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HHR23B is required for genotoxic-specific activation of p53 and apoptosis.

ABSTRACT: Rad23 proteins function in both DNA repair and protein stability regulation. As ubiquitinated forms of p53 are stabilized after DNA damage in concert with p53 functional activation, and human Rad23 proteins (hHR23A and B) regulate p53 stability in unstressed cells, the role of hHR23B in post-genotoxin regulation of p53 was investigated. Depletion of hHR23B by specific short interfering RNA before genotoxic exposure attenuated p53, p21 and bax induction, abrogated the accumulation of ubiquitinated p53 and suppressed apoptosis. Expression of ubiquitin derivatives with all lysines mutated except K48 or K63 demonstrated that K48-linked p53-ubiquitin conjugates were specifically induced after DNA damage. hHR23B, along with native and ubiquitinated p53, accumulated in chromatin after genotoxic exposure, and the accumulation of ubiquitinated p53 in chromatin was prevented by hHR23B depletion. Chromatin immunoprecipitation analysis demonstrated that hHR23B and p53 both localized to the p21 promoter shortly after DNA damage. hHR23B thus plays a critical role in the activation and function of p53 after specific genotoxic exposures.

SUBMITTER: Kaur M

PROVIDER: S-EPMC1804095 | biostudies-literature | 2007 Feb

REPOSITORIES: biostudies-literature

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hHR23B is required for genotoxic-specific activation of p53 and apoptosis.

Kaur M M Pop M M Shi D D Brignone C C Grossman S R SR

Oncogene 20060821 8

Rad23 proteins function in both DNA repair and protein stability regulation. As ubiquitinated forms of p53 are stabilized after DNA damage in concert with p53 functional activation, and human Rad23 proteins (hHR23A and B) regulate p53 stability in unstressed cells, the role of hHR23B in post-genotoxin regulation of p53 was investigated. Depletion of hHR23B by specific short interfering RNA before genotoxic exposure attenuated p53, p21 and bax induction, abrogated the accumulation of ubiquitinate ...[more]

PMID: 16924240

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Project description:The Sall2 transcription factor is deregulated in several cancers; however, little is known about its cellular functions, including its target genes. Recently, we demonstrated that p53 directly regulates Sall2 expression under genotoxic stress. Here, we investigated the role of Sall2 in the context of cellular response to genotoxic stress. In addition, we further examined the Sall2-p53 relationship during genotoxic stress in primary mouse embryo fibroblasts (MEFs), which are derived from Sall2 knockout mice separately, or in combination with the p53ERTAM knock-in mice. We found that the levels of Sall2 mRNA and protein are dynamically modulated in response to doxorubicin. At early times of stress, Sall2 is downregulated, but increases under extension of the stress in a p53-independent manner. Based on caspase-3/7 activities, expression of cleaved poly (ADP-ribose) polymerase, expression of cleaved caspase-3 and induction of proapoptotic proteins, Sall2 expression was correlated with cellular apoptosis. Consequently, Sall2-/- MEFs have decreased apoptosis, which relates with increased cell viability in response to doxorubicin. Importantly, Sall2 was required for apoptosis even in the presence of fully activated p53. Searching for putative Sall2 targets that could mediate its role in apoptosis, we identified proapoptotic NOXA/PMAIP1 (phorbol-12-myristate-13-acetate-induced protein 1). We demonstrated that Sall2 positively regulates Noxa promoter activity. Conserved putative Sall2-binding sites at the NOXA promoter were validated in vitro by electrophoretic mobility shift assay and in vivo by ChIP experiments, identifying NOXA as a novel Sall2 target. In agreement, induction of Noxa protein and mRNA in response to doxorubicin was significantly decreased in Sall2-/- MEFs. In addition, studies in leukemia Jurkat T cells support the existence of the Sall2/Noxa axis, and the significance of this axis on the apoptotic response to doxorubicin in cancer cells. Our study highlights the relevance of Sall2 in the apoptotic response to extended genotoxic stress, which is important for understanding its role in normal physiology and disease.

Project description:Chemicals often require metabolic activation to become genotoxic. Established test guidelines recommend the use of the rat liver S9 fraction or microsomes to introduce metabolic competence to in vitro cell-based bioassays, but the use of animal-derived components in cell culture raises ethical concerns and may lead to quality issues and reproducibility problems. The aim of the present study was to compare the metabolic activation of cyclophosphamide (CPA) and benzo[a]pyrene (BaP) by induced rat liver microsomes and an abiotic cytochrome P450 (CYP) enzyme based on a biomimetic porphyrine catalyst. For the detection of genotoxic effects, the chemicals were tested in a reporter gene assay targeting the activation of the cellular tumor protein p53. Both chemicals were metabolized by the abiotic CYP enzyme and the microsomes. CPA showed no activation of p53 and low cytotoxicity without metabolic activation, but strong activation of p53 and increased cytotoxicity upon incubation with liver microsomes or abiotic CYP enzyme. The effect concentration causing a 1.5-fold induction of p53 activation was very similar with both metabolization systems (within a factor of 1.5), indicating that genotoxic metabolites were formed at comparable concentrations. BaP also showed low cytotoxicity and no p53 activation without metabolic activation. The activation of p53 was detected for BaP upon incubation with active and inactive microsomes at similar concentrations, indicating experimental artifacts caused by the microsomes or NADPH. The activation of BaP with the abiotic CYP enzyme increased the cytotoxicity of BaP by a factor of 8, but no activation of p53 was detected. The results indicate that abiotic CYP enzymes may present an alternative to rat liver S9 fraction or microsomes for the metabolic activation of test chemicals, which are completely free of animal-derived components. However, an amendment of existing test guidelines would require testing of more chemicals and genotoxicity end points.

Dataset Information

HHR23B is required for genotoxic-specific activation of p53 and apoptosis.

Publications

hHR23B is required for genotoxic-specific activation of p53 and apoptosis.

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