Regulation of p14ARF Through Subnuclear Compartmentalization

The p53-mediated pathway cell cycle arrest and apoptosis is central to cancer and an important point of focus for therapeutics development. The p14ARF ("ARF") tumor suppressor induces the p53 pathway in response to oncogene activation or DNA damage. However, ARF is predominantly nucleolar in localization and engages in several interactions with nucleolar proteins, whereas p53 is nucleoplasmic. This raises the question as to how ARF initiates its involvement in the p53 pathway. We have found that UV irradiation of cells disrupts the interaction of ARF with two of its nucleolar binding partners, B23(NPM, nucleophosmin, NO38, numatrin) and topoisomerase I, and promotes an immediate and transient subnuclear redistribution of ARF to the nucleoplasm, where it can engage the p53 pathway (Lee et al, Cancer Research 65:9834-42; 2005). The results support a model in which the nucleolus serves as a p53 upstream sensor of cellular stress, and add to a growing body of evidence that nucleolar sequestration of ARF prevents activation of p53. The results also have therapeutic implications for therapies based on exploiting p53 and other cellular stress response pathways to suppress cancer.     

Signaling Crosstalk of FHIT,p53, and p38 in etoposide-induced apoptosis in MCF-7 cells

Fragile histidine trail (FHIT) is a tumor suppressor in response to DNA damage which has been deleted in various tumors. However, the signaling mechanisms and interactions of FHIT with regard to apoptotic proteins including p53 and p38 in the DNA damage-induced apoptosis are not well described. In the present study, we used etoposide-induced DNA damage in MCF-7 as a model to address these crosstalks. The time course study showed that the expression of FHIT, p53, and p38MAPK started after 1 hour following etoposide treatment. FHIT overexpression led to increase p53 expression, p38 activation, and augmented apoptosis following etoposide-induced DNA damage compared to wild-type cells. However, FHIT knockdown blocked p53 expression, delayed p38 activation, and completely inhibited etoposide-induced apoptosis. Inhibition of p38 activity prevented induction of p53, FHIT, and apoptosis in this model. Thus, activation of p38 upon etoposide treatment leads to increase in FHIT and p53 expression. In p53 knockdown MCF-7, the FHIT induction was hampered but p38 activation was induced in lower doses of etoposide. In p53 knockdown cells, inhibition of p38 induced FHIT expression and apoptosis. Our data demonstrated that the exposure of MCF-7 cells to etoposide increases apoptosis through a mechanism involving the activation of the p38-FHIT-p53 pathway. Moreover, our findings suggest signaling interaction for these pathways may represent a promising therapy for breast cancer.     

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.     

Change of the death pathway in senescent human fibroblasts in response to DNA damage is caused by an inability to stabilize p53

The cellular function of p53 is complex. It is well known that p53 plays a key role in cellular response to DNA damage. Moreover, p53 was implicated in cellular senescence, and it was demonstrated that p53 undergoes modification in senescent cells. However, it is not known how these modifications affect the ability of senescent cells to respond to DNA damage. To address this question, we studied the responses of cultured young and old normal diploid human fibroblasts to a variety of genotoxic stresses. Young fibroblasts were able to undergo p53-dependent and p53-independent apoptosis. In contrast, senescent fibroblasts were unable to undergo p53-dependent apoptosis, whereas p53-independent apoptosis was only slightly reduced. Interestingly, instead of undergoing p53-dependent apoptosis, senescent fibroblasts underwent necrosis. Furthermore, we found that old cells were unable to stabilize p53 in response to DNA damage. Exogenous expression or stabilization of p53 with proteasome inhibitors in old fibroblasts restored their ability to undergo apoptosis. Our results suggest that stabilization of p53 in response to DNA damage is impaired in old fibroblasts, resulting in induction of necrosis. The role of this phenomenon in normal aging and anticancer therapy is discussed.     

Palmdelphin,a novel target of p53 with Ser46 phosphorylation,controls cell death in response to DNA damage

The tumor suppressor gene p53 regulates apoptosis in response to DNA damage. Promoter selectivity of p53 depends on mainly its phosphorylation. Particularly, the phosphorylation at serine-46 of p53 is indispensable in promoting pro-apoptotic genes that are, however, poorly determined. In the current study, we identified palmdelphin as a pro-apoptotic gene induced by p53 in a phosphorylated serine-46-specific manner. Upregulation of palmdelphin was observed in wild-type p53-transfected cells, but not in serine-46-mutated cells. Expression of palmdelphin was induced by p53 in response to DNA damage. In turn, palmdelphin induced apoptosis. Intriguingly, downregulation of palmdelphin resulted in necroptosis-like cell death via ATP depletion. Upon DNA damage, palmdelphin dominantly accumulated in the nucleus to induce apoptosis. These findings define palmdelphin as a target of serine-46-phosphorylated p53 that controls cell death in response to DNA damage.     

