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Effect of different Trp53 allelic states on the response to etoposide treatment in ER-Hoxb8 cells

ABSTRACT: Effect of different Trp53 allelic states on the response to etoposide treatment in ER-Hoxb8 cells

PROVIDER: PRJNA1277311 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR33993712.fastq.gz	Fastqsanger.gz
	SRR33993713.fastq.gz	Fastqsanger.gz
	SRR33993714.fastq.gz	Fastqsanger.gz
	SRR33993715.fastq.gz	Fastqsanger.gz
	SRR33993716.fastq.gz	Fastqsanger.gz

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Effect of different Trp53 allelic states on the response to etoposide treatment in ER-Hoxb8 cells

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.

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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

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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.

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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.

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