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Home > Vol 5, No 18 >	Chen
Published in Oncotarget V5N18, Sep 30, 2014
Priority Research Papers:
TAp73 promotes cell survival upon genotoxic stress by inhibiting p53 activity
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Dongshi Chen _, Lihua Ming, Fangdong Zou, Ye Peng, Bennett Van Houten, Jian Yu and Lin Zhang
Dongshi Chen1,2, Lihua Ming1,3, Fangdong Zou1,2,4, Ye Peng1,2, Bennett Van Houten1,2, Jian Yu1,3, Lin Zhang1,2
1University of Pittsburgh Cancer Institute, Pittsburgh, PA, 15213, USA
2Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15213, USA
3Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15213, USA
4College of Life Sciences, Sichuan University, Chengdu, Sichuan, 610064, P.R. China
Dr. Lin Zhang, e-mail: zhanglx@upmc.edu
Received: July 18, 2014 Accepted: July 18, 2014 Published: September 22, 2014
p53 plays a key role in regulating DNA damage response by suppressing cell cycle progression or inducing apoptosis depending on extent of DNA damage. However, it is not clear why mild genotoxic stress favors growth arrest, whereas excessive lesions signal cells to die. Here we showed that TAp73, a p53 homologue thought to have a similar function as p53, restrains the transcriptional activity of p53 and prevents excessive activation of its downstream targets upon low levels of DNA damage, which results in cell cycle arrest. Extensive DNA damage triggers TAp73 depletion through ubiquitin/proteasome-mediated degradation of E2F1, leading to enhanced transcriptional activation by p53 and subsequent induction of apoptosis. These findings provide novel insights into the regulation of p53 function and suggest that TAp73 keeps p53 activity in check in regulating cell fate decisions upon genotoxic stress.
TAp73 promotes cell survival upon genotoxic stress by inhibiting p53 activity | Chen | Oncotarget
Mammalian cells have developed an intricate molecular network to deal with DNA damage inflicted by frequent environmental or endogenous insults [1]. Depending on various factors, DNA damage can trigger DNA repair, cell cycle arrest, or apoptosis [2]. The central regulator of DNA damage response is the tumor suppressor p53, which either inhibits cell growth by activating p21, 14-3-3σ, and other cell cycle regulators, or induces apoptosis through proapoptotic targets such as PUMA, Noxa and Bax [3]. DNA damage response is essential for maintenance of genomic integrity and functions as a guardian against oncogenic transformation [4]. Tumor cells are almost invariably defective in DNA damage response due to defects in the p53 and other DNA repair pathways [4]. Furthermore, ionizing radiation and chemotherapeutic drugs used in anticancer therapies often kill tumor cells by inducing toxic levels of DNA damage [5].
In addition to p53, several other p53 family members, such as p73 and p63, also play a significant role in DNA damage response [6]. p73 is expressed in two major isoform classes, including TAp73 and ΔNp73, which have distinct functions [7]. Similar to p53, TAp73 isoforms contain highly conserved DNA binding, transactivation, and oligomerization domains, whereas ΔNp73 lacks the transactivation domain but contains DNA-binding and oligomerization domains [7]. Following DNA damage, TAp73 can bind to the same set of p53-responsive elements and activate p53 target genes to arrest cell cycle or induce apoptosis [8]. Although TAp73 was shown to be a tumor suppressor [9, 10], it is rarely mutated in human tumors [11], and p73-deficient mice do not resemble p53-null mice in tumor phenotypes [9, 12]. Unlike p53, which is consistently proapoptotic, TAp73 can be proapoptotic or antiapoptotic [13, 14]. TAp73 expression can be either upregulated or downregulated in response to different DNA damaging agents [15]. These observations suggest that the function of p73 does not overlap with that of p53 in DNA damage response.
A fundamental and unresolved issue is how cells respond to different levels of stress. It is unclear why transient or low levels of DNA damage suppress cell growth, but extensive and persistent lesions often lead to apoptosis. Recent studies indicate that specific events can be triggered by excessive DNA damage to alert neighboring cells, or to eliminate the damaged cells by apoptosis [16]. However, little is known about how p53 activity is adjusted in response to different stress levels. In this study, we uncovered a function of TAp73 in restraining p53 activity in response to low levels of DNA damage. In the context of extensive DNA damage, depletion of TAp73 leads to enhanced proapoptotic activities of p53. Our results provide insight into cell fate determination through the interplay of p53 family members.
Downregulation of TAp73 following extensive DNA damage
There are at least 30 p73 transcript isoforms generated by two different promoters (TA and ΔN) and extensive alternative slicing. TAp73α is the most prominent and transcriptionally competent p73 isoform that resembles p53 [7]. To distinguish TAp73 from ΔNp73, a triple-Flag tag (3×Flag) was knocked into the N-terminus of TAp73 in p53-wildtype (WT) HCT116 colon cancer cells (Fig. S1, A and B). TAp73α (TAp73 hereafter) in the Flag-knock-in (Flag-KI) cells could be specifically detected by Flag western blotting (Fig. S1C). No other TAp73 isoforms, such as TAp73β, could be detected in HCT116 cells (Fig. S1, D and E). Knock-in of the Flag tag does not affect the sensitivity of HCT116 cells to DNA damage-induced apoptosis (Fig. S1F).
To investigate the role of TAp73 in DNA damage response, Flag-KI cells were treated with several DNA damaging agents at various concentrations. Both knock-in and endogenous forms of TAp73 were induced by a 24-hr treatment with either 12.5 μM cisplatin, 12.5 μM etoposide, 0.125 μM camptothecin (CPT), or 12.5 μg/ml 5-Fluorouracil (5-FU) (Fig. 1A, S1D and S1E), and TAp73 accumulation was detected through 36 hr after treatment with 12.5 μM cisplatin (Fig. 1B). These conditions induced relatively low levels of DNA damage indicated by histone 2Ax phosphorylation (γH2Ax), modest and delayed (after 24 hr) substantial induction of p53 target genes (Fig. 1A and 1B), and predominantly cell cycle arrest, but little apoptosis or caspase activation (Fig. 1, C-E). Treating cells with DNA damaging agents at escalated doses, including 50 μM cisplatin, 50 μM etoposide, 0.5 μM CPT, and 50 μg/ml 5-FU, led to higher levels of DNA damage, p53, and its target genes (Fig. 1, A and B), as well as strong apoptosis and caspase activation (Fig. 1, C-E), but surprisingly diminished expression of TAp73 (Fig. 1, A and B). Upon exposure to 50 μM cisplatin or 50 μg/ml 5-FU, TAp73 expression was initially increased up to 18 hr, but markedly declined afterwards, followed by enhanced expression of p53 target genes (Fig. 1B). Downregulation of TAp73 was not a consequence of caspase activation, as no TAp73 cleavage fragment was observed (Fig. S1, D and E), and caspase inhibition blocked apoptosis without affecting TAp73 depletion (Fig. S2). Furthermore, downregulation of TAp73 was also observed in p53-WT LOVO and RKO, and p53-mutant DLD1 colon cancer cells, as well as in mouse embryonic fibroblast (MEF) cells, following cisplatin treatment at a dose that induced substantial γH2Ax and apoptosis (Fig. 1F and data not shown). Therefore, TAp73 is induced by low levels of DNA damage, but is downregulated upon exposure to lethal doses of DNA damage.
TAp73 inhibits apoptosis and expression of p53 target genes following excessive DNA damage
We then determined whether TAp73 downregulation is involved in initiating apoptosis in response to high levels of DNA damage. TAp73 transfection markedly suppressed apoptosis and caspase activation induced by 50 μM cisplatin, slightly reduced low levels of apoptosis and caspase activation induced by 12.5 μM cisplatin, but had no effect on untreated cells (Fig. 2A and S3A). Enforced TAp73 expression also inhibited protein and mRNA induction of p53 downstream targets, including PUMA, Bax and p21, in cells exposed to 12.5 or 50 μM cisplatin (Fig. 2, B and C). Conversely, knockdown of p73 by siRNA sensitized cells to apoptosis induction (Fig. 2D), and enhanced the protein and mRNA expression of PUMA, Bax and p21 following cisplatin treatment at 12.5 or 50 μM (Fig. 2, E and F). These effects of TAp73 depletion were verified in HCT116 cells with stable knockdown of TAp73 by shRNA, which by itself did not affect the expression of ΔNp73 and p63 isoforms, or induce genotoxic stress or apoptosis (Fig. 2, G-J; Fig. S3B). Modulating TAp73 expression also did not affect the induction of p53 by cisplatin (Fig. 2, B, E and J). Furthermore, TAp73 transfection or knockdown had similar effects on the induction of apoptosis and p53 target genes by cisplatin in RKO colon cancer cells (Fig. S4, A-D), as well as that by 5-FU in HCT116 cells (Fig. S5, A-D). In contrast, TAp73 transfection or knockdown did not affect p53-independent induction of apoptosis and PUMA by the kinase inhibitor staurosporine [17], although TAp73 was also downregulated in response to staurosporine treatment (Fig. S6, A-E).
To determine whether the effects of TAp73 are mediated through p53, we analyzed p53-knockout (p53-KO) HCT116 cells. Treating p53-KO cells with 50 μM cisplatin downregulated TAp73, but did not induce the expression of PUMA, Bax, and p21 (Fig. 3A), and led to a lower level of apoptosis compared to WT HCT116 cells (Fig. 3B). TAp73 transfection or knockdown did not affect the induction of apoptosis and p53 target genes in p53-KO cells (Fig. 3, B-D), and in p53-mutant DLD1 cells (Fig. S4, E and F). PUMA plays a critical role in DNA damage-induced and p53-dependent apoptosis [18]. To determine whether the effect of TAp73 on apoptosis is mediated through PUMA, we analyzed PUMA-knockout (PUMA-KO) HCT116 cells, which are less sensitive to DNA damage-induced apoptosis than WT cells [18]. TAp73 transfection or knockdown also did not substantially affect cisplatin-induced apoptosis without PUMA (Fig. S7, A-D). Both p53 and TAp73 can bind to the p53 responsive elements in the PUMA promoter upon DNA damage [19, 20]. To determine whether the effects of TAp73 on PUMA expression and apoptosis are mediated by its action on the PUMA promoter, we analyzed HCT116 cells with a deletion of the p53 binding sites in the PUMA promoter (BS-KO) [17]. In the absence of the p53 binding sites, TAp73 transfection or depletion did not impact cisplatin-induced apoptosis and PUMA expression, but still suppressed p21 expression as in WT HCT116 cells (Fig. 3, E and F; Fig. S7, E and F). These results demonstrate specific inhibitory effects of TAp73 on DNA damage-induced apoptosis, which are meditated by p53 through its downstream target genes.
Figure 1: Downregulation of TAp73 in response to extensive DNA damage correlates with induction of apoptosis. (A) HCT116 human colon cancer cells with knock-in (KI) of Flag-tagged TAp73 were treated with cisplatin, etoposide, camptothecin (CPT), or 5-fluorouracil (5-FU) at indicated concentrations for 24 hr. Indicated proteins were analyzed by western blotting. KI TAp73 and En TAp73 indicate knock-in and endogenous forms of TAp73α, respectively. * indicates a non-specific band detected by the p73 antibody. (B) Time course of expression of indicated proteins in HCT116 cells treated with cisplatin (12.5 or 50 μM) or 5-FU (12.5 or 50 μg/ml) was analyzed by western blotting. (C) Following treatment of cells as in (A) for 24 hr, apoptosis was analyzed by counting cells with condensed and fragmented nuclei following staining with Hoechst 33258. (D) Caspase activity in HCT116 cells treated as in (A) for 24 hr was measured using the caspase-3/7 assay kit, as described in the Materials and Methods. Results were expressed as means ± s.d. of three independent experiments. (E) HCT116 cells were treated with cisplatin as indicated for 24 hr. Cell cycle was analyzed by propidium iodide (PI) staining followed by flow cytometry. (F) RKO, LOVO and MEF cells were treated with cisplatin at indicated concentrations for 24 hr. Indicated proteins were analyzed by western blotting. Arrowheads indicate TAp73 bands.
