TP53 Binding Site Research Articles

Optimisation of TP53 reporters by systematic dissection of synthetic TP53 response elements.

TP53 is a transcription factor that controls multiple cellular processes, including cell cycle arrest, DNA repairand apoptosis. The relation between TP53 binding site architecture and transcriptional output is still not fully understood. Here, we systematically examined in three different cell lines the effects of binding site affinity and copy number on TP53-dependent transcriptional output, and also probed the impact of spacer length and sequence between adjacent binding sites, and of core promoter identity. Paradoxically, we found that high-affinity TP53 binding sites are less potent than medium-affinity sites. TP53 achieves supra-additive transcriptional activation through optimally spaced adjacent binding sites, suggesting a cooperative mechanism. Optimally spaced adjacent binding sites have a ∼10-bp periodicity, suggesting a role for spatial orientation along the DNA double helix. We leveraged these insights to construct a log-linear model that explains activity from sequence features, and to identify new highly active and sensitive TP53 reporters.

Systematic dissection of the regulatory logic of transcriptional activation by TP53 binding sites

Systematic dissection of the regulatory logic of transcriptional activation by TP53 binding sites

Presence of mutant p53 increases stem cell frequency and is associated with reduced binding to classic TP53 binding sites in cell lines and primary AMLs

With an overall 5%-10% incidence rate in acute myeloid leukemia (AML), the occurrence of TP53 mutations is low compared with that in solid tumors. However, when focusing on high-risk groups including secondary AML (sAML) and therapy-related AMLs, the frequency of mutations reaches up to 35%. Mutations may include loss of heterozygosity (LOH) or deletion of the 17p allele, but are mostly missense substitutions that are located in the DNA-binding domain. Despite elaborate research on the effects of TP53 mutations in solid tumors, in hematological malignancies, the effects of TP53 mutations versus loss of TP53 remain unclear and under debate. Here, we compared the cellular effects of a TP53 mutant and loss of TP53 in human hematopoietic stem and progenitor cells (HSPCs). We found that when expressing TP53 mutant or loss of TP53 using siRNA, CD34+/CD38- cells have a significantly enhanced replating potential, which could not be demonstrated for the CD34+/CD38+ population. Using RNA-sequencing analysis, we found a loss of expression of p53 target genes in cells with TP53 knockdown. In contrast, an increased expression of a large number of genes was observed when expressing TP53 mutant, resulting in an increase in expression of genes involved in megakaryocytic differentiation, plasma membrane binding, and extracellular structure organization. When binding of p53 wild type and p53 mutant was compared in cell lines, we found that mutant p53 binds to a large number of binding sites genomewide, contrary to wild-type p53, for which binding is restricted to genes with a p53 binding motif. These findings were verified in primary AMLs with and without mutated TP53. In conclusion, in our models, we identified overlapping effects of TP53 mutant and loss of TP53 on in vitro stem cell properties but distinct effects on DNA binding and gene expression.

PD-L1 Expression and Gene Amplification in CD30+ Diffuse Large B-Cell Lymphoma (DLBCL): Significance of TP53 Genetic Impact and Clinicopathologic Characteristics

