P53-dependent Gene Expression Research Articles

A p53 score derived from TP53 CRISPR/Cas9 HMCLs predicts survival and reveals a major role of BAX in the response to BH3 mimetics

AbstractTo establish a strict p53-dependent gene-expression profile, TP53−/− clones were derived from TP53+/+ and TP53−/mut t(4;14) human myeloma cell lines (HMCLs) using CRISPR/Cas9 technology. From the 17 dysregulated genes shared between the TP53−/− clones from TP53+/+ HMCLs, we established a functional p53 score, involving 13 genes specifically downregulated upon p53 silencing. This functional score segregated clones and myeloma cell lines as well as other cancer cell lines according to their TP53 status. The score efficiently identified samples from patients with myeloma with biallelic TP53 inactivation and was predictive of overall survival in Multiple Myeloma Research Foundation–coMMpass and CASSIOPEA cohorts. At the functional level, we showed that among the 13 genes, p53-regulated BAX expression correlated with and directly affected the MCL1 BH3 mimetic S63845 sensitivity of myeloma cells by decreasing MCL1-BAX complexes. However, resistance to S63845 was overcome by combining MCL1 and BCL2 BH3 mimetics, which displayed synergistic efficacy. The combination of BH3 mimetics was effective in 97% of patient samples with or without del17p. Nevertheless, single-cell RNA sequencing analysis showed that myeloma cells surviving the combination had lower p53 score, showing that myeloma cells with higher p53 score were more sensitive to BH3 mimetics. Taken together, we established a functional p53 score that identifies myeloma cells with biallelic TP53 invalidation, demonstrated that p53-regulated BAX is critical for optimal cell response to BH3 mimetics, and showed that MCL1 and BCL2 BH3 mimetics in combination may be of greater effectiveness for patients with biallelic TP53 invalidation, for whom there is still an unmet medical need.

P53 Regulates the Extent of Fibroblast Proliferation and Fibrosis in Left Ventricle Pressure Overload.

Cardiomyopathy is characterized by the pathological accumulation of resident cardiac fibroblasts that deposit ECM (extracellular matrix) and generate a fibrotic scar. However, the mechanisms that control the timing and extent of cardiac fibroblast proliferation and ECM production are not known, hampering the development of antifibrotic strategies to prevent heart failure. We used the Tcf21 (transcription factor 21)MerCreMer mouse line for fibroblast-specific lineage tracing and p53 (tumor protein p53) gene deletion. We characterized cardiac physiology and used single-cell RNA-sequencing and in vitro studies to investigate the p53-dependent mechanisms regulating cardiac fibroblast cell cycle and fibrosis in left ventricular pressure overload induced by transaortic constriction. Cardiac fibroblast proliferation occurs primarily between days 7 and 14 following transaortic constriction in mice, correlating with alterations in p53-dependent gene expression. p53 deletion in fibroblasts led to a striking accumulation of Tcf21-lineage cardiac fibroblasts within the normal proliferative window and precipitated a robust fibrotic response to left ventricular pressure overload. However, excessive interstitial and perivascular fibrosis does not develop until after cardiac fibroblasts exit the cell cycle. Single-cell RNA sequencing revealed p53 null fibroblasts unexpectedly express lower levels of genes encoding important ECM proteins while they exhibit an inappropriately proliferative phenotype. in vitro studies establish a role for p53 in suppressing the proliferative fibroblast phenotype, which facilitates the expression and secretion of ECM proteins. Importantly, Cdkn2a (cyclin-dependent kinase inhibitor 2a) expression and the p16Ink4a-retinoblastoma cell cycle control pathway is induced in p53 null cardiac fibroblasts, which may eventually contribute to cell cycle exit and fulminant scar formation. This study reveals a mechanism regulating cardiac fibroblast accumulation and ECM secretion, orchestrated in part by p53-dependent cell cycle control that governs the timing and extent of fibrosis in left ventricular pressure overload.