Figure 2: TAp73 suppresses apoptosis and the expression of p53 downstream target genes following extensive DNA damage. (A) HCT116 cells with Flag-TAp73 KI were transfected with control or HA-TAp73α construct, and then treated with cisplatin at indicated concentrations for 24 hr. Left, analysis of apoptosis by nuclear staining; Right, analysis of caspase activity. (B) Following treatment as in (A), indicated proteins were analyzed by western blotting. TAp73 blots with short or long exposure (exp.) are presented to show transfected TAp73 and endogenous (En)/knock-in (KI) TAp73, respectively. (C) mRNA expression of PUMA, Bax, and p21 in cells treated as in (A) was analyzed by real-time RT-PCR and normalized to the control β-actin and untreated samples. (D) HCT116 cells with Flag-TAp73 KI were transfected with control or TAp73 siRNA, and treated with cisplatin at indicated concentrations for 24 hr. Left, analysis of apoptosis by nuclear staining; Right, analysis of caspase activity. (E) Following treatment as in (D), indicated proteins were analyzed by western blotting. (F) mRNA expression of PUMA, Bax and p21 in cells treated as in (D) was analyzed by real-time RT-PCR. (G) HCT116 cells with stable transfection of TAp73 or control shRNA were analyzed for the expression of TAp73, ΔNp73, TAp63 and ΔNp73 by RT-PCR. (H) HCT116 cells with stable transfection of TAp73 or control shRNA at inoculation (0 hr) or after culturing for 48 hr were analyzed for TAp73 and γH2Ax by western blotting. Lysates from cisplatin-treated HCT116 cells were used as a positive control (+) for γH2Ax. (I) HCT116 cells with stable transfection of TAp73 or control shRNA were treated with cisplatin at indicated concentrations for 24 hr. Apoptosis was analyzed by nuclear staining. (J) Following treatment as in (I), indicated proteins were analyzed by western blotting. Results in (A), (C), (D), (F) and (I) were expressed as means ± s.d. of three independent experiments. ***, P <0.001; **, P <0.01; *, P <0.05.
Figure 3: p53 is required for the effects of TAp73 on p53 target gene induction and apoptosis following extensive DNA damage. (A) Flag-KI and p53-knockout (p53-KO) HCT116 cells were treated with cisplatin at indicated concentrations for 24 hr. Indicated proteins were analyzed by western blotting. (B) Apoptosis in cells treated as in (A), (C) and (D) was analyzed by nuclear staining. **, P <0.01; NS, not significant. (C) p53-KO HCT116 cells transfected with control or HA-TAp73α construct were treated with cisplatin at indicated concentrations for 24 hr. Indicated proteins were analyzed by western blotting. Lysates from HCT116 cells were loaded as a control. (D) p53-KO HCT116 cells transfected with control or TAp73 siRNA were treated with cisplatin at indicated concentrations for 24 hr. Indicated proteins were analyzed by western blotting. Lysates from HCT116 cells were loaded as a control. (E) HCT116 cells with knockout of the p53 binding sites in the PUMA promoter (BS-KO) were transfected with control or HA-TAp73α expression construct, and then treated with cisplatin at indicated concentrations for 24 hr. Indicated proteins were analyzed by western blotting. (F) BS-KO cells were transfected with control or TAp73 siRNA, and then treated with cisplatin at indicated concentrations for 24 hr. Indicated proteins were analyzed by western blotting.
TAp73 inhibits DNA binding and transactivation by p53 following DNA damage
To determine a potential role of p53 and TAp73 occupancy on promoters of p53 target genes, we used chromatin immunoprecipitation (ChIP) to analyze the binding of p53 and TAp73 to several promoters in cisplatin-treated Flag-KI cells. In response to 12.5 μM cisplatin for 24 hr, p53 was recruited to its target gene promoters, while TAp73 was selectively recruited to the promoters of PUMA, p21 and 14-3-3σ (Fig. 4A and S8A). In response to 50 μM cisplatin for 24 hr, the binding of p53 to its target promoters markedly increased, while that of TAp73 decreased (Fig. 4A and S8A). The binding of p53 to its target gene promoters upon cisplatin exposure was markedly suppressed by transfection with TAp73 (Fig. 4B and S8B), while significantly increased upon knockdown of TAp73 (Fig. 4C and S8C). Furthermore, cisplatin dose escalation resulted in increased Histone H4 acetylation, a chromatin remodeling marker of transcriptional activation, in the PUMA and p21 promoter regions (Fig. 4D). TAp73 transfection almost completely abolished cisplatin-induced H4 acetylation, whereas p53 transfection had an opposite effect (Fig. 4D). TAp73 transfection also suppressed the activation of PUMA and p21 luciferase reporters by p53 in a dose-dependent manner in p53-KO cells (Fig. 4E). Together, these results indicate that upon sub-lethal DNA damage, TAp73 restricts p53 transcriptional activity, while excessive DNA damage downregulates TAp73 to unleash the full activity of p53 to drive apoptosis induction.
TAp73 and p53 inhibit each other in DNA binding and form protein complexes
Several possibilities may explain how TAp73 inhibits p53-mediated transactivation, such as competitive binding to the same promoter and inhibiting formation of transcription-competent p53 tetramers. We examined the former by an equilibrium binding assay using fluorescence polarization and GST-tagged recombinant p53 and TAp73 proteins. Recombinant p53 and TAp73 were found to bind to a synthetic PUMA promoter probe with an equilibrium binding constant (Kd) of 29.55 nM and 87.12 nM, respectively (Fig. 5A), suggesting that p73 is less efficient in DNA binding compared to p53 and may not directly compete out p53 at the p53 responsive elements. ChIP analysis showed that co-transfection of p53 and TAp73 attenuated the binding of both proteins to the promoters of PUMA, p21, Bax and Noxa, relative to transfection of p53 or TAp73 alone (Fig. 5B). Far-western blotting showed that co-expression of p53 and TAp73 in p53-KO cells abolished the in vitro binding of both proteins to the PUMA promoter probe (Fig. 5C). Furthermore, the binding of p53 or TAp73 to the PUMA promoter probe was inhibited by titration of TAp73 or p53 in fluorescence polarization assays, whereas the control GST-tagged X-linked IAP (XIAP) had no effect on the binding of p53 and TAp73 (Fig. 5D). Therefore, the presence of TAp73 can impair the ability of p53 to bind to DNA. Using gel filtration column chromatography, we found that both p53 and TAp73 peak fractions concordantly shifted to a higher molecular weight (MW) range in cells treated with 12.5 μM cisplatin (Fig. 5E), compared to untreated cells, suggesting formation of large and likely transcriptionally incompetent complexes. In contrast, the shifts of p53 and TAp73 fractions were less pronounced in cells treated with 50 μM cisplatin (Fig. 5E). TAp73 transfection led to similar shifts of p53 peak fractions as that caused by 12.5 μM cisplatin (Fig. 5F). Together, these results suggest that TAp73 inhibits p53 activity by forming protein complexes that are unable to efficiently bind to DNA.
Downregulation of p73 by ubiquitin/proteasome-mediated E2F1 degradation in response to excessive DNA damage
We next investigated the mechanism by which TAp73 expression is regulated in response to different levels of DNA damage. The mRNA expression of TAp73, but not that of ΔNp73, TAp63, and ΔNp63, was markedly decreased following treatment with 50 μM cisplatin (Fig. 6A and S9). The expression of E2F1, a key transcription factor that regulates TAp73 and DNA damage response [21, 22], as well as E2F1 bound to p73 promoter, were decreased upon 50 μM cisplatin (Fig. 6, B and C). E2F1 depletion was also detected in cells treated with 50 μg/ml 5-FU (Fig. S10A). E2F1 transfection blocked the downregulation of TAp73 protein and mRNA in cells treated with 50 μM cisplatin (Fig. 6, D and E). Conversely, E2F1 knockdown enhanced TAp73 depletion (Fig. 6, F and G). Similar effects of E2F1 on TAp73 expression were also observed in RKO colon cancer cells exposed to cisplatin (Fig. S10, B and C).
Figure 4: TAp73 inhibits p53 binding to its target gene promoters following excessive DNA damage. (A) HCT116 cells with Flag-TAp73 KI were treated with cisplatin at indicated concentrations for 24 hr. Chromatin immunoprecipitation (ChIP) followed by quantitative real-time PCR was used to analyze the binding of p53 and TAp73 to the promoters of PUMA, Bax and p21, using anti-p53 antibody and anti-Flag-conjugated beads, respectively. Results were normalized to those of IgG (for p53 ChIP) and parental HCT116 (for TAp73 ChIP), which were used as negative controls for ChIP, and plotted as fold of enrichment relative to the control. (B) Flag-KI HCT116 cells were transfected with control or HA-TAp73α construct, and then treated with cisplatin at indicated concentrations for 24 hr. Occupancy of p53 at the indicated promoters was analyzed by ChIP as in (A). (C) Flag-KI HCT116 cells were transfected with control or TAp73 siRNA, and then treated with cisplatin at indicated concentrations for 24 hr. Binding of p53 to the indicated promoters was analyzed as in (A). (D) HCT116 cells were transfected with p53 or TAp73α, and treated with cisplatin at indicated concentrations for 24 hr. Histone H4 acetylation, a marker of transcriptional activation, in PUMA and p21 promoters was analyzed by ChIP using an antibody specific for acetylated H4. The results were normalized to those of input. (E) p53-KO HCT116 cells were co-transfected with a PUMA or p21 luciferase reporter, along with indicated amounts of p53 and TAp73α expression vectors, or control empty vectors. Activation of the PUMA and p21 reporters was measured 24 hr after transfection, as described in the Materials and Methods. Results in (A)-(E) were expressed as means ± s.d. of three independent experiments. ***, P <0.001; **, P <0.01; *, P <0.05.
Figure 5: TAp73 inhibits the DNA binding of p53 through complex formation, rather than promoter competition. (A) Fluorescence polarization analysis of DNA binding by p53 and TAp73. Fluorescence polarization was performed by titrating GST-TAp73α and GST-p53 into 4 nM FAM-labeled PUMA promoter oligonucleotide. GST-XIAP, GST-Bcl-2 and a non-specific DNA sequence (NS) were used as negative control. Results were expressed as means ± s.d. of three independent experiments. ***, P<0.001. (B) p53-KO HCT116 cells were transfected with p53, HA-TAp73α, or both proteins at indicated amounts. Binding of p53 and TAp73 to the promoters of PUMA, p21, Bax and Noxa was analyzed by ChIP using anti-p53 antibody and anti-HA, respectively, followed by quantitative real-time PCR. Results were normalized to those of IgG, which was used as a negative control, and expressed as means ± s.d. of three independent experiments. **, P <0.01; *, P <0.05. (C) Far-western blotting analysis of p53 and TAp73 binding to the PUMA promoter. Biotinylated PUMA promoter probe was incubated with cell lysates from p53-KO HCT116 cells transfected with TAp73α, p53, or both, followed by pull-down with streptavidin-conjugated beads and analysis of p53 and TAp73 by western blotting. (D) Fluorescence polarization analysis of the interference of p53 and TAp73 on each other's DNA binding. Left, p53 was titrated into 4 nM of FAM-labeled PUMA promoter oligonucleotide to saturation, followed by the addition GST-TAp73 for titration. Right, TAp73 titration followed by p53 titration. GST-XIAP or blank binding buffer was used as negative control. (E)Whole cell extracts (~3 mg) were prepared from HCT116 cells treated with 0, 12.5 or 50 μM cisplatin, and then subjected to gel filtration column chromatography. An equal volume of eluted proteins in each fraction was analyzed by immunoblotting with the indicated antibodies. Molecular weight markers and three peak fractions for each sample are indicated. (F) HCT116 cells transfected with control or HA-TAp73α vector were analyzed as in (E).
