Human SIR2 deacetylates p53 and antagonizes PML/p53-induced cellular senescence

The yeast Sir2 protein mediates chromatin silencing through an intrinsic NAD-dependent histone deacetylase activity. Sir2 is a conserved protein and was recently shown to regulate lifespan extension both in budding yeast and worms. Here, we show that SIRT1, the human Sir2 homolog, is recruited to the promyelocytic leukemia protein (PML) nuclear bodies of mammalian cells upon overexpression of either PML or oncogenic Ras (Ha-rasV12). SIRT1 binds and deacetylates p53, a component of PML nuclear bodies, and it can repress p53-mediated transactivation. Moreover, we show that SIRT1 and p53 co-localize in nuclear bodies upon PML upregulation. When overexpressed in primary mouse embryo fibroblasts (MEFs), SIRT1 antagonizes PML-induced acetylation of p53 and rescues PML-mediated premature cellular senescence. Taken together, our data establish the SIRT1 deacetylase as a novel negative regulator of p53 function capable of modulating cellular senescence.     

Shifting senescence into quiescence by turning up p53

Comment on: Leontieva OV, et al. Cell Cycle 2010; 9:4323–7.     

Weak p53 permits senescence during cell cycle arrest

Cell cycle arrest coupled with hyper-active mTOR leads to cellular senescence. While arresting cell cycle, high levels of p53 can inhibit mTOR (in some cell lines), thus causing reversible quiescence instead of senescence. Nutlin-3a-induced p53 inhibited mTOR and thus caused quiescence in WI-38 cells. In contrast, while arresting cell cycle, the DNA-damaging drug doxorubicin (DOX) did not inhibit mTOR and caused senescence. Super-induction of p53 by either nutlin-3a or high concentrations of DOX (high-DOX) prevented low-DOX-induced senescence, converting it into quiescence. This explains why in order to cause senescence, DNA damaging drugs must be used at low concentrations, which arrest cell cycle but do not induce p53 at levels sufficient to suppress mTOR. Noteworthy, very prolonged treatment with nutlin-3a also caused senescence preventable by rapamycin. In RPE cells, low concentrations of nutlin-3a caused a semi-senescent morphology. Higher concentrations of nutlin-3a inhibited mTOR and caused quiescent morphology. We conclude that low p53 levels during prolonged cell cycle arrest tend to cause senescence, whereas high levels of p53 tend to cause either quiescence or cell death.     

Nek6 suppresses the premature senescence of human cancer cells induced by camptothecin and doxorubicin treatment

Cellular senescence plays an important role in tumor suppression. The mitotic kinase Nek6 has recently been shown to be overexpressed in various cancers and has been implicated in tumorigenesis. Previously, we reported that the down-regulation of Nek6 expression was required for p53-induced senescence. In this study, we examined the effect of Nek6 overexpression on the premature senescence of cancer cells induced by the anticancer drugs camptothecin (CPT) and doxorubicin (DOX). We found that CPT- and DOX-induced morphology changes and increases in senescence-associated β-galactosidase staining were significantly inhibited in EJ human bladder cancer cells and H1299 human lung cancer cells overexpressing HA-Nek6. DOX-induced G2/M cell cycle arrest and the reduction in cyclin B and cdc2 levels after DOX treatment were significantly reduced by Nek6 overexpression. In addition, an increase in the intracellular levels of ROS in response to DOX was also inhibited in cells overexpressing Nek6. These results suggest that the increased expression of Nek6 renders cancer cells resistant to premature senescence, and targeting Nek6 could be an efficient strategy for cancer treatment.     

SIRT1 overexpression antagonizes cellular senescence with activated ERK/S6k1 signaling in human diploid fibroblasts

