Direct Transcriptional Target Of P53 Research Articles

BH3-only proteins in apoptosis at a glance

Apoptosis is a genetically programmed process for the elimination of damaged or redundant cells by activation of caspases (aspartate-specific cysteine proteases). Caspases cleave vital proteins, leading the cell to fragment into vesicles that are rapidly engulfed by phagocytes (for reviews, see [

Abstract PR2: Functional characterization of p53-regulated long intervening non-coding RNAs (lincRNAs

Abstract LincRNAs (long intervening noncoding RNAs) constitute a novel class of long, noncoding RNAs that have been implicated in regulating gene expression in diverse biological processes by modulating the activity of chromatin-modifying complexes. In collaboration with the Rinn laboratory at the Broad Institute, we have identified a set of lincRNAs that appear to be direct transcriptional targets of p53. These lincRNAs contain p53 binding elements in their promoter regions and are strongly upregulated in a p53-dependent manner upon DNA damage and in response to oncogenic stress. We have hypothesized that these lincRNAs may be new components of the p53 network and may contribute to p53 tumor suppressor function by regulating the expression of genes in cancer-relevant pathways. Using a combination of molecular genetic studies, cell biology, biochemistry, as well as gene targeting in the mouse, our aim is to dissect the biological function of these lincRNAs in vivo and in isolated cells. To date, we have generated a conditional knockout mutant for lincRNA-p21, one of the p53-regulated lincRNAs, which was recently shown to mediate gene repression in the p53 transcriptional network. Using this genetic tool, we plan to characterize the consequences of lincRNA-p21 loss in mice and in isolated mouse embryonic fibroblasts. Our goal is to identify genes that are regulated by lincRNA-p21 and to map out interactions between lincRNA-p21 and the p53 transcriptional network. We will also focus on dissecting the mechanism by which lincRNA-p21 may regulate the expression of target genes. We have also identified a lincRNA, linc9, which is induced by DNA damage in a dose-dependent manner. Even though the expression of linc9 appears to be p53-independent, our preliminary experiments indicate that linc9 may influence the outcome of p53 activation by promoting apoptosis in response to high levels of DNA damage. In sum, by studying the function and mechanism of p53-regulated and DNA damage-induced lincRNAs, we hope to achieve a better understanding of the role of long noncoding RNAs in modulating the p53 pathway and more broadly in regulating cancer-related processes. This abstract is also presented as Poster B19. Citation Format: Nadya M. Dimitrova, Tyler Jacks. Functional characterization of p53-regulated long intervening non-coding RNAs (lincRNAs) [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr PR2.

Highlights of This Issue

Bortezomib is the first proteasome inhibitor approved for the treatment of cancer. The determinants of resistance to bortezomib, however, remain poorly defined. Here, Weniger and colleagues used gene expression profiling to investigate the cellular response to proteasome inhibition in mantle celllymphoma. They found that induction of a strong NRF2-regulated antioxidant response in tumor cells during bortezomib treatment identified patients with superior clinical response. In contrast, high basal antioxidant capacity was associated with a reduced stress response and inferior clinical outcome. These results indicate that high cellular antioxidant capacity contributes to bortezomib resistance and may represent a therapeutic target.Malignant pleural mesothelioma (MPM) is an aggressive tumor that has high resistance to chemotherapy and radiotherapy. Genetic alteration of TP53 is rare in MPM, even though MPM exhibits antiapoptosis and cell cycle alterations suggestive of functional p53 deficiency. The microRNA-34s (miR-34s) are direct transcriptional targets of p53. Here,Kubo and colleagues found that miR-34b/c expression was frequently silenced by DNA methylation in MPM, resulting in loss of tumorsuppressive p53 function. This study provides new insights into the molecular pathogenesis of MPM and suggests that miR-34b/c could be a potential therapeutic target.Preclinical data suggest human Vα24Vβ11 natural killer T cells (NKT) cells, activated by α-galactosylceramide presented by dendritic cells (DC), have antitumor potential. In a phase I clinical trial,Nicol and colleagues evaluated the effects of administration route (intravenous vs. intradermal) and α-galactosylceramide-pulsed DC dose on NKT cell stimulation. They found that both administration route and DC dose had a major impact on NKT cell responses and on secondary immune effects consequent on NKT cell activation. Inflammatory flares involving tumor deposits were frequently observed. These data provide important insights into the design of immunotherapy studies using DC-based NKT cell stimulation.BNC105P is an inhibitor of tubulin polymerization that has vascular disrupting and anti-proliferative effects. In this issue, Rischin and colleagues report on a first-in-human phase I trial of this agent. The experimental design incorporated a number of pharmacokinetic and pharmacodynamic assessments.Dynamic contrast enhanced MRI was used to assess tumor blood flow following exposure to drug, and a novel immunoblot densitometry assay was developed that assessed the level of polymerized tubulin in PBMCs isolated from trial subjects.BNC105P was associated with pharmacodynamic changes consistent with its mechanism of action and had a favorable toxicity profile at the recommended dose level.