Figure 6: Downregulation of E2F1-mediated TAp73 transcription following extensive DNA damage. (A) HCT116 cells were treated with cisplatin at indicated concentrations for 24 hr. TAp73 mRNA expression was analyzed by real-time RT-PCR. (B) Following treatment of HCT116 cells as in (A), indicated proteins were analyzed by western blotting. (C) HCT116 cells were treated with cisplatin at indicated concentrations for 24 hr. ChIP was performed using anti-E2F1 antibody, followed by PCR analysis using primers flanking the genomic region that contains the E2F1 binding sites in the TAp73 promoter. IgG was used as a negative control. Upper, analysis of PCR products by gel electrophoresis; Lower, quantification of the ChIP results by real-time PCR. (D) HCT116 cells were transfected with control or HA-E2F1 expression construct, and then treated with cisplatin at indicated concentrations for 24 hr. Indicated proteins were analyzed by western blotting. (E) Analysis of TAp73 mRNA expression in cells treated as in (D) by real-time RT-PCR. (F) HCT116 cells were transfected with control or E2F1 siRNA, and then treated with cisplatin at indicated concentrations for 24 hr. Indicated proteins were analyzed by western blotting. (G) Analysis of TAp73 mRNA expression in cells treated as in (F) by real-time RT-PCR. RT-PCR results in (A), (E) and (G) were normalized to the control β-actin and expressed as means ± s.d. of three independent experiments. *, P <0.05.
We further investigated the mechanism of E2F1 depletion. Unlike TAp73, the mRNA expression of E2F1 was not decreased, but slightly increased upon cisplatin exposure, compared to untreated cells (Fig. 7A), suggesting a post-transcriptional mechanism of E2F1 depletion upon 50 μM cisplatin. The increased E2F1 mRNA expression may account for the increased E2F1 protein expression upon 12.5 μM cisplatin (Fig. 6B). We then tested whether ubiquitin/proteasome-mediated protein degradation is involved in E2F1 depletion. The half-life of E2F1 was decreased from >9 hr in response to 12.5 μM cisplatin to 3–6 hr upon 50 μM cisplatin (Fig. 7B). Proteasome inhibitor MG132 treatment reverted the declined E2F1 expression in response to 50 μM of cisplatin (Fig. 7C). Ubiquitination of both endogenous and transfected E2F1 (analyzed in normalized cell lysates) was markedly enhanced following treatment with 50 μM cisplatin, compared to untreated cells or those treated with 12.5 μM cisplatin (Fig. 7, D and E). MG132 treatment also impeded the declined expression of p73 mRNA and protein (Fig. 7, C and F). Knockdown of E2F1 suppressed the effect of MG132 on TAp73 expression in response to 50 μM cisplatin (Fig. 7G). Since MG132 alone only slightly increased TAp73 level (Fig. 7C), the E2F1-dependent effect of MG132 on TAp73 expression suggests that downregulation of TAp73 is mediated by proteasomal degradation of E2F rather than TAp73 itself. However, the effects of MG132 cannot be directly correlated with the E2F1/p73 pathway, as proteasome inhibition has multiple cellular effects including severe cellular damage. To further understand the mechanism of E2F1 degradation, we probed ATM, an upstream regulator of DNA damage response [4]. The E2F1 depletion was found to be mediated by ATM, as co-treatment with the ATM inhibitor Ku55933 restored E2F1 expression upon 50 μM cisplatin (Fig. 7H). Together, these results demonstrate that lethal DNA damage triggers ubiquitin/proteasome-mediated E2F1 degradation and p73 downregulation to facilitate p53-dependent apoptosis.
Figure 7: E2F1 is degraded through the ubiquitin/proteasome pathway in response to excessive DNA damage. (A) HCT116 cells were treated with cisplatin at indicated concentrations for 18 hr. E2F1 mRNA expression was analyzed by real-time RT-PCR. Results were normalized to the control β-actin. ***, P <0.001. (B) HCT116 cells were treated with cisplatin at indicated concentrations along with 100 μM of the protein synthesis inhibitor cycloheximide (CHX). E2F1 and β-actin expression at indicated time points was analyzed by western blotting. Relative E2F1 expression of each sample, normalized to that of the loading control β-actin, is indicated, with that of the untreated cells arbitrarily set as 1.0. (C) HCT116 cells with or without cisplatin treatment at indicated concentrations for 18 hr were exposed to 5 μM of MG132 or the control DMSO for additional 6 hr. Western blotting was used to analyze indicated proteins. (D) HCT116 cells treated with cisplatin and/or MG132 as in (C) were analyzed by immunoprecipitation using anti-E2F1 antibody, followed by western blot analysis with anti-ubiquitin antibody. The IP inputs were normalized based on E2F1 expression. Arrowhead indicates E2F1 after pull-down, and asterisks indicate non-specific bands. (E) HCT116 cells were transfected with HA-E2F1, and then treated with cisplatin and/or MG132 as in (C). Immunoprecipitation was performed using anti-HA antibody, followed by western blot analysis with anti-ubiquitin antibody. (F) TAp73 mRNA expression in HCT116 cells treated as in (C) was analyzed by real-time RT-PCR. Results were normalized to the control β-actin. **, P <0.01. (G) HCT116 cells were transfected with control or E2F1 siRNA, treated with cisplatin and/or MG132 as in (C). Indicated proteins were analyzed by western blotting. (H) HCT116 cells were treated with cisplatin at indicated concentrations, along with 10 μM of the ATM inhibitor Ku55933 for 24 hr. Western blotting was used to analyze phospho-ATM (p-ATM; Ser1981) and E2F1. (I) A model of how p73 affects p53 activity in response to different levels of DNA damage.
Cell culture and drug treatment
Human colorectal cancer cell lines, including HCT116, RKO, LOVO, which contain WT p53, and DLD1, which contains mutant p53, were obtained from American Type Culture Collection (ATCC; Manassas, VA). p53-KO and PUMA-KO HCT116 cells were obtained from Dr. Bert Vogelstein (Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins). p53-binding site knockout (BS-KO) HCT116 cells and immortalized MEF cells were previously described [17]. Colon cancer cells were maintained in McCoy's 5A medium (Invitrogen). MEF cells were cultured using Dulbecco's modified Eagle's medium (BioWhittaker). All cell culture media were supplemented with 10% fetal bovine serum (HyClone), 100 U/ml of penicillin, and 100 μg/ml of streptomycin. Cells were maintained at 37 oC in 5 % CO2 atmosphere.
Cells were plated in 12-well plates at 40-50% density 24 hr before treatment. Cisplatin, etoposide, 5-Fluorouracil (5-FU), camptothecin and staurosporine (Sigma) were diluted to different concentrations in cell culture medium. In some experiments, cells were treated with DNA damaging drugs for 18 hr, and then with the proteasome inhibitor MG 132 (Sigma) for an additional 6 hr.
Knock-in of a triple-Flag tag in TAp73
Vectors for knocking-in Flag-tagged TAp73 were constructed using the pUSER-rAAV (recombinant adeno-associated virus) System [23]. Briefly, two homologous arms of ~1.32 kb and 0.995 kb, respectively, flanking the second intron of TAp73 were inserted between 2 USER sites in the AAV shuttle vector pTK-Neo-USER (Fig. S1A). Coding sequence of a triple-Flag tag was introduced by PCR into the left arm immediately 3′ following the initiation ATG codon of TAp73α, as previously described [24]. For gene targeting, HCT116 cells, in which TAp73 is the most prominently expressed p73 isoform [25], were infected with the targeting rAAV and selected by G418 (0.5 mg/mL; Mediatech) for 3 weeks. G418-resistant clones were pooled and screened by PCR for targeting events using the primer pair listed in Table S1. After gene targeting, Neo was excised by Cre adenovirus infection. Gene targeting was verified by sequencing of genomic DNA and analysis of Flag-TAp73 expression using western blotting.
Apoptosis and caspase activity assays
After treatment, adherent and floating cells were harvested and resuspended with PBS solution containing 3.7 % formaldehyde, 0.5 % Nonidet P-40, and 10 μg/ml Hoechst 33258 (Invitrogen). Apoptosis was assessed through microscopic visualization and counting of cells with condensed chromatin and micronucleations as described [26]. For each measurement, at least three independent experiments and a minimum of 300 cells were analyzed. For annexin V/propidium iodide (PI) staining, cells were stained by annexin Alexa-488/PI (Invitrogen) following the manufacturer's instruction, and then analyzed by flow cytometry. Caspase activity was measured using the SensoLyte Homogeneous AMC Caspase-3/7 Assay Kit (Anaspec). Briefly, 1.0×104 HCT116 cells were seeded in each well of a 96-well plate. After treatment, cells were incubated with the caspase substrate Ac-DEVD-AMC at room temperature for 40 min. Fluorescence was measured using a Wallac Victor 1420 Multilabel Counter (PerkinElmer), and the data are presented as relative fluorescence units.
Transfection and siRNA/shRNA knockdown
Transfection of expression constructs and small interfering RNA (siRNA) was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Expression constructs, including pCMV-Neo-Bam p53 WT (from Dr. Bert Vogelstein), HA-TAp73α-pcDNA3 (from Dr. William Kaelin) and pSG5L-HA-E2F1 (from Dr. William Sellers), were obtained from Addgene (Cambridge, MA). siRNA (Dharmacon) of 200 pmol each was transfected into cells 24 hr before drug treatment. The siRNA sequences have been previously described, including those for human TAp73 (5′-GGATTCCAGCATGGACGTCTT-3′) [27], and human E2F1 (5′-GGACCTTCGTAGCATTGCA-3′) [28]. Stable TAp73 knockdown cells were generated by transducing HCT116 cells with the pLKO.1-puro lentiviral vector (Addgene) expressing shRNA with the same targeting sequence as the TAp73 siRNA. Lentiviral particles were generated by co-transfecting 293T cells with the lentiviral vector, pMD2.G (VSVG), pMDLg/pRRE, and pRSV-REV (Addgene). Following lentiviral transduction, cells were plated in 96-well plates in the presence of puromycin (2 μg/ml; EMD/Millipore). TAp73 expression of the puromycin-resistant clones was then analyzed by Western blotting.
For analysis of promoter binding by p53 and E2F1, and histone H4 acetylation, ChIP was carried out by using the Chromatin Immunoprecipitation Assay Kit (Upstate/Millipore), as previously described [17]. Antibodies against p53 (SC-126), E2F1 (SC-251) (Santa Cruz Biotechnology), acetylated histone H4 (#06-598; Upstate/Millipore) were used. Isotype-matched IgG (R&D System) were used as control for IP. For analysis of promoter binding by TAp73, HCT116 cells with knock-in of Flag-TAp73 were treated and analyzed. IP was performed using EZview Red ANTI-FLAG M2 Affinity Gel (Sigma) according to manufacturer's instructions. Parental HCT116 cells were used as the negative control. After ChIP, the IP complex was analyzed by PCR using the primers listed in Table S1. In some experiments, cells were first transfected with HA-TAp73α, TAp73 siRNA, p53, or both HA-TAp73α and p53. Twenty-four hr after transfection, cells were treated with cisplatin and analyzed by ChIP for p53 using anti-p53, or for TAp73 using anti-HA antibody (Santa Cruz Biotechnology).
Luciferase reporter assay
For analysis of PUMA and p21 promoter activities, p53-KO HCT116 cells were transfected with a PUMA luciferase reporter containing the p53 binding site (Fragment A) [29] or p21 luciferase reporter (Addgene, Plasmid 16451), along with HA-p73α-pcDNA3 and/or pCMV-Neo-Bam p53 WT, and the control β-galactosidase reporter pCMVβ (Promega, Madison, WI, USA). Luciferase activities were measured and normalized to those of β-galactosidase, as previously described [30]. Experiments were performed in triplicate and repeated at least three times.