Involvement of the p53/p21 complex in p53-dependent gene expression

The p53 tumor suppressor regulates cell functions either by acting as a transcription factor or by interacting with other proteins. Previously, we reported that the non-transcriptional actions of p53 can be facilitated by the binding of p53 to p21. Herein, we investigated whether p53/p21 interaction influences the transcriptional activity of p53. We observed that the expression of the p53 promoter-based reporter gene is dependent on p21 levels. Moreover, using a p21 variant that is unable to bind p53, we showed that p53 promoter activity requires p53/p21 interaction. To investigate the possible role of p21 in regulating the expression of endogenous p53 targets, we analyzed mRNA levels of Puma, Mdm2, and Gadd45a in untreated control and γ-ray-irradiated cells. We observed that while Puma expression is dependent on p53 regardless of γ-irradiation, p53 mediates the expression of Mdm2 and Gadd45a only in irradiated cells. Notably, p53/p21 interaction is required only for the p53-dependent expression of the tested genes and not Mdm2 and Gadd45a in non-irradiated cells. Moreover, chromatin immunoprecipitation assay revealed that p21 is required for the binding of p53 to the promoters of Puma, Mdm2, and Gadd45a. Collectively, our data support the view that the p53/p21 complex is involved in regulating p53-dependent gene expression. These findings provide a new foundation for understanding the transcriptional action of p53.

Retraction Note to: Differential modulatory effects of GSK-3b and HDM2 on sorafenib-induced AIF nuclear translocation (programmed necrosis) in melanoma

GSK-3β phosphorylates numerous substrates that govern cell survival. It phosphorylates p53, for example, and induces its nuclear export, HDM2-dependent ubiquitination, and proteasomal degradation. GSK-3β can either enhance or inhibit programmed cell death, depending on the nature of the pro-apoptotic stimulus. We previously showed that the multikinase inhibitor sorafenib activated GSK-3β and that this activation attenuated the cytotoxic effects of the drug in various BRAF-mutant melanoma cell lines. In this report, we describe the results of studies exploring the effects of GSK-3β on the cytotoxicity and antitumor activity of sorafenib combined with the HDM2 antagonist MI-319. MI-319 alone increased p53 levels and p53-dependent gene expression in melanoma cells but did not induce programmed cell death. Its cytotoxicity, however, was augmented in some melanoma cell lines by the addition of sorafenib. In responsive cell lines, the MI-319/sorafenib combination induced the disappearance of p53 from the nucleus, the down modulation of Bcl-2 and Bcl-xL, the translocation of p53 to the mitochondria and that of AIF to the nuclei. These events were all GSK-3β-dependent in that they were blocked with a GSK-3β shRNA and facilitated in otherwise unresponsive melanoma cell lines by the introduction of a constitutively active form of the kinase (GSK-3β-S9A). These modulatory effects of GSK-3β on the activities of the sorafenib/MI-319 combination were the exact reverse of its effects on the activities of sorafenib alone, which induced the down modulation of Bcl-2 and Bcl-xL and the nuclear translocation of AIF only in cells in which GSK-3β activity was either down modulated or constitutively low. In A375 xenografts, the antitumor effects of sorafenib and MI-319 were additive and associated with the down modulation of Bcl-2 and Bcl-xL, the nuclear translocation of AIF, and increased suppression of tumor angiogenesis. Our data demonstrate a complex partnership between GSK-3β and HDM2 in the regulation of p53 function in the nucleus and mitochondria. The data suggest that the ability of sorafenib to activate GSK-3β and alter the intracellular distribution of p53 may be exploitable as an adjunct to agents that prevent the HDM2-dependent degradation of p53 in the treatment of melanoma.

Retraction Note to: Effects of HDM2 antagonism on sunitinib resistance, p53 activation, SDF-1 induction, and tumor infiltration by CD11b+/Gr-1+ myeloid derived suppressor cells