Abstract 4944: The impact of adenoviral gene transfer of microRNA-34b/c on malignant pleural mesotheliomas

Abstract Background: Altered expression of microRNA (miRNA) is strongly implicated in human malignancies. miRNAs are small non-coding RNAs that regulate translations of messenger RNAs of various genes. Similar to encoding genes, tumor suppressive miRNAs are silenced with DNA methylation. The miR-34b/c is direct transcriptional targets of p53 and has been reported to be down-regulated in human malignancies. Unlike other malignant tumors, mutation of TP53 gene is rare in malignant pleural mesotheliomas (MPMs). In our previous study, aberrant methylations of miR-34b/c were frequent in MPMs (−100%), resulting in the slicing of their expression. Ectopic expression of miR-34b/c by transit or stable plasmid transfection, that promoted physiological expression level of miR-34b/c, could inhibit the proliferation and invasion of MPM cells. In this study, we investigate the anti proliferative effect of adenoviral gene transfer of miR-34b/c on MPM cells to aim development of miR-34b/c gene therapy. Methods: We examined three MPM cell lines (H28, H290, and H2052) and a lung cancer cell line (H125). Three adenovirus vectors expressing miR-34b/c (Ad-miR-34b/c), p53 (Ad-p53) and luciferase (Ad-Luc) driven by CMV promoter were generated by homologous recombination and were infected to cell lines above. Expression of miR-34b/c was determined by quantitative PCR. Protein expression, cell proliferation, and cell cycle were analyzed by western blotting, MTS assay, and flow cytometry, respectively. Results: TP53 was wild-type and miR-34b/c was methylated in all three MPM cell lines, while TP53 was mutated and miR-34b/c was unmethylated in H125. After Ad-p53 transfer, p53 and p21 were up-regulated in all four cell lines. Of note, Ad-p53 transfer increased expression of miR-34b/c in H125 but not in all three MPM cell lines, resulting in a decrease of the cell viability in H125 but not in MPM cell lines. The expression of miR-34b and 34c were highly un-regulated after Ad-miR-34b/c transfer in all three MPM cell lines (100 to 2000 fold), that were much higher than physiological level. In addition, the cell viability was also decreased after Ad-miR-34b/c transfer compared with that after Ad-Luc transfer (P < 0.01) in MPM cell lines. Ad-miR-34b/c transfer promoted the strong down-regulation of Bcl-2 protein expression in MPM cell lines and caused an increase in the sub-G0-G1 DNA content, compared with that in cell lines infected with Ad-Luc, indicating the induction of apoptosis. In contrast, Ad-p53 transfer did not induce a drastic change in the sub-G0-G1 DNA content in MPM cell lines. Conclusion: Current study suggests that the adenoviral gene therapy of miR-34b/c, but not TP53, inhibits the cell proliferation of MPM cells by induction of apoptosis, indicating the possibility of miR-34b/c gene therapy in MPMs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4944. doi:10.1158/1538-7445.AM2011-4944

Abstract 3952: MicroRNA miR-34b/c is significantly down-regulated by aberrant promoter methylation and forced expression of miR-34b/c enhances the radiosensitivity in malignant pleural mesothelioma cells