Western blot and antibodies
Cells were lysed with 2×Laemmli sample buffer and subjected to western blotting, as previously described [31]. Antibodies used for western blotting included those against p73 (A300-136A; Bethyl Labs), PUMA [31], p53 (SC-126), HA (SC-805), Bax (SC-493), p21 (SC-397), 14-3-3σ (SC-100638) and E2F1 (SC-251) (Santa Cruz Biotechnology), Flag (F3165), β-Actin (A5441) (Sigma), phospho-ATM (Ser1981) (ab81292; Abcam) and ubiquitin (#550944; BD Transduction).
Real-time reverse transcriptase (RT) PCR
Total RNA was isolated by using Quick-RNA mini-prep kit (ZYMO Research, Irvine, CA) according to the manufacturer's instructions, and subjected to reverse transcription to generate cDNA. Real-time RT-PCR was performed on a CFX96 Real-Time PCR system (Bio-Rad, Hercules, CA) with SYBR Green (Invitrogen). Primers for RT-PCR are listed in Table S1. Agarose gel electrophoresis of the PCR products was used to verify the specificity of PCR amplification.
Ubiquitination Assay
To detect E2F1 ubiquitination following cisplatin treatment, HCT116 cells with or without transfection of pSG5L-HA-E2F1 were treated with cisplatin for 18 hr, and then with 5 μM MG 132 for an additional 6 hr. IP of endogenous and transfected E2F1 was performed using anti-E2F1 and anti-HA, respectively. IP complexes were pulled down using EZview Red Protein G Affinity Gel (Sigma) according to the manufacturer's instructions, lysed in 2×Laemmli sample buffer, and analyzed for ubiquitin by western blotting.
Analysis of DNA binding by p53 and TAp73
To analyze DNA binding by p53 and p73 using fluorescence anisotropy/polarization, aliquots of stock solutions of GST-p53 (2.5 μM) or GST-TAp73α (2.0 μM) (SignalChem) were added to HPLC-purified 6-carboxyfluorescein (6-FAM)-labeled DNA duplex (5′-/56-FAM/ CGCGCCTGCAAGTCCTGACTTGTCCGCGGC-3′; Integrated DNA Technologies) containing the p53 binding sites in the PUMA promoter at 4 nM in a 500-μL reaction volume at room temperature in a buffer containing 100 mM NaCl, 10 mM tris-HCl, PH7.5, 10% (vol/vol) glycerol, 5 mM DTT, and 0.2 mg/mL bovine serum albumin. After mixing by stirring for 10 sec and incubation for 30 sec, fluorescence anisotropy was recorded on a Varian Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies), with λex set at 480 nm and λem at 530 nm. A 6-FAM-labeled 26-bp random DNA duplex (5′-/56-FAM/AATGGAAATTTCCGGAAATTTCCATT-3′) was used as negative control with an initial concentration of 4 nM in 500-μL reaction volume as well. Data were analyzed as previous described [32].
To analyze DNA binding by p53 and TAp73 using far-western blotting, cell lysates prepared from p53-KO HCT116 cells transfected with p53, HA-TAp73α, or both proteins were incubated with 2 μg of the duplex PUMA promoter probe with or without 5′ biotin label for 90 min at room temperature, and then with streptavidin-conjugated magnetic beads (Pierce) at 4 oC for 2 hr. After isolation by centrifugation and two washes, the beads were boiled in 2×Laemmli sample buffer and subjected to western blotting for the transfected p53 and TAp73.
Gel filtration column chromatography
Gel filtration column chromatography was carried out as previously described [33]. In brief, 3 mg of whole cell extracts prepared in a column elution buffer (30 mM HEPES, pH 7.4; 150 mM NaCl; 10% glycerol; 0.1% NP-40; 5 μg/ml DNase I; and protease inhibitor cocktail [Roche]) were loaded on a column packed with Superose 6 prep grade gel (GE Healthcare), and 500-μl elution was collected in each fraction. Equal volumes of eluted fractions were subjected to western blotting. A mixture of protein markers containing blue dextran (2,000 kDa), β-amylase (200 kDa), bovine serum albumin (66 kDa), and cytochrome c (12.4 kDa) was used as the molecular weight standard.
In response to mild and repairable DNA damage, low levels of p53 induction tend to favor growth arrest, whereas severe, extended, or irrepairable DNA damage often leads to apoptosis. Our results indicate that TAp73 plays a critical role in controlling overall p53 activity and cell fate determination in response to DNA damage (Fig. 7I). TAp73 binds to p53 and inhibits p53-mediated transactivation following mild DNA damage, resulting in limited activation of p53 target genes to favor growth inhibition. Excessive DNA lesions lead to TAp73 depletion and enhanced p53 activation and trigger apoptosis. Although the expression of p53 target genes, such as PUMA and p21, is similarly modulated in response to different levels of DNA damage through TAp73 and p53, the impact of these changes on cell fates may be different due to differential threshold activities involved in different biological responses. While modest induction of p21 is probably sufficient to halt cell cycle progression, strong induction of PUMA and other apoptosis regulators may be necessary for efficient apoptosis induction. Following high levels of DNA damage, TAp73 expression sharply declined (Fig. 1A and B), but apoptosis induction was less dramatic and exhibited a dose response (Fig. 1C). This can probably be explained by the indirect and partial effects of TAp73 on apoptosis induction, which is regulated by TAp73 through p53 and its target genes, as well as other apoptosis regulators.
Differential p53 response can result from selective expression of p53 targets through several mechanisms. Variations in binding affinities of p53 for its target promoters can profoundly affect promoter choice and cell fate determination [34]. A number of p53 partner proteins have been implicated in modulating the selection of p53 targets, such as Brn3a and YB1, which favor growth arrest, and ASPP family members, p300, and CAS, which are proapoptotic [35, 36]. Hzf, a zinc finger protein that interacts with p53, can selectively induce p53 targets involved in growth arrest. Prolonged DNA damage exposure led to Hzf degradation and enhanced expression of the proapoptotic targets of p53 [37]. TAp73 appears to be different from Hzf as it regulates overall p53 activity rather than target selectivity. While TAp73 is modulated in response to altered extent of DNA damage, Hzf is altered upon changes in duration of DNA damage. Furthermore, different post-translational modifications of p53 also regulate selective expression of p53 targets [38]. In addition to p53, excessive DNA damage can also activate an NF-kB-dependent pathway to induce cytokine secretion and apoptosis [16]. Therefore, a set of well-orchestrated events including TAp73 depletion are set forth in response to lethal DNA damage to signal cells death through apoptosis.
TAp73 contains a highly-conserved DNA-binding domain and shares sequence specificity with p53 [39]. It is possible that TAp73 simply competes with p53 for DNA binding to prevent the activation of p53 target genes [40]. However, our data are inconsistent with this hypothesis. TAp73 binds less tightly than p53 to the PUMA promoter (Fig. 5A). TAp73 and p53 co-expression or co-incubation abolished DNA binding by both proteins in cells and in vitro binding assays (Fig. 5, B-D). Several p73 and p63 isoforms can physically interact with each other or with mutant p53 [41, 42]. In the context of mild DNA damage, p73 seems to antagonize p53 activity through the formation of large protein complexes (Fig. 5, E and F). These complexes might lack symmetric conformation and cooperativity required for binding to the p53 responsive elements. In support of our findings, DNA binding cooperativity of p53 has been shown to determine the extent of DNA damage-induced apoptosis [43]. It should be noted that the DNA binding and transactivation of both p53 and TAp73 are subjected to extensive modifications following DNA damage [44]. Our data cannot completely rule out the possibility that p53 and TAp73 compete with each other for DNA binding.
Despite numerous studies on p53 homologues and clarification of TAp73 as a tumor suppressor [9, 10], the precise function of TAp73 in DNA damage response and tumor suppression is not fully understood [45]. TAp73 is often proapoptotic, but could be antiapoptotic under certain circumstances [46, 47]. In contrast to our findings, TAp73 was shown to be proapoptotic in HCT116 cells in response to the MDM2 inhibitor Nutlin-3, which might be due to the effects of Nutlin-3 on p53 and other MDM2 targets [48]. The proapoptotic activity of TAp73 is regulated by DNA damage-dependent acetylation [49] and cellular localization [50]. Our results show for the first time that the interplay between TAp73 and p53 is dependent on extent of DNA damage and TAp73 levels. TAp73 can be antiapoptotic by antagonizing p53 when genotoxic stress is not severe enough to pose danger to an organism. However, TAp73 accumulation without concurrent p53 stabilization or in a p53-deficient background may allow TAp73 to induce p53 target genes and cell death [51], even though it is a less effective transactivator than p53. TAp73 also exhibits promoter selectivity and has a number of unique target genes that are distinct from those of p53 [52]. Antagonizing p53 by TAp73 in DNA damage response may also explain why TAp73 is not a classical tumor suppressor. TAp73 is rarely mutated in human tumors and even overexpressed in some tumors [45]. p73-deficient mice, including TAp73-knockout mice, are different from p53-null mice in both animal development and tumor incidence [9, 12, 53]. TAp73 was previously found to interfere with p53 in regulating telomerase activity [54]. In line with its pro-survival function, TAp73 has recently been shown to promote cell growth by activating AP-1 target genes [55], or through the pentose phosphate pathway [56]. It is likely that other p53 homologues, such as TAp73β, ΔNp73, and p63 isoforms, may help to further fine tune the activities of p53 and p73 in response to different stress conditions [57].
TAp73 expression is mainly regulated at the transcriptional level by E2F1 [22]. Upon sub-lethal DNA damage, E2F1 induction sustains TAp73 expression to keep the activity of p53 in check (Fig. 7I). Extensive DNA lesions trigger ubiquitin/proteasome-mediated E2F1 degradation (Fig. 7I), allowing p53 to fully activate its target genes and induce apoptosis. Similar to TAp73, E2F1 has been shown to promote apoptosis in a p53-deficient background [58]. The activity of TAp73 can also be regulated by a number of other proteins. For example, TAp73 can be phosphorylated by the tyrosine kinase c-Abl during apoptosis induced by cisplatin and other DNA damaging agents [59, 60]. TAp73 expression can also be regulated by p53 in response to oxidative stress [61]. A remaining question is how E2F1 degradation is triggered in response to lethal DNA damage, and our data suggest the involvement of ATM (Fig. 7H), which initiates a series of downstream signaling events following excessive DNA lesions [16]. E2F1 can also be methylated at lysine 185 by Set9 methyltransferase, which prevents DNA damage-induced E2F1 accumulation and p73 activation [28].
TAp73 and other p53 homologues can be activated by chemotherapeutic drugs and γ-irradiation, and implicated as a major determinant of chemotherapy sensitivity, particularly in p53-deficient tumors [62]. Our data suggest that the influence of TAp73 on p53 in both cancer and normal cells needs to be taken into consideration in the use of TAp73 as a therapeutic target. In conclusion, our study has uncovered that TAp73 helps to antagonize the action of p53. In response to high levels of DNA damage, the depletion of TAp73 through proteasomal degradation of E2F1 is a prerequisite to trigger p53-dependent apoptosis to eliminate cells harboring extensive genomic damage.
