Inhibition Of P300 Research Articles

Androgen receptor co-activators in the regulation of cellular events in prostate cancer

Androgen receptor (AR) action in benign and malignant tissue is potentiated by a number of co-regulatory proteins that may interact with one or more receptor domains. With improvement of research methodologies, it became possible to detect a number of co-activators whose expression is increased in prostate cancer tissue. Manuscripts describing prostate cancer-relevant regulation of cellular events by co-activators are selected and summarized. AR co-activators may regulate histone modification, proteasomal degradation, chaperones, sumoylation, chromatin remodeling, and cytoskeleton. Some of them (TIF-2) are up-regulated by androgens, whereas the expression of others increases during androgen ablation (p300, CBP, and Tip60). Most co-factors are important for the stimulation of cellular proliferation, although in some cases (ART-27), they act as tumor suppressors and are deleted in prostate cancer tissue. In addition to stimulating AR, some co-activators suppress apoptosis in prostate cancer cells that do not express the AR (p300 and SRC-3). It was recently shown that the inhibition of p300 slows down proliferation, stimulates apoptosis, and inhibits migration and invasion. Co-factors whose down-regulation results in the alterations of multiple cellular functions may be valid targets for novel therapies in advanced prostate cancer.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

The leukemogenicity of AML1-ETO is dependent on site-specific lysine acetylation.

The chromosomal translocations found in acute myelogenous leukemia (AML) generate oncogenic fusion transcription factors with aberrant transcriptional regulatory properties. Although therapeutic targeting of most leukemia fusion proteins remains elusive, the posttranslational modifications that control their function could be targetable. We found that AML1-ETO, the fusion protein generated by the t(8;21) translocation, is acetylated by the transcriptional coactivator p300 in leukemia cells isolated from t(8;21) AML patients, and that this acetylation is essential for its self-renewal-promoting effects in human cord blood CD34(+) cells and its leukemogenicity in mouse models. Inhibition of p300 abrogates the acetylation of AML1-ETO and impairs its ability to promote leukemic transformation. Thus, lysine acetyltransferases represent a potential therapeutic target in AML.

Paradoxical Enhancement of Fear Extinction Memory and Synaptic Plasticity by Inhibition of the Histone Acetyltransferase p300

It is well established that the coordinated regulation of activity-dependent gene expression by the histone acetyltransferase (HAT) family of transcriptional coactivators is crucial for the formation of contextual fear and spatial memory, and for hippocampal synaptic plasticity. However, no studies have examined the role of this epigenetic mechanism within the infralimbic prefrontal cortex (ILPFC), an area of the brain that is essential for the formation and consolidation of fear extinction memory. Here we report that a postextinction training infusion of a combined p300/CBP inhibitor (Lys-CoA-Tat), directly into the ILPFC, enhances fear extinction memory in mice. Our results also demonstrate that the HAT p300 is highly expressed within pyramidal neurons of the ILPFC and that the small-molecule p300-specific inhibitor (C646) infused into the ILPFC immediately after weak extinction training enhances the consolidation of fear extinction memory. C646 infused 6 h after extinction had no effect on fear extinction memory, nor did an immediate postextinction training infusion into the prelimbic prefrontal cortex. Consistent with the behavioral findings, inhibition of p300 activity within the ILPFC facilitated long-term potentiation (LTP) under stimulation conditions that do not evoke long-lasting LTP. These data suggest that one function of p300 activity within the ILPFC is to constrain synaptic plasticity, and that a reduction in the function of this HAT is required for the formation of fear extinction memory.

Transcriptional coactivators p300 and CBP stimulate estrogen receptor‐beta signaling and regulate cellular events in prostate cancer

Steroid receptor coactivators p300 and CBP are highly expressed in advanced prostate cancer. They potentiate activation of androgen receptor by androgens and anti-androgens. In the present study, we have addressed the question whether these coactivators enhance activity of estrogen receptor-beta (ER-β), which is variably expressed in prostate cancers. Expression levels of the coactivators p300 and CBP were manipulated by plasmid or siRNA transfections and activity of ER-β was measured by luciferase assays. Viability was measured by MTT assays and cellular migration was determined by wound-healing and Boyden chamber assays. High expression of ER-β was found in PC3 cells which were used for the experiments. p300 or CBP enhanced activation of ER-β by genistein. Antiestrogens did not acquire agonistic properties in the presence of increased concentrations of either coactivator. Inhibition of p300 or CBP decreased genistein stimulation of ER-β. Genistein reduced migration of PC3 prostate cancer cells and down-regulation of p300 potentiated this effect. p300 and CBP are implicated in regulation of ER-β activity and cellular migration in prostate cancer. These findings are important for understanding of action of ER-β in carcinoma of the prostate.

