Author response: NMNAT promotes glioma growth through regulating post-translational modifications of P53 to inhibit apoptosis
Year: 2021
Type: peer-review
Abstract: Article Figures and data Abstract Editor's evaluation eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Gliomas are highly malignant brain tumors with poor prognosis and short survival. NAD+ has been shown to impact multiple processes that are dysregulated in cancer; however, anti-cancer therapies targeting NAD+ synthesis have had limited success due to insufficient mechanistic understanding. Here, we adapted a Drosophila glial neoplasia model and discovered the genetic requirement for NAD+ synthase nicotinamide mononucleotide adenylyltransferase (NMNAT) in glioma progression in vivo and in human glioma cells. Overexpressing enzymatically active NMNAT significantly promotes glial neoplasia growth and reduces animal viability. Mechanistic analysis suggests that NMNAT interferes with DNA damage-p53-caspase-3 apoptosis signaling pathway by enhancing NAD+-dependent posttranslational modifications (PTMs) poly(ADP-ribosyl)ation (PARylation) and deacetylation of p53. Since PARylation and deacetylation reduce p53 pro-apoptotic activity, modulating p53 PTMs could be a key mechanism by which NMNAT promotes glioma growth. Our findings reveal a novel tumorigenic mechanism involving protein complex formation of p53 with NAD+ synthetic enzyme NMNAT and NAD+-dependent PTM enzymes that regulates glioma growth. Editor's evaluation The authors found that NMNAT binds to p53 and that p53 is post-translationally regulated to control apoptosis in glioma models. Work found that depletion of NMNAT1 and NMNAT2 inhibits and that overexpression of the enzymes promotes glioma growth. Furthermore, depletion of NMNAT1/2 increases apoptosis and overexpression of the enzymes inhibits apoptosis upon cisplatin treatment. This is an exciting mechanism that extends NAD biology, p53 regulation, and the field of glioma pathogenesis. https://doi.org/10.7554/eLife.70046.sa0 Decision letter eLife's review process eLife digest One of the most common types of brain cancer, glioma, emerges when harmful mutations take place in the ‘glial’ cells tasked with supporting neurons. When these genetically damaged cells are not fixed or eliminated, they can go on to multiply uncontrollability. A protein known as p53 can help to repress emerging tumors by stopping mutated cells in their tracks. Glioma is a highly deadly cancer, and treatments are often ineffective. Some of these approaches have focused on a protein involved in the creation of the coenzyme NAD+, which is essential to the life processes of all cells. However, these drugs have had poor outcomes. Instead, Liu et al. focused on NMNAT, the enzyme that participates in the final stage of the creation of NAD+. NMNAT is known to protect neurons, but it is unclear how it involved in cancer. Experiments in fruit flies which were then validated in human glioma cells showed that increased NMNAT activity allowed glial cells with harmful mutations to survive and multiply. Detailed molecular analysis showed that NMNAT orchestrates chemical modifications that inactivate p53. It does so by working with other molecular actors to direct NAD+ to add and remove chemical groups that control the activity of p53. Taken together, these results show how NMNAT can participate in the emergence of brain cancers. They also highlight the need for further research on whether drugs that inhibit this enzyme could help to suppress tumors before they become deadly. Introduction Glioma is the most common intrinsic tumor of the central nervous system (CNS) and derives from the neoplastic glial cells or neuroglia (Goodenberger and Jenkins, 2012). Based on pathological criteria, gliomas are classified from WHO grade I to IV, among which the high-grade gliomas generally have a much poorer prognosis (Wesseling and Capper, 2018). Several major cellular signaling pathways associated with glioma have been well studied, including RTK/Ras/PI3K, p53, and RB signaling pathways (Cancer Genome Atlas Research Network, 2008). In addition, metabolism factors, such as IDH1/2, were found to play important roles in glioma (Yan et al., 2009). IDH1 is an enzyme of tricarboxylic acid (TCA) cycle in glucose metabolism and the main producer of NADPH (Molenaar et al., 2014). However, drugs targeting these pathways showed a limited clinical response, indicating a critical need for the mechanistic understanding of the metabolic