Objective: P300/CBP Associated Factor (PCAF) is a histone acetyltransferase, and has been reported to interact with nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) and to stimulate cyclooxygenase-2 (COX-2) transcriptional activation. The aim of this study was to determine the role of PCAF in chronic pain modulation. / Method: In an in vitro experiment, PCAF small-interfering RNA (siRNA) was used to knock down PCAF. Interleukin-1 β (IL1β) was applied as COX-2 inducer in SK-N-SH neuroblastoma cells. Luciferase assay and chromatin immunoprecipitation (ChIP) were performed regarding COX-2 promoter region. / In an in vivo experiment, PCAF was examined for its distribution in dorsal horn of Spraque-Dawley rats. COX-2 level in the spinal cord was determined after inhibition of PCAF in rats with Complete Freund's Adjuvant (CFA)-induced pain. ChIP was also performed. / Finally, we tested whether genetic variations in the PCAF gene affected the risk of chronic pain in a gene association study of 267 surgical patients. The associations of pain with genotypes (58 single nucleotide polymorphisms, SNPs)/haplotypes were analyzed by χ² and Fisher exact tests. / Results: Knock-down of PCAF reduced COX-2 level and NF-κB activity. PCAF and acetylated histone 3 lysine 14 (H3K14) were enriched at COX-2 promoter when IL1β was applied. / PCAF was expressed in neurons at superficial layers of rat dorsal horn. In the in vivo experiment, COX-2 was reduced with the inhibition of PCAF in CFA rats. PCAF was enhanced at COX-2 promoter when CFA was injected. Anacardic acid and PCAF siRNA significantly alleviated thermal nociception and mechanical allodynia. / In the gene association study, 6 SNPs and 5 haplotypes were significantly associated with higher risk of severe chronic postsurgical pain. Multivariable analysis showed that patients with a SNP rs6763504 had a higher risk of developing severe chronic postsurgical pain (p = 0.001). / Conclusion: PCAF regulated COX-2 transcription and reduction or inhibition in PCAF resulted in a decrease in COX-2 and less chronic inflammatory pain. Genetic variations in the PCAF gene increased risk of severe chronic post-surgical pain in patients. / 實驗目的：P300/CBP相關蛋白（PCAF）是一種組蛋白乙酰化轉移酶。這種蛋白已被報道可以和核因子活化B細胞κ輕鏈增強子(NF-κB)相互作用，以及增進環氧合酶-2（COX-2）的轉錄激活。本實驗的目的在於研究PCAF在疼痛調節中的作用。 / 實驗方法：在細胞實驗中，我們使用了小干擾RNA（siRNA）來降低PCAF的含量。同時我們使用了白細胞介素-1β（IL1β）來作為SK-N-SH神經母細胞瘤細胞中COX-2的誘導劑。我們使用了熒光素酶試劑和染色質免疫沉澱來研究COX-2啟動子區域。 / 在體內實驗中，我們檢測了PCAF在大鼠脊椎背角部位的分佈。在弗氏完全佐劑（CFA）致痛的大鼠模型中，我們在PCAF抑制的情況下檢測脊髓中COX-2的水平。我們還進行了染色質免疫沉澱。 / 最後，在招募了267位手術患者的基因關聯試驗中，我們對PCAF基因中的基因變異對慢性疼痛風險的影響進行了分析。我們運用卡方檢驗和費雪精確性檢驗對疼痛與基因型（58個單核苷酸多態性）和單倍型的關係進行分析。 / 實驗結果：減少的PCAF降低了COX-2的水平以及NF-κB的活性。當添加了IL1β時PCAF和乙酰化第三組蛋白14號賴胺酸（H3K14）在COX-2啟動子處富集。 / PCAF在大鼠脊椎背角部位的淺表層（第一層和第二層）的神經細胞里表達。在動物實驗中，注射了CFA的大鼠顯示COX-2會隨著PCAF的抑制而下降。大鼠注射了CFA后PCAF在COX-2啟動子處有所增加。漆樹酸和PCAF siRNA顯著地減輕了熱痛和機械痛提高了機械痛閾值。 / 在該基因關聯試驗中，我們鑒定出六個單核苷酸多態性和五種單倍型與較高風險的嚴重術後慢性痛有顯著的相關性。多元回歸分析表明在PCAF基因上具有rs6763504遺傳變異的病人在術後發展嚴重慢性痛的幾率會較高(p = 0.001)。 / 結論：PCAF調節COX-2的轉錄，而且PCAF的減少或者抑制導致了COX-2的降低同時慢性炎症痛的減少。PCAF基因上的遺傳變異提高了術後病人嚴重慢性痛的風險。 / Meng, Zhaoyu. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 117-134). / Abstracts also in Chinese. / Title from PDF title page (viewed on 30, December, 2016). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

Thesis (Ph. D.)--Case Western Reserve University, 2006. / [School of Medicine] Department of Pharmacology. Includes bibliographical references. Available online via OhioLINK's ETD Center.