Abstract <Background> MicroRNA miR-34b/c, one of the direct transcriptional targets of p53, plays an important role in tumor-suppressor network. It is known that the miR-34b/c can induce cell cycle arrest by blocking CDK4, CDK6, CCND1, CCNE2, E2F, bcl-2 and promote apoptosis. On the contrary, loss of miR-34b/c attenuates p53-mediated cell death. We previously described the miR-34b/c was significantly down-regulated by heavy methylation in the promoter region in malignant pleural mesotheliomas(MPMs). It has been reported miR-34a enhanced the radiosensitivity in pancreatic cancer cells. But there is no report that demonstrates the relationship between miR-34b/c and radiosensitivity. We hypothesized that miR-34b/c might enhance the radiosensitivity by blocking cell cycle. Because miR-34b/c seems to play an important role in pathogenesis in MPMs, we examined the miR-34b/c-mediated radiosensitization in MPM cells. <Materials and Methods> To test this hypothesis, two MPM cell lines(H28, H2052) stably transfected with miR-34b/c were used in this study. A Real-time PCR for miR-34b and miR-34c was performed to confirm the expression of miR-34b/c in stable transfectants. A Western blotting for CDK4, CDK6, CCND1, CCNE2, E2F, bcl-2 was done in two MPM cell lines(H28, H2052). A clonogenic survival assay and a cell cycle analysis were performed. As a measure of radiation-induced DNA damage, gamma H2AX foci assay was performed at 1hour, 6hour and 24hour after 4Gy irradiation. <Results> The miR-34b expression was increased by 72.8-fold and 37.8-fold in H28 and H2052 stable miR-34b/c transfectants, respectively, compared to scramble microRNA stable transfectants. The miR-34c expression was increased by 6.7-fold and 15.3-fold in H28 and H2052 stable miR-34b/c transfectants, respectively. Western blotting showed CDK4, CDK6, CCND1, CCNE2 and E2F were down-regulated in stable miR-34b/c transfectants. Cell cycle analysis showed an increase in the G0-G1 fraction and a decrease in the G2-M and S fraction, indicating that miR-34b/c induced G1 cell cycle arrest. Clonogenic survival assay showed the miR-34b/c transfection significantly enhanced the radiosensitivity. Gamma H2AX foci assay demonstrated the average number of gamma H2AX foci at 24 hours after irradiation was 4.94/cell in stable miR-34b/c transfectants and 3.24/cell in stable scramble microRNA transfectants. <Conclusion> These results indicate miR-34b/c can enhance tumor cell radiosensitivity in MPM cells by inducing G1 cell cycle arrest and suggest that this effect may involve in an inhibition of DNA double strand break repair. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3952. doi:10.1158/1538-7445.AM2011-3952

Abstract 2630: Nicotinamide activates the p53/miR-34a/SIRT1 tumor suppressor network leading to apoptosis of chronic lymphocytic leukemia cells