Introduction: Regulation of the PD-1/PD-L1 immune checkpoint has been under intense investigation in the era of immunotherapy, and PD-1 blocκade has shown promising clinical responses in several neoplasms including classical Hodgkin lymphoma (HL). Frequent amplification of the PD-L1 / PD-L2 gene locus (9p24.1) are found in most HLs, however, in other lymphoma types such as diffuse large B-cell lymphomas (DLBCL), genetic alterations of 9p24.1 locus are detected only in a small subset of cases (< 20%). Therefore, other mechanisms including transcriptional regulation of the PD-L1 gene are currently under investigation. Previous studies have shown that in another CD30+ lymphoma type, namely anaplastic large cell lymphoma of T-cell origin, PD-L1 is highly expressed despite the absence of gene amplification or other chromosomal alterations of 9p24.1 locus, and transcriptional regulation is biologically involved. However, the PD-L1 expression levels, the genetic basis of PD-L1 expression and their clinical significance in CD30+ DLBCLs are not yet studied.Methods: From a large database of 2110 DLBCLs, 78 patients with CD30+ DLBCL (44 GCB and 34 ABC-type) diagnosed and treated at the University of Texas M.D. Anderson Cancer Center (USA) and Karolinska University Hospital (Sweden) were identified. All patients were treated with R-CHOP regimen and had available clinical data. PD-L1 expression was assessed by immunohistochemistry using a monoclonal antibody and autostainer systems (Dako or Ventana). PD-L1 gene amplification and chromosomal aberrations of its gene locus were assessed by FISH. Mutations of TP53 gene were confirmed by sequencing. Statistical associations of the PD-L1 expression level (% of positive tumor cells) among various patient subgroups were based on the non-parametrical Mann-Whitney and Kruskall-Wallis tests. Survival analyses were performed using the Kaplan-Meier method (log-rank test) and Cox regression models. The DLBCL in vitro system included 3 cell lines (RC-K8, OCI-Ly3, U2932), which were treated with increasing concentrations of Nutlin 3A to stabilize and activate wild-type TP53. Western blot analysis was used to assess the protein levels of p53 and PD-L1 and quantitative RT-PCR was utilized to assess the PD-L1 mRNA level in the cell lines.Results: Using FISH, PD-L1 gene amplification, chromosomal gains and polyploidy were observed in 6 (7.7%), 3 (3.8%) and 7 (9%) cases, respectively. Expression of PD-L1 protein was found in 66 (85%) CD30+ DLBCLs and the percentage of positive tumor cells ranged from 5 to 100% (median 30%) among the positive cases. In this cohort, TP53 mutations were detected in 14 (18%) CD30+ DLBCL and were not statistically associated with PD-L1 gene amplification or expression levels. However, all PD-L1 negative tumors (n=12) were associated with a wild-type TP53 gene. High expression levels of PD-L1 correlated with ABC phenotype (p=0.04), B symptoms (p=0.03), high serum LDH (p=0.04), ECOG performance status>1 (p=0.015), and the presence of >1 extranodal sites (p=0.04) but not with Ann Arbor stage. Patients who achieved complete remission showed significantly lower PD-L1 expression level (median 20% vs. 40%, p=0.03). Given the significant survival benefit of CD30 expression in DLBCL, PD-L1 expression in this subset abolished advantage of failure-free (FFS) and overall (OS) survival after 48 months of median follow-up. The in vitro data showed that stabilization and activation of p53 in DLBCL cell lines carrying a wild-type p53 gene led to concentration-dependent increase of the PD-L1 mRNA and protein levels. As in silico analysis did not demonstrate a possible TP53 binding site on the PD-L1 gene promoter, an indirect mechanism of p53-dependent regulation of PD-L1 is suggested.Conclusions: PD-L1 is expressed in most CD30+ DLBCLs, but amplification of the PD-L1 gene locus is not a common mechanism unlike Hodgkin lymphoma. Activation of wild-type TP53 indirectly upregulates PD-L1 in DLBCL and the underlying mechanisms merit further investigation. DisclosuresSmedby:Janssen: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees.

Alternative splicing of the tumor suppressor ASPP2 results in a stress-inducible, oncogenic isoform prevalent in acute leukemia.

BackgroundApoptosis-stimulating Protein of TP53-2 (ASPP2) is a tumor suppressor enhancing TP53-mediated apoptosis via binding to the TP53 core domain. TP53 mutations found in cancers disrupt ASPP2 binding, arguing for an important role of ASPP2 in TP53-mediated tumor suppression. We now identify an oncogenic splicing variant, ASPP2κ, with high prevalence in acute leukemia.MethodsAn mRNA screen to detect ASPP2 splicing variants was performed and ASPP2κ was validated using isoform-specific PCR approaches. Translation into a genuine protein isoform was evaluated after establishing epitope-specific antibodies. For functional studies cell models with forced expression of ASPP2κ or isoform-specific ASPP2κ-interference were created to evaluate proliferative, apoptotic and oncogenic characteristics of ASPP2κ.FindingsExon skipping generates a premature stop codon, leading to a truncated C-terminus, omitting the TP53-binding sites. ASPP2κ translates into a dominant-negative protein variant impairing TP53-dependent induction of apoptosis. ASPP2κ is expressed in CD34+ leukemic progenitor cells and functional studies argue for a role in early oncogenesis, resulting in perturbed proliferation and impaired induction of apoptosis, mitotic failure and chromosomal instability (CIN) – similar to TP53 mutations.Importantly, as expression of ASPP2κ is stress-inducible it defines a novel class of dynamic oncogenes not represented by genomic mutations.InterpretationOur data demonstrates that ASPP2κ plays a distinctive role as an antiapoptotic regulator of the TP53 checkpoint, rendering cells to a more aggressive phenotype as evidenced by proliferation and apoptosis rates – and ASPP2κ expression results in acquisition of genomic mutations, a first initiating step in leukemogenesis. We provide proof-of-concept to establish ASPP2κ as a clinically relevant biomarker and a target for molecule-defined therapy.FundUnrestricted grant support from the Wilhelm Sander Foundation for Cancer Research, the IZKF Program of the Medical Faculty Tübingen, the Brigitte Schlieben-Lange Program and the Margarete von Wrangell Program of the State Ministry Baden-Wuerttemberg for Science, Research and Arts and the Athene Program of the excellence initiative of the Eberhard-Karls University, Tübingen.