The studies reported herein were undertaken to determine if the angiostatic function of p53 could be exploited as an adjunct to VEGF-targeted therapy in the treatment of renal cell carcinoma (RCC). Nude/beige mice bearing human RCC xenografts were treated with various combinations of sunitinib and the HDM2 antagonist MI-319. Tumors were excised at various time points before and during treatment and analyzed by western blot and IHC for evidence of p53 activation and function. Sunitinib treatment increased p53 levels in RCC xenografts and transiently induced the expression of p21waf1, Noxa, and HDM2, the levels of which subsequently declined to baseline (or undetectable) with the emergence of sunitinib resistance. The development of resistance and the suppression of p53-dependent gene expression temporally correlated with the induction of the p53 antagonist HDMX. The concurrent administration of MI-319 markedly increased the antitumor and anti-angiogenic activities of sunitinib and led to sustained p53-dependent gene expression. It also suppressed the expression of the chemokine SDF-1 (CXCL12) and the influx of CD11b+/Gr-1+ myeloid-derived suppressor cells (MDSC) otherwise induced by sunitinib. Although p53 knockdown markedly reduced the production of the angiostatic peptide endostatin, the production of endostatin was not augmented by MI-319 treatment. The evasion of p53 function (possibly through the expression of HDMX) is an essential element in the development of resistance to VEGF-targeted therapy in RCC. The maintenance of p53 function through the concurrent administration of an HDM2 antagonist is an effective means of delaying or preventing the development of resistance.

Subcellular Distribution of p53 by the p53-Responsive lncRNA NBAT1 Determines Chemotherapeutic Response in Neuroblastoma.

Neuroblastoma has a low mutation rate for the p53 gene. Alternative ways of p53 inactivation have been proposed in neuroblastoma, such as abnormal cytoplasmic accumulation of wild-type p53. However, mechanisms leading to p53 inactivation via cytoplasmic accumulation are not well investigated. Here we show that the neuroblastoma risk-associated locus 6p22.3-derived tumor suppressor NBAT1 is a p53-responsive lncRNA that regulates p53 subcellular levels. Low expression of NBAT1 provided resistance to genotoxic drugs by promoting p53 accumulation in cytoplasm and loss from mitochondrial and nuclear compartments. Depletion of NBAT1 altered CRM1 function and contributed to the loss of p53-dependent nuclear gene expression during genotoxic drug treatment. CRM1 inhibition rescued p53-dependent nuclear functions and sensitized NBAT1-depleted cells to genotoxic drugs. Combined inhibition of CRM1 and MDM2 was even more effective in sensitizing aggressive neuroblastoma cells with p53 cytoplasmic accumulation. Thus, our mechanistic studies uncover an NBAT1-dependent CRM1/MDM2-based potential combination therapy for patients with high-risk neuroblastoma. SIGNIFICANCE: This study shows how a p53-responsive lncRNA mediates chemotherapeutic response by modulating nuclear p53 pathways and identifies a potential treatment strategy for patients with high-risk neuroblastoma.

P55PIK Regulates P53-Dependent Apoptosis in Cancer Cells by Interacting with P53 DNA-Specific Domain.

PurposePhosphatidylinositol 3-kinase (PI3K) plays an important role in tumorigenesis by cross-talking with several signaling pathways. p55PIK is a unique regulatory subunit of PI3K and contains an extra 24-residue N-terminal domain (N24). This study aimed to explore the interaction of p55PIK with p53 and the role of p55PIK in regulating p53-dependent apoptosis in cancer cells.Materials and MethodsThe expression of p55PIK was detected in cancer cells, and the interaction of p55PIK with p53 was examined by immunoprecipitation and pull-down assay. The expression of p53-dependent apoptosis-related genes was detected by PCR.ResultsN24 domain of p55PIK interacted with DNA-specific binding domain (DBD) of p53. The increase or decrease of p55PIK expression led to the change of the expression of p53 and p53-regulated genes in cancer cells. Moreover, N24 peptide led to the change of the expression of p53-regulated genes. Moreover, a membrane-permeable N24 peptide enhanced p53-dependent apoptosis induced by methyl methanesulfonate.ConclusionOur results reveal a novel mechanism that regulates p53-dependent apoptosis in cancer cells via p55PIK-p53 interaction.

Pulsatile MAPK Signaling Modulates p53 Activity to Control Cell Fate Decisions at the G2 Checkpoint for DNA Damage

SummaryCell-autonomous changes in p53 expression govern the duration and outcome of cell-cycle arrest at the G2 checkpoint for DNA damage. Here, we report that mitogen-activated protein kinase (MAPK) signaling integrates extracellular cues with p53 dynamics to determine cell fate at the G2 checkpoint. Optogenetic tools and quantitative cell biochemistry reveal transient oscillations in MAPK activity dependent on ataxia-telangiectasia-mutated kinase after DNA damage. MAPK inhibition alters p53 dynamics and p53-dependent gene expression after checkpoint enforcement, prolonging G2 arrest. In contrast, sustained MAPK signaling induces the phosphorylation of CDC25C, and consequently, the accumulation of pro-mitotic kinases, thereby relaxing checkpoint stringency and permitting cells to evade prolonged G2 arrest and senescence induction. We propose a model in which this MAPK-mediated mechanism integrates extracellular cues with cell-autonomous p53-mediated signals, to safeguard genomic integrity during tissue proliferation. Early steps in oncogene-driven carcinogenesis may imbalance this tumor-suppressive mechanism to trigger genome instability.

