RESUMEN
Survivin is a proto-oncogene that regulates cell division and apoptosis. It is a molecular marker of cancer. Recently, survivin has emerged as a feature of RA, associated with severe joint damage and poor treatment response. The present study examined if inhibition of survivin affects experimental arthritis, which was induced in mBSA-immunized mice by an injection of mBSA in the knee joint or developed spontaneously in collagen type II-immunized mice. The inhibition of survivin transcription by a lentivirus shRNA construct alleviated joint inflammation and reduced bone damage. The inhibition of survivin reduced the levels of metalloproteinases, ß-catenin, and vimentin, limiting the invasive capacity of synovia, while no inhibition of osteoclastogenesis could be found. The inhibition of survivin led to a p53-independent reduction of T cell proliferation and favored the transcription and activity of Blimp-1, which limited IL-2 production and facilitated formation of regulatory Foxp3(+)CD4(+) and effector CD8(+) T cells. The study shows that the inhibition of survivin is sufficient to reduce joint inflammation and bone damage in preclinical models of arthritis. Antiarthritic effects of survivin inhibition are related to p53-independent control of lymphocyte proliferation.
Asunto(s)
Artritis Experimental/inmunología , Artritis Reumatoide/inmunología , Proteínas Inhibidoras de la Apoptosis/inmunología , Proteínas Represoras/inmunología , Animales , Artritis Experimental/metabolismo , Artritis Reumatoide/metabolismo , Western Blotting , Regulación hacia Abajo , Femenino , Citometría de Flujo , Técnicas de Silenciamiento del Gen , Inmunohistoquímica , Proteínas Inhibidoras de la Apoptosis/biosíntesis , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos DBA , Proteínas Represoras/biosíntesis , SurvivinAsunto(s)
Transferasas Alquil y Aril/antagonistas & inhibidores , Inhibidores Enzimáticos/uso terapéutico , Genes ras , Trastornos Mieloproliferativos/tratamiento farmacológico , Transferasas Alquil y Aril/genética , Transferasas Alquil y Aril/fisiología , Animales , Modelos Animales de Enfermedad , Ratones , Trastornos Mieloproliferativos/enzimología , Fenotipo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológicoRESUMEN
In order to use adenovirus (Ad) type 5 (Ad5) for cancer gene therapy, Ad needs to be de-targeted from its native receptors and re-targeted to a tumor antigen. A limiting factor for this has been to find a ligand that (i) binds a relevant target, (ii) is able to fold correctly in the reducing environment of the cytoplasm and (iii) when incorporated at an optimal position on the virion results in a virus with a low physical particle to plaque-forming units ratio to diminish the viral load to be administered to a future patient. Here, we present a solution to these problems by producing a genetically re-targeted Ad with a tandem repeat of the HER2/neu reactive Affibody molecule (ZH) in the HI-loop of a Coxsackie B virus and Ad receptor (CAR) binding ablated fiber genetically modified to contain sequences for flexible linkers between the ZH and the knob sequences. ZH is an Affibody molecule specific for the extracellular domain of human epidermal growth factor receptor 2 (HER2/neu) that is overexpressed in inter alia breast and ovarian carcinomas. The virus presented here exhibits near wild-type growth characteristics, infects cells via HER2/neu instead of CAR and represents an important step toward the development of genetically re-targeted adenoviruses with clinical relevance.
Asunto(s)
Adenoviridae/genética , Antígenos de Neoplasias/genética , Terapia Genética/métodos , Vectores Genéticos/genética , Proteínas Recombinantes de Fusión/genética , Antígenos de Neoplasias/inmunología , Neoplasias de la Mama/terapia , Femenino , Humanos , Ligandos , Neoplasias Ováricas/terapia , Receptor ErbB-2/genética , Receptor ErbB-2/inmunología , Proteínas Recombinantes de Fusión/inmunología , Células Tumorales CultivadasRESUMEN
Most human carcinoma cell lines lack the high-affinity receptors for adenovirus serotype 5 (Ad5) at their surface and are nonpermissive to Ad5. We therefore tested the efficiency of retargeting Ad5 to alternative cellular receptors via immunoglobulin (Ig)-binding domains inserted at the extremity of short-shafted, knobless fibers. The two recombinant Ad5's constructed, Ad5/R7-Z(wt)-Z(wt) and Ad5/R7-C2-C2, carried tandem Ig-binding domains from Staphylococcal protein A (abbreviated Z(wt)) and from Streptococcal protein G (C2), respectively. Both viruses bound their specific Ig isotypes with the expected affinity. They transduced human carcinoma cells independently of the CAR pathway, via cell surface receptors targeted by specific monoclonal antibodies, that is, EGF-R on A549, HT29 and SW1116, HER-2/neu on SK-OV-3 and SK-BR-3, CA242 (epitope recognized by the monoclonal antibody C242) antigen on HT29 and SW1116, and PSMA (prostate-specific membrane antigen) expressed on HEK-293 cells, respectively. However, Colo201 and Colo205 cells were neither transduced by targeting CA242 or EGF-R nor were LNCaP cells transduced by targeting PSMA. Our results suggested that one given surface receptor could mediate transduction of certain cells but not others, indicating that factors and steps other than cell surface expression and virus-receptor interaction are additional determinants of Ad5-mediated transduction of tumor cells. Using penton base RGD mutants, we found that one of these limiting steps was virus endocytosis.