Abstract Nicotinamide, the amide form of the B-complex vitamin niacin, is the main precursor of NAD+ in most mammalian cells. Tumor transformation is generally accompanied by profound alterations in total cellular NAD+ and nicotinamide levels, and consequently in the activities of NAD+-dependent enzymes. Among these enzymes, sirtuins are class III histone deacetylases. SIRT1, the best characterized member, plays important roles in gene silencing, aging and longevity deacetylating histone and non-histone proteins, including p53. Several evidences indicate that SIRT1 may work as an oncogene by suppressing p53 functions, leading to genome instability and resistance to apoptosis. For these reasons, there is intense investigation in designing molecular tools that inhibit sirtuins. Because of a longstanding experience in the use of nicotinamide for the treatment of pellagra and because of the lack of reported side effects, nicotinamide could be in the pipeline of SIRT1 inhibitors to be tested in clinical settings. Our hypothesis is that nicotinamide affects human B cell homeostasis and that the effects might be more marked in a neoplastic context. Chronic lymphocytic leukemia (CLL), selected as disease model, is characterized by the slowly progressive expansion of a population of mature monoclonal B lymphocytes, intrinsically resistant to apoptosis. The results of this study confirm that CLL cells are characterized by increased expression and function of SIRT1, compared to normal B lymphocytes from peripheral blood of age- and sex-matched donors. Nicotinamide treatment directly leads to enzymatic activity inhibition of SIRT1 evident only in leukemic cells. Functional block of this enzyme is followed by re-activation of the p53 pathway marked by increased expression of p53-dependent genes, such as p21, NOXA and BAX and decreased of Mcl-1. The endpoint is a block of the cell cycle and induction of apoptosis in CLL cells with a wild type p53. Conversely CLL cells with a mutated/deleted p53 protein are partially resistant to nicotinamide-mediated effects. Our hypothesis is that the link between SIRT1 and p53 is represented by miR-34a, a direct transcriptional target of p53 that can bind SIRT1 mRNA and trigger its degradation. In line with this hypothesis, a combined treatment of CLL cells with nicotinamide and etoposide (a DNA-damaging chemotherapeutic) is followed by i) up-regulation of miR-34a expression, ii) marked down-modulation of SIRT1 expression and function, and iii) induction of expression and acetylation of p53. This positive feedback loop is operative in the CLL model, with the final outcome of a tumor cell apoptosis significantly enhanced. In conclusion this work suggests that nicotinamide represents a potentially useful adjunct in the treatment of selected forms of CLL, also considering that normal B lymphocytes are markedly less sensitive to nicotinamide effects. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2630. doi:10.1158/1538-7445.AM2011-2630

Akt-phosphorylated PIKE-A inhibits UNC5B-induced apoptosis in cancer cell lines in a p53-dependent manner

UNC5B acts as a tumor suppressor, and it induces apoptosis in the absence of its cognate ligand netrins. UNC5B is a direct transcriptional target of p53 upon UV stimulation. Here we show that Akt phosphorylates PIKE-A and regulates its association with UNC5B and inhibits UNC5B-provoked apoptosis in a p53-dependent manner. PIKE-A GTPase binds active Akt and stimulates its kinase activity in a guanine-nucleotide-dependent way. Akt feeds back and phosphorylates PIKE-A on Ser-472 and subsequently enhances its stimulatory effect on Akt kinase activity. Akt activity is significantly reduced in PIKE -/- Mouse Embryonic Fibroblast (MEF) cells as compared to wild-type cells. PIKE-A directly interacts with UNC5B, which is regulated by netrin-1-activated Akt. Overexpression of PIKE-A diminishes UNC5B expression through down-regulation of p53. Knocking down PIKE-A stabilizes p53, increases UNC5B, and escalates UV-triggered apoptosis. Depletion of Akt abrogates PIKE-A's inhibitory effect on both p53 and UNC5B. Hence our findings support the notion that Akt--phosphorylated PIKE-A inhibits UNC5B-elicited apoptosis and reduces its expression level through inactivation of p53.

Wild-type p53 enhances annexin IV gene expression in ovarian clear cell adenocarcinoma

The protein annexin IV (ANX4) is elevated specifically and characteristically in ovarian clear cell adenocarcinoma (CCA), a highly malignant histological subtype of epithelial ovarian cancer. On the basis of the hypothesis that the expression of ANX4 in CCA is regulated by a unique transcription mechanism, we explored the cis-elements involved in CCA-specific ANX4 expression using a luciferase reporter. We compared the transcriptional activities of the region from -1534 to +1010 relative to the ANX4 transcription start site in CCA and non-CCA-type cell lines, and found that two repeated binding motifs for the tumor suppressor protein, p53, in the first intron of ANX4 were involved in CCA-specific transcriptional activity. Furthermore, chromatin immunoprecipitation showed that endogenous p53 bound to this site in CCA cell lines. Moreover, the use of short interference RNA to silence the p53 gene decreased the transcriptional activity and mRNA expression of ANX4 in CCA cell lines. Thus, the ANX4 gene is, at least in part, regulated by p53 in CCA cells. Mutations in the p53 gene were absent and levels of p53 target genes were higher in several CCA-derived cell lines. Although the expression of ANX4 is typically low in these non-CCA cell lines, ANX4 levels were elevated more than three-fold by the overexpression of wild-type but not mutant p53. Therefore, we conclude that the ANX4 gene is a direct transcriptional target of p53, and its expression is enhanced by wild-type p53 in CCA cells.