For 30 years, the tumor suppressor p53 has been a subject of intense research in nearly every discipline of scientific inquiry. While numerous surprising roles for p53 in health and disease are uncovered each year, the central role of its activation in preventing neoplastic transformation has been and will remain at the forefront of p53 research as investigators work to address an unexpectedly complex question—precisely how does p53 integrate upstream stress signals to coordinate activation of its target genes in response to stress? One manner in which to address this question is at the level of transcription initiation—after upstream signals converge on p53 and produce a number of pools of post-transcriptionally modified p53, how exactly are specific target promoters activated in such a sensitive, context-specific manner? The work presented herein aims to address the role of histone acetylation at the p21 promoter—a critical mediator of G1/S arrest—by the P/CAF acetyltransferase in response to a variety of p53-activating stresses. We show that depletion of P/CAF strongly inhibits p21 expression in response to a variety of stresses, despite normal stabilization of p53 and recruitment to target promoters. This defect in p21 expression correlates closely with abrogation of stress-induced cell-cycle arrest. Strikingly, a p53 allele lacking putative P/CAF acetylation sites was still able to direct p21 expression, which was still dependent upon P/CAF. We show further that histone acetylation at H3K14 at the p21 promoter following stress is dependent upon P/CAF. Rescue of p21 expression with wild-type P/CAF or a ∆HAT point mutant indicates that P/CAF requires an intact HAT domain, suggesting that histone acetylation at H3K14 is catalyzed by P/CAF HAT activity, not the molecular bridging of a heterologous HAT by P/CAF. Furthermore, RNA polymerase II (RNAP II) was present at the p21 proximal promoter under all basal and stress conditions, but elongation of RNAP II after stress required the presence of P/CAF. These data indicate that H3K14 acetylation by P/CAF closely correlates with the activation status of the p21 promoter, and may be necessary for activation of a larger subset of p53-responsive promoters. In addition to its critical role in p21 expression, we noted that p53 stabilization and cell-cycle arrest in response to p14ARF, but not other p53-stabilizing stresses, were also dependent on P/CAF. Cell-cycle arrest induced by p16INK4A was intact after P/CAF ablation, indicating a role for P/CAF in cell-cycle arrest specific to p14ARF-p53 signaling. Basal MDM2 levels were unaffected by P/CAF knockdown, as were p53- MDM2 and ARF-MDM2 complexes. A preliminary analysis of MDM2 localization was inconclusive, due to vastly different quantities of MDM2 in different conditions making analysis of subcellular localization difficult; however, the role of P/CAF in the relocalization of MDM2 to the nucleolus by p14ARF could potentially explain the defect in p53 stabilization, and should be explored further. These observations, underscored by recent reports that P/CAF undergoes loss of heterozygosity in several tumor types, suggest that P/CAF plays a critical role in p53-mediated cell-cycle arrest through multiple, independent mechanisms. Further study should clarify whether P/CAF is lost in tumors maintaining wild-type p53, and whether its reintroduction into these tumors confers any potential therapeutic benefit.

p53, known as the “guardian of the genome”, is the most well-characterized tumor suppressor gene. The central role of p53 is to prevent genome instability. p53 is the central node in an incredibly elaborate genome defense network for receiving various input stress signals and controlling diverse cellular responses. The final output of this network is determined not only by the p53 protein itself, but also by other p53 cooperating proteins. p300 and CBP (CREB-Binding Protein) act as multifunctional regulators of p53 via acetylase and ubiquitin ligase activities. Prior work in vitro has shown that the N-terminal 595 aa of p300 encode both generic ubiquitin ligase (E3) and p53-directed E4 functions. Analysis of p300 or CBP-deficient cells revealed that both coactivators were required for endogenous p53 polyubiquitination and the normally rapid turnover of p53 in unstressed cells. Unexpectedly, p300/CBP ubiquitin ligase activities were absent in nuclear extracts and exclusively cytoplasmic. In the nucleus, CBP and p300 exhibited differential regulation of p53 gene target expression, C-terminal acetylation, and biologic response after DNA damage. p300 activated, and CBP repressed, PUMA expression, correlating with activating acetylation of p53 C-terminal lysines by p300, and a repressive acetylation of p53 lysine-320 induced by CBP. Consistent with their gene expression effects, CBP deficiency augmented, and p300 deficiency blocked, apoptosis after doxorubicin treatment. Subcellular compartmentalization of p300/CBP’s ubiquitination and transcription activities reconciles seemingly opposed functions—cytoplasmic p300/CBP E4 activities ubiquitinate and destabilize p53, while nuclear p300/CBP direct p53 acetylation, target gene activation, and biological outcome after genotoxic stress. p53 is a prominent tumor suppressor gene and it is mutated in more than 50% of human tumors. Reactivation of endogenous p53 is one therapeutic avenue to stop cancer cell growth. In this thesis, we have identified S5as a critical regulator of p53 degradation and activity. S5a is a non-ATPase subunit in the 19S regulatory particle of the 26S proteasome. Our preliminary data indicates that S5a is required for p53 instability and is a negative regulator of p53 tranactivation. As a negative regulator of p53, S5a may therefore also represent a new target for cancer drug development against tumors that specifically maintain wild type p53.