requirement for glioma tumorigenesis. Nicotinamide adenine dinucleotide (NAD+) is an essential signaling cofactor that regulates cancer metabolism through its co-enzymatic function for many bioenergetic pathways, including glycolysis, TCA cycle, and oxidative phosphorylation (Hanahan and Weinberg, 2011). Multiple processes associated with NAD+ signaling are dysregulated in cancer, including DNA repair, cell proliferation, differentiation, and apoptosis (Chiarugi et al., 2012). Inherited polymorphisms and epigenetic repression of DNA damage repair genes are significantly correlated with the risk of gliomas, indicating that abnormal DNA damage repair plays important roles in glioma formation and progression (Chen et al., 2010; Qi et al., 2017). One of the key initiation events of DNA damage response is poly (ADP-ribose) polymerase (PARP)-mediated poly(ADP-ribosyl)ation (PARylation), the main process that consumes nuclear NAD+ (Amé et al., 2004). Moreover, NAD+-dependent SIRTs-mediated deacetylation regulates many oncogenes and tumor suppressor genes in cancer cells (Brooks and Gu, 2009). Consistently, a high level of NAD+ is observed in gliomas (Reddy et al., 2008; Tso et al., 2006), and 90% of gliomas are susceptible to NAD+ depletion (Tateishi et al., 2015). Therefore, it is critical for rapidly proliferating glioma cells to replenish the NAD+ pool for survival. In the past years, targeting NAD+ metabolism has been considered for cancer therapy, and most efforts have been focused on nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of the NAD+ salvage pathway, whose expression is increased in multiple types of cancer (Garten et al., 2015; Lucena-Cacace et al., 2018; Ohanna et al., 2018; Pylaeva et al., 2019). Disappointingly, several clinical trials of NAMPT inhibitors have failed due to low efficacy and high toxicities (Sampath et al., 2015), which demands the urgent consideration of an alternative target in the NAD+ metabolic pathway. Nicotinamide mononucleotide adenylyltransferase (NMNAT), the last enzyme in the NAD+ salvage synthetic pathway, has recently emerged as a potential candidate (Chiarugi et al., 2012). NMNAT has three isoforms in mammals with distinct subcellular localizations: NMNAT1, in the nucleus; NMNAT2, in the cytosol; and NMNAT3, in the mitochondria (Berger et al., 2005). Dysregulations of both NMNAT1 and NMNAT2 have been implicated in cancer. For example, NMNAT1 is considered a poor prognostic marker for renal cancer (Uhlén et al., 2015; Uhlen et al., 2017). Decreased NMNAT1 expression leads to epigenetic silencing of tumor suppressor genes (Henderson et al., 2017). Inhibition of NMNAT1 delays DNA repair and increases rRNA transcription (Song et al., 2013). In colorectal cancer, NMNAT2 upregulation correlates with the cancer invasive depth and TNM stage (Cui et al., 2016; Qi et al., 2018). In non-small cell lung cancer (NSCLC), NMNAT2 enzymatic activity is upregulated by SIRT3-mediated deacetylation process or p53 signaling (Li et al., 2013; Pan et al., 2014). Moreover, the depletion of NMNAT2 inhibits cell growth indirectly by reducing glucose availability in neuroblastoma cells (Ryu et al., 2018). These observations indicate the regulatory link between compartmentalized NAD+ synthesis and cellular metabolism and rapid cancer cell growth, and further underscore the potential of NMNAT as a viable alternative target in NAD+ synthetic pathway, given their aberrant regulation and critical role in cancer metabolism. In this report, to address the knowledge gap regarding the role of NMNAT in glioma, we adapted an in vivo glial neoplasia in Drosophila (Read et al., 2009) and discovered a genetic requirement for NMNAT in glioma growth. Combined with human glioma cell culture models, we characterized the mechanism of NMNAT in gliomagenesis. Our results identified the upregulation of enzymatically active NMNAT as an essential metabolic regulator for promoting gliomagenesis and revealed that NMNAT-sustained PARylation and deacetylation of p53 results in suppression of apoptosis, a key tumor-inhibitory response. Results NMNAT is upregulated in oncogenic Rasv12 induced glial neoplasia The Ras/Raf/ERK signaling cascade is one of the most conserved pathways both in Drosophila and human, and a major component of the MAP kinase signaling stress-response network (Morrison, 2012). RAS mutations are the most commonly found oncogenic alteration in human cancers, most frequently observed in KRAS (85%), and to a lesser degree in NRAS (12%) and HRAS (3%) (Simanshu et al., 2017). Upregulated RAS and mutant RAS have been detected in gliomas (Arvanitis et al., 1991; Guha et al., 1997; Knobbe et al., 2004; Rajasekhar et al., 2003), and activation of Ras has been used to model human glioma in Drosophila (Read, 2011; Read et al., 2009). Ras oncogene at 85D (Ras85D) is the Drosophila orthologue of human RAS. The constitutively active Ras85D mutation (G12V), Rasv12, has been suggested to be analogous to human oncogenic RAS mutation and used to induce tumor (Barbacid, 1987; Wu et al., 2010). We established a Drosophila glial neoplasia model by overexpressing Rasv12 in glial cells, driven by the pan-glial driver repo-GAL4 (Read et al., 2009). Green fluorescent protein (GFP) was co-expressed as a reporter to mark the Ras expressing cells. Under normal conditions, the Drosophila CNS is wrapped by perineurial, subperineurial, and ensheathing glia (Freeman, 2015). Powered with high-resolution quantitative brain morphology analysis (Brazill et al., 2018b), we analyzed glial neoplasia tissue using three criteria, (i) tissue double-positive for GFP and endogenous Repo expression; (ii) tissue mass consists of multiple layers of glia of at least 400 cells, and (iii) tissue mass volume greater than 12.4 × 103 μm3 (Figure 1—figure supplement 1). When Rasv12 was expressed in glia, numerous glial neoplasia tissues marked by GFP and Repo in the brain and ventral nerve cord (VNC) were detected as early as 100 hr after egg laying (AEL), and the volumes of glial neoplasia increased with age (Figure 1A, B and G). The brain tumors caused early lethality in pupal stage and greatly reduced survival rate (Figure 1H). Notably, compared with the normal brain (Figure 1C and E), we found significantly increased endogenous NMNAT in glial cells at both 100 and 150 hr AEL. NMNAT was most prominently increased in the nuclear region (Figure 1D and F), suggesting a possible role for NMNAT1, the nuclear isoform, in Rasv12-induced glial neoplasia formation in Drosophila. Figure 1 with 1 supplement see all Download asset Open asset NMNAT is upregulated in Rasv12-induced glial neoplasia in Drosophila. (A, B) Larval CNS at 100 AEL with glial expression of GFP+ GFP or Rasv12+ GFP was probed for F-actin (white), Repo (red), and DAPI (blue). The yellow dashed lines mark the boundary of glial neoplasia. The third and fourth rows show the boxed area of the first and second rows. (C–F) Larval CNS at 100 (C, D) and 150 (E, F) AEL. The second to fourth rows show the boxed areas in the first row. (C–E) Brains were probed for Nmnat (gray), Repo (red), and DAPI (blue). (F) Brains were probed for HRP (magenta), Nmnat (gray), F-actin (white), and DAPI (blue). Yellow dashed lines mark the glial neoplasia boundaries. (G) Quantification of the total glial neoplasia volumes in each fly. Data are presented as mean ± s.d., n=4. Significance level was established by one-way ANOVA post hoc Bonferroni test. (H) Survival rate. Data are presented as mean ± s.d., n≥3. Significance level was established by Chi-square test. (I–J) Nmnat intensity at 100 and 150 AEL. Data are presented as median ± quartiles, n≥3. Significance level was established by one-way ANOVA post hoc Bonferroni test. ***p≤0.001; ****p≤0.0001. Scale bars, 30 µm. AEL, after egg laying; CNS, central nervous system; NMNAT, nicotinamide mononucleotide adenylyltransferase. NMNAT is required for glial neoplasia development in Drosophila To determine whether increased NMNAT is required for glial neoplasia development, we used the RNAi approach to downregulate NMNAT expression in Rasv12-induced glial neoplasia cells (Brazill et al., 2018a). NMNAT RNAi-mediated knockdown in Rasv12 overexpression cells reduced NMNAT expression level to around 36% of wild-type flies (Figure 2—figure supplement 1). Interestingly, knocking down Nmnat drastically reduced both the volume and the number of individual Rasv12-expressing glial cells in the brain and VNC at 100 hr AEL (Figure 2A, C and D), demonstrating a strong antitumor effect of NMNAT inhibition in vivo. We further analyzed RNAi-mediated knockdown of NMNAT in normal glial cells (without Rasv12 expression) and found no growth inhibition (Figure 2—figure supplement 2), suggesting NMNAT is not essential for normal cell survival. Figure 2 with 3 supplements see all Download asset Open asset NMNAT is required for glial neoplasia growth in Drosophila. (A, B) Larval CNS at 100 AEL with glial expression of Rasv12+lacZ, Rasv12+NmnatRNAi, lacZ+Rasv12, PC+Rasv12, PCWR+Rasv12, and