Rad is a p53 direct transcriptional target that inhibits cell migration and is frequently silenced in lung carcinoma cells

The p53 tumor suppressor exerts its function mainly as a transcriptional activator. Here we show that the Ras-related small GTPase Rad, an inhibitor of Rho kinase, is a direct transcriptional target of p53. Expression of Rad messenger RNA (mRNA) and protein was induced by DNA damage in a p53-dependent manner. The -2934/-2905-bp Rad promoter region, to which p53 bound, was required for p53-mediated Rad gene activation. Treatment by DNA damaging agents increased p53 occupancy and histone acetylation in the region of Rad promoter containing the p53-binding site. Expression of Rad diminished the inhibitory phosphorylation at Ser3 of cofilin, a regulator of actin dynamics, and suppressed migration and invasiveness of cancer cells. Knockdown of Rad promoted cell migration and alleviated the p53-mediated migration suppression. Frequent loss of Rad mRNA and protein expression was observed in non-small cell lung carcinoma tissues. Together our results reveal a mechanism that p53 may inhibit cell migration by disrupting actin dynamics via Rad activation and implicate a tumor suppressor role of Rad in lung cancer.

P53-dependent repression of polo-like kinase-1 (PLK1)

PLK1 is a critical mediator of G2/M cell cycle transition that is inactivated and depleted as part of the DNA damage-induced G2/M checkpoint. Here we show that down-regulation of PLK1 expression occurs through a transcriptional repression mechanism and that p53 is both necessary and sufficient to mediate this effect. Repression of PLK1 by p53 occurs independently of p21 and of arrest at G1/S where PLK1 levels are normally repressed in a cell cycle-dependent manner through a CDE/CHR element. Chromatin immunoprecipitation analysis indicates that p53 is present on the PLK1 promoter at two distinct sites termed p53RE1 and p53RE2. Recruitment of p53 to p53RE2, but not to p53RE1, is stimulated in response to DNA damage and/or p53 activation and is coincident with repression-associated changes in the chromatin. Down-regulation of PLK1 expression by p53 is relieved by the histone deacetylase inhibitor, trichostatin A, and involves recruitment of histone deacetylase to the vicinity of p53RE2, further supporting a transcriptional repression mechanism. Additionally, wild type, but not mutant, p53 represses expression of the PLK1 promoter when fused upstream of a reporter gene. Silencing of PLK1 expression by RNAi interferes with cell cycle progression consistent with a role in the p53-mediated checkpoint. These data establish PLK1 as a direct transcriptional target of p53, independently of p21, that is required for efficient G2/M arrest.

Abstract 2081: Frequent silencing of tumor suppressive MicroRNA-34b/c by aberrant methylation in malignant mesothelioma

