Polyploidy mitigates the impact of DNA damage while simultaneously bearing its burden
Hayashi, Kazuki
;
Horisaka, Kisara
;
Harada, Yoshiyuki
, et al.
Cell Death Discov.,2024,10,436.
DOI:
10.1038/s41420-024-02206-w
PubMed ID:
39397009
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Abstract: Polyploidy is frequently enhanced under pathological conditions, such as tissue injury and cancer in humans. Polyploidization is critically involved in cancer evolution, including cancer initiation and the acquisition of drug resistance. However, the effect of polyploidy on cell fate remains unclear. In this study, we explored the effects of polyploidization on cellular responses to DNA damage and cell cycle progression. Through various comparisons based on ploidy stratifications of cultured cells, we found that polyploidization and the accumulation of genomic DNA damage mutually induce each other, resulting in polyploid cells consistently containing more genomic DNA damage than diploid cells under both physiological and stress conditions. Notably, despite substantial DNA damage, polyploid cells demonstrated a higher tolerance to its impact, exhibiting delayed cell cycle arrest and reduced secretion of inflammatory cytokines associated with DNA damage-induced senescence. Consistently, in mice with ploidy tracing, hepatocytes with high ploidy appeared to potentially persist in the damaged liver, while being susceptible to DNA damage. Polyploidy acts as a reservoir of genomic damage by mitigating the impact of DNA damage, while simultaneously enhancing its accumulation.
Purchased from AmBeed:
104206-65-7
A novel enzymatic assay for the identification of 4-hydroxyphenylpyruvate dioxygenase modulators
Parkins, Andrew
;
Pantouris, Georgios
;
STAR protoc.,2023,4(2):102300.
DOI:
10.1016/j.xpro.2023.102300
PubMed ID:
37167058
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Abstract: 4-hydroxyphenylpyruvate dioxygenase (HPPD) is a key enzyme involved in the pathogenesis of tyrosinemia III and cancer. Herein, we describe a spectroscopy-based assay to detect HPPD dioxygenase activity in the presence or absence of small-mol. modulators. We describe steps for transformation, expression, and purification of HPPD and preparation of the assay plate. We detail initiation and completion of the enzymic reaction followed by detection of remaining substrate in the form of enol-HPP/borate complex. This assay is applicable for high-throughput screening. For complete details on the use and execution of this protocol, please refer to Parkins et al.1
Purchased from AmBeed:
104206-65-7
Self-cleaving guide RNAs enable pharmacological selection of precise gene editing events in vivo
Tiyaboonchai, Amita
;
Vonada, Anne
;
Posey, Jeffrey
, et al.
Nat. Commun.,2022,13(1):7391.
DOI:
10.1038/s41467-022-35097-5
PubMed ID:
36450762
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Abstract: Expression of guide RNAs in the CRISPR/Cas9 system typically requires the use of RNA polymerase III promoters, which are not cell-type specific. Flanking the gRNA with self-cleaving ribozyme motifs to create a self-cleaving gRNA overcomes this limitation. Here, we use self-cleaving gRNAs to create drug-selectable gene editing events in specific hepatocyte loci. A recombinant Adeno Associated Virus vector targeting the Albumin locus with a promoterless self-cleaving gRNA to create drug resistance is linked in cis with the therapeutic transgene. Gene expression of both are dependent on homologous recombination into the target locus. In vivo drug selection for the precisely edited hepatocytes allows >30-fold expansion of gene-edited cells and results in therapeutic levels of a human Factor 9 transgene. Importantly, self-cleaving gRNA expression is also achieved after targeting weak hepatocyte genes. We conclude that self-cleaving gRNAs are a powerful system to enable cell-type specific in vivo drug resistance for therapeutic gene editing applications.
Purchased from AmBeed:
104206-65-7
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