Induction of the human CDC45 gene promoter activity by natural compound trans­resveratrol.

GGAA motifs in the human TP53 and HELB gene promoters play a part in responding to trans­resveratrol (Rsv) in HeLa S3 cells. This sequence is also present in the 5'­upstream region of the human CDC45 gene, which encodes a component of CMG DNA helicase protein complex. The cells were treated with Rsv (20 µM), then transcripts and the translated protein were analyzed by quantitative RT­PCR and western blotting, respectively. The results showed that the CDC45 gene and protein expression levels were induced after the treatment. To examine whether they were due to the activation of transcription, a 5'­upstream 556­bp of the CDC45 gene was cloned and inserted into a multi­cloning site of the Luciferase (Luc) expression vector. In the present study, various deletion/point mutation­introduced Luc expression plasmids were constructed and they were used for the transient transfection assay. The results showed that the GGAA motif, which is included in a putative RELB protein recognizing sequence, plays a part in the promoter activity with response to Rsv in HeLa S3 cells.

Trimebutine suppresses Toll-like receptor 2/4/7/8/9 signaling pathways in macrophages.

Because of the critical roles of Toll-like receptors (TLRs) and receptor for advanced glycation end-products (RAGE) in the pathophysiology of various acute and chronic inflammatory diseases, continuous efforts have been made to discover novel therapeutic inhibitors of TLRs and RAGE to treat inflammatory disorders. A recent study by our group has demonstrated that trimebutine, a spasmolytic drug, suppresses the high mobility group box 1‒RAGE signaling that is associated with triggering proinflammatory signaling pathways in macrophages. Our present work showed that trimebutine suppresses interleukin-6 (IL-6) production in lipopolysaccharide (LPS, a stimulant of TLR4)-stimulated macrophages of RAGE-knockout mice. In addition, trimebutine suppresses the LPS-induced production of various proinflammatory cytokines and chemokines in mouse macrophage-like RAW264.7 cells. Importantly, trimebutine suppresses IL-6 production induced by TLR2-and TLR7/8/9 stimulants. Furthermore, trimebutine greatly reduces mortality in a mouse model of LPS-induced sepsis. Studies exploring the action mechanism of trimebutine revealed that it inhibits the LPS-induced activation of IL-1 receptor-associated kinase 1 (IRAK1), and the subsequent activations of extracellular signal-related kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and nuclear factor-κB (NF-κB). These findings suggest that trimebutine exerts anti-inflammatory effects on TLR signaling by downregulating IRAK1‒ERK1/2‒JNK pathway and NF-κB activity, thereby indicating the therapeutic potential of trimebutine in inflammatory diseases. Therefore, trimebutine can be a novel anti-inflammatory drug-repositioning candidate and may provide an important scaffold for designing more effective dual anti-inflammatory drugs that target TLR/RAGE signaling.

Human microRNA hsa-miR-15b-5p targets the RNA template component of the RNA-dependent RNA polymerase structure in severe acute respiratory syndrome coronavirus 2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a pandemic of the coronavirus disease in 2019. RNA-dependent RNA polymerase (RdRp) plays an essential role in RNA replication and transcription in SARS-CoV-2. In this study, we focused on the RNA template component of viral RdRp structure and analyzed human microRNAs (miRNAs) targeting specific sequences in this RNA. By examining miRNA databases and using an in vitro RNA-RNA interaction assay, we observed hsa-miR-15b-5p interacting with the RNA component of viral RdRp. Our findings provide evidence that hsa-miR15b-5p may suppresses viral infection and proliferation by targeting the RNA template component of SARS-CoV-2 RdRp.

A 3-styrylchromone converted from trimebutine 3D pharmacophore possesses dual suppressive effects on RAGE and TLR4 signaling pathways.

Receptor for advanced glycation end-products (RAGE) and Toll-like receptors (TLRs) are potential therapeutic targets in the treatment of acute and chronic inflammatory diseases. We previously reported that trimebutine, a spasmolytic drug, suppresses RAGE pro-inflammatory signaling pathway in macrophages. The aim of this study was to convert trimebutine to a new small molecule using in silico 3D pharmacophore similarity search, and dissect the mechanistic anti-inflammatory basis. Of note, a unique 3-styrylchromone (3SC), 7-methoxy-3-trimethoxy-SC (7M3TMSC), converted from trimebutine 3D pharmacophore potently suppressed both high mobility group box 1-RAGE and lipopolysaccharide-TLR4 signaling pathways in macrophage-like RAW264.7 cells. More importantly, 7M3TMSC inhibited the phosphorylation of extracellular signaling-regulated kinase 1 and 2 (ERK1/2) and downregulated the production of cytokines, such as interleukin-6. Furthermore, 3D pharmacophore-activity relationship analyses revealed that the hydrogen bond acceptors of the trimethoxy groups in a 3-styryl moiety and the 7-methoxy-group in a chromone moiety in this compound are significant in the dual anti-inflammatory activity. Thus, 7M3TMSC may provide an important scaffold for the development of a new type of anti-inflammatory dual effective drugs targeting RAGE/TLR4-ERK1/2 signaling.

Trimebutine attenuates high mobility group box 1-receptor for advanced glycation end-products inflammatory signaling pathways.