Mutational inhibition of c-Myb or p300 ameliorates treatment-induced thrombocytopenia

The transcription factor c-Myb and coregulator p300 have a key role in maintaining production of controlled numbers of megakaryocytes and platelets. In mice, mutations in c-Myb or p300 cause thrombocytosis in otherwise wild-type animals and can ameliorate the thrombocytopenia in mice lacking the thrombopoietin receptor, c-Mpl, a model for human congenital amegakaryocytic thrombocytopenia. To examine whether inhibition of c-Myb/p300 is effective in other models of thrombocytopenia, the effect of the c-Myb(Plt4) mutation on thrombocytopenia associated with reduced platelet life span in Bcl-X(Plt20/Plt20) mice was assessed, as were responses in c-Myb(Plt4) and/or p300(Plt6) mutant mice to thrombocytopenia associated with antiplatelet antibodies, chemotherapy, or bone marrow transplantation. Homozygosity of the c-Myb(Plt4) allele ameliorated thrombocytopenia associated with reduced platelet life span, and c-Myb(Plt4/+) mice exhibited more rapid than normal recovery from thrombocytopenia caused by antiplatelet serum or bone marrow transplantation. Recovery to pretreatment platelet levels was unaltered in 5-fluorouracil-treated c-Myb(Plt4/+) mice relative to wild-type controls, but enhanced platelet production during subsequent thrombocytosis was evident. More modest enhancement of platelet recovery after 5-fluorouracil or bone marrow transplantation was also evident in p300(Plt6/+) animals. The data suggest potential utility of c-Myb/p300 as a target for therapeutic intervention in thrombocytopenia of diverse origins.

Curcumin prevents diabetes-associated abnormalities in the kidneys by inhibiting p300 and nuclear factor-κB

Objective Diabetic nephropathy is a debilitating disease that leads to end-stage renal failure in the Western world. Hyperglycemia is the initiating factor in several chronic diabetic complications which mediates increased oxidative stress and eventually the increased production of vasoactive factors and extracellular matrix proteins. We hypothesized that curcumin, a potent antioxidant, might be beneficial in preventing the development of diabetic nephropathy because this compound has been shown to inhibit p300, a histone acetyltransferase that plays a role in regulating gene expression through its interaction with the transcription factor nuclear factor-κB. Methods To test this hypothesis, male Sprague-Dawley rats were injected with streptozotocin to induce diabetes. These animals were subsequently treated with curcumin for a period of 1 mo. Results Real-time reverse transcriptase polymerase chain reaction analyses showed that diabetes-induced upregulation of vasoactive factors (endothelial nitric oxide synthase and enothelin-1), transforming growth factor-β1 and extracellular matrix proteins (fibronectin and extradomain-B–containing fibronectin) in the kidneys. These changes were associated with increased oxidative stress, mesangial expansion, and p300 and nuclear factor-κB activity that were prevented with curcumin treatment. Conclusion These beneficial effects of curcumin were mediated through the inhibition of p300 and nuclear factor-κB.

Abstract 3385: Cardiac Stem Cell Survival During DNA Damage Is Regulated By Modulation Of Histone Acetyl Transferase p300

Background: Many sources of oxidative stress, including ischemia/reperfusion and chemotherapy, can induce DNA damage in the heart, leading to loss of cardiomyocytes and development of heart failure. Cardiac stem cells (CSCs) may serve to regenerate injured myocardium. However, ischemic injury has been associated with senescence and apoptosis in CSCs, potentially limiting their contribution to tissue repair. The histone acetyltransferase p300 has been shown to be an important regulator of genome stability and is involved in cell survival in response to DNA damage. We hypothesized that p300 could play a role in CSC response to oxidant stress. Methods: c-kit+, sca-1+ CSCs were isolated and cloned by serial dilution from adult mouse hearts. CSC clones were stably transfected with lentivirus vectors carrying shRNAmir against p300 or a non-silencing RNA (NS) and maintained under puromycin selection prior to treatment. CSCs were exposed to doxorubicin (DOX), a DNA-damaging agent, for defined intervals. Levels of p300 were monitored by Western analysis and expression of DNA damage-regulated genes was assessed using multiplex realtime QPCR. CSCs were treated with 1 uM DOX and survival was assessed over a period of 72h. Results: Levels of p300 increased rapidly in CSCs within 4h after exposure to doxorubicin and were sustained for at least 24h. CSCs expressing p300 shRNAmir (p300 KD) contained ~20% of the amount of p300 in CSCs transfected with the NS sequence (NS CSCs). QPCR analysis of DNA repair and cell cycle control genes indicated that 44 were significantly expressed in CSCs; 13 of these were up-regulated by p300 loss, and 5 were down-regulated, indicative of a DNA damage response to p300 loss. Both cell proliferation rates and progression through the cell cycle were slower in p300 KD vs. NS CSCs. Surprisingly, following 48h treatment with DOX, p300 KD CSCs had 32% improved survival vs. NS CSCs. Conclusions: Under acute genotoxic stress, p300 accumulates in CSCs. Reducing p300 levels induces the expression of genes involved in the DNA damage response, probably as a compensatory response, and slows CSC proliferation. Loss or inhibition of p300 is likely to cause multifactorial growth arrest in CSCs and thereby reduce their vulnerability to genotoxic agents. This research has received full or partial funding support from the American Heart Association, AHA National Center.

