F. MG132 treatment method did not direct to a statistically important increase in the proportion of apoptotic cells in p53+ bone marrow at 4 and 6 hours post-remedy with MG132 on your own or with MG132 and amsacrine. Apoptotic cells had been established from counting the proportion of energetic caspase-three-good cells in bone marrow from immunohistochemistry-stained sections and light-weight microscopy. 1675201-83-8The share of constructive cells per total cell depend (five hundred cells) was compared in between treated and untreated cells the benefits are expressed as the mean SD (n = 3) drastically over represented was glycolysis I, followed by creatine-phosphate biosynthesis, sucrose degradation V, and gluconeogenesis I (P < 0.05). The disease and disorder analysis showed the strongest correlation with hematological disease, followed by immunological disease and inflammatory disease (P < 0.05). For validation of proteins in the thymus, western blotting showed increased protein abundance for 8 proteins in p53+ treated cells (Table 1 and examples given in S2B and S2C Fig.). These proteins included those involved in metabolism, such as ATP5B, protein biosynthesis and protein turnover EEF2, PSMA1, PSMA3, and PSMB8, and those involved in cell cycle progression such as lamin B1. Proteins differentially expressed in thymus cells fitted into three networks using Ingenuity software. All proteins are targets of ubiquitin C (score 31). Proteins altered in p53+ treated cells compared with untreated cells formed a network of four proteins centred on myc and tumor necrosis factor (TNF) nodes (S2D Fig.). A comparison of proteins altered in p53+ treated cells compared with p53-, mpro, and 122p53 treated cells were centred on a network of YWHAZ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide) signaling (score 41). The most significant canonical pathway in this network was p70s6k signaling, followed by P13K/AKT signaling, and cell cycle: G2/M DNA damage checkpoint regulation (P < 0.05). The disease and disorder analysis showed the strongest correlation with cancer (P < 0.05). Reduced alpha-enolase as part of a p53+ response was further investigated. The decrease in alpha-enolase in p53+ treated bone marrow and PBMCs was not due to reduced expression of ENO1 as determined by real-time PCR (Fig. 1C). There was no difference in ENO1 expression between p53+, and 122p53 untreated and treated cells (Fig. 1C). Reduced alpha-enolase in p53+ treated bone marrow was also evident from immunohistochemistry of bone marrow and PBMCs following amsacrine treatment (Fig. 1D). The percentage of alpha-enolase positive cells was decreased in p53+ treated bone marrow (P < 0.0001) and PBMCs (P < 0.0001) compared with p53+ untreated cells and treated and untreated p53- and 122p53 cells. Representative photomicrographs to illustrate a reduction in alpha-enolase positive cells in p53+ treated bone marrow compared to p53+ untreated bone marrow are shown in S3 Fig. To investigate the importance of ubiquitin-associated proteasome degradation for reduced abundance of alpha-enolase, bone marrow from p53+ and p53- mice was pre-treated with the ubiquitin-associated proteasome inhibitor MG132 prior to amsacrine treatment and the abundance of alpha-enolase determined by western blotting. The addition of MG132 to p53+ cells effectively stabilized alpha-enolase at 4 and 6 hours post-amsacrine treatment compared to p53+ cells that were treated with amsacrine alone (Fig. 1E). No effect on alpha-enolase was evident in p53- cells in the presence of MG132 (Fig. 1E). Treatment with MG132 can increase apoptosis [33]. To test whether an increase in apoptosis with MG132 treatment was a confounding factor, the percentage of active caspase-3-positive cells was counted in p53+ bone marrow following treatment with MG132 alone or the combined treatment of MG132 and amsacrine. No significant differences were found in apoptotic cells after 4 or 6 hours with MG132 alone or when combined with amsacrine in comparison with untreated cells cultured for 4 hours. There was a trend toward increased apoptotic cells at 6 hours with MG132 and amsacrine treatment however, the difference was not statistically significant (Fig. 1F). In summary, the reduction in proteins upon a p53+ response to DNA damage is at least in part due to ubiquitin-associated proteasome degradation.Although 122p53 bone marrow, thymus, and lung showed no global changes in protein abundance compared with the other mutants and p53+ untreated cells, individual proteins showed aberrant abundance (Table 1). Seven of these proteins were validated by western blotting to identify possible mechanisms by which 122p53 promotes tumorigenesis. In bone marrow -enolase was increased. In thymocytes (S2C Fig.) TKT and TPT1 were increased in untreated and treated 122p53 cells. Heterogeneous nuclear ribonucleoprotein K was increased in 122p53 untreated cells (S2C Fig.). In the lung valosin-containing protein (VCP) was increased in 122p53 cells (data not shown).Other studies have established a link between overexpression of -enolase and