We thank Dr. Bert Vogelstein, Dr. Monica E. Buchanan, and our lab members for critical reading, and Dr. Chris Bakkenist for help with ATM inhibition experiment. This work is supported by NIH grants CA106348, CA121105, CA172136, American Cancer Society grant RSG-07-156-01-CNE (L.Z.), and NIH grant CA129829 and RGS-10-124-01-CCE (J.Y.). This project used the UPCI shared glassware and cell imaging facilities that were supported in part by award P30CA047904.
The authors declare no conflict of interest and all authors have agreed on the submission.
LZ designed and supervised all experiments. DC, LM, FZ, YP designed and performed experiments. DC, BV, and LZ analyzed data. DC, JY and LZ wrote the paper.
1. Weinert T. DNA damage and checkpoint pathways: molecular anatomy and interactions with repair. Cell. 1998; 94(5):555–558.
2. Evan G and Littlewood T. A matter of life and cell death. Science. 1998; 281(5381):1317–1322.
3. Vogelstein B, Lane D and Levine AJ. Surfing the p53 network. Nature. 2000; 408(6810):307–310.
4. Jackson SP and Bartek J. The DNA-damage response in human biology and disease. Nature. 2009; 461(7267):1071–1078.
5. Lord CJ and Ashworth A. The DNA damage response and cancer therapy. Nature. 2012; 481(7381):287–294.
6. Levine AJ, Tomasini R, McKeon FD, Mak TW and Melino G. The p53 family: guardians of maternal reproduction. Nat Rev Mol Cell Biol. 2011; 12(4):259–265.
7. Dotsch V, Bernassola F, Coutandin D, Candi E and Melino G. p63 and p73, the ancestors of p53. Cold Spring Harb Perspect Biol. 2010; 2(9):a004887
8. Jost CA, Marin MC and Kaelin WG. p73 is a human p53-related protein that can induce apoptosis. Nature. 1997; 389(6647):191–194.
9. Tomasini R, Tsuchihara K, Wilhelm M, Fujitani M, Rufini A, Cheung CC, Khan F, Itie-Youten A, Wakeham A, Tsao MS, Iovanna JL, Squire J, Jurisica I, Kaplan D, Melino G, Jurisicova A and Mak TW. TAp73 knockout shows genomic instability with infertility and tumor suppressor functions. Genes Dev. 2008; 22(19):2677–2691.
10. Flores ER, Sengupta S, Miller JB, Newman JJ, Bronson R, Crowley D, Yang A, McKeon F and Jacks T. Tumor predisposition in mice mutant for p63 and p73: evidence for broader tumor suppressor functions for the p53 family. Cancer Cell. 2005; 7(4):363–373.
11. Kaghad M, Bonnet H, Yang A, Creancier L, Biscan JC, Valent A, Minty A, Chalon P, Lelias JM, Dumont X, Ferrara P, McKeon F and Caput D. Monoallelically expressed gene related to p53 at 1p36, a region frequently deleted in neuroblastoma and other human cancers. Cell. 1997; 90(4):809–819.
12. Yang A, Walker N, Bronson R, Kaghad M, Oosterwegel M, Bonnin J, Vagner C, Bonnet H, Dikkes P, Sharpe A, McKeon F and Caput D. p73-deficient mice have neurological, pheromonal and inflammatory defects but lack spontaneous tumours. Nature. 2000; 404(6773):99–103.
13. Pozniak CD, Radinovic S, Yang A, McKeon F, Kaplan DR and Miller FD. An anti-apoptotic role for the p53 family member, p73, during developmental neuron death. Science. 2000; 289(5477):304–306.
14. Flores ER, Tsai KY, Crowley D, Sengupta S, Yang A, McKeon F and Jacks T. p63 and p73 are required for p53-dependent apoptosis in response to DNA damage. Nature. 2002; 416(6880):560–564.
15. Irwin MS, Kondo K, Marin MC, Cheng LS, Hahn WC and Kaelin WG. Chemosensitivity linked to p73 function. Cancer Cell. 2003; 3(4):403–410.
16. Biton S and Ashkenazi A. NEMO and RIP1 control cell fate in response to extensive DNA damage via TNF-alpha feedforward signaling. Cell. 2011; 145(1):92–103.
17. Wang P, Yu J and Zhang L. The nuclear function of p53 is required for PUMA-mediated apoptosis induced by DNA damage. Proc Natl Acad Sci U S A. 2007; 104(10):4054–4059.
18. Yu J, Wang Z, Kinzler KW, Vogelstein B and Zhang L. PUMA mediates the apoptotic response to p53 in colorectal cancer cells. Proc Natl Acad Sci U S A. 2003; 100(4):1931–1936.
19. Yu J, Zhang L, Hwang PM, Kinzler KW and Vogelstein B. PUMA induces the rapid apoptosis of colorectal cancer cells. Mol Cell. 2001; 7(3):673–682.
20. Melino G, Bernassola F, Ranalli M, Yee K, Zong WX, Corazzari M, Knight RA, Green DR, Thompson C and Vousden KH. p73 Induces apoptosis via PUMA transactivation and Bax mitochondrial translocation. J Biol Chem. 2004; 279(9):8076–8083.
21. Irwin M, Marin MC, Phillips AC, Seelan RS, Smith DI, Liu W, Flores ER, Tsai KY, Jacks T, Vousden KH and Kaelin WG. Role for the p53 homologue p73 in E2F-1-induced apoptosis. Nature. 2000; 407(6804):645–648.
22. Stiewe T and Putzer BM. Role of the p53-homologue p73 in E2F1-induced apoptosis. Nat Genet. 2000; 26(4):464–469.
23. Zhang X, Guo C, Chen Y, Shulha HP, Schnetz MP, LaFramboise T, Bartels CF, Markowitz S, Weng Z, Scacheri PC and Wang Z. Epitope tagging of endogenous proteins for genome-wide ChIP-chip studies. Nat Methods. 2008; 5(2):163–165.
24. Li H, Wang P, Sun Q, Ding WX, Yin XM, Sobol RW, Stolz DB, Yu J and Zhang L. Following cytochrome c release, autophagy is inhibited during chemotherapy-induced apoptosis by caspase 8-mediated cleavage of Beclin 1. Cancer Res. 2011; 71(10):3625–3634.
25. Yu J, Zhang L, Hwang PM, Rago C, Kinzler KW and Vogelstein B. Identification and classification of p53-regulated genes. Proc Natl Acad Sci U S A. 1999; 96(25):14517–14522.
26. Kohli M, Yu J, Seaman C, Bardelli A, Kinzler KW, Vogelstein B, Lengauer C and Zhang L. SMAC/Diablo-dependent apoptosis induced by nonsteroidal antiinflammatory drugs (NSAIDs) in colon cancer cells. Proc Natl Acad Sci U S A. 2004; 101(48):16897–16902.
27. Rocco JW, Leong CO, Kuperwasser N, DeYoung MP and Ellisen LW. p63 mediates survival in squamous cell carcinoma by suppression of p73-dependent apoptosis. Cancer Cell. 2006; 9(1):45–56.
28. Kontaki H and Talianidis I. Lysine methylation regulates E2F1-induced cell death. Mol Cell. 2010; 39(1):152–160.
29. Ming L, Sakaida T, Yue W, Jha A, Zhang L and Yu J. Sp1 and p73 Activate PUMA Following Serum Starvation. Carcinogenesis. 2008; 29:1878–1884.
30. Dudgeon C, Wang P, Sun X, Peng R, Sun Q, Yu J and Zhang L. PUMA induction by FoxO3a mediates the anticancer activities of the broad-range kinase inhibitor UCN-01. Mol Cancer Ther. 2010; 9(11):2893–2902.
31. Ming L, Wang P, Bank A, Yu J and Zhang L. PUMA dissociates Bax and BCL-XL to induce apoptosis in colon cancer cells. J Biol Chem. 2006; 281(23):16034–16042.
32. Weinberg RL, Veprintsev DB and Fersht AR. Cooperative binding of tetrameric p53 to DNA. J Mol Biol. 2004; 341(5):1145–1159.
33. Samanta AK, Chakraborty SN, Wang Y, Schlette E, Reddy EP and Arlinghaus RB. Destabilization of Bcr-Abl/Jak2 Network by a Jak2/Abl Kinase Inhibitor ON044580 Overcomes Drug Resistance in Blast Crisis Chronic Myelogenous Leukemia (CML). Genes & cancer. 2010; 1(4):346–359.
34. Vousden KH and Lu X. Live or let die: the cell's response to p53. Nat Rev Cancer. 2002; 2(8):594–604.
35. Aylon Y and Oren M. Living with p53, dying of p53. Cell. 2007; 130(4):597–600.
36. Tanaka T, Ohkubo S, Tatsuno I and Prives C. hCAS/CSE1L associates with chromatin and regulates expression of select p53 target genes. Cell. 2007; 130(4):638–650.
37. Das S, Raj L, Zhao B, Kimura Y, Bernstein A, Aaronson SA and Lee SW. Hzf Determines cell survival upon genotoxic stress by modulating p53 transactivation. Cell. 2007; 130(4):624–637.
38. Vousden KH and Prives C. Blinded by the Light: The Growing Complexity of p53. Cell. 2009; 137(3):413–431.
39. Lokshin M, Li Y, Gaiddon C and Prives C. p53 and p73 display common and distinct requirements for sequence specific binding to DNA. Nucleic Acids Res. 2007; 35(1):340–352.
40. Vikhanskaya F, D'Incalci M and Broggini M. p73 competes with p53 and attenuates its response in a human ovarian cancer cell line. Nucleic Acids Res. 2000; 28(2):513–519.
41. Li Y and Prives C. Are interactions with p63 and p73 involved in mutant p53 gain of oncogenic function?. Oncogene. 2007; 26(15):2220–2225.
42. Stiewe T, Theseling CC and Putzer BM. Transactivation-deficient Delta TA-p73 inhibits p53 by direct competition for DNA binding: implications for tumorigenesis. J Biol Chem. 2002; 277(16):14177–14185.
43. Schlereth K, Beinoraviciute-Kellner R, Zeitlinger MK, Bretz AC, Sauer M, Charles JP, Vogiatzi F, Leich E, Samans B, Eilers M, Kisker C, Rosenwald A and Stiewe T. DNA binding cooperativity of p53 modulates the decision between cell-cycle arrest and apoptosis. Mol Cell. 2010; 38(3):356–368.
44. Dai C and Gu W. p53 post-translational modification: deregulated in tumorigenesis. Trends Mol Med. 2010; 16(11):528–536.
45. Deyoung MP and Ellisen LW. p63 and p73 in human cancer: defining the network. Oncogene. 2007; 26(36):5169–5183.
46. Nyman U, Sobczak-Pluta A, Vlachos P, Perlmann T, Zhivotovsky B and Joseph B. Full-length p73alpha represses drug-induced apoptosis in small cell lung carcinoma cells. J Biol Chem. 2005; 280(40):34159–34169.
47. Tebbi A, Guittet O, Cottet MH, Vesin MF and Lepoivre M. TAp73 induction by nitric oxide: regulation by checkpoint kinase 1 (CHK1) and protection against apoptosis. J Biol Chem. 2011; 286(10):7873–7884.
48. Kitagawa M, Aonuma M, Lee SH, Fukutake S and McCormick F. E2F-1 transcriptional activity is a critical determinant of Mdm2 antagonist-induced apoptosis in human tumor cell lines. Oncogene. 2008; 27(40):5303–5314.
49. Costanzo A, Merlo P, Pediconi N, Fulco M, Sartorelli V, Cole PA, Fontemaggi G, Fanciulli M, Schiltz L, Blandino G, Balsano C and Levrero M. DNA damage-dependent acetylation of p73 dictates the selective activation of apoptotic target genes. Mol Cell. 2002; 9(1):175–186.
50. Dobbelstein M, Strano S, Roth J and Blandino G. p73-induced apoptosis: a question of compartments and cooperation. Biochem Biophys Res Commun. 2005; 331(3):688–693.
51. Rana S, Gupta K, Gomez J, Matsuyama S, Chakrabarti A, Agarwal ML, Agarwal A, Agarwal MK and Wald DN. Securinine induces p73-dependent apoptosis preferentially in p53-deficient colon cancer cells. Faseb J. 2010; 24(6):2126–2134.
52. Harms K, Nozell S and Chen X. The common and distinct target genes of the p53 family transcription factors. Cell Mol Life Sci. 2004; 61(7-8):822–842.
53. Donehower LA, Harvey M, Slagle BL, McArthur MJ, Montgomery CA, Butel JS and Bradley A. Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature. 1992; 356(6366):215–221.
54. Toh WH, Kyo S and Sabapathy K. Relief of p53-mediated telomerase suppression by p73. J Biol Chem. 2005; 280(17):17329–17338.
55. Vikhanskaya F, Toh WH, Dulloo I, Wu Q, Boominathan L, Ng HH, Vousden KH and Sabapathy K. p73 supports cellular growth through c-Jun-dependent AP-1 transactivation. Nat Cell Biol. 2007; 9(6):698–705.
56. Du W, Jiang P, Mancuso A, Stonestrom A, Brewer MD, Minn AJ, Mak TW, Wu M and Yang X. TAp73 enhances the pentose phosphate pathway and supports cell proliferation. Nat Cell Biol. 2013; 15(8):991–1000.
57. Yang A and McKeon F. P63 and P73: P53 mimics, menaces and more. Nat Rev Mol Cell Biol. 2000; 1(3):199–207.
58. Wikonkal NM, Remenyik E, Knezevic D, Zhang W, Liu M, Zhao H, Berton TR, Johnson DG and Brash DE. Inactivating E2f1 reverts apoptosis resistance and cancer sensitivity in Trp53-deficient mice. Nat Cell Biol. 2003; 5(7):655–660.
59. Gong JG, Costanzo A, Yang HQ, Melino G, Kaelin WG, Levrero M and Wang JY. The tyrosine kinase c-Abl regulates p73 in apoptotic response to cisplatin-induced DNA damage. Nature. 1999; 399(6738):806–809.
60. Yuan ZM, Shioya H, Ishiko T, Sun X, Gu J, Huang YY, Lu H, Kharbanda S, Weichselbaum R and Kufe D. p73 is regulated by tyrosine kinase c-Abl in the apoptotic response to DNA damage. Nature. 1999; 399(6738):814–817.
61. Wang J, Liu YX, Hande MP, Wong AC, Jin YJ and Yin Y. TAp73 is a downstream target of p53 in controlling the cellular defense against stress. J Biol Chem. 2007; 282(40):29152–29162.
62. Lunghi P, Costanzo A, Mazzera L, Rizzoli V, Levrero M and Bonati A. The p53 family protein p73 provides new insights into cancer chemosensitivity and targeting. Clin Cancer Res. 2009; 15(21):6495–6502.


