be located within 1 kb of a DHS (p=10(-43)). Further, this correlation was statistically independent from the association with TSS. The preference for DHS far exceeds that seen for other hallmarks of gammaretroviral VIS, including TSS, and may help explain several aspects of gammaretroviral vector biology, including the mechanism of VIS selection, as well as the relative frequency and underlying biology of gammaretroviral vector-mediated genotoxicity.


Assuntos
Cromatina/genética , Desoxirribonuclease I/genética , Fibrossarcoma/virologia , Gammaretrovirus/genética , Integração Viral , Linhagem Celular Tumoral , Mapeamento Cromossômico , Fibrossarcoma/genética , Fibrossarcoma/patologia , Vetores Genéticos , Humanos , Sequências Reguladoras de Ácido Nucleico , Análise de Sequência de DNA , Sítio de Iniciação de Transcrição , Transcrição Gênica
10.
Cancer Gene Ther ; 16(10): 776-85, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19390568

RESUMO

Interleukin 23 (IL-23) is a member of the IL-12 family of heterodimeric cytokines, composed of p19 and p40 subunits, which exhibits immunostimulatory properties similar to IL-12. IL-23 has been shown to possess potent antitumor activities in several establishment models of cancer and a few therapeutic models, but the efficacy of local, adenoviral-mediated expression of IL-23 in established tumors has yet to be investigated. Here we have examined the antitumor activity of adenovirally delivered IL-23 in a day-7 MCA205 murine fibrosarcoma tumor model. Three intratumoral injections of adenovirus expressing IL-23 (Ad.IL-23) significantly increased animal survival and resulted in complete rejection of 40% of tumors, with subsequent generation of protective immunity and MCA205-specific cytotoxic T lymphocytes. In addition, we have shown that the antitumor activity of IL-23 is independent of IL-17, perforin and Fas ligand, but dependent on interferon-gamma, CD4(+) and CD8(+) T cells. These results demonstrate that direct intratumoral injection of adenovirus expressing IL-23 results in enhanced survival, tumor eradication and generation of protective immunity by generation of a Th1-type immune response.


Assuntos
Adenoviridae/genética , Fibrossarcoma/terapia , Terapia Genética/métodos , Interleucina-23/genética , Animais , Linfócitos T CD4-Positivos/imunologia , Linhagem Celular Tumoral , Proteína Ligante Fas/imunologia , Feminino , Fibrossarcoma/genética , Fibrossarcoma/imunologia , Fibrossarcoma/virologia , Técnicas de Transferência de Genes , Interferon gama/imunologia , Interleucina-17/biossíntese , Interleucina-23/biossíntese , Interleucina-23/imunologia , Estimativa de Kaplan-Meier , Camundongos , Camundongos Endogâmicos C57BL , Perforina/imunologia , Linfócitos T Citotóxicos/imunologia , Transdução Genética
11.
J Gen Virol ; 89(Pt 1): 138-147, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18089737

RESUMO

Equine sarcoids are fibrosarcoma-like skin tumours with a prevalence of approximately 1-2 %. Strong evidence exists for a causative role of bovine papillomavirus (BPV) type 1 or type 2 in the development of sarcoids. No effective treatment of equine sarcoid is available and after surgical excision relapse of the tumours is very frequent. We developed chimeric virus-like particles (CVLPs) of BPV 1 L1-E7 for the immunotherapy of equine sarcoid. In a phase I clinical trial 12 horses suffering from equine sarcoid with an average number of more than 22 tumours per animal were vaccinated in a dose-escalation setting. The animals were followed-up for 63 days, eight of the twelve horses were followed-up for more than a year and side-effects, humoral immune responses and tumour appearance were recorded. BPV DNA was detected in tumours of 11 cases. CVLPs were well tolerated in all dose groups, a robust anti-L1 antibody response was induced in all but one of the horses. Anti-E7 antibodies were detected in five of the 12 animals at low titres. Two animals showed a clear improvement of the clinical status after treatment, i.e. the number of the tumours per horse was reduced. In another horse regression of five sarcoids was observed; three of them relapsed during the study. Two animals showed tumour regression as well as growth of new sarcoids. In two horses the clinical status remained unchanged, in another two horses growth of existing tumours or growth of additional tumours was observed. The remaining three animals showed simultaneously regression and growth of existing tumours. Neither the humoral immune responses nor the observed effects on the tumours was correlated with the dose group.