2003P - Expression of mutant p53 affects cancer cell sensitivity to topotecan

BackgroundInactivation of tumor suppressor p53 is a common event in tumor progression. In almost 50% of cases p53 inactivation is caused by mutations that primarily affect DNA-binding domain. Oncogenic missense mutation Y220C is the ninth most common for p53 and is annually observed in ∼100,000 new diagnosed cancer cases worldwide. Presence of this mutation disturbs tertiary structure of the p53 DNA-binding domain that further leads to destabilization of the whole protein, its partial denaturation and loss of transcriptional activity. In some cases, p53 mutations result in more aggressive cancer cells and alter drug sensitivity. MethodsWe employ CRISPR-Cas9 gene editing technology to generate p53 knock-out and Y220C mutant MCF-7 cell lines. Quantitative analysis of alterations in intracellular protein levels were performed by immunoblotting, analysis of p53-dependent gene expression by quantitative real-time reverse transcription PCR; cell proliferation and chemotherapy cytotoxicity by MTS test. Statistical analysis was performed using ANOVA and Holm-Sidak method for multiple comparison, p≤0.05 was considered to be statistically significant. ResultsWe have confirmed that p53 mutation Y220C leads to p53 inactivation. Proliferation rate of p53 knockout and mutant MCF-7 cell lines was 15% higher than wild type. We have shown statistically significant decrease in topotecan cytotoxicity towards knockout and mutant cells compared to wild-type – 14% and 26%, respectively. ConclusionsDecline of topotecan cytotoxicity in mutant and knockout cells can be explained by topotecan-mediated induction of p53 that leads to higher levels of cell death in wild-type MCF-7. References Bulatov, E., Zagidullin, A., Valiullina, A., Sayarova, R., Rizvanov, A. (2018). Small molecule modulators of RING-type E3 ligases: MDM and Cullin families as targets. Frontiers in pharmacology, 9, 450. Legal entity responsible for the studyAlbert Rizvanov. FundingGrant of the President of the Russian Federation (MK-4253.2018.4). DisclosureAll authors have declared no conflicts of interest.

Abstract 511: Regulation of p53 Protein Levels Drives Activation of Cardiac Fibroblasts in Response to Pressure Overload

Heart disease is accompanied by the accumulation of resident cardiac fibroblasts (CF) that subsequently become myofibroblasts and secrete copious amounts of extracellular matrix (ECM), impeding cardiac function and driving the progression of heart failure. Understanding the mechanisms coordinating CF accumulation and myofibroblast activation may reveal novel therapeutic strategies to block pathologic fibrosis. We recently found that s mall pr oline r ich protein 2b (SPRR2B) drives CF proliferation in response to pathological cues by facilitating MDM2-dependent proteasomal degradation of p53. Surprisingly, although Sprr2b gene deletion or targeted mutation of the USP7/MDM2 interacting domain in mice ( Sprr2b-KO and Sprr2b-USP m , respectively) stimulated the expression of p53-dependent cell cycle arrest genes, mutant animals also developed excessive fibrosis in LV pressure overload. The development of fibrosis in Sprr2b mutant mice was traced primarily to a more robust myofibroblast activation response, providing evidence that CF accumulation and myofibroblast activation may be mutually antagonistic. To investigate the contribution p53 to CF accumulation and fibrosis in mouse heart disease models, we deleted floxed p53 alleles specifically in adult CF using the tamoxifen-inducible Tcf21 MerCreMer mouse line (called p53-CKO). Surprisingly, while p53-CKO animals display exaggerated accumulation of Tcf21 + /PDGFRα + CF in response to left ventricle pressure overload, we observed a biphasic physiological response; initially, p53-CKO animals are resistant to systolic functional decline, only developing more severe fibrosis and functional decline than littermate controls at later time points. Time course studies using primary adult mouse CF revealed that p53 positively correlates with myofibroblast activation, while reduction in p53 levels correlates with accelerated cell cycle and the suppression of myofibroblast activation until the subsequent induction of p16/19 - Rb-mediated cell cycle arrest. Taken together, this study offers detailed insight into the transition of CF from a proliferative to an activated state that may accelerate the development of anti-fibrotic strategies.