PD+Rasv12 was probed for F-actin (green), Repo (red), DAPI (blue), and Nmnat (gray). Each individual glial neoplasia is marked with dashed lines and numbered. The second and third rows show the high magnification of glial neoplasia areas in the first row. Scale bars, 30 µm. (C) Quantification of glial neoplasia volume in each fly. Data are presented as mean ± s.d., n≥7. Significance level was established by one-way ANOVA post hoc Bonferroni test. (D) Quantification of glial neoplasia number in each fly. Data are presented as mean ± s.d., n≥7. Significance level was established by one-way ANOVA post hoc Bonferroni test. (E) Survival rate of flies with glial expression of Rasv12 together with lacZ, PC, PCWR, or PD. Data are presented as mean ± s.d., n≥3. Significance level was established by chi-square test. *p≤0.05; **p≤0.01; ***p≤0.001; ****p≤0.0001. AEL, after egg laying; NMNAT, nicotinamide mononucleotide adenylyltransferase. Next, we tested whether upregulating NMNAT can promote glial neoplasia formation and growth. Drosophila has one Nmnat gene, expressing two protein isoforms through alternative splicing, a nuclear isoform Nmnat-PC and a cytosolic isoform Nmnat-PD. The Nmnat-PC (nuclear) and Nmnat-PD (cytoplasmic) isoforms share similar enzymatic activity but are differentially regulated under stress conditions (Ruan et al., 2015). In Rasv12-induced glial neoplasia, dramatically increased Nmnat is mainly observed in the nuclear region (Figure 1D and F), likely to be the Nmnat-PC (nuclear) isoform. To further evaluate the compartment-specific role of NMNAT during glial neoplasia formation, we generated flies expressing Rasv12 together with Nmnat-PC (nuclear) or Nmnat-PD (cytoplasmic). Consistent with the previous report, Nmnat-PC (nuclear) is highly enriched in the nucleus and colocalizes with the nuclear marker Repo, while Nmnat-PD is predominantly cytoplasmic (Ruan et al., 2015). Interestingly, overexpression of Nmnat-PC (nuclear), but not Nmnat-PD (cytoplasmic), significantly increased the total volumes of glial neoplasia (Figure 2B and C), while the number of glial neoplasia showed no significant difference among the groups (Figure 2D). The lethality of the flies (Figure 2E) was positively correlated with glial neoplasia size and overexpression of Nmnat-PC (nuclear) significantly increased the lethality. To determine whether the enzyme activity of NMNAT is required for glial neoplasia tumorigenesis, we generated flies expressing an enzyme inactive mutant Nmnat-PC (nuclear) isoform (PCWR) where two key residues for substrate binding were mutated (Figure 2—figure supplement 3; Zhai et al., 2006). We found that Nmnat-PCWR (nuclear) overexpression did not significantly affect glial neoplasia volumes or numbers or survival outcome when compared to the control (Figure 2C–E). These results suggest that nuclear enzymatically active NMNAT promoted glial neoplasia growth. NMNAT is essential to the proliferation of human glioma cells We next examined the function of NMNAT in human glioma cell proliferation, specifically human NMNAT1 (nuclear) and NMNAT2 (cytoplasmic) (Berger et al., 2005). Since approximately 51% of glioma are mutated for p53, we included two glioma cell lines with different p53 status, U87MG with wild-type p53, and T98G with a gain-of-function M237I mutation (Van Meir et al., 1994), to dissect common mechanisms of the role of NMNAT in glioma cell growth. We determined NMNAT1 and NMNAT2 protein levels in human glioma cells and normal astroglia cells (SVG p12). Compared to SVG p12 cells, NMNAT1 and NMNAT2 are increased in both glioma cells T98G and U87MG (Figure 3—figure supplement 3). Next, we manipulated the expression of NMNAT by siRNA-mediated knockdown and plasmid-mediated overexpression in T98G cells and monitored real-time cell growth using the xCELLigence platform (Ke et al., 2011). Interestingly, we found T98G cell proliferation was drastically inhibited when either NMNAT1 or NMNAT2 was knocked down (Figure 3A). This observation was confirmed and extended in an MTT assay (van Meerloo et al., 2011), where NMNAT1 or NMNAT2 knockdown reduced cell proliferation (Figure 3—figure supplement 1). In contrast, overexpressing NMNAT1 or NMNAT2 promoted cell growth (Figure 3D). Moreover, we used a plate colony formation assay to determine clonogenic survival (Franken et al., 2006), and found that knockdown of NMNAT1 or NMNAT2 reduced the colony numbers of T98G, while overexpression of NMNAT1 or NMNAT2 