Assuntos
Papillomavirus Bovino 1/genética , Papillomavirus Bovino 1/isolamento & purificação , Fibrossarcoma/virologia , Doenças dos Cavalos/virologia , Infecções por Papillomavirus/veterinária , Sarcoidose/veterinária , Sarcoidose/virologia , Animais , Anticorpos Antivirais/análise , Formação de Anticorpos , Biópsia , Quimera , DNA Viral/genética , DNA Viral/isolamento & purificação , Ensaio de Imunoadsorção Enzimática , Feminino , Fibrossarcoma/imunologia , Fibrossarcoma/cirurgia , Fibrossarcoma/veterinária , Doenças dos Cavalos/imunologia , Doenças dos Cavalos/cirurgia , Cavalos , Imunoterapia , Masculino , Infecções por Papillomavirus/imunologia , Infecções por Papillomavirus/cirurgia , Recidiva , Sarcoidose/imunologia , Sarcoidose/cirurgia
12.
Mol Cancer Ther ; 6(9): 2487-95, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17876046

RESUMO

The thymidine analogue 4-thiothymidine (S(4)TdR) is a photosensitizer for UVA radiation. The UV absorbance spectrum of S(4)TdR and its incorporation into DNA suggests that it might act synergistically with nonlethal doses of UVA to selectively kill hyperproliferative or cancerous skin cells. We show here that nontoxic concentrations of S(4)TdR combine with nonlethal doses of UVA to kill proliferating cultured skin cells. Established cell lines with a high fraction of proliferating cells were more sensitive than primary keratinocytes or fibroblasts to apoptosis induction by S(4)TdR/UVA. Although S(4)TdR plus UVA treatment induces stabilization of p53, cell death, as measured by apoptosis or clonal survival, occurs to a similar extent in both p53 wild-type and p53-null backgrounds. Furthermore, different types of human papilloma virus E6 proteins, which protect against UVB-induced apoptosis, have little effect on killing by S(4)TdR/UVA. S(4)TdR/UVA offers a possible therapeutic intervention strategy that seems to be applicable to human papilloma virus-associated skin lesions.


Assuntos
Apoptose , Fibrossarcoma/terapia , Papillomaviridae/genética , Infecções por Papillomavirus/virologia , Neoplasias Cutâneas/terapia , Timidina/análogos & derivados , Raios Ultravioleta , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Western Blotting , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Células Cultivadas , Terapia Combinada , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/efeitos da radiação , Fibroblastos/virologia , Fibrossarcoma/tratamento farmacológico , Fibrossarcoma/radioterapia , Fibrossarcoma/virologia , Citometria de Fluxo , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/efeitos da radiação , Queratinócitos/virologia , Camundongos , Pessoa de Meia-Idade , Octreotida/análogos & derivados , Pele/citologia , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/radioterapia , Neoplasias Cutâneas/virologia , Timidina/uso terapêutico , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/fisiologia
13.
Oncogene ; 26(28): 4124-34, 2007 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-17213803

RESUMO

Reovirus shows considerable potential as an oncolytic agent for Ras-activated tumors and is currently in clinical trials. Here we ask whether such tumor cell lines can acquire resistance to reoviral oncolysis. We challenged human HT1080 fibrosarcoma cells that carry a Ras mutation by prolonged exposure to reovirus, thereby yielding highly virus-resistant HTR1 cells. These cells are persistently infected with reovirus, exhibit high Ras activity and retain the original Ras gene mutation, showing that resistance to reovirus can be displayed in cells with active Ras. The HTR1 cells also exhibit reduced cellular cathepsin B activity, which normally contributes to viral entry and activation. Persistently infected HTR1 cells were not tumorigenic in vivo, whereas immunologically cured virus-free HTR1 cells were highly tumorigenic. Thus, acquisition of resistance to reovirus may constrain therapeutic strategies. To determine whether reoviral resistance is associated with a general reduction in apoptotic potential, we challenged the HTR1 cells with apoptotic inducers and E1B-defective adenovirus, resulting in significant apoptosis and cell death following both approaches. Therefore, even if resistance to reoviral oncolysis should arise in tumor cells in vivo, other therapeutic strategies may nevertheless remain effective.