Sir2, a NAD-dependent deacetylase, modulates lifespan in yeasts, worms and flies. The SIRT1, mammalian homologue of Sir2, regulates signaling for favoring survival in stress. But whether SIRT1 has the function to influence cell viability and senescence under non-stressed conditions in human diploid fibroblasts is far from unknown. Our data showed that enforced SIRT1 expression promoted cell proliferation and antagonized cellular senescence with the characteristic features of delayed Senescence-Associated beta-galactosidase (SA-beta-gal) staining, reduced Senescence-Associated Heterochromatic Foci (SAHF) formation and G1 phase arrest, increased cell growth rate and extended cellular lifespan in human fibroblasts, while dominant-negative SIRT1 allele (H363Y) did not significantly affect cell growth and senescence but displayed a bit decreased lifespan. Western blot results showed that SIRT1 reduced the expression of p16(INK4A) and promoted phosphorylation of Rb. Our data also exposed that overexpression of SIRT1 was accompanied by enhanced activation of ERK and S6K1 signaling. These effects were mimicked in both WI38 cells and 2BS cells by concentration-dependent resveratrol, a SIRT1 activator. It was noted that treatment of SIRT1-.transfected cells with Rapamycin, a mTOR inhibitor, reduced the phosphorylation of S6K1 and the expression of Id1, implying that SIRT1-induced phosphorylation of S6K1 may be partly for the decreased expression of p16(INK4A) and promoted phosphorylation of Rb in 2BS. It was also observed that the expression of SIRT1 and phosphorylation of ERK and S6K1 was declined in senescent 2BS. These findings suggested that SIRT1-promoted cell proliferation and antagonized cellular senescence in human diploid fibroblasts may be, in part, via the activation of ERK/ S6K1 signaling.     

p53: Guardian of reprogramming

The reprogramming of somatic cells to induced pluripotent stem (iPS) cells is one of the major discoveries of recent years. The development and application of patient specific iPS lines could potentially revolutionize cell-based therapy, facilitating the treatment of a wide range of diseases. Despite the numerous technological advancements in the field, an in-depth mechanistical understanding of the pathways involved in reprogramming is still lacking. Several groups have recently provided a mechanistical insight into the role of the p53 tumor suppressor pathway in reprogramming. The repercussions of these findings are profound and reveal an unexpected role of p53 as a "guardian of reprogramming", ensuring genomic integrity during reprogramming at the cost of a reduced efficiency of the process. Here we analyze the latest findings in the field and discuss their relevance for future applications of iPS cell technology.     

2-Deoxyglucose combined with wild-type p53 overexpression enhances cytotoxicity in human prostate cancer cells via oxidative stress

Overexpression of the tumor suppressor gene, wild-type p53 (wtp53), using adenoviral vectors (Adp53) has been suggested to kill cancer cells by hydroperoxide-mediated oxidative stress [1,2] and nutrient distress induced by the glucose analog, 2-deoxyglucose (2DG), has been suggested to enhance tumor cell killing by agents that induce oxidative stress via disrupting hydroperoxide metabolism [3,4]. In the current study clonogenic cell killing of PC-3 and DU-145 human prostate cancer cells (lacking functional p53) mediated by 4 h exposure to 50 plaque forming units (pfus)/cell of Adp53 (that caused the enforced overexpression of wtp53) was significantly enhanced by treatment with 2DG. Accumulation of glutathione disulfide was found to be significantly greater in both cell lines treated with 2DG+Adp53 and both cell lines treated with 2DG+Adp53 showed a approximately 2-fold increases in dihydroethidine (DHE) and 5-(and-6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate (CDCFH(2)) oxidation, indicative of increased steady-state levels of O(2)(.-) and hydroperoxides, respectively. Finally, overexpression of catalase or glutathione peroxidase using adenoviral vectors partially, but significantly, protected DU-145 cells from the toxicity induced by 2DG+Adp53 treatment. These results show that treatment of human prostate cancer cells with the combination of 2DG (a nutrient stress) and overexpression of the tumor suppressor gene, wtp53, enhances clonogenic cell killing by a mechanism that involves oxidative stress as well as allowing for the speculation that inhibitors of glucose and hydroperoxide metabolism can be used in combination with Adp53 gene therapy to enhance therapeutic responses.     