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Scotland's Digital Future outlines the steps that are required to ensure Scotland is well placed to take full advantage of all the economic, social and environmental opportunities offered by the digital age.
The Government Economic Strategy (GES)
Reaffirms the Scottish Government's commitment to delivering faster sustainable economic growth with opportunities for all to flourish.
Provides a narrative of Scotland's current economic performance and economic issues relevant to the Scottish economy in light of global and UK economic conditions.
Economic Development Reading List
Suggested reading list on economic development-related topics. Kindly provided by Professor Alan McGregor of University of Glasgow.
Luath Publishing: Established in 1981, Luath Press is a leading independent book publisher with over 300 books in print
Other Scottish Economy Publications

redpajama_set_name: RedPajamaCommonCrawl


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text:
Isabel Garcia Canet (Pego, 1981) és una poetessa valenciana.

Llicenciada en Filologia Catalana per la Universitat de València, la seva tasca literaria l'ha desenvolupat dins el gènere de la poesia. Així, ha escrit diversos poemes i poemaris com Pell de serp (Viena Edicions, 2003) –finalista en el XXVII Premi de Haikus Joan Teixidor de la ciutat d'Olot–; Claustre (3i4, 2007) –guardons en el XXVI Premi Senyoriu d'Ausiàs March de Beniarjó i, en la modalitat de poesia al País Valencià, el VIII Premi de la Crítica dels Escriptors Valencians (AELC)– o L'os de la música (Bromera, 2011) –guanyador del XII Premi de Poesia Vicent Andrés Estellés de Burjassot el 2010–. Tanmateix, els seus poemes han estat inclosos a antologies com Eròtiques i despentinades (Arola Editors, 2008); Pedra foguera. Antologia de poesia jove dels Països Catalans (Documenta Balear, 2008), For sale o 50 veus de la terra (Edicions 96, 2010), i han estat traduïts a nombrosos idiomes com l'anglès, francès, l'italià, portuguès, castellà i noruec.

Col·labora habitualment a la revista literària Caràcters, en diversos recitals de poesia i també ha treballat en el darrer disc de Rafa Xambó, Andanes.

Obres
 Llibre de Poemes L'os de la música, XVII Premi Vicent Andrés de Burjassot; ed.Bromera (2010).Isabel Garcia-Canet és la primera jove i dona que ha guanyat aquest premi.
 Poemari Claustre, ed. Tres i Quatre (2007)
 Recull de Haikus Pell de serp, Viena edicions (2003)

 Espectacles i col·laboracions -textos i rapsòdia-veu i música':
 Poemes i cançons (2008) espectacle en companyia del músic Rafa Xambó

 Poemes musicats:
 Poema Ancorada de Claustre, en Andanes de Rafa Xambó.
 Poema L'os de la música del llibre amb el mateix títol, en Nipenthí (2013) del grup Krama Premis
 2020: Premi Ciutat de Torrent en la categoria de poesia, per La llum del buit''
 2010: XII premi Vicent Andrés Estellés de Burjassot de poesia
 2008: XVIII Premis de la Crítica dels Escriptors Valencians
 2006: XXVI premi Senyoriu d'Ausiàs March de Beniarjó
 2003: XXVII Premi de Haikus Joan Teixidor de la ciutat d'Olot

Referències

Enllaços externs
 Blog: Entre dos mons: l'arrel
 Antologia en català, castellà, anglès, portugués i francés
 Isabel García i Canet Poemes accessibles en línia.

Pegolins
Poetes valencians contemporanis en català
Alumnes de la Universitat de València
Escriptors de la Marina Alta
Filòlegs valencians contemporanis

redpajama_set_name: RedPajamaWikipedia


text:
La Rift Valley  oppure Great Rift Valley (dall'inglese rift valley, "fossa tettonica") o anche Grande fossa tettonica nei testi in italiano, è una vasta frattura geologica che si estende per quasi 6000 km in direzione nord-sud, dal sud-ovest dell'Asia nell'attuale Siria all'est dell'Africa in Mozambico. La valle varia in larghezza dai 30 ai 100 km e in profondità da qualche centinaio a parecchie migliaia di metri. Si è creata dalla separazione delle placche tettoniche africana e araba, e dalla separazione dell'Africa dell'est dal resto dell'Africa, processo iniziato 35 milioni di anni fa e che continua tutt'oggi. Il nome fu dato dall'esploratore John Walter Gregory.

Geografia
La parte settentrionale della Rift Valley forma la valle del fiume Giordano, che scorre verso sud attraverso il Lago Hula e il Mar di Galilea, in Israele, fino al Mar Morto. A sud del Mar Morto, la Rift Valley è occupata dal wadi Araba e ancora più a Sud dal golfo di Aqaba e dal Mar Rosso.

Nella parte più meridionale del mar Rosso la Rift Valley si dirama in due direzioni diverse, verso est e verso sud. La zona della diramazione è chiamata triangolo di Afar o depressione della Dancalia, si trova in Etiopia e si è formata in corrispondenza della tripla giunzione di Afar, una tripla giunzione geologica di tipo R-R-R. La diramazione verso est forma il golfo di Aden, e da questo punto in poi la Rift Valley continua come dorsale oceanica fino ad unirsi, in corrispondenza della zona di frattura di Owen, con la dorsale di Carlsberg, estremità nord-occidentale della dorsale medio-indiana. La diramazione verso sud è spesso indicata come sistema di rift dell'Africa orientale e fino a poco fa veniva chiamata essa stessa Great Rift Valley. Tra la depressione dell'Afar e la depressione del lago Turkana, in Kenya, si sviluppa la cosiddetta Rift Valley Etiopica, che separa l'altopiano etiopico, a ovest, da quello somalo, a est. Più a sud il rift dell'Africa orientale si divide in due rami, il ramo orientale e il ramo occidentale.

Il ramo occidentale della Rift Valley è chiamato faglia albertina (in inglese Albertine Rift) e si estende dall'estremità settentrionale del Lago Alberto all'estremità meridionale del Lago Tanganica. È delimitato da alcune delle montagne più alte dell'Africa, inclusi i Monti Virunga, i Monti Mitumba e Ruwenzori e contiene alcuni dei grandi laghi africani, che includono laghi tra i più profondi del mondo, come il lago Tanganica, profondo fino a 1.470 metri. Al contrario il Lago Vittoria non è direttamente parte del sistema della Rift Valley, anche se in realtà è posizionato tra i rami orientale e occidentale.