Chromatin-remodeling factor, RSF1, controls p53-mediated transcription in apoptosis upon DNA strand breaks

Remodeling and spacing factor 1 (RSF1), which is one of chromatin-remodeling factors, has been linked to the DNA damage response (DDR) and DNA repair. However, the biological consequence of RSF1 deficiency in DDR in vivo and its molecular mechanisms remain unknown. Because defective DDR is related to neuropathological phenotypes, we developed neural-specific Rsf1 knockout mice. Rsf1 deficiency did not result in any neuropathological abnormalities, but prevented neural apoptosis triggered by excessive DNA strand breaks during neurogenesis. Likewise, cell death was significantly reduced in RSF1 deficient human cell lines after DNA damage, and the global transcriptome of these cells revealed that the expressions of p53 downstream genes were significantly reduced upon DNA strand breaks. Inactivation of these genes resulted from decreased binding of p53/p300 complex and subsequent reduction of H3 acetylation at their promoters. Our data show that RSF1 is necessary for p53-dependent gene expression in response to DNA strand breaks via controlling the accessibility of p53/p300 complex to its target genes and contributes to the maintenance of cellular integrity.

Thyroxine inhibits resveratrol-caused apoptosis by PD-L1 in ovarian cancer cells.

Thyroid hormone, l-thyroxine (T4), has been shown to promote ovarian cancer cell proliferation via a receptor on plasma membrane integrin αvβ3 and to induce the activation of ERK1/2 and expression of programmed death-ligand 1 (PD-L1) in cancer cells. In contrast, resveratrol binds to integrin αvβ3 at a discrete site and induces p53-dependent antiproliferation in malignant neoplastic cells. The mechanism of resveratrol action requires nuclear accumulation of inducible cyclooxygenase (COX)-2 and its complexation with phosphorylated ERK1/2. In this study, we examined the mechanism by which T4 impairs resveratrol-induced antiproliferation in human ovarian cancer cells and found that T4 inhibited resveratrol-induced nuclear accumulation of COX-2. Furthermore, T4 increased expression and cytoplasmic accumulation of PD-L1, which in turn acted to retain inducible COX-2 in the cytoplasm. Knockdown of PD-L1 by small hairpin RNA (shRNA) relieved the inhibitory effect of T4 on resveratrol-induced nuclear accumulation of COX-2- and COX-2/p53-dependent gene expression. Thus, T4 inhibits COX-2-dependent apoptosis in ovarian cancer cells by retaining inducible COX-2 with PD-L1 in the cytoplasm. These findings provide new insights into the antagonizing effect of T4 on resveratrol's anticancer properties.

Abstract 58: Therapeutically targeting the cell cycle in head and neck cancer

Abstract Head and neck squamous cell carcinomas (HNSCC) have a complex mutational spectrum with tumor suppressor and cell cycle dysregulation as the most prominent features. Additionally, HPV infection has been shown to be a cause of some HNSCC, particularly oropharyngeal cancer, and it is increasing in the US. Our recent studies have established WEE1 inhibition as a new therapeutic strategy in HNSCC. WEE1 inhibition not only leads to premature mitosis by weakening the G2/M checkpoint but also causes replication stress in S-phase particularly in tumors with deficient TP53. Given that HPV infection causes genetic instability by inactivating tumor suppressor pathways (TP53 and Rb) and impairing proper DNA damage repair (DDR) response, we aim to explore targets that will synergize with WEE1 and decrease reliance on chemotherapy. To this end, we performed a q-PCR array profiling p53 dependent gene expression in a pair of isogenic HNSCC cell lines for p53 knockdown (vs wildtype control). Using a similar profiling technique in an isogenic pair of immortalized human oral keratinocytes (OKF4 cells) over expressing HPV16 E6/E7 proteins, we profiled the expression of DDR genes in response to DNA damage in wildtype vs. E6E7 expressing keratinocytes. A functional high-throughput siRNA library screen for DDR genes was performed to functionally validate the pathways identified. We looked for DDR genes that specifically sensitized p53 deficient HNSCC cells as well as E6/E7 expressing cells to WEE1 inhibition. TP53 deficient cells exhibited elevated levels of DDR related genes in response to DNA damage when compared to isogenic wildtype. We identified potential survival DDR pathways for E6E7- mediated transformation, targeting of which represents a novel therapeutic strategy. The DDR siRNA screen with WEE1 inhibition provided a functional validation for our findings. Gene ontology analysis of top 10% of WEE1 sensitizers identified BRCA1 related DNA damage response genes as top sensitizers for WEE1 inhibition in p53 deficient cells. Collectively, these results would help develop a new therapeutic paradigm for HPV-positive HNSCC by providing tailored mechanism-based therapies. Citation Format: Ahmed Diab, Xu Chang, Michael Kao, Bruce Clurman, Denise Galloway, Mendez Eduardo. Therapeutically targeting the cell cycle in head and neck cancer [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 58.

