RESUMO
BACKGROUND: Dynamic transitions of tumour cells along the epithelial-mesenchymal axis are important in tumorigenesis, metastasis and therapy resistance. METHODS: In this study, we have used cell lines, 3D spheroids and tumour samples in a variety of cell biological and transcriptome analyses to highlight the cellular and molecular dynamics of OSCC response to ionising radiation. RESULTS: Our study demonstrates a prominent hybrid epithelial-mesenchymal state in oral squamous cell carcinoma cells and tumour samples. We have further identified a key role for levels of E-cadherin in stratifying the hybrid cells to compartments with varying levels of radiation response and radiation-induced epithelial-mesenchymal transition. The response to radiation further entailed the generation of a new cell population with low expression levels of E-cadherin, and positive for Vimentin (ECADLow/Neg-VIMPos), a phenotypic signature that showed an enhanced capacity for radiation resistance and invasion. At the molecular level, transcriptome analysis of spheroids in response to radiation showed an initial burst of misregulation within the first 30 min that further declined, although still highlighting key alterations in gene signatures. Among others, pathway analysis showed an over-representation for the Wnt signalling pathway that was further confirmed to be functionally involved in the generation of ECADLow/Neg-VIMPos population, acting upstream of radiation resistance and tumour cell invasion. CONCLUSION: This study highlights the functional significance and complexity of tumour cell remodelling in response to ionising radiation with links to resistance and invasive capacity. An area of less focus in conventional radiotherapy, with the potential to improve treatment outcomes and relapse-free survival.
Assuntos
Carcinoma de Células Escamosas/patologia , Transição Epitelial-Mesenquimal , Neoplasias Bucais/patologia , Tolerância a Radiação/genética , Carcinoma de Células Escamosas/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Transição Epitelial-Mesenquimal/genética , Transição Epitelial-Mesenquimal/efeitos da radiação , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Genes de Troca/fisiologia , Genes de Troca/efeitos da radiação , Estudos de Associação Genética , Humanos , Neoplasias Bucais/genética , Invasividade Neoplásica , Fenótipo , Radiação Ionizante , Transcriptoma/efeitos da radiação , Via de Sinalização Wnt/genética , Via de Sinalização Wnt/efeitos da radiaçãoRESUMO
Engineered cell-based therapies have emerged as a new paradigm in modern medicine, with several engineered T cell therapies currently approved to treat blood cancers and many more in clinical development. Tremendous progress in synthetic biology over the past two decades has allowed us to program cells with sophisticated sense-and-response modules that can effectively control therapeutic functions. In this review, we highlight recent advances in mammalian synthetic gene switches, focusing on devices designed for therapeutic applications. Although many gene switches responding to endogenous or exogenous molecular signals have been developed, the focus is shifting towards achieving remote-controlled production of therapeutic effectors by stimulating implanted engineered cells with traceless physical signals, such as light, electrical signals, magnetic fields, heat or ultrasound.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Mamíferos , Animais , Terapia Baseada em Transplante de Células e Tecidos/métodos , Genes de Troca/efeitos da radiação , Genes Sintéticos/efeitos da radiação , Humanos , Mamíferos/genética , Neoplasias/genética , Neoplasias/patologia , Neoplasias/terapia , Biologia Sintética/métodosRESUMO
Site directed RNA editing is an engineered tool for the posttranscriptional manipulation of RNA and proteins. Here, we demonstrate the inclusion of additional N- and C-terminal protein domains in an RNA editing-dependent manner to switch between protein isoforms in mammalian cell culture. By inclusion of localization signals, a switch of the subcellular protein localization was achieved. This included the shift from the cytoplasm to the outer-membrane, which typically is inaccessible at the protein-level. Furthermore, the strategy allows to implement photocaging to achieve spatiotemporal control of isoform switching. The strategy does not require substantial genetic engineering, and might well complement current optogenetic and optochemical approaches.