Abstract Background: Altered expression of microRNA (miRNA) is strongly implicated in human malignancies. Some miRNAs are classified as oncogenic or tumor suppressive miRNAs according to their function in cellular transformation. Similar to encoding genes, tumor suppressive miRNAs are silenced with DNA methylation. The miR-34a and b/c are direct transcriptional targets of p53 and sometimes down-regulated in malignancies. Malignant pleural mesothelioma (MPM) is a highly aggressive tumor with a dismal prognosis. Unlike other malignant tumors, mutation of P53 gene is rare and p53 expression is generally intact in MPM. In this study, we examined methylation and expression status of miR-34a and b/c in MPM. We also investigated the effect of miR-34b/c induction into MPM cells. Methods: We examined 6 MPM cell lines (H28, H290, 211H, H2052, H2452, and HP1), 20 primary MPMs and 7 non-malignant mesotheial tissues. Methylation and expression status of miR-34a and b/c were determined with methylation-specific PCR and quantitative PCR, respectively. Colony formation assay was applied to examine the anti-proliferative effect by induction of miR-34b/c and scramble sequence control with plasmid vector. Protein expression and cell cycle were analyzed with western blotting and flow cytometry, respectively. Results: Aberrant methylation was found in 2 (33.3%) of 6 MPM cell lines and 6 (33.3%) of 20 primary MPMs in miR-34a and in all 6 MPM cell lines (100%) and 18 (90%) of 20 primary MPMs in miR-34b/c. No methylation was found in 7 non-malignant mesothelial tissues. Expression of miR-34a and b/c in all methylated cell lines was silenced and restored with 5-Aza-CdR treatment, indicating that methylation caused gene silencing. Because the frequency of methylation was particularly high in miR-34b/c, we focused on miR-34b/c in following study. Ectopic induction of miR-34b/c gene in examined 3 MPM cell lines (H28, H290, and 211H) showed significant inhibition of colony formation compared with induction of control vector, demonstrating anti-proliferative effect of miR-34b/c on MPM. Western blotting analysis for all 3 MPM cell lines after induction of miR-34b/c showed suppression of total and phospho-c-met, cdk4, cdk6, and cyclin E2 collated genes of cell proliferation, suggesting that these genes were interfering targets of miR-34b/c. Cell cycle analysis showed that G2-M fractions were reduced with flow cytometry in all 3 MPM cell lines with miR-34b/c induction. Conclusion: Our results demonstrated that miR-34b/c methylation is frequent in MPM and resulting miR-34b/c silencing plays an important role in pathogenesis of MPM. The miR-34b/c can be a promising candidate as a therapeutic miRNA for MPM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2081.

RFWD3–Mdm2 ubiquitin ligase complex positively regulates p53 stability in response to DNA damage

In unstressed cells, the tumor suppressor p53 is maintained at low levels by ubiquitin-mediated proteolysis mainly through Mdm2. In response to DNA damage, p53 is stabilized and becomes activated to turn on transcriptional programs that are essential for cell cycle arrest and apoptosis. Activation of p53 leads to accumulation of Mdm2 protein, a direct transcriptional target of p53. It is not understood how p53 is protected from degradation when Mdm2 is up-regulated. Here we report that p53 stabilization in the late phase after ionizing radiation correlates with active ubiquitination. We found that an E3 ubiquitin ligase RFWD3 (RNF201/FLJ10520) forms a complex with Mdm2 and p53 to synergistically ubiquitinate p53 and is required to stabilize p53 in the late response to DNA damage. This process is regulated by the DNA damage checkpoint, because RFWD3 is phosphorylated by ATM/ATR kinases and the phosphorylation mutant fails to stimulate p53 ubiquitination. In vitro experiments suggest that RFWD3 is a p53 E3 ubiquitin ligase and that RFWD3-Mdm2 complex restricts the polyubiquitination of p53 by Mdm2. Our study identifies RFWD3 as a positive regulator of p53 stability when the G(1) cell cycle checkpoint is activated and provides an explanation for how p53 is protected from degradation in the presence of high levels of Mdm2.

A newly identified dependence receptor UNC5H4 is induced during DNA damage-mediated apoptosis and transcriptional target of tumor suppressor p53

UNC5H4 is a netrin-1 receptor UNC5H family member. In this study, we found that UNC5H4 is a direct transcriptional target of p53. During adriamycin (ADR)-mediated apoptosis, UNC5H4 was significantly induced in p53-proficient U2OS cells but not in p53-deficient H1299 cells. Enforced expression of p53 induced UNC5H4. Consistent with these results, siRNA-mediated knockdown of p53 in U2OS cells attenuated ADR-dependent induction of UNC5H4. Indeed, we found four putative p53-responsive elements within intron 1 of UNC5H4 gene. Luciferase reporter assay and ChIP analysis demonstrated that, among them, two tandem elements respond to exogenous p53 which is efficiently recruited onto them. Furthermore, enforced expression of UNC5H4 remarkably reduced number of drug-resistant colonies in p53-proficient cells but not in p53-deficient cells, suggesting that UNC5H4-induced apoptosis is dependent on p53 status. siRNA-mediated knockdown of UNC5H4 rendered U2OS cells resistant to ADR. Collectively, our present results suggest that UNC5H4 amplifies p53-dependent apoptotic response.