Il ramo orientale, chiamato Rift di Gregory, si sviluppa in Kenya e Tanzania; ad esso è associata la montagna più alta d'Africa, il Kilimangiaro ed altri rilievi principali come il monte Kenya. I laghi del ramo orientale sono meno profondi rispetto a quelli del ramo occidentale e sono caratterizzati da un'alta concentrazione di sali minerali dovuta alle piogge, che portano i sali minerali dai vicini vulcani, e alla forte evaporazione dell'acqua. Il lago Magadi, ad esempio, ha la sua superficie completamente coperta di carbonato di sodio cristallizzato, e i laghi Elmenteita, Baringo, Bogoria e Nakuru sono fortemente alcalini, mentre il Lago Naivasha ha una grande varietà biologica grazie alle sorgenti di acqua dolce che lo alimentano.

I due rami si riuniscono nella parte meridionale terminale, formando il Lago Niassa, il terzo corpo d'acqua dolce più profondo del mondo, con profondità massima di 706 metri e disperdendosi poi nella valle dello Zambesi, nel Mozambico centrale.

L'attività geotermica e l'allargamento della faglia ha causato un assottigliamento della litosfera fino a uno spessore di soli 20 km, quando per i continenti lo spessore tipico è di 100 km. Tra qualche milione di anni, la litosfera potrebbe spaccarsi e l'Africa orientale potrebbe dividersi dal resto del continente.

L'attività vulcanica e la inusuale concentrazione di punti caldi ha prodotto le montagne vulcaniche del Kilimangiaro, monte Kenya, Karisimbi, Nyiragongo, monte Meru, monte Elgon e il vulcano Ol Doinyo Lengai, che è l'unico vulcano natrocarbonitico del mondo (cioè con presenza di carbonati).
Il 3 aprile 2018 codesta faglia ha procurato una frattura lunga 100 km e larga 20 m nel Kenya.

Scoperte paleoantropologiche
La Rift Valley è stata una ricca sorgente di scoperte paleoantropologiche. Gli abbondanti sedimenti della valle, provenienti dalla rapida erosione degli altopiani circostanti, crearono un ambiente favorevole alla preservazione dei resti umani. Sono infatti state trovate numerose ossa di ominidi, antenati della moderna specie umana, tra cui anche quelle della cosiddetta "Lucy", uno scheletro quasi completo di australopiteco, che fu scoperto dall'antropologo Donald Johanson. La famosa coppia di antropologi Louis e Mary Leakey operarono principalmente in queste zone.

Recentemente sono stati ritrovati i resti di due antenati ominidi: una scimmia antropomorfa risalente a 10 milioni di anni fa e chiamata Chororapithecus abyssinicus, trovata nel triangolo di Afar, nell'Etiopia orientale, e il Nakalipithecus nakayamai, risalente anch'esso a 10 milioni di anni fa.

Energia geotermica

La Great Rift Valley ha un grande potenziale per la produzione di energia elettrica da fonte geotermica. Il Kenya nel 2013 aveva a disposizione una potenza installata di 212MW geotermici e diversi nuovi impianti sono in costruzione; nel 2021 risulta produrre per via geotermica il 38% dell'energia che utilizza.

Note

Altri progetti

Collegamenti esterni

Valli dell'Asia
Valli dell'Africa
Valley


How bout we invite THIS GUY! Takashi Amano Takashi Amano (天野尚 Amano Takashi?, born July 18, 1954) is a photographer, designer and aquarist. His interest in aquaria led him to create the Japanese company Aqua Design Amano. Amano is the author of Nature Aquarium World (TFH Publications, 1994), a three-book series on aquascaping and freshwater aquarium plants and fish. He has also published the book "Aquarium Plant Paradise" (T.F.H. Publications, 1997). A species of freshwater shrimp is named the "Amano shrimp" or "Yamato shrimp" (Caridina multidentata; previously Caridina japonica) after him. After discovering this species' ability to eat large quantities of algae, Amano asked a local distributor to special order several thousand of them. They have since become a staple in the freshwater planted aquarium hobby. He has also developed a line of aquarium components that are known as ADA, and his "Nature Aquarium" article series appears monthly in both Practical Fishkeeping magazine in the UK, and Tropical Fish Hobbyist magazine in the US. <excerpt from his Wiki entry> Maybe Partner with the National Aquarium to get him to visit. He is kind of the Miyazaki or Otomo of Fishtanks.
I have offered To PAY for John Ringo to show up. Personally out of pocket. I tried to enlist his publisher to send him.. (HI TONI!) John goes to Dragon Con and By the Time of Otakon he is in full on prep for DC. Same for David Webber and Mike Z williamson. (Except mike is at Pennisc those two weeks then is gearing up for Dragon Con. ) Larry Corriea would show up but as a Parent to his daughter. He isn't keen on DC or Baltimore as a Place in general. the Problem is that Authors tend to go to Litterary cons NOT anime conventions. which is why Dragon Con is more Prefered for those guys. ANYway I dont know that you would enjoy the politics that goes along with those particular authors when they show up they tend to not quite give a POOP about Politically correct.
Sadaharu Oh - Life time Home Run king for Professional Baseball.
Chef Naomichi Yasuda <cribing notes from Anthony Burdain> Naomichi Yasuda is my master, my mentor as far as all things related to sushi, and my friend. For almost two decades, he was the man around whom the eponymous Sushi Yasuda in New York City revolved; one of the first, greatest and most important sushi chefs in America. Over many epically delicious meals at his restaurant, he taught me everything I know about sushi. How to eat it. Where it comes from. Which is more important, the fish or the rice? Is fresher necessarily better? Everything. He is also a fan of classic cinema, an intellectual, a deep thinker, and a lifelong practitioner of and competitor in bare-knuckle (Kyokushin) karate -- both sanctioned fights and underground matches. His massive fists, enlarged knuckles (from years of pounding cinderblock walls) and terrifying forearms are, to say the least -- unusual for a sushi chef. He is unusual in other ways as well. He was among the first to employ female sushi chefs at his restaurant, a diversion from accepted practice once unthinkable. (Women's hands were believed, by old school dudes, to be "too hot" to handle sushi without "ruining" it). He was among the first real masters to employ Westerners behind the bar. He was certainly the first acclaimed sushi chef I know of who not only admitted, but proudly boasted of freezing some of his fish, (much of the fish you eat in sushi bars is, in fact, at one point, frozen), giving the dates or "vintage" of each fish he'd blast frozen in a medical freezer to "cure" it in a desirable way. Many varieties of fish, Yasuda taught me, are in fact, improved by freezing. Perhaps the most unconventional thing he did was disappear. A few years ago, at the top of his game, his always-packed restaurant considered among the best -- if not THE best -- sushi bar in New York, he announced he would, in his fifties, be leaving for Japan, to start all over again, at the bottom, opening a tiny, modest, low-overhead sushi bar in Tokyo, where he could prove himself anew, show Japanese that what he had done in America, he could do in Japan. Sushi Yasuda still runs quite nicely in New York. It still bears his name. It is still excellent. Naomichi Yasuda, incredibly enough, though, says he has nothing to do with it. He is a fascinating subject and a great chef with an engrossing story. In this episode, we get to know him a bit, and explore where his unique style comes from. Can the very different disciplines of fighting and sushi-making be said to connect? You will be surprised, I think, at the answer.
come on wouldn't it be cool to have the Prime Minister of a NATION or the EMPEROR of a Nation Do the opening ceremonies? and we have had plenty of non anime guests over the years The Two Sumo wrestlers this year the Samisen players a few years before that not to mention all the various live action movies that are shown (more this year than any year I can remember) 2 years ago we had a guest do a HUGE pannel on Tea and Tea ceremony, This year we had a series of well attended (to the point where I couldn't get in) panels on Drinking culture, and Whiskey and Sake.. (3 pannels on alcohol on the 21st birthday of Otakon no less) We have any of a few different folks from non animated action shows (We have had at various points the Red, green and White Rangers in attendance from Power Rangers) There is a LOT more to Japan than anime mate and there is a lot more interesting stuff out there than anime given the fact that much of the anime you are watching has tie ins to the culture itself especially the stories and traditional kinds of story telling that are done in Anime. Much of those come out of the traditions of Kabuki and Noh theater. The concept of Manga is a modern outgrowth of kibyôshi from the 18th and early 19th centuries and even some of the syle queues still come from that era of Ukiyo-e (Look at the limited backgrounds in many scenes in for instance Naruto or Bleach where the foreground is detailed but the deeper you get into the background the detail sort of mists out) But Hey what the F do I know I'm just a douche in a Mario costme 3 days a year.
come on wouldn't it be cool to have the Prime Minister of a NATION or the EMPEROR of a Nation Do the opening ceremonies? and we have had plenty of non anime guests over the years The Two Sumo wrestlers this year the Samisen players a few years before that not to mention all the various live action movies that are shown (more this year than any year I can remember) 2 years ago we had a guest do a HUGE pannel on Tea and Tea ceremony, This year we had a series of well attended (to the point where I couldn't get in) panels on Drinking culture, and Whiskey and Sake.. (3 pannels on alcohol on the 21st birthday of Otakon no less) We have any of a few different folks from non animated action shows (We have had at various points the Red, green and White Rangers in attendance from Power Rangers) There is a LOT more to Japan than anime mate and there is a lot more interesting stuff out there than anime given the fact that much of the anime you are watching has tie ins to the culture itself especially the stories and traditional kinds of story telling that are done in Anime.


text:
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    #include <windows.h>
    typedef HANDLE mxrd_t;
#else
    #define __DEF_MUTEX_POSIX
    #include <pthread.h>
	typedef pthread_mutex_t mxrd_t;
#endif

#endif

redpajama_set_name: RedPajamaGithub


Gaterman Mfg. Co., Inc. has been in business since 1903 in the Manitowoc, Wisconsin area. The company, fourth generation and family owned, is dedicated to fulfilling your steel fabricating needs. We offer full sheet metal fabrication services to the Manitowoc area and surrounding counties.
We specialize on short-run contract sheet metal fabrication, prototyping, sheet metal repairs, and plant services fabrication. Our skilled and experienced employees are committed to getting the job done right, as witnessed by numerous quality awards from our customers. We have an extensive materials inventory and the equipment to take the job from concept to delivery. We pride ourselves on our personalized customer service, flexibility, and timely deliveries. details . . .
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Charge More Money and Get More Business.
In the retail world, price only stands for the price of a product. But in the marketing world price stands for so much more. Because once your brand is established and your customer is interested in purchasing your product or service then price has as much to do with the ultimate purchase decision as anything.
The intuitive logic is that customers will always buy at a lower price. But don't you believe this old assumption because it simply is not true.
The value of many products actually increases with an increasing price. Consultants, luxury goods, and engagement rings are all examples of this seemingly backward pricing phenomenon.
Imagine the disaster when the romantic but frugal boyfriend wheels in a 55-gallon drum on a hand truck. "I know you love that fancy French perfume honey, but this smells exactly the same and now you'll have enough for a lifetime." If our hapless suitor presented his gift on the evening of Valentine's Day we can just imagine who he'll be with later that night — nobody.
By establishing a powerful brand that is laser-focused on your buyer you can actually use pricing — and higher pricing — to make your product more desirable than the competition. And with the extra money you earn you'll be able to put more time, effort, and resources into doing an even better job for your customer which creates a win-win-win situation for everybody.
And if you order before midnight tonight, you can get your choice of some great deals.
These deals end at Midnight tonight so do not delay. Order now and charge more money tomorrow.


