Regulation of the DNA damage response by p53 cofactors

The selective expression of p53-targeted genes is central to the p53-mediated DNA damage response. It is affected by multiple factors including posttranslational modifications and cofactors of p53. Here, we proposed an integrated model of the p53 network to characterize how the cellular response is regulated by key cofactors of p53, Hzf and ASPP. We found that the sequential induction of Hzf and ASPP is crucial to a reliable cell-fate decision between survival and death. After DNA damage, activated p53 first induces Hzf, which promotes the expression of p21 to arrest the cell cycle and facilitate DNA repair. The cell recovers to normal proliferation after the damage is repaired. If the damage is beyond repair, Hzf is effectively degraded, and activated E2F1 induces ASPP, which promotes the expression of Bax to trigger apoptosis. Furthermore, interrupting the induction of Hzf or ASPP remarkably impairs the cellular function. We also proposed two schemes for the production of the unknown E3 ubiquitin ligase for Hzf degradation: it is induced by either E2F1 or p53. In both schemes, the sufficient degradation of Hzf is required for apoptosis induction. These results are in good agreement with experimental observations or are experimentally testable.     

MYCN sensitizes human neuroblastoma to apoptosis by HIPK2 activation through a DNA damage response

MYCN amplification occurs in approximately 20% of human neuroblastomas and is associated with early tumor progression and poor outcome, despite intensive multimodal treatment. However, MYCN overexpression also sensitizes neuroblastoma cells to apoptosis. Thus, uncovering the molecular mechanisms linking MYCN to apoptosis might contribute to designing more efficient therapies for MYCN-amplified tumors. Here we show that MYCN-dependent sensitization to apoptosis requires activation of p53 and its phosphorylation at serine 46. The p53(S46) kinase HIPK2 accumulates on MYCN expression, and its depletion by RNA interference impairs p53(S46) phosphorylation and apoptosis. Remarkably, MYCN induces a DNA damage response that accounts for the inhibition of HIPK2 degradation through an ATM- and NBS1-dependent pathway. Prompted by the rare occurrence of p53 mutations and by the broad expression of HIPK2 in our human neuroblastoma series, we evaluated the effects of the p53-reactivating compound Nutlin-3 on this pathway. At variance from other tumor histotypes, in MYCN-amplified neuroblastoma, Nutlin-3 further induced HIPK2 accumulation, p53(S46) phosphorylation, and apoptosis, and in combination with clastogenic agents purged virtually the entire cell population. Altogether, our data uncover a novel mechanism linking MYCN to apoptosis that can be triggered by the p53-reactivating compound Nutlin-3, supporting its use in the most difficult-to-treat subset of neuroblastoma.     

Downregulation of VRK1 by p53 in response to DNA damage is mediated by the autophagic pathway

Human VRK1 induces a stabilization and accumulation of p53 by specific phosphorylation in Thr18. This p53 accumulation is reversed by its downregulation mediated by Hdm2, requiring a dephosphorylated p53 and therefore also needs the removal of VRK1 as stabilizer. This process requires export of VRK1 to the cytosol and is inhibited by leptomycin B. We have identified that downregulation of VRK1 protein levels requires DRAM expression, a p53-induced gene. DRAM is located in the endosomal-lysosomal compartment. Induction of DNA damage by UV, IR, etoposide and doxorubicin stabilizes p53 and induces DRAM expression, followed by VRK1 downregulation and a reduction in p53 Thr18 phosphorylation. DRAM expression is induced by wild-type p53, but not by common human p53 mutants, R175H, R248W and R273H. Overexpression of DRAM induces VRK1 downregulation and the opposite effect was observed by its knockdown. LC3 and p62 were also downregulated, like VRK1, in response to UV-induced DNA damage. The implication of the autophagic pathway was confirmed by its requirement for Beclin1. We propose a model with a double regulatory loop in response to DNA damage, the accumulated p53 is removed by induction of Hdm2 and degradation in the proteasome, and the p53-stabilizer VRK1 is eliminated by the induction of DRAM that leads to its lysosomal degradation in the autophagic pathway, and thus permitting p53 degradation by Hdm2. This VRK1 downregulation is necessary to modulate the block in cell cycle progression induced by p53 as part of its DNA damage response.     

The methyltransferase Set7/9 (Setd7) is dispensable for the p53-mediated DNA damage response in vivo

p53 is the central regulator of cell fate following genotoxic stress and oncogene activation. Its activity is controlled by several posttranslational modifications. Originally defined as a critical layer of p53 regulation in human cell lines, p53 lysine methylation by Set7/9 (also called Setd7) was proposed to fulfill a similar function in?vivo in the mouse, promoting p53 acetylation, stabilization, and activation upon DNA damage (Kurash et?al., 2008). We tested the physiological relevance of this circuit in an independent Set7/9 knockout mouse strain. Deletion of Set7/9 had no effect on p53-dependent cell-cycle arrest or apoptosis following sublethal or lethal DNA damage induced by radiation or genotoxic agents. Set7/9 was also dispensable for p53 acetylation following irradiation. c-myc oncogene-induced apoptosis was also independent of Set7/9, and analysis of p53 target genes showed that Set7/9 is not required for the p53-dependent gene expression program. Our data indicate that Set7/9 is dispensable for p53 function in the mouse.     