Mutation Burden in Multiple Myeloma Is Captured By Gene Expression Profiles

Mutation Burden in Multiple Myeloma Is Captured By Gene Expression Profiles

Oncogenic Splicing of the Apoptosis-Stimulating Protein of p53-2 (ASPP2) Is an Initiating Step in Leukemogenesis Facilitating Acquisition of Structural Genomic Mutations

ASPP2 is an independent haploinsufficient tumor suppressor which initiates induction of apoptosis after cellular damage in a TP53-dependent manner. We recently reported the identification of two C-terminally truncated splice variants of ASPP2 with high frequency in acute leukemias. Tantalizingly, these variants lack the functionally relevant TP53 binding sites, suggesting a dominant-negative, oncogenic function. The most prevalent isoform lacking exon 17 (ASPP2k) is detected in up to 60% of acute leukemias. A cell sort for the CD34+ stem cell fraction confirmed ASPP2k-expression in the leukemia-initiating clone. Immunoblotting revealed translation of ASPP2k into a genuine protein isoform.Generation of isoform-specific antibodies allows to discriminate ASPP2k from the wildtype isoforms. Interestingly, ASPP2k is the sole isoform expressed in some native leukemia samples. Other samples reveal a heterozygous expression pattern suggesting a dominant-negative function of the C-terminally truncated ASPP2k isoform.Forced expression of ASPP2k using a HisMax plasmid vector in murine IL3-dependent pro-B Ba/F3 cells induced a more aggressive phenotype with mitotic failure and perturbed cellular proliferation compared to the empty vector controls. Of note, IL3 was successfully weaned in the ASPP2k cells - indicating that autoactivating genomic alterations must have occurred upholding cellular integrity and viability. Fractionated gamma irradiation accelerated this process. In contrast, the empty vector strains did not survive IL3 weaning. Chromosomal analyses of IL3-independent Ba/F3 ASPP2kcells revealed structural alterations including monosomies and additional marker chromosomes.Furthermore, we demonstrate that ASPP2k is stress-inducible. Changing culture conditions (e.g. temperature) of cell lines or ex vivo native AML samples resulted in a dramatic increase of the ASPP2k isoform. Consecutive treatment of cells with daunorubicin lead to relative resistance of pre-stressed cells compared to their counterparts cultured under standard conditions. Vice versa, specific knock-down of ASPP2k resulted in a significant increase in induction of apoptosis upon chemotherapy compared to empty vector controls.Our data demonstrate that ASPP2k plays a distinctive role as an anti-apoptotic regulator of the TP53 checkpoint rendering cells to a more aggressive phenotype as evidenced by proliferation and apoptosis rates - and facilitates acquisition of genomic mutations, a first initiating step in leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.

Abstract 2890: ERCC5 variant rs2296147 T-allele creates a predicted TP53 binding site and up-regulates transcript abundance in normal bronchial epithelial cells, while rs17655 C-allele is linked to miRNA binding site variant and down regulates