increased -enolase on the cell surface [24]. This link, and the finding of increased -enolase in 122p53 cells, was the basis for determining whether -enolase was increased on the surface of 122p53 PBMCs. PBMCs were separated into cytosol and cell membrane fractions and the amount of alphaenolase in each fraction measured using western blotting. Increased alpha-enolase was present in the membrane and cytosolic fractions of 122p53 cells (Fig. 2A) compared with p53+ and p53- cells in 3 separate experiments. p53+ and p53- cells showed minimal alpha-enolase in the cell membrane (Fig. 2A). These results suggested that the 122p53 allele led to increased alpha-enolase in the cytosol and cell membrane.Increased alpha-enolase on the cell surface of 122p53 PBMCs would be expected to elicit a pro-inflammatory response to plasminogen stimulation [246]. To determine if this occurs, 122p53, p53+, and p53- PBMCs were pre-treated with plasminogen, and the concentration of the pro-inflammatory cytokine TNF-alpha released into culture media measured by ELISA (Fig. 2B). 122p53 PBMCs had increased TNF-alpha concentrations compared with p53+ and p53- cells with and without plasminogen induction. In the vehicle control treated cells the concentration of TNF-alpha was 5.6-fold higher in 122p53 PBMCs compared to that in p53+ (P < 0.01) and 2-fold higher compared to p53- cells (P < 0.05). Following plasminogen treatment the concentration of TNF-alpha increased 3.3-fold in 122p53 PBMCs compared to that treated with the vehicle control alone (from 317 76 pg/mL without plasminogen to 1033 252 pg/mL with plasminogen treatment, P < 0.01). The induction of TNF-alpha following plasminogen treatment in 122p53 PBMCs required plasminogen to plasmin activation, as evidenced by a reduction in plasminogen-induced TNF-alpha expression upon co-treatment with the plasmin activation inhibitor TXA (Fig. 2B, P < 0.01). In p53+ cells, plasminogen treatment did not induce TNF-alpha compared with cells treated with the vehicle control. In p53- cells a trend toward increased TNF-alpha occurred that did not reach a statistically significant level.Increased alpha-enolase on the 122p53 PBMC cell membrane. A. Increased alpha-enolase is present on the cell membrane of 122p53 PBMCs compared to that on p53- and p53+ PBMCs. The cell membrane and cytosolic fractions of untreated PBMCs from p53+, p53-, and 122p53 mice were separated and subjected to western blotting with an antibody to alpha-enolase. -actin, FAK, and CD45 were used as loading controls for total protein, cytosolic, and cell membrane fractions, respectively. B. Increased TNFalpha was released from 122p53 PBMCs following plasminogen stimulation compared with that from p53+ and p53- PBMCs. PBMCs were pre-incubated with plasminogen (lys-plasminogen) with or without the NFB inhibitor BAY 11082 (2.5 M for 90 minutes), or the inhibitor to plasmin activation (TXA, 10 mM), or vehicletreated only (VC). Following pre-incubation and prior to TNF-alpha measurement, tissue plasminogen activator (3 nM) was added, and the amount of TNF-alpha secreted in culture media was measured by ELISA. The results represent the mean SD (n = 3 for each measurement). , P < 0.001, , P < 0.01, P < 0.05.The induction of TNF-alpha by plasminogen can involve NF-kappaB signaling [25]. To test whether the increased TNF- in 122p53 PBMCs was due to NF-B signalling, 122p53 PBMCs were pre-treated with the NF-B inhibitor BAY11082. Results showed that the increase in TNF-alpha concentration following plasminogen treatment was obliterated upon NF-B inhibition, P < 0.001 (Fig. 2B). Overall, these data suggest that increased TNF-alpha expression in 122p53 cells involved NF-B signaling. When a similar experiment was performed with p53+ cells incubation with BAY11082 did not reduce TNF-alpha concentrations, which remained similar to those in cells treated either with the vehicle control or plasminogen (Fig. 2B). In p53- cells BAY11082 treatment did not alter TNF-alpha concentrations compared with those treated with the vehicle control only. However, BAY11082 treatment did reduce TNF-alpha concentrations in cells treated with plasminogen (P < 0.01). This suggests that NF-B induction of TNF-alpha suppressed most robustly by a wild-type p53 response. Alpha-enolase may not be the only plasminogen receptor increased at the 122p53 cell surface. Western blots for an alternative plasminogen receptor, histone H2B, using the separated transmembrane cellular component showed a slight increase in histone H2B at the 122p53 compared to that on the p53+ PBMC cell membrane (Fig. 3A) [34]. To determine that the increased TNF- alpha in 122p53 PBMCs following plasminogen stimulation was due to alphaenolase, the TNF-alpha measurements were repeated using 122p53 PBMCs incubated with LPS in addition to plasminogen to increase the amount of alpha-enolase on the 122p53 PBMC cell surface and in separate incubations antibodies toward alpha-enolase where included to block plasminogen binding. The addition of LPS did lead to increased alpha-enolase on the 122p53 PBMCs cell surface (Fig. 3B), and