Mechanisms of action of nonpeptide hormones on resveratrol-induced antiproliferation of cancer cells.

Nonpeptide hormones, such as thyroid hormone, dihydrotestosterone, and estrogen, have been shown to stimulate cancer proliferation via different mechanisms. Aside from their cytosolic or membrane-bound receptors, there are receptors on integrin αv β3 for nonpeptide hormones. Interaction between hormones and integrin αv β3 can induce signal transduction and eventually stimulate cancer cell proliferation. Resveratrol induces inducible COX-2-dependent antiproliferation via integrin αv β3 . Resveratrol and hormone-induced signals are both transduced by activated extracellular-regulated kinases 1 and 2 (ERK1/2); however, hormones promote cell proliferation, while resveratrol induces antiproliferation in cancer cells. Hormones inhibit resveratrol-stimulated phosphorylation of p53 on Ser15, resveratrol-induced nuclear COX-2 accumulation, and formation of p53-COX-2 nuclear complexes. Subsequently, hormones impair resveratrol-induced COX-2-/p53-dependent gene expression. The inhibitory effects of hormones on resveratrol action can be blocked by different antagonists of specific nonpeptide hormone receptors but not integrin αv β3 blockers. Results suggest that nonpeptide hormones inhibit resveratrol-induced antiproliferation in cancer cells downstream of the interaction between ligand and receptor and ERK1/2 activation to interfere with nuclear COX-2 accumulation. Thus, the surface receptor sites for resveratrol and nonpeptide hormones are distinct and can induce discrete ERK1/2-dependent downstream antiproliferation biological activities. It also indicates the complex pathways by which antiproliferation is induced by resveratrol in various physiological hormonal environments. .

Abstract 1: Sprr2b Drives Proliferation of Cardiac Fibroblasts by Relieving p53-mediated Cell Cycle Inhibition

Pathological cardiac remodeling is initially a compensatory attempt to increase cardiac output, but ultimately leads to the development of fibrosis, a form of scarring that contributes to heart failure (HF). In contrast, physiological cardiac remodeling in response to exercise is not associated with the development of fibrosis and typically remains compensatory. Understanding how cardiac fibroblasts (CF), the primary source of extracellular matrix in the heart, respond to pathological and physiological cues might lead to novel approaches to limit the maladaptive effects of pathological cardiac remodeling. We performed RNA sequencing to define genes that are differentially regulated in CF during physiological (swimming) or pathological (pressure overload) remodeling. This study revealed that cardiac expression of the s mall pr oline r ich 2b ( Sprr2b) gene is restricted to CFs and is significantly elevated in disease and lost in exercise. We demonstrate that SPRR2B drives CF proliferation, but not myofibroblast differentiation, in response to pathological cues. SPRR2B facilitates an interaction between MDM2 and USP7, a nuclear deubiquitinase that leads to proteasomal degradation of p53. SPRR2B-USP7-MDM2 complex formation and p53 degradation is at least partially dependent upon phosphorylation of SPRR2B by Src-family NRTKs. SPRR2B thus relieves p53-mediated constraints on cell cycle progression in response to Src-dependent signaling, leading to CF accumulation. Importantly, SPRR2B expression is elevated in cardiac tissue from human HF patients relative to individuals without heart disease and positively correlates with a proliferative, activated gene expression profile in HF patient CF. Treatment of human HF fibroblasts with IGF-1/H 2 O 2 to mimic physiological cues significantly abrogated SPRR2B expression and increased expression of p53-dependent cell cycle checkpoint genes, which correlated with a less activated phenotype. Taken together, this study defines a unique tissue-specific role of Sprr2b in driving pathological CF cell cycle progression that may underlie the development of cardiac fibrosis.