increased colony formation (Figure 3B–F). These results are consistent with the genetic dependency on NMNAT observed in the fly glial neoplasia models, suggesting the conservation of NMNAT function in promoting glioma cell growth and proliferation. Figure 3 with 3 supplements see all Download asset Open asset NMNAT expression is essential to the proliferation of human GBM cells. (A, D) The xCELLigence real-time cell analysis assay was used to monitor the growth index of T98G cells after NMNAT knockdown by transfecting siNMNAT1 or siNMNAT2, or after NMNAT overexpression by transfecting NMNAT1 or NMNAT2 plasmid. Cells transfected with siRNA control or DsRed were used as controls. (B, E) Colony formation assay was used to measure the colony formation capabilities of T98G cells after NMNAT knockdown by transfecting siNMNAT1 or siNMNAT2, or after NMNAT overexpression by transfecting NMNAT1 or NMNAT2. Cells transfected with siRNA control or DsRed were used as controls. (C, F) Quantification of the colony number in (B, E). Data are presented as mean ± s.d. n=3. Significance level was established by one-way ANOVA post hoc Bonferroni test. (G) T98G cell apoptosis was detected by flow cytometry after NMNAT knockdown. (H) Quantification of apoptotic cells rate of siRNA control, siNMNAT1-1, siNMNAT1-2, siNMNAT2-1, and siNMNAT2-2. The sum of Q2 and Q4 was quantified as apoptotic cells. Data are presented as mean ± s.d. n=4. Significance level was established by t-test. *p≤0.05; **p≤0.01; ***p≤ 0.001. GBM, glioblastoma multiforme; NMNAT, nicotinamide mononucleotide adenylyltransferase. Figure 3—source data 1 siRNA sequences for NMNAT1/2 knockdown and primer sequences for PCR. https://cdn.elifesciences.org/articles/70046/elife-70046-fig3-data1-v1.doc Download elife-70046-fig3-data1-v1.doc To further determine whether NMNAT is involved in glioma cell survival, we carried out a flow cytometric apoptosis detection assay through flow cytometry. We transfected siRNA targeting NMNAT into T98G cells and then analyzed Annexin V-FITC/PI by flow cytometric 72 hr Interestingly, we found that knockdown of NMNAT, at the knockdown rate of for NMNAT1 or at for NMNAT2, significantly increased the of apoptotic cells, including early apoptotic and apoptotic cells (Figure and We also examined the cell cycle of these cells. The cell cycle assay showed was increased in T98G cells with NMNAT1 knockdown (Figure 3—figure supplement These results suggest that NMNAT promotes glioma cell growth mainly through cell of NMNAT activation in glioma The are by to apoptosis and 2009). is the main a number of during revealed that nuclear and of play a critical role in the progression of apoptosis et al., 2018). The pathway is highly conserved in and Drosophila and 2011; Figure supplement To the role of pathway in Drosophila glial neoplasia, we examined tumor growth in flies with of the of In these glial neoplasia volume was significantly increased (Figure supplement and C), suggesting the important role of apoptosis in Drosophila glial neoplastic growth. To whether NMNAT regulates this we determined the and protein levels of in the glial with overexpression of different Nmnat We used Repo and DAPI to the region and observed a significant of levels in glial that Nmnat-PC (nuclear), compared with overexpressing lacZ, Nmnat-PCWR (nuclear), or Nmnat-PD (cytoplasmic) (Figure and In addition, when we knocked down Nmnat in Rasv12-expressing glial cells, we observed significant nuclear of (Figure and These results suggest that NMNAT is a regulator of glial neoplastic cell apoptosis in Drosophila. Figure with 1 supplement see all Download asset Open asset of NMNAT activation in glial neoplasia. neoplasia from expressing lacZ, PC, PCWR, or were probed for Repo (red), F-actin (green), DAPI (blue), and (gray). The the glial neoplasia The second and third rows are the high magnification of the boxed areas in the first row. Yellow dashed lines indicate the nuclear neoplasia from flies expressing or Nmnat RNAi were probed for Repo (red), F-actin (green), DAPI (blue), and (gray). Yellow lines indicate glial neoplasia boundary in the Yellow dashed lines indicate the of the nucleus and Scale bars, µm. (C) Quantification of the of nuclear intensity glial neoplasia. Data are presented as mean ± s.d. n≥3. Significance level was established by one-way ANOVA post hoc Bonferroni test. (D) Quantification of the nuclear glial Data are presented as median ± quartiles, n≥3. Significance level was established by one-way