to increased TNF-alpha compared with 122p53 PBMCs incubated without LPS (Fig. 3C, P < 0.01). A reduction in TNF-alpha was also found in the presence of alpha-enolase antibodies in 122p53 PBMCs, with no reduction in TNF-alpha found with the IgG control (Fig. 3C, P < 0.001). These results are consistent with increased alpha-enolase function as a plasminogen receptor on the 122p53 cell membrane.Increased plasmin activity followed by extracellular matrix degradation as a result of increased alpha-enolase on the 122p53 tumor cell surface would provide an explanation for why 122p53 sarcomas metastasize more rapidly compared to those from p53- mice. The cytosolic and membrane fractions were separated in 6 sarcomas, 3 from 122p53 and 3 from p53- mice. The amount of alpha-enolase in each fraction was measured using western blotting. Increased alpha-enolase was present in the membrane fraction of all 3 tumors from 122p53 mice compared with those from p53- mice. The results from 2 tumors per genotype are shown in Fig. 4. The finding of increased alpha-enolase on the 122p53 tumor cell surface is supportive of alpha-enolase having a role in tumor invasion in the 122p53 model.The results of the complete proteome in response to DNA damage obtained from this study highlight p53-directed protein changes rather than transcript responses. This has enabled us to discover more about how p53 responds to stress and show for the first time that p53 function increased -enolase function on the 122p53 cell membrane. A. Alpha-enolase was not the only plasminogen receptor increased on the 122p53 cell membrane. The cell membrane and cytosolic fractions of untreated PBMCs from p53+ and 122p53 mice were separated and subjected to western blotting with an antibody to histone H2B and alpha-enolase. -actin, FAK, and CD18 were used as loading controls for total protein, cytosolic, and cell membrane fractions, respectively. B. LPS was added to enhance the amount of alpha-enolase on the 122p53 PBMCs cell membrane. The cell membrane and cytosolic fractions of untreated 122p53 PBMCs or those incubated with LPS (5 g/mL for 6 hours) were separated and subjected to western blotting with an antibody to alpha-enolase. FAK and CD18 were used as loading controls for cytosolic and cell membrane fractions, respectively. C. TNF-alpha released from 122p53 following pre-incubation with lys-plasminogen and LPS (5 g/mL for 6 hours) with or without two anti-alphaenolase antibodies (each at 15 g/mL) to block plasminogen binding or rabbit IgG as a control (30 g/mL). Prior to TNF-alpha measurement, tissue plasminogen activator (3 nM) was added and the amount of TNFalpha secreted in culture media was measured by ELISA. The results represent the mean SD (n = 3 for each measurement) , P < 0.001, , P < 0.01 affects the amount of alpha-enolase in the cell. A wild-type p53 stress response led to a reduction in alpha-enolase. A mimic for the human 133p53 isoform showed that this isoform might increase alpha-enolase, leading to plasminogen activation and increased pro-inflammatory cytokine production. Alpha-enolase is found in almost all tissues and is one of the most abundantly expressed proteins in the cytosol, where it is best known for its role in glycolysis converting 2-phosphoglycerate to phosphoenolpyruvate [35]. It is commonly associated with differential expression in proteomic studies, which reveal that alpha-enolase is often altered when normal and diseased tissues are compared [36]. The results of this study suggest alpha-enolase is often found in proteomic studies due to different p53 states being compared. Restricting alpha-enolase activity is consistent with the well-demonstrated role of wild-type p53 as a tumor suppressor. 22560567Alpha-enolase provides a metabolic advantage to compensate for hypoxia and is overexpressed in many cancer types [27,370]. The reduction in alpha-enolase following a wild-type p53 DNA damage response was eliminated on inhibition of ubiquitin C, suggesting p53 reduces alpha-enolase by targeting it toward ubiquitin C-mediated degradation. In the current study the protein reported as alpha-enolase using western blotting corresponded to a size of approximately 48 kDa, which is consistent with the size of alpha-enolase and not that of the smaller-sized protein, myc-binding protein-1. That protein is also encoded by the enolase 1 alpha (ENO1) gene and has been shown by others to be degraded by ubiquitin-dependent degradation [41,42]. The amsacrine dose and the time point following treatment were chosen specifically to identify changes that occur before p53-directed apoptosis or cell cycle arrest was evident. Consistent with this, the increased -enolase on the 122p53 tumor cell membrane. Tumors from 122p53 mice had increased alpha-enolase at the cell surface compared to tumors from p53- mice. Sarcomas were dissected from 122p53 and p53- mice at necropsy, the cytosolic and cell membrane fractions were separated, and these fractions were subjected to western blotting using an antibody to alpha-enolase.
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