The Transcriptional Coactivator p300 Plays a Critical Role in the Hypertrophic and Protective Pathways Induced by Phenylephrine in Cardiac Cells but Is Specific to the Hypertrophic Effect of Urocortin

Anacardic acid is an alkylsalicylic acid obtained from cashew-nut-shell liquid, and is a potent inhibitor of p300 histone acetyl-transferase (HAT) activity. We have used anacardic acid to prevent the induction of hypertrophy in isolated neonatal rat cardiomyocytes. Hypertrophy was detected as an increase in cell size, the rearrangement of sarcomeres into a striated pattern, and the induction of embryonic genes beta-MHC and ANF. p300 inhibition was equally effective at preventing hypertrophy whether it was induced by treatment with the alpha1-adrenergic agonist, phenylephrine, or by treatment with urocortin, a member of the corticotrophin-releasing-factor family, which stimulates specific G protein-coupled receptors. Spiruchostatin A is a natural-product inhibitor of histone deacetylases (HDAC) similar to the depsipeptide FK228 molecule. We have recently synthesized spiruchostatin A and now show that, although HDACs act in opposition to HATs, spiruchostatin A has the same effect as anacardic acid, that is, it prevents the induction of hypertrophy in response to phenylephrine or urocortin. Pretreatment with either phenylephrine or urocortin reduced the extent of death observed after the exposure of isolated cardiomyocytes to simulated ischaemia and reoxygenation. Inhibition of p300 or HDAC activity eliminated the protection conferred by phenylephrine; however, it did not affect the protection conferred by urocortin. Therefore, it might eventually be possible to use chemical inhibitors such as these in a therapeutic setting to dissociate the protective effect and hypertrophic effect of urocortin, enhancing the survival of cardiomyocytes exposed to transient ischemia, while inhibiting the hypertrophic pathway that would otherwise be induced concurrently.

Ethanol selectively modulates inflammatory activation signaling of brain microglia

In spite of well-known deleterious effects of alcohol on the nervous system in general, its specific effect on the brain immune system remains poorly understood. In order to better understand the effect of alcohol consumption on the innate immunity and inflammatory responses in the central nervous system (CNS), we sought to determine how ethanol influences inflammatory activation of microglia that function as the resident immune defense system of the brain. After treatment of BV-2 mouse microglial cells or rat primary microglia cultures with various stimuli, nitric oxide (NO) production was measured as an indicator of microglial activation. Pretreatment of the cells with ethanol (10–100 mM) for 1 h resulted in a significant decrease in lipopolysaccharide (LPS)-induced, but not interferon-gamma (IFNγ)-induced, NO production, indicating that ethanol specifically inhibits LPS-induced inflammatory activation of microglia. This was further supported by the ethanol inhibition of LPS-induced IL-1β expression. In addition, ethanol pretreatment selectively regulated LPS-induced NF-κB signaling pathway without affecting IFNγ-induced signal transducer and activator of transcription 1 (STAT1) phosphorylation, interferon regulatory factor-1 (IRF-1) induction or IFNγ-inducible IP-10 expression. The modulation of LPS-induced NF-κB by ethanol was due to the inhibition of coactivator p300. Altogether, these results suggest that acute ethanol exposure may selectively modulate signal transduction pathways associated with inflammatory activation of microglia, which may lead to derangement of CNS immune and inflammatory responses.

Transcriptional Coactivators CBP and p300 Cooperatively Enhance HNF-1 -Mediated Expression of the Albumin Gene in Hepatocytes

Transcriptional coactivators, CREB-binding protein (CBP) and p300, exhibit high homology in structure and similar functions. In the present study, we analyzed the function of CBP and p300 proteins as transcriptional coactivators in the expression of albumin in hepatocytes. The expression levels of CBP and p300 were high in fetal hepatocytes, but low in adult ones. Immunoprecipitation assays showed that both CBP and p300 interacted with hepatocyte nuclear factor-1alpha (HNF-1alpha) in primary hepatocytes. Furthermore, CBP and p300 were co-precipitated without HNF-1alpha. Chromatin immunoprecipitation (ChIP) assays revealed that both CBP and p300 are located in the albumin promoter region in hepatocytes. These results suggested that HNF-1alpha, CBP and p300 were incorporated into a preinitiation complex of RNA polymerase II at the albumin promoter. Luciferase reporter assays showed that CBP and p300 cooperatively triggered HNF-1alpha-mediated transcription of the albumin promoter. In addition, inhibition of CBP or p300 using small interfering RNAs (siRNAs) resulted in a reduction in albumin expression. These results suggest that both CBP and p300 are required for enhanced expression of albumin.