No / Resveratrol is a polyphenolic phytoestrogen that has been shown to exhibit potent anti-oxidant, anti-inflammatory, and anti-catabolic properties. Increased osteoclastic and decreased osteoblastic activities result in bone resorption and loss of bone mass. These changes have been implicated in pathological processes in rheumatoid arthritis and osteoporosis. Receptor activator of NF-kappaB ligand (RANKL), a member of the TNF superfamily, is a major mediator of bone loss. In this study, we investigated the effects of resveratrol on RANKL during bone morphogenesis in high density bone cultures in vitro. Untreated bone-derived cell cultures produced well organized bone-like structures with a bone-specific matrix. Treatment with RANKL induced formation of tartrate-resistant acid phosphatase-positive multinucleated cells that exhibited morphological features of osteoclasts. RANKL induced NF-kappaB activation, whereas pretreatment with resveratrol completely inhibited this activation and suppressed the activation of IkappaBalpha kinase and IkappaBalpha phosphorylation and degradation. RANKL up-regulated p300 (a histone acetyltransferase) expression, which, in turn, promoted acetylation of NF-kappaB. Resveratrol inhibited RANKL-induced acetylation and nuclear translocation of NF-kappaB in a time- and concentration-dependent manner. In addition, activation of Sirt-1 (a histone deacetylase) by resveratrol induced Sirt-1-p300 association in bone-derived and preosteoblastic cells, leading to deacetylation of RANKL-induced NF-kappaB, inhibition of NF-kappaB transcriptional activation, and osteoclastogenesis. Co-treatment with resveratrol activated the bone transcription factors Cbfa-1 and Sirt-1 and induced the formation of Sirt-1-Cbfa-1 complexes. Overall, these results demonstrate that resveratrol-activated Sirt-1 plays pivotal roles in regulating the balance between the osteoclastic versus osteoblastic activity result in bone formation in vitro thereby highlighting its therapeutic potential for treating osteoporosis and rheumatoid arthritis-related bone loss.

No / Vitiligo is characterized by a patchy loss of inherited skin color affecting approximately 0.5% of individuals of all races. Despite the absence of the protecting pigment and the overwhelming evidence for hydrogen peroxide (H(2)O(2))-induced oxidative stress in the entire epidermis of these patients, there is neither increased photodamage/skin aging nor a higher incidence for sun-induced nonmelanoma skin cancer. Here we demonstrate for the first time increased DNA damage via 8-oxoguanine in the skin and plasma in association with epidermal up-regulated phosphorylated/acetylated p53 and high levels of the p53 antagonist p76(MDM2). Short-patch base-excision repair via hOgg1, APE1, and polymerasebeta DNA repair is up-regulated. Overexpression of Bcl-2 and low caspase 3 and cytochrome c levels argue against increased apoptosis in this disease. Moreover, we show the presence of high epidermal peroxynitrite (ONOO(-)) levels via nitrotyrosine together with high nitrated p53 levels. We demonstrate by EMSA that nitration of p53 by ONOO(-) (300 x 10(-6) M) abrogates DNA binding, while H(2)O(2)-oxidized p53 (10(-3) M) enhances DNA binding capacity and prevents ONOO(-)-induced abrogation of DNA binding. Taken together, we add a novel reactive oxygen species to the list of oxidative stress inducers in vitiligo. Moreover, we propose up-regulated wild-type p53 together with p76(MDM2) as major players in the control of DNA damage/repair and prevention of photodamage and nonmelanoma skin cancer in vitiligo.