Abstract Background: Excision repair cross-complementation group 5 (ERCC5) gene plays an important role in nucleotide excision repair (NER) and dysregulation of ERCC5 is associated with increased lung cancer risk. This study was conducted to characterize cis-acting genetic variants responsible for inter-individual variation in ERCC5 transcript regulation in normal bronchial epithelial cells (NBEC). Methods: We determined genotypes at putative ERCC5 cis-regulatory single nucleotide polymorphic sites (SNP) rs751402 and rs2296147, and marker SNPs rs1047768 and rs17655. Using a recently developed targeted sequencing method, ERCC5 allele-specific transcript abundance was assessed in NBEC RNA from 55 individuals heterozygous for rs1047768 and 21 subjects heterozygous for rs17655. Syntenic relationships among alleles at rs751402, rs2296147 and rs1047768 were assessed by allele-specific PCR followed by Sanger sequencing. We assessed association of NBEC ERCC5 allele-specific expression at rs1047768 with haplotype and diplotype structure at putative ERCC5 promoter cis-regulatory SNPs rs751402 and rs2296147. Results: Genotype analysis revealed higher inter-individual variation in allelic ratios in cDNA samples relative to matched gDNA samples at both rs1047768 and rs17655 (p&amp;lt;0.0001 and p = 0.0005 respectively). By haplotype analysis, mean expression was higher at the rs1047768 alleles syntenic with rs2296147 T allele compared to rs2296147 C allele (p = 0.0030). Sequence analysis predicts that T allele at SNP rs2296147 creates a TP53 binding site. Mean expression was higher at rs17655 G allele (p&amp;lt;0.0001) which is syntenic with G allele at a linked SNP rs873601 (r2 = 0.74). C allele at SNP rs873601 is predicted to create a miRNA binding site. Conclusions: These data support the conclusion that T allele at SNP rs2296147 creates a TP53 binding site and up-regulates ERCC5 while C allele at SNP rs873601 creates a miRNA binding site and down-regulates ERCC5. Variation in ERCC5 transcript abundance associated with allelic variation at these SNPs is likely associated with variation in NER function in NBEC and lung cancer risk. Citation Format: Xiaolu Zhang, Erin L. Crawford, Thomas M. Blomquist, Sadik A. Khuder, Jiyoun Yeo, Albert M. Levin, James C. Willey. ERCC5 variant rs2296147 T-allele creates a predicted TP53 binding site and up-regulates transcript abundance in normal bronchial epithelial cells, while rs17655 C-allele is linked to miRNA binding site variant and down regulates. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2890.

Multiplex enhancer-reporter assays uncover unsophisticated TP53 enhancer logic

Transcription factors regulate their target genes by binding to regulatory regions in the genome. Although the binding preferences of TP53 are known, it remains unclear what distinguishes functional enhancers from nonfunctional binding. In addition, the genome is scattered with recognition sequences that remain unoccupied. Using two complementary techniques of multiplex enhancer-reporter assays, we discovered that functional enhancers could be discriminated from nonfunctional binding events by the occurrence of a single TP53 canonical motif. By combining machine learning with a meta-analysis of TP53 ChIP-seq data sets, we identified a core set of more than 1000 responsive enhancers in the human genome. This TP53 cistrome is invariably used between cell types and experimental conditions, whereas differences among experiments can be attributed to indirect nonfunctional binding events. Our data suggest that TP53 enhancers represent a class of unsophisticated cell-autonomous enhancers containing a single TP53 binding site, distinct from complex developmental enhancers that integrate signals from multiple transcription factors.

Mutant tp53 Causes a Gain of Function Increase in Sensitivity to Reactive Oxygen Species in Erythroid Precursors