An inducible long noncoding RNA amplifies DNA damage signaling.

Long noncoding RNAs (lncRNAs) are prevalent genes with frequently exquisite regulation but mostly unknown functions. Here we demonstrate a role of lncRNAs in guiding organismal DNA damage response. DNA damage activates transcription of DINO (Damage Induced NOncoding) via p53. DINO is required for p53-dependent gene expression, cell cycle arrest, and apoptosis in response to DNA damage, and DINO expression suffice to activate damage signaling and cell cycle arrest in the absence of DNA damage. DINO binds to and promotes p53 protein stabilization, mediating a p53 auto-amplification loop. Dino knockout or promoter inactivation in mice dampens p53 signaling and ameliorates acute radiation syndrome in vivo. Thus, inducible lncRNA can create a feedback loop with its cognate transcription factor to amplify cellular signaling networks.

Benzo(a)pyrene Induced p53 Mediated Male Germ Cell Apoptosis: Synergistic Protective Effects of Curcumin and Resveratrol.

Benzo(a)pyrene (B(a)P) is an environmental toxicant that induces male germ cell apoptosis. Curcumin and resveratrol are phytochemicals with cytoprotective and anti-oxidative properties. At the same time resveratrol is also a natural Aryl hydrocarbon Receptor (AhR) antagonist. Our present study in isolated testicular germ cell population from adult male Wistar rats, highlighted the synergistic protective effect of curcumin and resveratrol against B(a)P induced p53 mediated germ cell apoptosis. Curcumin-resveratrol significantly prevented B(a)P induced decrease in sperm cell count and motility, as well as increased serum testosterone level. Curcumin-resveratrol co-treatment actively protected B(a)P induced testicular germ cell apoptosis. Curcumin-resveratrol co-treatment decreased the expression of pro-apoptotic proteins like cleaved caspase 3, 8 and 9, cleaved PARP, Apaf1, FasL, tBid. Curcumin-resveratrol co-treatment decreased Bax/Bcl2 ratio, mitochondria to cytosolic translocation of cytochrome c and activated the survival protein Akt. Curcumin-resveratrol decreased the expression of p53 dependent apoptotic genes like Fas, FasL, Bax, Bcl2, and Apaf1. B(a)P induced testicular reactive oxygen species (ROS) generation and oxidative stress were significantly ameliorated with curcumin and resveratrol. Curcumin-resveratrol co-treatment prevented B(a)P induced nuclear translocation of AhR and CYP1A1 (Cytochrome P4501A1) expression. The combinatorial treatment significantly inhibited B(a)P induced ERK 1/2, p38 MAPK and JNK 1/2 activation. B(a)P treatment increased the expression of p53 and its phosphorylation (p53 ser 15). Curcumin-resveratrol co-treatment significantly decreased p53 level and its phosphorylation (p53 ser 15). The study concludes that curcumin-resveratrol synergistically modulated MAPKs and p53, prevented oxidative stress, regulated the expression of pro and anti-apoptotic proteins as well as the proteins involved in B(a)P metabolism thus protected germ cells from B(a)P induced apoptosis.