Regulation of Hematopoietic Stem Cell Quiescence - A Novel Role for p53.

Although the p53 tumor suppressor can elicit cell-cycle arrest or apoptosis in hematopoietic cells upon DNA damage, its function during steady-state hematopoiesis is largely unknown. We demonstrated that the Ets transcription factor MEF/ELF4 regulates both HSC proliferation/self-renewal and quiescence, as Mef null mice exhibit greater numbers of hematopoietic stem cells and the Mef null HSCs are more quiescent than normal. As such, the hematopoietic compartment of Mef null mice shows significant resistance to chemotherapy and radiation (Lacorazza et al., Cancer Cell, 2006). In this study, we have investigated the mechanisms utilized by MEF/ELF4 to regulate the quiescence and self-renewal of hematopoietic stem cells, identifying p53 as a key regulator. We have recently found that Mef null mouse embryonic fibroblasts (mefs) accumulate p53 and undergo premature senescence; MEF appears to surpress the expression of p53 by directly upregulating Mdm2 (G. Sashida et al., submitted). We hypothesized that p53 may play a role in the enhanced stem cell quiescence or the increased HSC frequency seen in Mef null mice. To examine this, we generated p53−/− Mef −/− mice and compared HSC biology in the double knock out mice (p53−/− Mef −/−) vs p53 null mice, Mef null mice and wt mice. Loss of p53 decreased the fraction of Pyronin Ylow Lin-Sca-1+ cells, suggesting fewer quiescent HSCs, and staining of CD34-LSK cells for the proliferation marker Ki67 also showed enhanced HSC proliferation in the absence of p53 (with fewer quiescent cells present). These data suggest that p53 promotes quiescence in HSCs, and in the absence of p53, HSCs more readily enter the cell cycle. When we analyzed the DKO (p53−/− Mef −/−) mice, we observed that the percentage of G0 HSCs returned to normal, indicating that p53 is essential for maintaining the enhanced quiescence of MEF null HSCs. p21 is a major target gene of p53 in cells, and has been shown to play an important role in maintaining HSC quiescence. As expeceted, we found elevated levels of p21 mRNA in MEF null LSK cells and reasoned that p21 may account for their enhanced quiescence. We generated p21 −/− Mef −/− mice, which are viable, born at normal mendelian frequency and appear grossly normal. However, cell cycle analysis of HSCs obtained from p21 −/− Mef −/− mice showed that the enhanced quiescence in MEF null HSCs did not depend on p21, indicating that other p53 target genes play an important role in maintaining stem cell quiescence. We therefore utilized transcript profiling (Microarray studies and quantitative PCR analysis) to identify potential p53-regulated genes that may be differentialy expressed in LSK cells isolated from wild type, p53−/−, Mef −/−, and p53−/− Mef −/− mice. By ChiP and luciferase reporter assays, we show for the first time that Gfi-1 and Necdin are direct transcriptional targets of p53 in HSCs and both Gfi-1 and Necdin regulate the enhanced quiescence exhibited in MEF null HSCs. Taken together, our work defines a novel role for p53 in the maintenance of HSC quiescence. Furthermore, HSC quiescence and self-renewal appear to be mediated by different p53 target genes during steady state hematopoiesis.

MicroRNAs join the p53 network — another piece in the tumour-suppression puzzle

Several recent studies have found a conserved microRNA (miRNA) family, the miR-34s, to be direct transcriptional targets of p53. miR-34 activation can recapitulate elements of p53 activity, including induction of cell-cycle arrest and promotion of apoptosis, and loss of miR-34 can impair p53-mediated cell death. These data reinforce the growing awareness that non-coding RNAs are key players in tumour development by placing miRNAs in a central role in a well-known tumour-suppressor network.