TP53 is mutated in approximated 10 – 15% of cases of leukemia and myelodysplastic syndrome. It is recently appreciated that oxidative stress plays a role in the dysplastic processes that drives these diseases. Animal modeling of oxidative stress has been a challenge as many of the central genes in the oxidative stress response pathway are necessary for life and knockout animals die as embryos due to overwhelming oxidative stress. The zebrafish model allows pro-oxidant exposure early in hematopoietic development, and gata1DsRed1 transgenic animals allow clear identification of erythroid precursors. We capitalized on these advantages to interrogate the effects of oxidative stress on erythroid precursors. The gata1DsRed1 erythroid precursors showed a dose-responsive increase in reactive oxygen species (ROS) generation after naphthol exposure. Pro-oxidant exposure significantly up-regulated several anti-oxidant genes including: hypoxia-inducible factor (hif1a), nuclear factor (erythroid derived)-like 2 (nrf2), ferritin heavy chain (fth1a), thioredoxin (txn), and heme oxygenase 1 (hmox1); (p < 0.05) shown by qRT-PCR. In silico promoter analysis of these genes revealed several tp53 binding sites within 4 kb upstream of the first exon in each gene. Pro-oxidant exposure was able to induce tp53 expression 3-fold over baseline by qRT-PCR. We next took advantage of the tp53 mutant line tp53M214K/M241K which has a mutation in the DNA binding region of tp53 rendering it non-functional. We found that tp53M214K/M124K fish were highly sensitive to pro-oxidant exposure with 80% of embryos showing severe to moderate anemia and cardiac edema after 72 hours of exposure to naphthol (versus 25% in wild-type control animals, n = 100/group; p < 0.001). There was a 3-fold decrease in the number of hemoglobin producing cells in naphthol treated tp53M214K/M124K animals as shown by o-dianisidine staining (versus control animals, n = 10/group; p < 0.01). A dose-response between the amount of pro-oxidant exposure and severity of anemia/edema also existed as determined by correlation of pro-oxidant concentration to an edema severity scale. We next measured the amount of ROS generated after naphthol exposure using CellROX detection assays and found that tp53M214K/M124K animals showed a 5 – 10-fold increase in ROS generated compared to wild-type (n = 30/group, p < 0.01). This increased ROS was maintained even in the heterozygous states of tp53M214K/wt suggesting a gain-of-function phenomenon. ROS generation could be completely reversed by treatment with the anti-oxidant n-acetylcysteine. When we completely abrogated mutant tp53 by morpholino knockdown, ROS generation and hemolysis were significantly decreased, providing further evidence that the ROS generation is a gain-of-function phenomenon of mutated tp53. Finally, uncoupling of mitochondrial respiration using oligomycin also decreased ROS generation by 50% (p < 0.01) implicating a dysregulatory process ongoing in mitochondria. In addition to the recently appreciated increased tumorigenicity caused by mutated tp53, our data suggest that cells harboring mutant tp53 also have increased ROS generation and increased sensitivity with pro-oxidant exposure. Understanding the mechanisms by which the anti-oxidant response is regulated will allow us to potentially find more effective druggable targets to treat the oxidative stress that accompanies the dysplastic process. DisclosuresNo relevant conflicts of interest to declare.

TP53 engagement with the genome occurs in distinct local chromatin environments via pioneer factor activity.

Despite overwhelming evidence that transcriptional activation by TP53 is critical for its tumor suppressive activity, the mechanisms by which TP53 engages the genome in the context of chromatin to activate transcription are not well understood. Using a compendium of novel and existing genome-wide data sets, we examined the relationship between TP53 binding and the dynamics of the local chromatin environment. Our analysis revealed three distinct categories of TP53 binding events that differ based on the dynamics of the local chromatin environment. The first class of TP53 binding events occurs near transcriptional start sites (TSS) and is defined by previously characterized promoter-associated chromatin modifications. The second class comprises a large cohort of preestablished, promoter-distal enhancer elements that demonstrates dynamic histone acetylation and transcription upon TP53 binding. The third class of TP53 binding sites is devoid of classic chromatin modifications and, remarkably, fall within regions of inaccessible chromatin, suggesting that TP53 has intrinsic pioneer factor activity and binds within structurally inaccessible regions of chromatin. Intriguingly, these inaccessible TP53 binding sites feature several enhancer-like properties in cell types within the epithelial lineage, indicating that TP53 binding events include a group of “proto-enhancers” that become active enhancers given the appropriate cellular context. These data indicate that TP53, along with TP63, may act as pioneer factors to specify epithelial enhancers. Further, these findings suggest that rather than following a global cell-type invariant stress response program, TP53 may tune its response based on the lineage-specific epigenomic landscape.