Ser18 and Ser23 Phosphorylation Plays Synergistic Roles in Activating p53-Dependent Neuronal Apoptosis

Tumor suppressor p53 is required for the neuronal apoptosis in response to DNA double-stranded break (DSB) damage. Posttranslational modifications such as phosphorylation play important roles in activating p53-dependent apoptosis after DNA damage. In support of this notion, our recent studies indicate that Ser18 and Ser23 phosphorylation together plays critical roles in activating p53 apoptotic activities in vivo. Thymocytes derived from p53S18/23A mice are essentially resistant to p53-dependent apoptosis after DNA DSB damage. In addition, identical to p53-deficiency, p53S18/23A knock-in mutation completely rescues the embryonic lethality of XRCC4-/- mice, which die of the massive p53-dependent apoptosis of embryonic neurons likely as a result of accumulated endogenous DNA damage. To dissect the contribution of Ser18 and Ser23 phosphorylation to p53-dependent neuronal apoptosis, we report here that neither p53S18A nor p53S23A mutation alone can rescue the embryonic lethality of XRCC4-/- mice. Therefore, Ser18 and Ser23 phosphorylation plays synergistic and critical roles in activating p53-dependent neuronal apoptosis.     

Sentinels of DNA integrity in stem cells: Safeguarding future generations of cells

Integrated into the somatic cell cycle are multi-faceted mechanisms to protect genomic fidelity from genotoxic threats occurring during cell division or cellular quiescence. How embryonic stem cells respond to an array of attacks on genomic integrity has been uncertain, particularly in light of embryonic-like rapid cell cycle phases versus adult cells and the lack of an effective G1/S checkpoint. Whether a DNA damage response is activated similarly to somatic cells or apoptotic pathways used to purge damaged cells are important questions, since the longevity of embryonic stem cells provides opportunities for accumulated mutations and a source for carcinogenic cells. In this issue, Chuyikin et al. investigate the timing and sensitivity of the DNA damage response pathway to double strand breaks (DSBs) in mouse embryonic stem cells (ESCs), validating its responsiveness and providing a comprehensive view of key signaling events.

DNA DSBs are potently mutagenic lesions incurring chromosome breaks, potential rearrangements, mutation and loss of information.¹ The cellular response is immediate, sensitive and persistent, occurring within 30 seconds of damage upon detection of as little as 8 DSBs per cell. The response can be fully active in 15 minutes and persist for hours. Repair is preferred and may elicit checkpoint delays to cell cycle progression, with extreme genotoxic conditions initiating apoptotic pathways. The majority of DSB proteins are activated by PI-3 like kinases, with the primary mammalian response to DSBs occurring via the ATM kinase that is able to respond directly to DSBs. Phosphorylated downstream targets include the uncommon histone, H2AX. This histone provides a cytological platform at DSB sites for the recruitment of DSB mediator and effector proteins such as MDC1 and NBS1. To this scaffold further DSB proteins are recruited, amplifying the signal. NBS1 is part of the MRN complex that includes MRE11 and Rad50 and mediates nuclear localization of the complex to the DNA for stabilizing chromatin ends. The nucleolytic processing of DNA ends by MRE11 resection triggers a second pathway modulated by ATR, that responds to RPA coated ssDNA. Chuyikin et al., used antibodies to phosphorylated ATM and H2AX (pATM, pH2AX) as sensitive temporal markers of DNA repair foci that form at DSBs and followed these events through the cell cycle.

In fast proliferating undifferentiated cells an increase in single strand DNA breaks (SSBs) is typically observed, attributed to ongoing DNA replication, and not generally considered mutagenic. Chuyikin et al. used sensitive comet assays along with pH2AX and pATM antibodies to confirm the presence of SSBs in mESCs and a low background of pH2AX positive/pATM absent poised foci. Upon γ-irradiation to induce DSBs, dramatic detection of DNA repair foci including both pH2AX and pATM occurs. FACs analysis indicated no cell cycle arrest at G1/S from γ-irradiation, although a slight delay at G2/M. Chuyikin et al. did find that mESCs have an active spindle assembly checkpoint allowing cells to be blocked at G2/M with nocodazole and then released synchronously through the cell cycle. The key to their detection of this checkpoint was a six hour treatment with drug, versus longer timepoints. Indeed Reider and Maiato² have shown that in mammalian cells, spindle assembly checkpoint duration is variable and need not be satisfied to be overridden by adaptation, slippage or leakage, quite unlike the tight cell cycle arrest observed in fungi. Therefore longer treatments with nocodazole to arrest mESCs at this stage would be expected to simply be ineffective and promote further polyploidy by attenuating the mitotic mechanism. The authors detailed analysis of induction of DNA repair foci in all cell cycle stages revealed that all stages generate foci, including metaphase chromosomes in mitosis, although foci were most prominent in G1, G2 phases. Thus the primary response by the ATM pathway in these cells is not limited by cell cycle phase.