Assuntos
Fibrossarcoma/patologia , Proteína Oncogênica p21(ras)/fisiologia , Reoviridae/fisiologia , Sequência de Bases , Catepsina B/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Primers do DNA , Fibrossarcoma/virologia , Humanos , Mutação , Proteína Oncogênica p21(ras)/genética
14.
J Biol Chem ; 281(45): 34064-71, 2006 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-16973618

RESUMO

Human P54 and P56 proteins are tetratricopeptide proteins that are encoded by two closely related genes, ISG54 and ISG56. These genes are induced strongly but transiently when cells are treated with interferons or double-stranded RNA or infected with a variety of viruses. We observed that, although double-stranded RNA or Sendai virus infection induced the two genes with similar kinetics, their induction kinetics in response to interferon-beta were quite different. The induction kinetics by virus infection were also different between two cell lines. Functionally the two proteins were similar. Like P56, P54 bound to the translation initiation factor eIF3 and inhibited translation. However, unlike P56, P54 bound to both the "e" and the "c" subunits of eIF3. Consequently, P54 inhibited two functions of eIF3. Like P56, it inhibited the ability of eIF3 to stabilize the eIF2 x GTP x Met-tRNA(i) ternary complex. But in addition, it also inhibited the formation of the 48 S pre-initiation complex between the 40 S ribosomal subunit and the 20 S complex composed of eIF3, ternary complex, eIF4F, and mRNA. Thus, although similar in structure, the human P54 and P56 proteins are induced differently and function differently.


Assuntos
Fator de Iniciação 3 em Eucariotos/metabolismo , Biossíntese de Proteínas , Proteínas/metabolismo , Fatores de Transcrição/biossíntese , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Reguladoras de Apoptose , Células Cultivadas/metabolismo , Células Cultivadas/virologia , Fator de Iniciação 3 em Eucariotos/genética , Fibrossarcoma/tratamento farmacológico , Fibrossarcoma/metabolismo , Fibrossarcoma/virologia , Humanos , Imunoprecipitação , Interferon beta/farmacologia , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/virologia , Modelos Biológicos , Plasmídeos , RNA de Cadeia Dupla/fisiologia , RNA de Transferência de Metionina/metabolismo , Proteínas de Ligação a RNA , Proteínas Recombinantes , Ribonucleases , Ribossomos/metabolismo , Vírus Sendai/fisiologia , Transfecção
15.
Cancer Res ; 66(15): 7694-700, 2006 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-16885371

RESUMO

Cancer cells secrete matrix metalloproteinases (MMP) that degrade the extracellular matrix and are responsible for some hallmarks of malignant cancer. Many viruses, including a few currently used in oncolytic virotherapy clinical trials, depend on intracellular proteases to process their proteins and activate their particles. We show here for measles virus (MV) that particle activation can be made dependent of proteases secreted by cancer cells. The MV depends on the intracellular protease furin to process and activate its envelope fusion (F) protein. To make F protein activation cancer cell specific, we introduced hexameric sequences recognized by an MMP and identified the mutant proteins most effective in fusing MMP-expressing human fibrosarcoma cells (HT1080). We showed that an MMP inhibitor interferes with syncytia formation elicited by mutant F proteins and confirmed MMP-dependent cleavage by Edman degradation sequence analysis. We generated recombinant MVs expressing the modified F proteins in place of furin-activated F. These viruses spread only in cells secreting MMP. In nude mice, an MMP-activated MV retarded HT1080 xenograft growth as efficiently as the furin-activated MV vaccine strain. In MV-susceptible mice, the furin-activated virus caused lethal encephalitis upon intracerebral inoculation, whereas the MMP-activated did not. Thus, MV particle activation can be made dependent of proteases secreted by cancer cells, enhancing safety. This study opens the perspective of combining targeting at the particle activation, receptor recognition, and selective replication levels to improve the therapeutic index of MV and other viruses in ongoing clinical trials of oncolysis.


Assuntos
Metaloproteinases da Matriz/metabolismo , Vírus do Sarampo/fisiologia , Terapia Viral Oncolítica/métodos , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Chlorocebus aethiops , Fibrossarcoma/enzimologia , Fibrossarcoma/terapia , Fibrossarcoma/virologia , Furina/metabolismo , Humanos , Inibidores de Metaloproteinases de Matriz , Vírus do Sarampo/metabolismo , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Terapia Viral Oncolítica/efeitos adversos , Células Vero , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo , Ativação Viral
16.
Cancer Res ; 66(9): 4835-42, 2006 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-16651439

