Project description:Trp53 is an essential tumor suppressor gene that coordinates the response to cellular stress, including exposure to chemotherapeutic agents. We aimed to characterize the effect of various Trp53 allelic states (i.e. wild-type, monoallelic mutations and biallelic mutations) on the modulation of downstream pathways in the context of treatment with the chemotherapeutic agent etoposide. In order to obtain a cell model that represents key characteristics of the pre-malignant condition of clonal hematopoiesis, we utilized ER-Hoxb8 cells, which represent an immortalized, but otherwise genetically normal hematopoietic progenitor cell line. Generation of this cell model from a mouse strain carrying the inducible Trp53 missense mutation R245W in the endogenous locus and coupled to a GFP reporter, allows therefore to study the impact of the various allelic states of Trp53 on cellular consequences in a context that models clonal hematopoiesis.
Project description:We investigated how the Trp53 allelic state influences the response of hematopoietic stem and progenitor cells (HSPCs) to γ-irradiation. We used a transgenic mouse model harboring hematopoietic cell–specific, tamoxifen-inducible Trp53-R245W missense mutations linked to a GFP reporter. In mice with biallelic transgenes, tamoxifen treatment generates three GFP-defined populations: GFP-negative cells (Trp53 wild-type), GFP-low cells (monoallelic Trp53 mutation, due to recombination of only one transgenic allele), and GFP-high cells (biallelic Trp53 mutation). Flow sorting of these populations enables direct comparison of all three allelic states in HSPCs. Using this system, we sought to define the transcriptional responses of HSPCs with different Trp53 genotypes following γ-irradiation.
Project description:We aimed to study the effect of non-mutational p53 inactivation on the response of hematopoietic progenitor cells to genotoxic treatment. In order to study this in vitro, we generated HSPC lines by enforced ER-Hoxb8 expression in bone marrow cells derived from transgenic Trp53-fl-R245W-GFP mice. In the absence of Cre-mediated recombination, these cells are wild-type for Trp53. We additionally transduced these cells lentivirally with a construct overexpressing murine Mdm2 or a control vector. This leads to non-mutational inactivation of functional p53. Thereby, we aimed to compare the transcriptional response to genotoxic treatment in Trp53 wild-type cells with or without non-mutational p53 inactivation through Mdm2 overexpression
Project description:We aimed to study the effect of mutational and non-mutational p53 inactivation on the response of hematopoietic progenitor cells to genotoxic treatment. In order to study this in vitro, we generated HSPC lines by enforced ER-Hoxb8 expression in bone marrow cells derived from transgenic Trp53-fl-R245W-GFP mice (in hetero- or homozygous state). Upon induction of the Trp53-R245W missense mutation, these cells carry mutational mono- or biallelic loss of wild-type p53. In the genetic background of mono- or biallelic Trp53 mutations, we additionally transduced these cells lentivirally with a construct overexpressing murine Mdm2 or a control vector. This leads to non-mutational inactivation of functional p53. Thereby, we aimed to compare the transcriptional response to genotoxic treatment in cells carrying mono- or biallelic Trp53 mutations with or without non-mutational p53 inactivation through Mdm2 overexpression.
Project description:Gene expression profiling of progenitor and neutrophil-derived HoxB8-ER cells (WT and Ezh2-/-) and neutrophil-derived HOXB8-ER treated with GSK343 and vehicle for 9 days. Ezh2 loss might lead to effective cytopenias caused by dysfunctional mature cells. To test this hypothesis, we examined Ezh2-null murine myeloid cell lines capable of neutrophilic differentiation. Bone marrow cells from Ezh2-/- and controls were immortalized with a HoxB8-estrogen receptor fusion and could be differentiated into mature neutrophils by removing estrogen from the culture medium.Gene expression profiling showed upregulation of oxidative phosphorylation pathway genes and ectopic expression of Gata1 and its associated erythroid gene targets. Functional and gene expression differences observed in Ezh2-/- derived neutrophils could be replicated in controls by treatment with the Ezh2-specific inhibitor GSK343. In conclusion, we demonstrate that Ezh2 loss allows for differentiation of normal appearing, but dysfunctional mature neutrophils characterized by ectopic gene expression and elevated levels of reactive oxygen species. These defects could contribute to the inflammatory bone marrow microenvironment and clinical phenotypes observed in patients with MDS.
Project description:Analysis of the different genotype tumors revealed enhanced chromosomal aberrations in all tumors. The most chromosomal aberrations were found in the krt18-/- tumors.
Project description:We used an ex vivo system of murine ER-Hoxb8 neutrophils that phenotypically and morphologically recapitulate neutrophil maturation stages during five days of in vitro culture in the presence of G-CSF. To investigate the development of the ROS system in neutrophils during their development, we compared transcriptomes of D0, D1, D3 and D5 Hoxb8 neutrophils.