The Transcriptional Co-activators CREB-binding Protein (CBP) and p300 Play a Critical Role in Cardiac Hypertrophy That Is Dependent on Their Histone Acetyltransferase Activity

The CBP and p300 proteins are transcriptional co-activators that are involved in a variety of transcriptional pathways in development and in response to specific signaling pathways. We have previously demonstrated that the ability of both these factors to stimulate transcription is greatly enhanced by treatment of cardiac cells with the hypertrophic agent phenylephrine (PE). Here, we show that inhibition of either CBP or p300 with antisense or dominant negative mutant constructs inhibits PE-induced hypertrophy as assayed by atrial naturetic protein production, cardiac cell protein:DNA ratio and cell size. Furthermore, we show that overexpression of CBP or p300 can induce hypertrophy and that this effect requires their histone acetyltransferase (HAT) activity. Moreover, we show that PE can directly enhance CBP HAT activity and that artificial enhancement of HAT activity is sufficient to induce hypertrophy. Hence, CBP and p300 play an essential role in hypertrophy induced by PE, and this effect is mediated via PE-induced enhancement of their HAT activity. This is the first time a role for these factors, and their HAT activity, in hypertrophy has been directly demonstrated.

Neuralization of theXenopusEmbryo by Inhibition of p300/ CREB-Binding Protein Function

p300/ CREB-binding protein (CBP) is a transcriptional coactivator for a plethora of transcription factors and plays critical roles in signal transduction pathways. We report that the inhibition of p300/CBP function in the Xenopus embryo abolishes non-neural tissue formation and, strikingly, initiates neural induction and primary neurogenesis in the entire embryo. The observed neuralization is achieved in the absence of anterior or posterior gene expression, suggesting that neural fate activation and anterior patterning may represent distinct molecular events. We further demonstrate that the neuralizing and anteriorizing activities of chordin and noggin are separable properties of these neural inducers. This study reveals that all embryonic cells possess intrinsic neuralizing capability and that p300/CBP function is essential for embryonic germ layer formation and neural fate suppression during vertebrate embryogenesis.

A Viral Mechanism for Inhibition of p300 and PCAF Acetyltransferase Activity

Nucleosomal histone modification is believed to be a critical step in the activation of RNA polymerase II–dependent transcription. p300/CBP and PCAF histone acetyltransferases (HATs) are coactivators for several transcription factors, including nuclear hormone receptors, p53, and Stat1 α, and participate in transcription by forming an activation complex and by promoting histone acetylation. The adenoviral E1A oncoprotein represses transcriptional signaling by binding to p300/CBP and displacing PCAF and p/CIP proteins from the complex. Here, we show that E1A directly represses the HAT activity of both p300/CBP and PCAF in vitro and p300-dependent transcription in vivo. Additionally, E1A inhibits nucleosomal histone modifications by the PCAF complex and blocks p53 acetylation. These results demonstrate the modulation of HAT activity as a novel mechanism of transcriptional regulation.

Suppression of the p300-dependent mdm2 negative-feedback loop induces the p53 apoptotic function.

The p53 tumor suppressor gene product interacts with the p300 transcriptional coactivator that regulates the transactivation of p53-inducible genes. The adenovirus E1A protein has been shown to bind to p300 and inhibit its function. E1A inhibits p53 transactivation and also promotes p53 accumulation by a p300-dependent mechanism. Murine double minute 2 (Mdm2) is a transcriptional target of p53 that binds to p53 and inhibits its transcriptional activity. E1A inhibited mdm2 transactivation without affecting the expression of p21(WAF1) or Bax, which resulted in high levels of p53 accumulation and apoptosis. Ectopic expression of p300 restored Mdm2 levels and inhibited p53-dependent apoptosis, as did ectopic expression of Mdm2. Thus, p300 is required for mdm2 induction by p53 and the subsequent inhibition of p53 stabilization. Inhibition of p300 by E1A results in stabilization of p53 and causes apoptosis. Moreover, E1B 19K or Bcl-2 expression in E1A-transformed cells abrogated p53-dependent apoptosis by restoring mdm2 transactivation by p53. Hence, p300 regulation of mdm2 expression controls apoptotic activity of p53, and 19K or Bcl-2 bypass E1A inhibition of p300 transactivation of Mdm2.