p53 acetylation enhances Taxol-induced apoptosis in human cancer cells

Microtubule inhibitors (MTIs) such as Taxol have been used for treating various malignant tumors. Although MTIs have been known to induce cell death through mitotic arrest, other mechanisms can operate in MTI-induced cell death. Especially, the role of p53 in this process has been controversial for a long time. Here we investigated the function of p53 in Taxol-induced apoptosis using p53 wild type and p53 null cancer cell lines. p53 was upregulated upon Taxol treatment in p53 wild type cells and deletion of p53 diminished Taxol-induced apoptosis. p53 target proteins including MDM2, p21, BAX, and β-isoform of PUMA were also upregulated by Taxol in p53 wild type cells. Conversely, when the wild type p53 was re-introduced into two different p53 null cancer cell lines, Taxol-induced apoptosis was enhanced. Among post-translational modifications that affect p53 stability and function, p53 acetylation, rather than phosphorylation, increased significantly in Taxol-treated cells. When acetylation was enhanced by anti-Sirt1 siRNA or an HDAC inhibitor, Taxol-induced apoptosis was enhanced, which was not observed in p53 null cells. When an acetylation-defective mutant of p53 was re-introduced to p53 null cells, apoptosis was partially reduced compared to the re-introduction of the wild type p53. Thus, p53 plays a pro-apoptotic role in Taxol-induced apoptosis and acetylation of p53 contributes to this pro-apoptotic function in response to Taxol in several human cancer cell lines, suggesting that enhancing acetylation of p53 could have potential implication for increasing the sensitivity of cancer cells to Taxol.     

C-terminal peptides of p53 molecules enhance radiation-induced apoptosis in human mutant p53 cancer cells

We propose here a novel p53-targeting radio-cancer therapy using p53 C-terminal peptides for patients having mutated p53. Hoechst 33342 staining showed that X-ray irradiation alone efficiently induced apoptotic bodies in wild-type p53 (wt p53) human head and neck cancer cells transfected with a neo control vector (SAS/neo cells), but hardly induced apoptotic bodies in mutation-type p53 (m p53) cells transfected with a vector carrying the m p53 gene (SAS/m p53). In contrast, transfection of p53 C-terminal peptides (amino acid residues 361-382 or 353-374) via liposomes caused a remarkable increase of apoptotic bodies in X-ray-irradiated SAS/m p53 cells, but did not enhance apoptotic bodies in X-ray-irradiated SAS/neo cells. In immunocytochemical analysis, positively stained cells for active type caspase-3 were observed at high frequency after X-ray irradiation in the SAS/m p53 cells pre-treated with p53 C-terminal peptides. In SAS/neo cells, positively stained cells for active type caspase-3 were observed with X-ray irradiation alone. Furthermore, protein extracts from X-ray-irradiated SAS/m p53 cells showed higher DNA-binding activity of p53 to p53 consensus sequence when supplemented in vitro with p53 C-terminal peptides than extracts from non-irradiated SAS/m p53 cells. These results suggest that radiation treatment in the presence of p53 C-terminal peptides is more effective for inducing p53 -mediated apoptosis than radiation treatment alone or p53 C-terminal peptide treatment alone, especially in m p53 cancer cells. This novel tool for enhancement of apoptosis induction in m p53 cells might be useful for p53-targeted radio-cancer therapy.     

ErbB2 overexpression in p53-inactivated mammary epithelial cells

Functional loss of p53 and ErbB2 overexpression are the frequent genetic alterations in human breast carcinomas. Here, we found that ErbB2 expression was upregulated in primary cultured mammary epithelial cells (MECs) isolated from mice with a defect in exons 5 and 6 of the p53 gene (p53(Delta5,6)). The reporter gene activity in the p53(Delta5,6) MECs transfected with the -756bp flanking region of the hErbB2 gene was higher than the wild type MECs. p53 inactivation selectively increased the level of AP-2alpha, but not AP-2beta and AP-2gamma and a mutation of the two AP-2 binding sites completely inhibited the reporter activity.     

The associations of p53 overexpression with p53 codon 72 genetic polymorphism in esophageal cancer

Variations in p53 codon 72 have been identified as significant predisposing factors for various cancers, but molecular mechanisms remain unclear. We investigated associations of p53 overexpression with codon 72 variants and other factors with esophageal cancer. Status of p53 overexpression was determined by immunohistochemical staining. Codon 72 polymorphisms and mutation of p53 was identified by PCR-RFLP and direct sequencing from exons 4 to 9, respectively. We evaluated 126 patients who underwent esophagectomy in the National Taiwan University Hospital, and found that the status of p53 overexpression was significantly influenced by presence of codon 72 polymorphisms. After adjustment for other possible confounders, the incidence of p53 overexpression was significantly decreased in patients with Pro/Pro genotype with an odds ratio (OR) of 0.21 (95% CI: 0.067-0.64) (p = 0.0065) compared with incidence in patients with Arg/Arg genotype. The incidence of p53 overexpression was additively increased with environmental exposure to cigarette smoke, alcohol, and areca quid. When compared with individuals exposed to only one of these environmental risk factors, patients who had exposure to two or three risk factors had ORs of 6.11 (95% CI: 1.80-20.75) and 6.22 (95% CI: 1.81-21.34) for p53 overexpression, respectively. Elderly patients (age >70 years) were also more likely to have p53 overexpression, with an OR of 5.63 (95% CI: 1.53-20.64) compared with overexpression among patients aged less than 55 years. Forty-one patients received further evaluation of p53 mutation. There was also a higher incidence of, but without reaching a statistical significance, p53 mutation in patients with p53 overexpression (OR[95% CI]: 2.18 [0.52-9.6]) and codon 72 Arg/Arg genotype (OR [95% CI] of 0.8 [0.13-4.2], comparing genotypes of Pro/Pro and Arg/Pro with Arg/Arg). Our data provide the first observations that the presence of p53 codon 72 variants can be a significant factor influencing p53 overexpression in esophageal cancer, with overexpression also influenced by combined or prolonged environmental exposures.     