The Oxidative Stress Response In Erythroid Precursors Is Mediated By tp53

Oxidative stress plays a key role in acute and especially chronic anemia as well red blood cell storage. Recent findings suggest that both erythroid precursors as well as mature red cells have increased sensitively to oxidative stress. Modeling oxidative stress in animals has been a challenge as many of the central genes in the oxidative stress response pathway are necessary for life and knockout animals die of overwhelming oxidative stress early in life. The zebrafish model allows pro-oxidant exposure early in hematopoietic development, and gata1DsRed1 transgenic animals allow clear identification of erythroid precursors. We capitalized on these advantages of the zebrafish to interrogate the effects of oxidative stress on erythroid precursors. After 72 hours of exposure to the strong pro-oxidant naphthol at 10 – 30 µg, embryonic zebrafish up-regulated several anti-oxidant genes including: hypoxia-inducible factor (hif1a), nuclear factor (erythroid derived)-like 2 (nrf2), ferritin heavy chain (fth1a), thioredoxin (txn), and heme oxygenase 1 (hmox1); 1.5, 2.3, 2.5, and 3.0-fold respectively (p < 0.05) as shown by qRT-PCR. To understand if a common pathway was driving anti-oxidant gene expression, we performed an in silico promoter analysis of these genes and discovered several tp53 binding sites within 4 kb upstream of the first exon in each gene. We showed naphthol was able to induce tp53 expression 3-fold over baseline by qRT-PCR. We next took advantage of the tp53 mutant line tp53M214K which has a mutation in the DNA binding region of tp53 rendering it non-functional similar to a complete knockout. We found that tp53M214K fish were highly sensitive to pro-oxidant exposure with 80% of embryos showing severe to moderate anemia and cardiac edema after 72 hours of exposure to naphthol (versus 25% in wild-type control animals, n = 100/group; p < 0.001). There was also a 3-fold decrease in the number of hemoglobin staining cells in naphthol treated tp53M214K animals as shown by o-dianisidine staining (versus control animals, n = 10/group; p < 0.01). A dose-response between the amount of pro-oxidant exposure and severity of anemia/edema also existed as determined by correlation of pro-oxidant concentration to an edema severity scale. We next measured the amount of reactive oxygen species (ROS) generated after naphthol exposure using CellROX detection assays and found that tp53M214K animals showed a doubling in ROS generated compared to wild-type (n = 30/group, p < 0.01) in whole animals. To disable tp53 by an alternative manner, we employed a known tp53 inhibitor, pifithrin, to inactivate tp53. Exposure of animals to pifithrin simultaneous with naphthol recapitulated the finding that inhibition of tp53 increased sensitivity to ROS as 90% of exposed embryos displayed moderate to severe anemia induced cardiac edema (versus 30% in controls, n = 100/group; p < 0.01). Although pifithrin combined with naphthol exposure caused no increase in ROS above that seen with naphthol alone. Our hypothesis is that the anemic phenotype is largely caused by hemolysis in erythroid precursors. The gata1DsRed1 zebrafish has labeled erythroid precursors, and using our experimental system we were able to specifically measure a dose responsive induction of ROS in erythroid precursors after naphthol exposure. Furthermore, gata1DsRed1 animals harboring tp53M214K showed a 20-fold increase in ROS after naphthol exposure (versus tp53+/+ animals, n = 10/group; p < 0.01). Accompanying the increase ROS is apoptosis of erythroid precursors as shown by flow cytometry for gata1DsRed1 cells. In conclusion, we show that amongst the many functions of tp53, providing an anti-oxidant response is also a mechanism though which erythroid precursors metabolize ROS after exposure to pro-oxidants. Understanding the mechanisms by which the anti-oxidant response is regulated will allow us to potentially find more effective drug-able targets to treat the oxidative stress that accompanies acute and chronic anemia's. Disclosures:No relevant conflicts of interest to declare.

Ionizing radiation-induced gene expression changes in TP53 proficient and deficient glioblastoma cell lines

The genetic heterogeneity presented by different cell lines derived from glioblastoma (GBM) seems to influence their responses to antitumoral agents. Although GBM tumors present several genomic alterations, it has been assumed that TP53, frequently mutated in GBM, may to some extent be responsible for differences in cellular responses to antitumor agents, but this is not clear yet. To directly determine the impact of TP53 on GBM response to ionizing radiation, we compared the transcription profiles of four GBM cell lines (two with wild-type (WT) TP53 and two with mutant (MT) TP53) after 8Gy of gamma-rays. Transcript profiles of cells analyzed 30min and 6h after irradiation showed that WT TP53 cells presented a higher number of modulated genes than MT TP53 cells. Our findings also indicate that there are several pathways (apoptosis, DNA repair/stress response, cytoskeleton organization and macromolecule metabolic process) in radiation responses of GBM cell lines that were modulated only in WT TP53 cells (30min and 6h). Interestingly, the majority of differentially expressed genes did not present the TP53 binding site, suggesting secondary effects of TP53 on transcription. We conclude that radiation-induced changes in transcription profiles of irradiated GBM cell lines mainly depend on the functional status of TP53.