ANOVA post hoc Bonferroni test. ****p≤0.0001. NMNAT, nicotinamide mononucleotide adenylyltransferase. Next, we examined apoptosis and the activation of in human glioma cells. We found that knockdown of NMNAT to increased nuclear (Figure and analysis showed a of of (Figure indicating the activation of apoptosis and To the effect of overexpressing NMNAT on apoptosis, we cisplatin to induce apoptosis as the level of apoptosis in T98G glioma cells is low et al., significantly increased nuclear levels as Interestingly, overexpression of either NMNAT1 or NMNAT2 reduced nuclear in apoptosis (Figure and E), specifically the as shown by analysis (Figure Taken together, these results suggest that NMNAT promotes glioma growth by Figure with 2 supplements see all Download asset Open asset NMNAT activation in human glioma cells. T98G cells were transfected with siNMNAT1 or and with DAPI and T98G cells were transfected with DsRed (red), (red), or (red), with cisplatin hr after and with DAPI and (gray). The second and third rows are the high magnification of the boxed areas in the first row. In the third the intensity of is by a Scale bars, µm. (C) Quantification of nuclear intensity in Data are presented as median ± quartiles, Significance level was established by one-way ANOVA post hoc Bonferroni test. (E) Quantification of nuclear intensity in Data are presented as median ± quartiles, Significance level was established by one-way ANOVA post hoc Bonferroni test. F) were from T98G cells transfected with siRNA and with cisplatin for hr (F) for was considered as was used as an ****p≤0.0001. NMNAT, nicotinamide mononucleotide adenylyltransferase. Figure data 1 NMNAT activation in human glioma cells. Download of NMNAT increases DNA damage and nuclear p53 in glial neoplasia DNA is one of the of cancer. common cancer cells to the cell apoptosis by DNA damage are DNA damage repair, and cell apoptosis initiation and 2004). Since NAD+ plays important regulatory roles in both DNA damage repair and cell apoptosis, and NAD+ synthase activity is required for glial neoplasia growth (Figure 2), we next examined the effect of NMNAT on the DNA damage pathway in We first determined DNA damage by using a to a marker for DNA et al., 2013). We observed a significant of in Nmnat-PC (nuclear) overexpressing compared to that in Nmnat-PD (cytoplasmic), Nmnat-PCWR (nuclear), or overexpressing (Figure suggesting DNA damage level is in glial neoplasia with Nmnat-PC (nuclear) Figure Download asset Open asset Nmnat-PC inhibits DNA p53 activation in glial neoplasia. neoplasia from flies expressing lacZ, PC, PCWR, or were with (red), Repo (green), and F-actin The second and third rows are high magnification of the boxed areas in the first row. In the third the intensity of is by a neoplasia from flies expressing lacZ, PC, PCWR, or were with p53, Repo (green), F-actin (magenta), and DAPI (blue). The second and third rows are high magnification of the boxed areas in the first row. In the third the intensity of p53 is by a Yellow dashed lines indicate the nuclear Scale bars, µm. (C) Quantification of intensity in cells. The dashed the to the is as the Data are presented as median ± quartiles, n≥3. Significance level was established by one-way ANOVA post hoc Bonferroni test. (D) Quantification of nuclear p53 Data are presented as mean ± s.d., n≥3. Significance level was established by t-test. **p≤0.01; ***p≤0.001; ****p≤0.0001. We next examined the of endogenous p53 in glial neoplasia and found that while in control glial neoplasia cells p53 was with of p53 in the a significantly reduced nuclear p53 pool was found in Nmnat-PC (nuclear) overexpressing glial neoplasia cells (Figure and with the observation of DNA damage levels in Nmnat-PC (nuclear) overexpressing glial neoplasia cells, these results indicate that Nmnat-PC (nuclear) expression regulates p53 response to DNA to to DNA p53 is a key cell in response to DNA DNA repair when is limited DNA and induce apoptosis when DNA damage is and 2013). To the role of p53 in glial neoplasia development in we examined the effect of a p53 is to inhibit of p53 and affect et al., et al., 2004; et al., 2004). We analyzed glial neoplasia tissue volume with GFP and DAPI in the CNS of flies (Figure The glial neoplasia volume was significantly increased in flies compared to that in flies
Source: eLife
Language: English
Subfield: Genetics
Field: Medicine
Domain: Health Sciences
Open Access status: gold
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