p53 mutants that lack DNA-binding activities, and therefore, transcriptional activities, are among the most common mutations in human cancer. Recently, a new role for p53 has come to light, as the tumour suppressor also functions in DNA repair and recombination. In cooperation with its function in transcription, the transcription-independent roles of p53 contribute to the control and efficiency of DNA repair and recombination.

Diverse functions, including DNA replication, recombination and repair, occur during S phase of the eukaryotic cell cycle. It has been proposed that p53 and BLM help regulate these functions. We show that p53 and BLM accumulated after hydroxyurea (HU) treatment, and physically associated and co-localized with each other and with RAD51 at sites of stalled DNA replication forks. HU-induced relocalization of BLM to RAD51 foci was p53 independent. However, BLM was required for efficient localization of either wild-type or mutated (Ser15Ala) p53 to these foci and for physical association of p53 with RAD51. Loss of BLM and p53 function synergistically enhanced homologous recombination frequency, indicating that they mediated the process by complementary pathways. Loss of p53 further enhanced the rate of spontaneous sister chromatid exchange (SCE) in Bloom syndrome (BS) cells, but not in their BLM-corrected counterpart, indicating that involvement of p53 in regulating spontaneous SCE is BLM dependent. These results indicate that p53 and BLM functionally interact during resolution of stalled DNA replication forks and provide insight into the mechanism of genomic fidelity maintenance by these nuclear proteins.

p53-mediated apoptosis may involve the induction of redox-controlling genes, resulting in the production of reactive oxygen species. Microarray expression analysis of doxorubicin exposed, related human lymphoblasts, p53 wild-type (WT) Tk6, and p53 mutant WTK1 identified the p53-dependent up-regulation of manganese superoxide dismutase (MnSOD) and glutathione peroxidase 1 (GPx). Consensus p53 binding sequences were identified in human MnSOD and GPx promoter regions. A 3-fold increase in the MnSOD promoter activity was observed after the induction of p53 in Li-Fraumeni syndrome (LFS) fibroblast, TR9-7, expressing p53 under the control of a tetracycline-regulated promoter. An increased protein expression of endogenous MnSOD and GPx also positively correlated with the level of p53 induction in TR9-7 cells. However, catalase (CAT) protein expression remained unaltered after p53 induction. We also examined the expression of MnSOD, GPx, and CAT in a panel of normal or LFS fibroblasts, containing either WT or mutant p53. We found increased MnSOD enzymatic activity, MnSOD mRNA expression, and MnSOD and GPx protein in LFS fibroblasts carrying a WT p53 allele when compared with homozygous mutant p53 isogenic cells. The CAT protein level was unchanged in these cells. We observed both the release of cytochrome C and Ca 2؉ from the mitochondria into the cytoplasm and an increased frequency of apoptotic cells after p53 induction in the TR9-7 cells that coincided with an increased expression of MnSOD and GPx, and the level of reactive oxygen species. The increase in apoptosis was reduced by the antioxidant N-acetylcysteine. These results identify a novel mechanism of p53-dependent apoptosis in which p53-mediated up-regulation of MnSOD and GPx, but not CAT, produces an imbalance in antioxidant enzymes and oxidative stress.

ChaC1 is a mammalian proapoptic protein of unknown function induced during endoplasmic reticulum stress. We show using in vivo studies and novel in vitro assays that the ChaC family of proteins function as c-glutamyl cyclotransferases acting specifically to degrade glutathione but not other c-glutamyl peptides. The overexpression of these proteins (but not the catalytically dead E4Q mutants) led to glutathione depletion and enhanced apoptosis in yeast. The ChaC family is conversed across all phyla and represents a new pathway for glutathione degradation in living cells, and the first cytosolic pathway for glutathione degradation in mammalian cells.

ING2 is a candidate tumor suppressor gene that can activate p53 by enhancing its acetylation. Here, we demonstrate that ING2 is also involved in p53-mediated replicative senescence. ING2 protein expression increased in late-passage human primary cells, and it colocalizes with serine 15-phosphorylated p53. ING2 and p53 also complexed with the histone acetyltransferase p300. ING2 enhanced the interaction between p53 and p300 and acted as a cofactor for p300-mediated p53 acetylation. The level of ING2 expression directly modulated the onset of replicative senescence. While overexpression of ING2 induced senescence in young fibroblasts in a p53-dependent manner, expression of ING2 small interfering RNA delayed the onset of senescence. Hence, ING2 can act as a cofactor of p300 for p53 acetylation and thereby plays a positive regulatory role during p53-mediated replicative senescence.

Bloom's syndrome is a rare autosomal recessive genetic disorder characterized by chromosomal aberrations, genetic instability, and cancer predisposition, all of which may be the result of abnormal signal transduction during DNA damage recognition. Here, we show that BLM is an intermediate responder to stalled DNA replication forks. BLM colocalized and physically interacted with the DNA damage response proteins 53BP1 and H2AX. Although BLM facilitated physical interaction between p53 and 53BP1, 53BP1 was required for efficient accumulation of both BLM and p53 at the sites of stalled replication. The accumulation of BLM/53BP1 foci and the physical interaction between them was independent of γ-H2AX. The active Chk1 kinase was essential for both the accurate focal colocalization of 53BP1 with BLM and the consequent stabilization of BLM. Once the ATR/Chk1- and 53BP1-mediated signal from replicational stress is received, BLM functions in multiple downstream repair processes, thereby fulfilling its role as a caretaker tumor suppressor.