RESUMO

We have previously developed an oncolytic herpes simplex virus-1 based on a clinical virus isolate, which was deleted for ICP34.5 to provide tumor selected replication and ICP47 to increase antigen presentation as well as tumor selective virus replication. A phase I/II clinical trial using a version of this virus expressing granulocyte macrophage colony-stimulating factor has shown promising results. The work reported here aimed to develop a version of this virus in which local tumor control was further increased through the combined expression of a highly potent prodrug activating gene [yeast cytosine deaminase/uracil phospho-ribosyltransferase fusion (Fcy::Fur)] and the fusogenic glycoprotein from gibbon ape leukemia virus (GALV), which it was hoped would aid the spread of the activated prodrug through the tumor. Viruses expressing the two genes individually or in combination were constructed and tested, showing (a) GALV and/or Fcy::Fur expression did not affect virus growth; (b) GALV expression causes cell fusion and increases the tumor cell killing at least 30-fold in vitro and tumor shrinkage 5- to 10-fold in vivo; (c) additional expression of Fcy::Fur combined with 5-fluorocytosine administration improves tumor shrinkage further. These results indicate, therefore, that the combined expression of the GALV protein and Fcy::Fur provides a highly potent oncolytic virus with improved capabilities for local tumor control. It is intended to enter the GALV/Fcy::Fur expressing virus into clinical development for the treatment of tumor types, such as pancreatic or lung cancer, where local control would be anticipated to be clinically advantageous.


Assuntos
Fibrossarcoma/terapia , Flucitosina/farmacocinética , Vírus da Leucemia do Macaco Gibão/genética , Glicoproteínas de Membrana/genética , Terapia Viral Oncolítica/métodos , Simplexvirus/fisiologia , Animais , Biotransformação , Fusão Celular , Terapia Combinada , Citosina Desaminase/biossíntese , Citosina Desaminase/genética , Citosina Desaminase/metabolismo , Fibrossarcoma/genética , Fibrossarcoma/metabolismo , Fibrossarcoma/virologia , Flucitosina/farmacologia , Fluoruracila/farmacocinética , Fluoruracila/farmacologia , Humanos , Vírus da Leucemia do Macaco Gibão/metabolismo , Vírus da Leucemia do Macaco Gibão/fisiologia , Glicoproteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Pentosiltransferases/biossíntese , Pentosiltransferases/genética , Pentosiltransferases/metabolismo , Pró-Fármacos/farmacocinética , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Simplexvirus/genética , Replicação Viral , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Intervirology ; 48(5): 292-6, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-15956796

RESUMO

We studied the sensitivity of several human cancer cell strains (HeLa, HT1080, SPC-A1, and ACHN) and normal cell strains (MRC-5 and Wish) to mumps virus (MuV) S79, a live attenuated vaccine strain. These cells exhibited a differential sensitivity to infection with MuV, and the susceptible sequences were ACHN > HeLa > HT1080 > SPC-A1 > Wish > MRC-5. In experiments in vivo, nude mice with HT1080 fibrosarcoma xenografts were randomly divided into three groups for intratumoral treatment with MuVS79, UV-inactivated MuVS79, and PBS. At 10(7) PFU, live MuVS79 injection caused in 7 of 9 mice complete regression by day 15 while rapid tumor growth occurred in all 9 mice treated with PBS. Rapid tumor progression also occurred in all 8 mice treated with UV-inactivated virus; however, tumor growth was delayed in the logarithmic phase relative to the PBS-treated tumors.


Assuntos
Fibrossarcoma/terapia , Vírus da Caxumba/fisiologia , Neoplasias/terapia , Neoplasias/virologia , Animais , Apoptose , Linhagem Celular Tumoral , Sobrevivência Celular , Fibrossarcoma/virologia , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neoplasias/patologia , Replicação Viral
18.
Cancer Res ; 64(14): 4919-26, 2004 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-15256464