The rest of my Thanksgiving preparations went really well. And what I really mean to say is… after the debacle that was my pumpkin pie baking Wednesday night, I managed to cook everything else perfectly.
Okay, let me tell you what happened! Remember how I tested pies ALL MONTH until I came up with the perfect pie recipe? Well, I screwed up when I doubled the recipe, and didn't put the right amount of pumpkin in, and in the second batch, forgot the salt to boot! (I'm good at math, it was just a stupid mistake!) I didn't realize it until was adding the doubling instructions to my recipe that I had goofed! Then I tried not to lose my mind. We tasted pieces from both of the batches, and they were still good. They still tasted like pumpkin pie, they just weren't as good, so I was a bit disappointed in myself, especially after all that hard work! I did have half of my perfect pie left from the night before, so I served that one first. The one I made crustless was a success, however. I added that info to the recipe post too.
Everything else came out great. The Field Roast Hazelnut Cranberry Roast en Croute was a nice addition to the meal. Both of my parents really enjoyed it and so did we. I was still glad I made the Tofurky though, because the field roast was more like a pastry wrapped sausage. The Tofurky filled the turkey void, and even made the house smell like Thanksgiving, according to Thomas : ) More people might have tried my fake meats had my aunt not shooed them away by saying, "that's made from tofu, you won't like that!" It was actually seitan, but I didn't feel like getting into it. More delicious fake meat for me!
Out of the oven! I misted both the pan & the roast with a little olive oil before baking.
The brussel sprouts were a big hit even though they got a bit overdone from being reheated at my mom's sans foil, too close to the element. No one seemed to notice. As an aside, I did think that the medium sized ones, halved, that I used for my pre-Thanksgiving trial dinner had a better texture than these mini ones left whole.
Here's a pic of the crustless pumpkin pie I made.
I lightly greased the pie plate with some oil on a paper towel and poured in the filling. It actually sliced and served surprisingly well, and only 101 calories and 0.8g fat!
This was actually a picture of my breakfast plate, the next day!
Ah, the beauty of leftovers. This was brunch the next day. Cornbread, Tofurky and roasted veggies, mashed potatoes, field roast, stuffing, cranberry sauce and sweet potatoes with pecan topping. SO GOOD! The only non-vegan dishes that were served ended up being the ones my aunt brought, which were turkey, green bean casserole and squash. So I had plenty to eat. She did take home a pie though, and texted me later that it was delicious. I only wish I had been able to send everyone off with the correct version of the recipe, but hey, there's always next year!
Hope you and yours had a great one!
I have one of those Field Roasts set aside for Christmas… I can hardly wait! I'm still trying to track down a Tofurky. Trader Joes didn't have any last time I checked. Your Thanksgiving looked delicious!


Gear up, space tourists! Richard Branson's Virgin Galactic is just weeks away from reaching space.
In a recent interview with CNBC, Branson revealed the company is planning to take its VSS Unity spaceplane into orbit as part of a test flight.
The move, a first for Virgin Galactic, will be a major step towards enabling quick manned space trips in coming months.
Jeff Bezos' affair with TV host led to divorce?
Virgin Galactic weeks away from first-ever human spaceflight?
Virgin Galactic hopes to use VSS Unity to enter the nascent market of space tourism and send paying passengers on quick orbital trips.
The flight, including the ascent and descent, would take nearly two and a half hours, while the microgravity of space will be experienced for a few minutes.
VSS Unity will hit speeds faster than sound during flight and then glide back.
However, in order to make that happen, Virgin Galactic has been conducting a series of extensive tests with the spaceplane.
It has already conducted high-altitude glides and powered flights, leaving powered spaceflight to be conducted.
If this happens in a matter of weeks (as suggested by Branson), Virgin will be pretty close to taking humans into space.
What will happen in the upcoming spaceflight?
In the upcoming test, the pilots of VSS Unity will try to burn its rocket boosters for a longer period.
The move will not just increase the speed of the spaceplane (estimated to be faster than Mach 3 or 4,000km/hr), but also take it higher than ever and ultimately into orbit, some 110km above Earth.
After this, they'll glide back to the ground.
Within a few months of completing the test spaceflight, Branson will also head on a space voyage.
Speaking to CNBC, he stated he's been working and training to spend time in microgravity.
"We will be in space with people not too long after that so we have got a very, very exciting couple of months ahead," he added in the statement.
What would be the cost of Virgin Galactic's space trip?
Though it remains unclear when exactly Virgin Galactic's first passenger trip will happen, but it is imperative to note the tickets for the same have already been sold, for $250,000.
Some 800 people have purchased the tickets since they went on sale 15 years ago.
Branson has said the price won't come down any time soon, but could down to $40,000 in 10 years.
Virgin Galactic started its mission all the way back in 2004.
However, its years of progress was jeopardized when the first spaceplane, VSS Enterprise, disintegrated during a test flight in 2014 and crashed in the Mojave desert, killing one pilot and injuring the other.
The incident disrupted Virgin's efforts, but they continued and came back stronger with the introduction of VSS Unity in 2016.
That said, it is important to note Virgin Galactic isn't the only player heading into space tourism.
Elon Musk's SpaceX, which has mastered the art of reusability with its Falcon 9, has already booked a passenger for a round trip to Moon, while Jeff Bezos' Blue Origin has also been conducting tests to fly paying passengers to orbit and back.
Days after revealing a major data breach, social media giant Facebook has finally confirmed the information hackers stole during the attack - and it's pretty scary.
Reliance Infrastructure's Mumbai Metro has partnered with PayPal, one of the biggest platforms in the world for global online payments.


text:
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Home / All About Baseball / Major Leagues / Grandy-Man Heading West: Mets Deal Outfielder To Dodgers
Grandy-Man Heading West: Mets Deal Outfielder To Dodgers
By David Conde
Photo by Gabe Rodriguez
The New York Mets are continuing with their fire sale as Curtis Granderson was dealt late Friday night to the Los Angeles Dodgers for a player to be named later.
Granderson came over to the Mets in December 2013, after spending his first 10 seasons in the majors with the Detroit Tigers and then the New York Yankees. He didn't put up the huge power numbers that he did with the Yankees, but he was a consistent force on the Mets especially when he heated up during the season.
In four seasons with the Mets, starting back in 2014, Granderson batted a combined .239, with 95 home runs, and 247 RBIs. In 2015, he helped lead the Mets into the World Series against the Kansas City Royals, and he led the team with three home runs in the series.
Not only was Granderson a presence on the field, he was also a positive influence off the field. He always gave time to all the fans, signing autographs before the game and showcased his many accomplishments off the field with the Grand Kids Foundation, a charity geared towards aiding positive youth development via education, physical fitness and nutrition. His site also states that it provides the tools and resources necessary for educational and societal advancement.
But Granderson was also a major figure in the Mets clubhouse and made sure to be there for the younger players. Outfielder Michael Conforto spoke to the media about what Granderson has meant to him, "It's tough to see him go. He's a huge part of this team, as far as on the field and off the field, he plays a huge part of who we are. We'll definitely remember the way he goes about his business and hopefully try to do some things like he does."
The Dodgers have gained a veteran that has peaked over the last few weeks. Back in June, Grandy hit .316 with eight home runs and 15 RBIs, but then he had a horrible July where he hit only .159 with one home run and 4 RBIs. But August has been good to Granderson, as he is hitting .256 in the first 19 days, and has six home runs and 14 RBIs.
Granderson goes from a team in the rebuilding stages to a team who is running away with the NL West Title and huge favorites to win the World Series. Granderson has yet to win a title appearing in the Fall Classic twice in his career and coming up on the short end of the stick twice. But now at the tail end of his career, the 35-year-old takes his hot bat and goes West with the best chance to win it all and finally end his drought.
Good Luck Grandy-Man!!!
Co-Founder/Executive Editor at Legends On Deck®
David is the Founder and Executive Editor of Legends On Deck®. He was a Senior Editor for MetsMerizedOnline, MetsMinors.net and Contributor for Hardballchat.com. David's idea forLegends on Deck is to be a site where passionate fans of the game have a place to read about their favorite baseball teams. The site is also for people who had a talent to write or even take photographs and have the platform to share. The passion of following the Minor Leagues can be seen all over the site as the players journey's are what what drives the LOD team to share their content. David's passion is making available a site that people can have fun sharing and reading.
You can also reach out to David at daconde@legendsondeck.com
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Peter Jackson's Middle-Earth Movies Get Gigantic 31-Disc Box Set
Matt Singer Published: September 9, 2021
It's only fitting that two of the biggest film series in history are getting maybe the most epic box set ever. Just in time for the 20th anniversary of The Lord of the Rings: The Fellowship of the Ring, all six of Peter Jackson's J.R.R. Tolkien adaptations — The Lord of the Rings: The Fellowship of the Ring, The Two Towers, and The Return of the King, and The Hobbit: An Unexpected Journey, The Desolation of Smaug, and The Battle of the Five Armies — are getting a new combined 31-disc box set.
No, that's not a typo. It's six movies on 31 discs. 31! (Plus digital codes!) You're going to have to buy a new DVD shelf just to store this thing.
What's being billed as the "Middle-Earth: Ultimate Collector's Edition" set contains both the theatrical and extended versions of all six of the films on 4K Ultra HD and Blu-ray. (All six films have also been remastered from the 4K master.) According to the press release, the extras included in the set include "the original Cannes Film Festival presentation reel, and footage from the recent Alamo Drafthouse cast reunion featuring director Peter Jackson along with cast members Sean Astin, Cate Blanchett, Orlando Bloom, Billy Boyd, Ian McKellan, Dominic Monaghan, Viggo Mortenson, Andy Serkis, Liv Tyler, and Elijah Wood."
The set comes packaged in a huge "puzzle box" that includes a 64-page booklet, art cards, posters, and more. You can see how the whole thing unfolds below:
There isn't a hardcore Lord of the Rings fan alive that doesn't already own all of these films, and likely on multiple home video formats. So it's not like these films have been unavailable for the last 20 years. Still, the extensiveness of the package will probably prove tough to resist — provided you can scrounge up the $249.99 asking price. The set goes on sale on October 26.
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Source: Peter Jackson's Middle-Earth Movies Get Gigantic 31-Disc Box Set
Filed Under: J.R.R. Tolkien, Peter Jackson, The Hobbit, The Lord of the Rings
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text:
/*
 * config.h
 *
 *  Created on: 02.09.2010
 */

#ifndef CONFIG_H_
#define CONFIG_H_

#include <string>

struct MainConfig
{
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		int windowHeight;
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		float minFrameRate;
		int renderMethod;
	} video;
	float widgetsPosZ;
	int mapTileSize;
	std::string mapDefaultTile;
	int dayLength;
	int actionInterval;
	int maxSpawn;
	int maxEdibles;
	int playerDies;
	std::string default_image;
	struct {
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		std::string scripts;
		std::string image;
		std::string normals;
		std::string fonts;
		std::string proto;
		std::string shaders;
		std::string meshes;
	} path;

	float _tileAngle;
};

bool loadMainConfig();

#endif /* CONFIG_H_ */