Project description:The airways of the human lung are lined by an epithelium made up of ciliated and secretory luminal cells and undifferentiated p63+ Krt5+ progenitors. The integrity of this epithelium and its ability to act as a selective barrier are critical for normal lung function. In other epithelia there is evidence that transcription factors of the evolutionarily conserved grainyheadlike (GRHL) family play key roles in co-ordinating the expression of numerous proteins required for epithelial morphogenesis, differentiation, remodeling and repair. However, little is known about their function in the adult lung. We used Affymetrix microarray analysis to compare transcripts in lentivirus transfected primary human bronchial epithelial (HBE) cells expressing either EGFP or DN-GRHL2 to help identify GRHL2 target genes and their functions in HBE cells.
Project description:The airways of the human lung are lined by an epithelium made up of ciliated and secretory luminal cells and undifferentiated p63+ Krt5+ progenitors. The integrity of this epithelium and its ability to act as a selective barrier are critical for normal lung function. In other epithelia there is evidence that transcription factors of the evolutionarily conserved grainyheadlike (GRHL) family play key roles in co-ordinating the expression of numerous proteins required for epithelial morphogenesis, differentiation, remodeling and repair. However, little is known about their function in the adult lung. We used Affymetrix microarray analysis to compare transcripts in lentivirus transfected primary human bronchial epithelial (HBE) cells expressing either EGFP or DN-GRHL2 to help identify GRHL2 target genes and their functions in HBE cells. Primary HBE cells from three donors were infected with modified TripZ lentivirus designed to express EGFP or DN-GRHL2 in response to Dox. Dox was added to cells cultured at the air-liquid interface from day 7 to 14.
Project description:The airways of the human lung are lined by an epithelium made up of ciliated and secretory luminal cells and undifferentiated p63+ Krt5+ basal cells. The integrity of this epithelium and its ability to act as a selective barrier are critical for normal lung function. In other epithelia there is evidence that transcription factors of the evolutionarily conserved grainyheadlike (GRHL) family play key roles in co-ordinating the expression of numerous proteins required for epithelial morphogenesis, differentiation, remodeling and repair. However, little is known about their function in the adult lung. We use Affymetrix microarray analysis to compare transcripts in lentivirus transfected primary human bronchial epithelial (HBE) cells expressing either EGFP or DN-GRHL2 for 48h when the transepithelial electrical resistance (TER) reached a threshold level. The goal is to identify direct target genes of GRHL2 and early events in the uncoupling of junctional interactions, including those regulating transepithelial resistance.
Project description:The airways of the human lung are lined by an epithelium made up of ciliated and secretory luminal cells and undifferentiated p63+ Krt5+ basal cells. The integrity of this epithelium and its ability to act as a selective barrier are critical for normal lung function. In other epithelia there is evidence that transcription factors of the evolutionarily conserved grainyheadlike (GRHL) family play key roles in co-ordinating the expression of numerous proteins required for epithelial morphogenesis, differentiation, remodeling and repair. However, little is known about their function in the adult lung. We use Affymetrix microarray analysis to compare transcripts in lentivirus transfected primary human bronchial epithelial (HBE) cells expressing either EGFP or DN-GRHL2 for 48h when the transepithelial electrical resistance (TER) reached a threshold level. The goal is to identify direct target genes of GRHL2 and early events in the uncoupling of junctional interactions, including those regulating transepithelial resistance. Primary HBE cells from three donors were infected with EGFP or DN-GRHL2 expression lentivirus. Dox was added for 48h to induce the expression of either EGFP or DN-GRHL2 when TER reached a threshold level in ALI culture.
Project description:Glioblastoma multiforme (GBM) is the most malignant and most common tumor of the central nervous system characterized by rapid growth and extensive tissue infiltration. GBM results in more years of life lost than any other cancer type. Notch signaling has been implicated in GBM pathogenesis through several modes of action. Inhibition of Notch leads to a reduction of cancer-initiating cells in gliomas and reduces proliferation and migration. Deltex1 (DTX1) is part of an alternative Notch signaling pathway distinct from the canonical MAML1/RBP-Jκ-mediated cascade. In this study, we show that DTX1 activates both the RTK/PI3K/PKB as well as the MAPK/ERK pathway. Moreover, we found the anti-apoptotic factor Mcl-1 to be induced by DTX1. In accordance with this, the clonogenic potential and proliferation rates of glioma cell lines correlated with DTX1 levels. DTX1 knock down mitigated the tumorigenic potential in vivo, and overexpression of DTX1 increased cell migration and invasion of tumor cells accompanied by an elevation of the pro-migratory factors PKBβ and Snail1. Microarray gene expression analysis identified a DTX1-specific transcriptional program - including microRNA-21 - which is distinct from the canonical Notch signaling. We propose the alternative Notch pathway via DTX1 as oncogenic factor in malignant glioma and found low DTX1 expression levels to correlate with prolonged survival of GBM and early breast cancer patients in open source databases. We generated human glioma U373 cell lines stably expressing Enhanced Green Fluorescent Protein (EGFP), human Deltex1-Myc Tag (DTX1-myc), or Master Mind Like 1 - dominant negative (MAML1-dn) to compare differences in overall gene expression. We included 3x EGFP control samples, 3x DTX1-myc, and 3 MAML1-dn samples.