RESUMO
Treatment options of glioblastoma multiforme are limited due to the blood-brain barrier (BBB). In this study, we investigated the utility of intranasal (IN) delivery as a means of transporting stem cell-based antiglioma therapeutics. We hypothesized that mesenchymal stem cells (MSCs) delivered via nasal application could impart therapeutic efficacy when expressing TNF-related apoptosis-inducing ligand (TRAIL) in a model of human glioma. ¹¹¹In-oxine, histology and magnetic resonance imaging (MRI) were utilized to track MSCs within the brain and associated tumor. We demonstrate that MSCs can penetrate the brain from nasal cavity and infiltrate intracranial glioma xenografts in a mouse model. Furthermore, irradiation of tumor-bearing mice tripled the penetration of (¹¹¹In)-oxine-labeled MSCs in the brain with a fivefold increase in cerebellum. Significant increase in CXCL12 expression was observed in irradiated xenograft tissue, implicating a CXCL12-dependent mechanism of MSCs migration towards irradiated glioma xenografts. Finally, MSCs expressing TRAIL improved the median survival of irradiated mice bearing intracranial U87 glioma xenografts in comparison with nonirradiated and irradiated control mice. Cumulatively, our data suggest that IN delivery of stem cell-based therapeutics is a feasible and highly efficacious treatment modality, allowing for repeated application of modified stem cells to target malignant glioma.
Assuntos
Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/terapia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Animais , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Movimento Celular , Rastreamento de Células , Quimiocina CXCL12/genética , Modelos Animais de Doenças , Raios gama , Expressão Gênica , Glioma/mortalidade , Glioma/patologia , Glioma/terapia , Humanos , Imageamento por Ressonância Magnética , Camundongos , Compostos Organometálicos , Oxiquinolina/análogos & derivados , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The olfactory epithelium is a sensory neuroepithelium that supports adult neurogenesis and tissue regeneration following injury, making it an excellent model for investigating neural stem cell regulation in vivo. Previous studies have identified the horizontal basal cell (HBC) as the neural stem cell of the postnatal olfactory epithelium. However, the molecules and pathways regulating HBC self-renewal and differentiation are unknown. In the present study, we demonstrate that the transcription factor p63, a member of the p53 tumor suppressor gene family known to regulate stem cell dynamics in other epithelia, is highly enriched in HBCs. We show that p63 is required cell autonomously for olfactory stem cell renewal and further demonstrate that p63 functions to repress HBC differentiation. These results provide critical insight into the genetic regulation of the olfactory stem cell in vivo and more generally provide an entrée toward understanding the coordination of stem cell self-renewal and differentiation.
Assuntos
Diferenciação Celular/genética , Regulação da Expressão Gênica/genética , Neurogênese/genética , Bulbo Olfatório/citologia , Fosfoproteínas/metabolismo , Células-Tronco/fisiologia , Transativadores/metabolismo , Animais , Animais Recém-Nascidos , Proteínas de Bactérias/genética , Citometria de Fluxo , Perfilação da Expressão Gênica , Queratina-15 , Queratina-5/genética , Proteínas Luminescentes/genética , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Mucosa Olfatória/citologia , Análise de Sequência com Séries de Oligonucleotídeos , Fosfoproteínas/genética , Proteínas/genética , RNA não Traduzido , Transativadores/genéticaRESUMO
A fundamental question in developmental biology is how morphogenesis is coordinated with cell cycle progression. In Caulobacter crescentus, each cell cycle produces morphologically distinct daughter cells, a stalked cell and a flagellated swarmer cell. Construction of both the flagellum and stalk requires the alternative sigma factor RpoN (sigma(54)). Here we report that a sigma(54)-dependent activator, TacA, is required for cell cycle regulated stalk biogenesis by collaborating with RpoN to activate gene expression. We have also identified the first histidine phosphotransferase in C. crescentus, ShpA, and show that it too is required for stalk biogenesis. Using a systematic biochemical technique called phosphotransfer profiling we have identified a multicomponent phosphorelay which leads from the hybrid histidine kinase ShkA to ShpA and finally to TacA. This pathway functions in vivo to phosphorylate and hence, activate TacA. Finally, whole genome microarrays were used to identify candidate members of the TacA regulon, and we show that at least one target gene, staR, regulates stalk length. This is the first example of a general method for identifying the connectivity of a phosphorelay and can be applied to any organism with two-component signal transduction systems.
Assuntos
Caulobacter crescentus/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Caulobacter crescentus/citologia , Proteínas de Ligação a DNA/metabolismo , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Fosfotransferases/química , Fosfotransferases/metabolismo , Homologia de Sequência de Aminoácidos , Transativadores/metabolismoRESUMO
Two-component signal transduction systems, comprised of histidine kinases and their response regulator substrates, are the predominant means by which bacteria sense and respond to extracellular signals. These systems allow cells to adapt to prevailing conditions by modifying cellular physiology, including initiating programs of gene expression, catalyzing reactions, or modifying protein-protein interactions. These signaling pathways have also been demonstrated to play a role in coordinating bacterial cell cycle progression and development. Here we report a system-level investigation of two-component pathways in the model organism Caulobacter crescentus. First, by a comprehensive deletion analysis we show that at least 39 of the 106 two-component genes are required for cell cycle progression, growth, or morphogenesis. These include nine genes essential for growth or viability of the organism. We then use a systematic biochemical approach, called phosphotransfer profiling, to map the connectivity of histidine kinases and response regulators. Combining these genetic and biochemical approaches, we identify a new, highly conserved essential signaling pathway from the histidine kinase CenK to the response regulator CenR, which plays a critical role in controlling cell envelope biogenesis and structure. Depletion of either cenK or cenR leads to an unusual, severe blebbing of cell envelope material, whereas constitutive activation of the pathway compromises cell envelope integrity, resulting in cell lysis and death. We propose that the CenK-CenR pathway may be a suitable target for new antibiotic development, given previous successes in targeting the bacterial cell wall. Finally, the ability of our in vitro phosphotransfer profiling method to identify signaling pathways that operate in vivo takes advantage of an observation that histidine kinases are endowed with a global kinetic preference for their cognate response regulators. We propose that this system-wide selectivity insulates two-component pathways from one another, preventing unwanted cross-talk.