Assuntos
Genes de Troca/genética , Genes de Troca/efeitos da radiação , Mutagênese Sítio-Dirigida/métodos , Proteínas/metabolismo , Edição de RNA/genética , Edição de RNA/efeitos da radiação , Frações Subcelulares/metabolismo , Células HEK293 , Humanos , Luz , Proteínas/genéticaRESUMO
Recently, we demonstrated that ultrasound-based hyperthermia can activate cells containing a heat-activated and ligand-inducible gene switch in a spatio-temporally controlled manner. These engineered cells can be incorporated into hydrogel scaffolds (e.g., fibrin) for in vivo implantation, where ultrasound can be used to non-invasively pattern transgene expression. Due to their high water content, the acoustic attenuation of fibrin scaffolds is low. Thus, long ultrasound exposures and high acoustic intensities are needed to generate sufficient hyperthermia for gene activation. Here, we demonstrate that the attenuation of fibrin scaffolds and the resulting hyperthermia achievable with ultrasound can be increased significantly by doping the fibrin with hydroxyapatite (HA) nanopowder. The attenuation of a 1% (w/v) fibrin scaffold with 5% (w/v) HA was similar to soft tissue. Transgene activation of cells harboring the gene switch occurred at lower acoustic intensities and shorter exposures when the cells were encapsulated in HA-doped fibrin scaffolds versus undoped scaffolds. Inclusion of HA in the fibrin scaffold did not affect the viability of the encapsulated cells.
Assuntos
Durapatita/química , Genes de Troca/genética , Sonicação/métodos , Células-Tronco/fisiologia , Alicerces Teciduais , Transgenes/genética , Animais , Células Cultivadas , Preparações de Ação Retardada/administração & dosagem , Preparações de Ação Retardada/química , Preparações de Ação Retardada/efeitos da radiação , Relação Dose-Resposta à Radiação , Durapatita/efeitos da radiação , Genes de Troca/efeitos da radiação , Ondas de Choque de Alta Energia , Temperatura Alta , Camundongos , Doses de Radiação , Células-Tronco/efeitos da radiação , Transgenes/efeitos da radiaçãoRESUMO
In Schizosaccharomyces pombe 11 different switching genes (swi1 to swi10 and rad22) are known which are involved in mating-type (MT) switching. Mutations in swi5, swi9, swi10 and rad22 also cause an increased radiation sensitivity. We tested whether the survival of these mutants after UV irradiation is influenced by caffeine. We included rad1 and rad13 mutants in our experiments which do not affect MT switching. Several double and triple mutants were constructed. We were able to assign the switching genes to different repair pathways: swi9 and swi10 are involved in excision repair, rad22 has a function in recombination repair, while swi5 appears to be involved in a hitherto unknown pathway. This 'swi5 pathway' is stimulated (!) by caffeine. Previously it was found that the swi5 mutation also reduces meiotic recombination. As to rad genes, we found a few inconsistencies with previous reports in the literature.
Assuntos
Cafeína/farmacologia , Reparo do DNA/efeitos dos fármacos , Genes Fúngicos/efeitos dos fármacos , Genes Fúngicos Tipo Acasalamento , Genes de Troca/efeitos dos fármacos , Mutação , Schizosaccharomyces/genética , Alelos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , DNA Fúngico/efeitos dos fármacos , DNA Fúngico/efeitos da radiação , Genes Fúngicos/efeitos da radiação , Genes de Troca/efeitos da radiação , Schizosaccharomyces/efeitos dos fármacos , Schizosaccharomyces/efeitos da radiação , Raios Ultravioleta/efeitos adversosRESUMO
Three chloroplast genes, chlL, chlN and chlB, encoding the light-independent protochlorophyllide oxidoreductase (LIPOR) in Chlorella protothecoides CS-41 growing either photoautotrophically, mixotrophically or heterotrophically, were all transcribed constitutively independent of illumination and presence of glucose. Steady-state amounts of all three transcripts in the light-grown cells were, however, approximately two- to three-fold greater than those in the dark-grown cells. In addition, Western blotting demonstrated that approximately the same amount of protein was present in cultures grown mixtrophically or heterotrophically both containing glucose. However, much less protein was in photoautotrophic cells. These results suggest that LIPOR activity depends on post-transcriptional and post-translational regulation. Moreover, the fact that LIPOR accumulates in the light-grown cells indicates that LIPOR, which was thought to work only in darkness, may partially account for the protochlorophyllide photoreduction in light. Therefore, LIPOR switches on both in light and in darkness.