SummaryMutations in RECQL4 helicase are associated with Rothmund-Thomson syndrome (RTS). A subset of RTS patients is predisposed to cancer and is sensitive to DNA damaging agents. The enhanced sensitivity of cells from RTS patients correlates with the accumulation of transcriptionally active nuclear p53. We found that in untreated normal human cells these two nuclear proteins, p53 and RECQL4, instead colocalize in the mitochondrial nucleoids. RECQL4 accumulates in mitochondria in all phases of the cell cycle except S phase and physically interacts with p53 only in the absence of DNA damage. p53-RECQL4 binding leads to the masking of the nuclear localization signal of p53. The N-terminal 84 amino acids of RECQL4 contain a mitochondrial localization signal, which causes the localization of RECQL4-p53 complex to the mitochondria. RECQL4-p53 interaction is disrupted after stress, allowing p53 translocation to the nucleus. In untreated normal cells RECQL4 optimizes de novo replication of mtDNA, which is consequently decreased in fibroblasts from RTS patients. Wild-type RECQL4-complemented RTS cells show relocalization of both RECQL4 and p53 to the mitochondria, loss of p53 activation, restoration of de novo mtDNA replication and resistance to different types of DNA damage. In cells expressing D84 RECQL4, which cannot translocate to mitochondria, all the above functions are compromised. The recruitment of p53 to the sites of de novo mtDNA replication is also regulated by RECQL4. Thus these findings elucidate the mechanism by which p53 is regulated by RECQL4 in unstressed normal cells and also delineates the mitochondrial functions of the helicase.

The glucocorticoid receptor (GR) and the tumor supressor p53 mediate different stress responses. We have studied the mechanism of their mutual inhibition in normal endothelial cells (HUVEC) in response to hypoxia, a physiological stress, and mitomycin C, which damages DNA. Dexamethasone (Dex) stimulates the degradation of endogenous GR and p53 by the proteasome pathway in HUVEC under hypoxia and mitomycin C treatments, and also in hepatoma cells (HepG2) under normoxia. Dex inhibits the functions of p53 (apoptosis, Bax, and p21 WAF1/CIP1 expression) and GR (PEPCK and G-6-Pase expression). Endogenous p53 and GR form a ligand-dependent trimeric complex with Hdm2 in the cytoplasm. Disruption of the p53-HDM2 interaction prevents Dex-induced ubiquitylation of GR and p53. The ubiquitylation of GR requires p53, the interaction of p53 with Hdm2, and E3 ligase activity of Hdm2. These results provide a mechanistic basis for GR and p53 acting as opposing forces in the decision between cell death and survival. The development of most tumors is associated with loss of function of the tumor suppressor p53. Different physiological stress, including DNA damage and hypoxia, activate p53. p53 is a transcription factor that regulates genes involved in growth arrest and apoptosis. p53 is down-regulated by its target gene product Mdm2 (human homolog Hdm2). Mdm2 forms an autoregulatory loop with p53 by binding to its N-terminal domain, inhibiting its transcriptional activity and increasing its degradation by the ubiquitin proteasome pathway (for reviews, see Jimenez et al. 1999;Lakin and Jackson 1999;Sionov and Haupt 1999). Mdm2 is a RING finger-dependent ubiquitin protein ligase for p53 and itself (Argentini et al. 2000;Fang et al. 2000;Honda and Yasuda 2000). Mdm2 also inhibits p53 by nuclear export through a mechanism involving either the nuclear export signal (NES) of Mdm2 (Tao and Levine 1999) or the RING finger of Mdm2 and the NES of p53 (Boyd et al. 2000;Geyer et al. 2000). The NES of p53 is masked in the transcriptionally active heterodimer, but is exposed in the monomeric form of p53 ). MdmX, a Mdm2 homolog that lacks a NES, stabilizes p53 by retention in the nucleus (Jackson and Berberich 2000;Stad et al. 2000). Nuclear p53 levels can also be maintained by ARF, which blocks nucleo-cytoplasmic shuttling of Mdm2 (Sherr and Weber 2000). Most studies of p53 involve DNA damaging drugs and radiation. Much less is known about the physiological stress, hypoxia. Under hypoxic conditions, p53 is stabilized by mitochondria through a redox-dependent mechanism (Chandel et al. 2000) and by HIF-1␣ , whereas p53 induces degradation of HIF-1␣ (Ravi et al. 2000). The response to hypoxia in vivo also involves the glucocorticoid receptor (GR) (Bauer et al. 1999).GR is a member of the steroid receptor superfamily that mediates physiological processes controlled by glucocorticoids. In the unbound state GR is located in the cytoplasm bound to chaperones. Upon ligand binding the chaperones dissociate, exposing the NLS that enables GR to enter the n...