RESUMO

Live attenuated Edmonston B strain of measles virus (MV-Edm) is a potent and specific oncolytic agent, but the mechanism underlying its tumor selectivity is unknown. The virus causes cytopathic effects (CPEs) of extensive syncytial formation in tumor cells but minimal damage or cell killing in normal cells. The CPE is dependent on expression of viral proteins and the presence of CD46, the major cellular receptor of MV-Edm. Using a virally encoded soluble marker peptide to provide a quantitative readout of the level of viral gene expression, we determined that tumor cells and normal cells expressed comparable levels of viral proteins. CD46 mediates virus attachment, entry, and virus-induced cell-to-cell fusion. Using engineered cells expressing a range of CD46 densities, we determined that whereas virus entry increased progressively with CD46 density, cell fusion was minimal at low receptor densities but increased dramatically above a threshold density of CD46 receptors. It is well established that tumor cells express abundant CD46 receptors on their surfaces compared with their normal counterparts. Thus, at low CD46 densities typical of normal cells, infection occurs, but intercellular fusion is negligible. At higher densities typical of tumor cells, infection leads to extensive cell fusion. Intercellular fusion also results in enhancement of viral gene expression through recruitment of neighboring uninfected cells into the syncytium, further amplifying the CPE. Discrimination between high and low CD46 receptor density provides a compelling basis for the oncolytic specificity of MV-Edm and establishes MV-Edm as a promising CD46-targeted cancer therapeutic agent.


Assuntos
Antígenos CD/metabolismo , Vírus do Sarampo/fisiologia , Glicoproteínas de Membrana/metabolismo , Neoplasias/metabolismo , Neoplasias/virologia , Receptores Virais/metabolismo , Animais , Antígenos CD/genética , Células CHO , Linhagem Celular Tumoral , Chlorocebus aethiops , Cricetinae , Efeito Citopatogênico Viral , Feminino , Fibrossarcoma/genética , Fibrossarcoma/metabolismo , Fibrossarcoma/terapia , Fibrossarcoma/virologia , Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/terapia , Neoplasias Pulmonares/virologia , Proteína Cofatora de Membrana , Glicoproteínas de Membrana/genética , Neoplasias/genética , Neoplasias/terapia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/terapia , Neoplasias Ovarianas/virologia , Receptores Virais/genética , Transfecção , Células Vero
19.
J Virol Methods ; 107(1): 9-14, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12445932

RESUMO

A plaque assay for detecting, isolating and titrating avian pneumoviruses using a Japanese quail fibrosarcoma cell line (QT-35) is described. Plaques are produced after application of either an agarose or carboxymethyl-cellulose (CMC) overlay onto cell monolayers infected with representative avian metapneumoviruses which belong to subgroup A, B or C. Virus plaques can be easily visualized by light microscopy or after staining. The parameters affecting plaque appearance include: cell seeding concentration, virus strain, overlay composition and incubation time following infection. Optimal conditions for plaquing involve seeding QT-35 cells at 40,000 cells per cm(2) when using a 1.5% CMC overlay or 100,000 cells per cm(2) when using a 1.0 or 0.8% agarose overlay. In both cases, cell monolayers are infected with virus 24 h post-seeding and clearly visible plaques develop in 6 days. Due to the robust nature of these cells, the incubation time can be extended to a maximum of 13 days after infection in order to produce larger plaques.


Assuntos
Metapneumovirus/crescimento & desenvolvimento , Ensaio de Placa Viral/métodos , Animais , Coturnix , Fibrossarcoma/virologia , Metapneumovirus/isolamento & purificação , Células Tumorais Cultivadas
20.
J Med Virol ; 68(2): 278-84, 2002 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12210420

RESUMO

Amphotropic murine leukemia virus (MLV) replicates in cells from various mammalian species including humans and is a potential contaminant in MLV vector preparations for human gene transfer studies. In general, MLV replication depends on the expression of viral genes under the control of 75 bp enhancer elements in the long terminal repeat. However, in specific human fibrosarcoma and lymphoma lines replication of amphotropic MLV is possible without these enhancers. Fibrosarcomas are malignant tumors of fibroblast origin. To test the replication potential of intact and enhancerless amphotropic MLV in untransformed cells, infection studies with these viruses were carried out in three types of primary human fibroblasts. Replication of amphotropic MLV is observed in two of three tested fibroblast strains. None of these primary human fibroblasts is permissive for enhancer-deficient MLV, suggesting that replication of this virus may be limited to transformed cells.


Assuntos
Fibroblastos/virologia , Fibrossarcoma/virologia , Vírus da Leucemia Murina/genética , Vírus da Leucemia Murina/fisiologia , Animais , Sequência de Bases , Células Cultivadas , DNA Viral/genética , Elementos Facilitadores Genéticos , Humanos , Vírus da Leucemia Murina/patogenicidade , Camundongos , RNA Viral/genética , RNA Viral/isolamento & purificação , Células Tumorais Cultivadas , Replicação Viral
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