Pre-clinical efficacy and synergistic potential of the MDM2-p53 antagonists, Nutlin-3 and RG7388, as single agents and in combined treatment with cisplatin in ovarian cancer

Ovarian cancer is the fifth leading cause of cancer-related female deaths. Due to serious side effects, relapse and resistance to standard chemotherapy, better and more targeted approaches are required. Mutation of the TP53 gene accounts for 50% of all human cancers. In the remaining malignancies, non-genotoxic activation of wild-type p53 by small molecule inhibition of the MDM2-p53 binding interaction is a promising therapeutic strategy. Proof of concept was established with the cis-imidazoline Nutlin-3, leading to the development of RG7388 and other compounds currently in early phase clinical trials. This preclinical study evaluated the effect of Nutlin-3 and RG7388 as single agents and in combination with cisplatin in a panel of ovarian cancer cell lines. Median-drug-effect analysis showed Nutlin-3 or RG7388 combination with cisplatin was additive to, or synergistic in a p53-dependent manner, resulting in increased p53 activation, cell cycle arrest and apoptosis, associated with increased p21WAF1 protein and/or caspase-3/7 activity compared to cisplatin alone. Although MDM2 inhibition activated the expression of p53-dependent DNA repair genes, the growth inhibitory and pro-apoptotic effects of p53 dominated the response. These data indicate that combination treatment with MDM2 inhibitors and cisplatin has synergistic potential for the treatment of ovarian cancer, dependent on cell genotype.

P53 Pulses Diversify Target Gene Expression Dynamics in an mRNA Half-Life-Dependent Manner and Delineate Co-regulated Target Gene Subnetworks

The transcription factor p53 responds to DNA double-strand breaks by increasing in concentration in a series of pulses of fixed amplitude, duration, and period. How p53 pulses influence the dynamics ofp53 target gene expression is not understood. Here, we show that, in bulk cell populations, patterns of p53 target gene expression cluster into groups with stereotyped temporal behaviors, including pulsing and rising dynamics. These behaviors correlate statistically with the mRNA decay rates of target genes: short mRNA half-lives produce pulses of gene expression. This relationship can be recapitulated by mathematical models of p53-dependent gene expression in single cells and cell populations. Single-cell transcriptional profiling demonstrates that expression of a subset of p53 target genes is coordinated across time within single cells; p53 pulsing attenuates this coordination. These results help delineate how p53 orchestrates the complex DNA damage response and give insight into the function of pulsatile signaling pathways.

Murine Gammaherpesvirus 68 LANA and SOX Homologs Counteract ATM-Driven p53 Activity during Lytic Viral Replication.

Tumor suppressor p53 is activated in response to numerous cellular stresses, including viral infection. However, whether murine gammaherpesvirus 68 (MHV68) provokes p53 during the lytic replication cycle has not been extensively evaluated. Here, we demonstrate that MHV68 lytic infection induces p53 phosphorylation and stabilization in a manner that is dependent on the DNA damage response (DDR) kinase ataxia telangiectasia mutated (ATM). The induction of p53 during MHV68 infection occurred in multiple cell types, including splenocytes of infected mice. ATM and p53 activation required early viral gene expression but occurred independently of viral DNA replication. At early time points during infection, p53-responsive cellular genes were induced, coinciding with p53 stabilization and phosphorylation. However, p53-related gene expression subsided as infection progressed, even though p53 remained stable and phosphorylated. Infected cells also failed to initiate p53-dependent gene expression and undergo apoptosis in response to treatment with exogenous p53 agonists. The inhibition of p53 responses during infection required the expression of the MHV68 homologs of the shutoff and exonuclease protein (muSOX) and latency-associated nuclear antigen (mLANA). Interestingly, mLANA, but not muSOX, was necessary to prevent p53-mediated death in MHV68-infected cells under the conditions tested. This suggests that muSOX and mLANA are differentially required for inhibiting p53 in specific settings. These data reveal that DDR responses triggered by MHV68 infection promote p53 activation. However, MHV68 encodes at least two proteins capable of limiting the potential consequences of p53 function. Gammaherpesviruses are oncogenic herpesviruses that establish lifelong chronic infections. Defining how gammaherpesviruses overcome host responses to infection is important for understanding how these viruses infect and cause disease. Here, we establish that murine gammaherpesvirus 68 induces the activation of tumor suppressor p53. p53 activation was dependent on the DNA damage response kinase ataxia telangiectasia mutated. Although active early after infection, p53 became dominantly inhibited as the infection cycle progressed. Viral inhibition of p53 was mediated by the murine gammaherpesvirus 68 homologs of muSOX and mLANA. The inhibition of the p53 pathway enabled infected cells to evade p53-mediated cell death responses. These data demonstrate that a gammaherpesvirus encodes multiple proteins to limit p53-mediated responses to productive viral infection, which likely benefits acute viral replication and the establishment of chronic infection.