Acetyltransferase p300 regulates NBS1-mediated DNA damage response

The role of p300 in DNA damage response is unclear. To understand how ATM-dependent phosphorylation of p300 affects its function in response to DNA damage, we present evidence that S106 of p300, which is phosphorylated by ATM, regulates stability of NBS1 and recruitment into damaged DNA, possibly leading to regulation of DNA repair. Non-phosphorylatable p300 (S106A) destabilized NBS1 and decreased NBS1–p300 interaction. The recruitment of NBS1 into damaged DNA was impaired in the presence of S106A. Also, a dominant negative p300 lacking enzymatic activity induced destabilization of NBS1, suggesting that its acetyltransferase is required for NBS1 stability. These results indicate that phosphorylation of p300 can regulate NBS1-mediated DNA damage response, and that these events occur in an acetylation-dependent manner.

Structured summary

MINT-8058074, MINT-8058083: p300 (uniprotkb:Q09472) physically interacts (MI:0915) with NBS1 (uniprotkb:O60934) by anti bait coimmunoprecipitation (MI:0006)MINT-8058111: p300 (uniprotkb:Q09472) and NBS1 (uniprotkb:O60934) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-8058657: p300 (uniprotkb:Q09472) physically interacts (MI:0915) with NBS1 (uniprotkb:O60934) by two hybrid (MI:0018)MINT-8058093: p300 (uniprotkb:Q09472) physically interacts (MI:0915) with NBS1 (uniprotkb:O60934) by anti tag coimmunoprecipitation (MI:0007)     

Constant rate of p53 tetramerization in response to DNA damage controls the p53 response

The dynamics of the tumor suppressor protein p53 have been previously investigated in single cells using fluorescently tagged p53. Such approach reports on the total abundance of p53 but does not provide a measure for functional p53. We used fluorescent protein-fragment complementation assay (PCA) to quantify in single cells the dynamics of p53 tetramers, the functional units of p53. We found that while total p53 increases proportionally to the input strength, p53 tetramers are formed in cells at a constant rate. This breaks the linear input–output relation and dampens the p53 response. Disruption of the p53-binding protein ARC led to a dose-dependent rate of tetramers formation, resulting in enhanced tetramerization and induction of p53 target genes. Our work suggests that constraining the p53 response in face of variable inputs may protect cells from committing to terminal outcomes and highlights the importance of quantifying the active form of signaling molecules in single cells.Quantification of the dynamics of p53 tetramers in single cells using a fluorescent protein-fragment complementation assay reveals that, while total p53 increases proportionally to the DNA damage strength, p53 tetramers are formed at a constant rate.     

DNA damage,p53 gene expression and p53 protein level in the rat brain aging

The aging induces free radicals leading to DNA damage (8‐oxo‐2′‐deoxyguanosine, 8‐oxo2dG). DNA injury causes increased expression of p53 gene and p53 protein. Levels of 8‐oxo2dG (HPLC), p53 mRNA (PCR) and p53 protein (Western blot) were estimated in gray matter (GM), white matter (WM), cerebellum (C) and medulla oblongata (MO) of control, 12‐ and 24‐month‐old rats. The level of 8‐oxo2dG increased with age in C (P < 0.05 in 12‐month‐old and P < 0.01 in 24‐month‐old rats) and MO. In 12‐month‐old animals the level of 8‐oxo2dG in GM and WM was higher than in controls. In 12‐month‐old animals p53 gene expression decreased while amounts of p53 protein increased, depending on the oxidative DNA damage. In 24‐month‐old rats, expression of p53 increased in all structures (P ≤ 0.05) while p53 protein showed decreased levels in most of structures of central nervous system (WM, C, MO). Aging leads to increased 8‐oxo2dG and augmented p53 gene expression, accompanied by a lowered expression of p53 protein.     

Telomerase-immortalized human fibroblasts retain UV-induced mutagenesis and p53-mediated DNA damage responses

Immortalized cells frequently have disruptions of p53 activity and lack p53-dependent nucleotide excision repair (NER). We hypothesized that telomerase immortalization would not alter p53-mediated ultraviolet light (UV)-induced DNA damage responses. DNA repair proficient primary diploid human fibroblasts (GM00024) were immortalized by transduction with a telomerase expressing retrovirus. Empty retrovirus transduced cells senesced after a few doublings. Telomerase transduced GM00024 cells (tGM24) were cultured continuously for 6 months (>60 doublings). Colony forming ability after UV irradiation was dose-dependent between 0 and 20J/m2 UVC (LD50=5.6J/m2). p53 accumulation was UV dose- and time-dependent as was induction of p48(XPE/DDB2), p21(CIP1/WAF1), and phosphorylation on p53-S15. UV dose-dependent apoptosis was measured by nuclear condensation. UV exposure induced UV-damaged DNA binding as monitored by electrophoretic mobility shift assays using UV irradiated radiolabeled DNA probe was inhibited by p53-specific siRNA transfection. p53-Specific siRNA transfection also prevented UV induction of p48 and improved UV survival measured by colony forming ability. Strand-specific NER of cyclobutane pyrimidine dimers (CPD) within DHFR was identical in tGM24 and GM00024 cells. CPD removal from the transcribed strand was nearly complete in 6h and from the non-transcribed strand was 73% complete in 24h. UV-induced HPRT mutagenesis in tGM24 was indistinguishable from primary human fibroblasts. These wide-ranging findings indicate that the UV-induced DNA damage response remains intact in telomerase-immortalized cells. Furthermore, telomerase immortalization provides permanent cell lines for testing the immediate impact on NER and mutagenesis of selective genetic manipulation without propagation to establish mutant lines.