A microRNA component of the p53 tumour suppressor network

A global decrease in microRNA (miRNA) levels is often observed in human cancers, indicating that small RNAs may have an intrinsic function in tumour suppression. To identify miRNA components of tumour suppressor pathways, we compared miRNA expression profiles of wild-type and p53-deficient cells. Here we describe a family of miRNAs, miR-34a-c, whose expression reflected p53 status. Genes encoding miRNAs in the miR-34 family are direct transcriptional targets of p53, whose induction by DNA damage and oncogenic stress depends on p53 both in vitro and in vivo. Ectopic expression of miR-34 induces cell cycle arrest in both primary and tumour-derived cell lines, which is consistent with the observed ability of miR-34 to downregulate a programme of genes promoting cell cycle progression. The p53 network suppresses tumour formation through the coordinated activation of multiple transcriptional targets, and miR-34 may act in concert with other effectors to inhibit inappropriate cell proliferation.

P53 aerobics: The major tumor suppressor fuels your workout

In addition to its role as the central regulator of the cellular stress response, p53 can regulate aerobic respiration via the novel transcriptional target SCO2, a critical regulator of the cytochrome c oxidase complex (Matoba et al., 2006). Loss of p53 results in decreased oxygen consumption and aerobic respiration and promotes a switch to glycolysis, thereby reducing endurance during physical exercise.

Siah1b, a Direct Transcriptional Target of P53, Is an Oocyte-Specific Potential Regulator for Ovarian Folliculogenesis

Siah is murine homologue of the Drosophila seven in absentia gene (sina) and has three homologues (siah1a, siah1b, and siah2). To elucidate the unique function of Siah family members, we investigated the differential expression of the three members during ovarian folliculogenesis.

Siah-1b is a direct transcriptional target of p53: identification of the functional p53 responsive element in the siah-1b promoter.

Siah proteins are E3 ubiquitin ligases. They are homologues of the Drosophila seven in absentia (Sina), a protein required for the R7 photoreceptor development. We have previously found that the expression of human siah-1 and its mouse homologue siah-1b are induced by p53 during apoptosis and tumor reversion. So far, no evidence that the siah-1b gene is a direct transcriptional target of p53 has been provided. In the present study we investigate this issue. Northern blot analysis with a specific probe demonstrates an increase in siah-1b transcription on activation of endogenous and inducible exogenous p53. To explore whether this effect is directly mediated by p53 we analyzed 20 kb of chromosome X DNA, containing the siah-1b locus. A p53-binding site was identified in the siah-1b promoter, located at nucleotides -2155/-2103 relative to the translational start site. This site is composed of two half-sites, conforming to the p53-binding consensus sequence but separated by a nonclassical 33-bp spacer. In luciferase assays, p53 induces a substantial increase in siah-1b promoter activity. Gel shift and DNase-I-footprinting studies, combined with mutational analysis and chromatin immunoprecipitation, indicate that p53 effectively binds the siah-1b promoter in vitro and in vivo. Thus, the siah-1b gene is a direct transcriptional target of p53.

Caspase 10 Levels are Increased Following DNA Damage in a p53-Dependent Manner

p53 plays a central role in the cellular response to stressful stimuli, including DNA damage, aberrant growth signals, and hypoxia. Upon activation, it binds to a specific DNA binding motif and functions to transcriptionally activate or repress different groups of genes. The biologic outcome of these events can lead to cell-cycle arrest, DNA repair, or apoptosis. While many downstream targets of p53 have been identified, the factors that determine which of these outcomes will occur in an individual cell are less clear. In this study, we show that caspase 10, an initiator caspase, is induced in response to DNA damaging chemotherapeutic agents in a p53-dependent manner. Following treatment with etoposide or adriamycin, induction of caspase 10 occurred at both the mRNA and protein level. In contrast, DNA damage did not affect levels of caspase 8, another initiator caspase with significant structural and functional homology to caspase 10. By chromatin immunoprecipitation assay, p53 bound in vivo to multiple p53-specific binding sites located within the caspase 10 gene locus, suggesting that caspase 10 is a direct transcriptional target of p53. Upregulation of caspase 10 may represent one factor that determines how p53 activation leads to cellular apoptosis following DNA damage.