The maintenance of genomic fidelity in ESCs may require more enhanced DNA repair³ as well as alternative mechanisms to DNA repair, such as increased apoptosis. Chuyikin et al. observed increased caspase activity triggered after γ-irradiation of mESCs, but found no significant increase in cell death. They also found that protein levels of p53, a downstream target of the ATM kinase that is important for the G1/S checkpoint as well as p53-dependent apoptosis, were comparable to fibroblast cells, however p53 lacked activating phosphorylation. Both of these observations help to explain an ineffective G1/S checkpoint and the need for p53-independent apoptosis.

Additional alternate mechanisms for maintaining genomic integrity ESCs have been reported and contribute. This includes a 100X reduction in mutations versus somatic cells and resistance to oxidative stress. Asymmetry mechanisms,⁴ that are a commonly used means of cellular signaling and polarity from yeast to man may also apply, as in the Cairns immortal strand hypothesis. In 1975 Cairns proposed that stem cells might minimize mutations to their genomes from DNA replication by asymmetric segregation of their DNA. Retention of parental strands in the stem cell and segregation of potential mutation carrying DNAs into non-stem cell or differentiating daughters could reduce the mutation potential.4 Such asymmetric sister chromatid strand segregation is still controversial despite having been observed during mitosis in several stem cell populations. Continued elegant studies, such at that by Chuyikin et al, that define which pathways are present and examine the crosstalk in pathways used to detect, signal, repair and protect genomic integrity will continue to provide exciting new systemic views into stem cells. Our therapeutic use of stem cells in the future including understanding of cellular differentiation and cancer depends on it.

ReferencesRiches LC, et al. Mutagenesis 2008; In press.Rieder CL, et al. Dev Cell 2004; 7:637-51.Maynard S, et al. Stem Cells 2008; In press. Doxsey S, et al. Annu Rev Cell Dev Biol 2005; 21:411-34.Cairns J. Genetics 2006; 174:1069-72.Chuykin I, et al. Cell Cycle 2008; 7:In this issue.     

DNA damage- and stress-induced apoptosis occurs independently of PIDD

The p53-induced protein with a death domain, PIDD, was identified as a p53 target gene whose main role is to execute apoptosis in a p53-dependent manner. To investigate the physiological role of PIDD in apoptosis, we generated PIDD-deficient mice. Here, we report that, although PIDD expression is inducible upon DNA damage, PIDD-deficient mice undergo apoptosis normally not only in response to DNA damage, but also in response to various p53-independent stress signals and to death receptor (DR) engagement. This indicates that PIDD is not required for DNA damage-, stress-, and DR-induced apoptosis. Also, in the absence of PIDD, both caspase-2 processing and activation occur in response to DNA damage. Our findings demonstrate that PIDD does not play an essential role for all p53-mediated or p53-independent apoptotic pathways. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

ATR-Chk2 signaling in p53 activation and DNA damage response during cisplatin-induced apoptosis

Cisplatin is one of the most effective anti-cancer drugs; however, the use of cisplatin is limited by its toxicity in normal tissues, particularly injury of the kidneys. The mechanisms underlying the therapeutic effects of cisplatin in cancers and side effects in normal tissues are largely unclear. Recent work has suggested a role for p53 in cisplatin-induced renal cell apoptosis and kidney injury; however, the signaling pathway leading to p53 activation and renal apoptosis is unknown. Here we demonstrate an early DNA damage response during cisplatin treatment of renal cells and tissues. Importantly, in the DNA damage response, we demonstrate a critical role for ATR, but not ATM (ataxia telangiectasia mutated) or DNA-PK (DNA-dependent protein kinase), in cisplatin-induced p53 activation and apoptosis. We show that ATR is specifically activated during cisplatin treatment and co-localizes with H2AX, forming nuclear foci at the site of DNA damage. Blockade of ATR with a dominant-negative mutant inhibits cisplatin-induced p53 activation and renal cell apoptosis. Consistently, cisplatin-induced p53 activation and apoptosis are suppressed in ATR-deficient fibroblasts. Downstream of ATR, both Chk1 and Chk2 are phosphorylated during cisplatin treatment in an ATR-dependent manner. Interestingly, following phosphorylation, Chk1 is degraded via the proteosomal pathway, whereas Chk2 is activated. Inhibition of Chk2 by a dominant-negative mutant or gene deficiency attenuates cisplatin-induced p53 activation and apoptosis. In vivo in C57BL/6 mice, ATR and Chk2 are activated in renal tissues following cisplatin treatment. Together, the results suggest an important role for the DNA damage response mediated by ATR-Chk2 in p53 activation and renal cell apoptosis during cisplatin nephrotoxicity.