Klotho RNAi induces premature senescence of human cells via a p53/p21 dependent pathway

Klotho has recently emerged as a regulator of aging. To investigate the role of Klotho in the regulation of cellular senescence, we generated stable MRC-5 human primary fibroblast cells knockdown for Klotho expression by RNAi. Downregulation of Klotho dramatically induces premature senescence with a concomitant upregulation of p21. The upregulation of p21 is associated with cell cycle arrest at G1/S boundary. Knockdown of p53 in the Klotho attenuated MRC-5 cells restores normal growth and replicative potential. These results demonstrate that Klotho normally regulates cellular senescence by repressing the p53/p21 pathway. Our findings implicate Klotho as a regulator of aging in primary human fibroblasts.     

Cooperation between p21 and Akt is required for p53‐dependent cellular senescence

Cellular senescence has been implicated in normal aging, tissue homeostasis, and tumor suppression. Although p53 has been shown to be a central mediator of cellular senescence, the signaling pathway by which it induces senescence remains incompletely understood. In this study, we have shown that both Akt and p21 are required to induce cellular senescence in response to p53 expression. In a p53‐induced senescence model, we found that Akt activation was essential for inducing a cellular senescence phenotype. Surprisingly, Akt inhibition did not abolish p53‐induced cell cycle arrest, but it suppressed the increase in intracellular reactive oxygen species (ROS) levels. The results of the cell cycle and morphological analysis suggest that p53 induced quiescence, not senescence, following Akt inhibition. Conversely, the inhibition of p21 induction abolished cell cycle arrest but did not affect the p53‐induced increase in ROS levels. Additionally, p21 and Akt separately controlled cell cycle arrest and ROS levels, respectively, during H‐Ras‐induced senescence in human normal fibroblasts. The mechanistic analysis revealed that Akt increased ROS levels through NOX4 induction, and increased Akt‐dependent NF‐κB binding to the NOX4 promoter is responsible for NOX4 induction upon p53 expression. We further showed that Akt activation upon p53 expression is mediated by mammalian target of rapamycin complex 2. In addition, p53‐mediated IL6 and IL8 induction was abrogated by Akt inhibition, suggesting that Akt activation is also required for the senescence‐associated secretory phenotype. Collectively, these results suggest that p53 simultaneously controls multiple pathways to induce cellular senescence through p21 and Akt.     

Actin-binding doliculide causes premature senescence in p53 wild type cells

Addressing the actin cytoskeleton as future anticancer target can be an innovative chemotherapeutic approach to combat malignancies. Doliculide is a potent stabilizer of actin filaments and can be used as tool and therapeutic lead in cancer research. Though a variety of molecules are known to bind to actin and lead to either its over- or depolymerization little is known about the pharmacological consequences of these effects within the cancer cell. In this work we used p53 wild-type cells to dissect the reaction of these cells towards subtoxic doses of doliculide. We could show that doliculide leads to a transient change in actin cytoskeleton dynamics that are reversible. The cells react towards the treatment with the induction of premature senescence, an established anti-cancer mechanism, in concentrations that are not cytotoxic. Furthermore, we investigated the signaling pathways that are involved in the induction and maintenance of senescence by a pathway directed mRNA PCR-array. This analysis revealed that under doliculide treatment up to 13% of senescence related genes are altered. Taken together, our data provide evidence for an antitumoral potential of actin binding agents in p53 wild type cells and brings the strategy of targeting the actin cytoskeleton closer to clinical application.