Identification of a pH‐responsive DNA region upstream of the transcription start site of human NBCe1‐B

In rodent incisors two distinct stages of enamel formation can be identified visually based on cell morphology: the secretory stage and the maturation stage. The expression profiles of many genes characterize both stages, including the bicarbonate transport protein NBCe1. Bicarbonate is a requirement for the mineralizing enamel matrix to buffer excessive protons that form as a consequence of hydroxyapatite formation. NBCe1-B mRNA is up-regulated during the maturation stage of amelogenesis, where hydroxyapatite formation predominates. In this study, a presumed 572-bp NBCe1-B promoter region was subcloned into a reporter construct, and within this 572-bp region of DNA we characterized a 285-bp segment that shows an increase of ≈ 2.3-fold in gene-transcription activity when transfected into ameloblast-like cells and cultured in medium maintained at pH 6.8 (vs. pH 7.4). A presumed pH-responsive transcriptional factor-binding domain(s) thus resides in the 285-bp NBCe1-B promoter region where candidate domains include the nuclear factor of kappa light polypeptide gene enhancer in B-cells1(NFKB1), jun proto-oncogene (JUN), and tumor protein p53(TP53)-binding sites. Mutagenesis studies identify that both the NFKB1- and TP53-binding sites are responsive to changes in the extracellular pH. These data help to explain how ameloblasts respond to the altered extracellular milieu of protons by changing their gene-expression profile throughout the stages of amelogenesis.

Reduced levels of miR‐34a in neuroblastoma are not caused by mutations in the TP53 binding site

Neuroblastoma (NB) is the most common extracranial solid tumor in children below the age of 5 years. miR-34a, located in chromosome band 1p36, has been recently implicated as a tumor suppressor gene in NB. In addition, it has been shown that miR-34a is activated by TP53 by binding to a TP53 binding site upstream to the mature miR-34a. We studied NB tumors from 57 patients for miR-34a expression levels, 1p status, mutations in the TP53 coding region and mutations of the TP53 binding site. Reduced expression levels of miR-34a were identified in tumors harboring 1p36.3 Loss (P = 0.028). No mutations were identified in the coding region of TP53, or in the TP53 binding site. Thus, mutations in the binding site are not an additional mechanism for the inactivation of miR-34a in NB. Other regulatory mechanisms controlling miR-34a expression and its relationship to TP53 should be further explored.

Ionizing Radiation Enhances the Expression of the Nonsteroidal Anti-inflammatory Drug-Activated Gene (NAG1) by Increasing the Expression of TP53 in Human Colon Cancer Cells

The induction of apoptosis in cells of human colon cancer cell lines after gamma irradiation was investigated to determine whether apoptosis was mediated by TP53 and the subsequent expression of its downstream target, the NSAID-activated gene (NAG1). HCT116 (TP53(+/+)), HCT15 (TP53 mutant) and TP53 null HCT116 (TP53(-/-)) cells were irradiated with gamma rays, and apoptosis was measured at various times after irradiation. In HCT116 TP53(+/+) cells, apoptosis was increased after irradiation; the increase was dependent on the time after treatment and the dose of gamma rays. However, in HCT15 TP53 mutant cells and HCT116 TP53(-/-) cells, there were no remarkable changes in apoptosis. The expression of TP53 protein in HCT116 cells was increased after irradiation and was followed by an increase in the expression of NAG1 protein. In contrast, the expression of NAG1 protein in TP53 mutant cells and TP53(-/-) cells was not increased by the radiation treatment, suggesting that NAG1 was required for apoptosis. The expression of NAG1 increased apoptosis in HCT116 cells, but radiation treatment did not further increase apoptosis. The transfection of a NAG1 siRNA into HCT116 cells suppressed radiation-induced apoptosis and inhibited the induction of NAG1 protein without altering the expression of TP53. a NAG1 luciferase promoter construct that included both of the TP53 binding sites, was activated by radiation in dose-dependent manner, while the promoters lacking one or both of the TP53 binding sites in the NAG1 promoter activity either was less responsive or did not respond. The findings reported here indicate that gamma radiation activates the TP53 tumor suppressor, which then increases the expression of NAG1. NAG1 mediates the induction of apoptosis in human colorectal cells.