We previously identified papaverine as an inhibitor of receptor for advanced glycation end-products (RAGE) and showed its suppressive effect on high mobility group box 1 (HMGB1)-mediated responses to inflammation. Here, we found trimebutine to be a 3D pharmacophore mimetics of papaverine. Trimebutine was revealed to have more potent suppressive effects on HMGB1-induced production of pro-inflammatory cytokines, such as interleukin-6 and tumor necrosis factor-α in macrophage-like RAW264.7 cells and mouse bone marrow primarily differentiated macrophages than did papaverine. However, the inhibitory effect of trimebutine on the interaction of HMGB1 and RAGE was weaker than that of papaverine. Importantly, mechanism-of-action analyses revealed that trimebutine strongly inhibited the activation of RAGE downstream inflammatory signaling pathways, especially the activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2), which are mediator/effector kinases recruited to the intracellular domain of RAGE. Consequently, the activation of Jun amino terminal kinase, which is an important effector kinase for the up-regulation of pro-inflammatory cytokines, was inhibited. Taken together, these results suggest that trimebutine may exert its suppressive effect on the HMGB1-RAGE inflammatory signal pathways by strongly blocking the recruitment of ERK1/2 to the intracellular tail domain of RAGE in addition to its weak inhibition of the extracellular interaction of HMGB1 with RAGE. Thus, trimebutine may provide a unique scaffold for the development of novel dual inhibitors of RAGE for inflammatory diseases.

Structural Basis of Beneficial Design for Effective Nicotinamide Phosphoribosyltransferase Inhibitors.

Inhibition of nicotinamide phosphoribosyltransferase (NAMPT) is an attractive therapeutic strategy for targeting cancer metabolism. So far, many potent NAMPT inhibitors have been developed and shown to bind to two unique tunnel-shaped cavities existing adjacent to each active site of a NAMPT homodimer. However, cytotoxicities and resistances to NAMPT inhibitors have become apparent. Therefore, there remains an urgent need to develop effective and safe NAMPT inhibitors. Thus, we designed and synthesized two close structural analogues of NAMPT inhibitors, azaindole-piperidine (3a)- and azaindole-piperazine (3b)-motif compounds, which were modified from the well-known NAMPT inhibitor FK866 (1). Notably, 3a displayed considerably stronger enzyme inhibitory activity and cellular potency than did 3b and 1. The main reason for this phenomenon was revealed to be due to apparent electronic repulsion between the replaced nitrogen atom (N1) of piperazine in 3b and the Nδ atom of His191 in NAMPT by our in silico binding mode analyses. Indeed, 3b had a lower binding affinity score than did 3a and 1, although these inhibitors took similar stable chair conformations in the tunnel region. Taken together, these observations indicate that the electrostatic enthalpy potential rather than entropy effects inside the tunnel cavity has a significant impact on the different binding affinity of 3a from that of 3b in the disparate enzymatic and cellular potencies. Thus, it is better to avoid or minimize interactions with His191 in designing further effective NAMPT inhibitors.

Low Levels of Brain-Derived Neurotrophic Factor Trigger Self-aggregated Amyloid ß-Induced Neuronal Cell Death in an Alzheimer's Cell Model.

Alzheimer's disease (AD) is pathologically characterized by accumulation of amyloid ß (Aß) and hyperphosphorylated tau, and thereby induction of neuronal cell death. The Aß-induced neuronal cell death has been shown to occur by several modes, such as apoptosis, necrosis, and necroptosis. Interestingly, in AD patients, the brain and serum levels of brain-derived neurotrophic factor (BDNF) have been reported to be significantly decreased. However, the relationship between Aß and BDNF in the onset of AD remains to be fully understood. Here, we used neuron-like differentiated human neuroblastoma SH-SY5Y (ndSH-SY5Y) cells to study the neurotoxicity of self-aggregated Aß1-42 peptide under different concentrations of BDNF in the culture medium. Importantly, decreasing levels of BDNF caused a considerable suppression in the extension of neurite length. Furthermore, only under low levels of BDNF, the aggregated Aß was revealed to induce neurite fragmentation and neuronal cell death in ndSH-SY5Y cells. Notably, the aggregated Aß and low levels of BDNF-induced neuronal cell death was characterized at least as caspase-6 dependent cell death and necroptosis. These results indicate that our ndSH-SY5Y cell system, cultured under decreasing levels of BDNF and aggregated Aß, has the potential to be applied in the analysis of the molecular mechanisms of the progressive neurodegenerative processes of AD and the discovery of neuroprotective drug candidates.

Effect of the natural compound trans­resveratrol on human MCM4 gene transcription.

trans­Resveratrol (Rsv) is a natural compound contained in red wine and grape skins that has various beneficial effects for organisms such as lengthening of their life span. Rsv induces expression of the human TP53 and HELB genes, which are involved in the regulation of DNA maintenance. In the present study, a luciferase expression vector containing 309 bp of the 5' upstream end of the human MCM4 gene was transfected into HeLa S3 cells. A luciferase assay revealed that Rsv treatment increased the minichromosome maintenance 4 (MCM4) gene promoter activity by GC­box and GGAA (TTCC) motifs. Electro phoretic mobility shift assay revealed that the specific binding factor (complex) contains PU.1 (SPI1). Quantitative reverse transcription­polymerase chain reaction analysis indicated that MCM4 gene expression was transiently induced by Rsv. Moreover, western blotting revealed that the SP1/PU.1 ratio markedly increased after Rsv treatment, indicating that a balance or profile of these transcription factors may control Rsv­inducible initiation of transcription. These observations indicated that the beneficial effects of Rsv can be attributed to induction of the chromosomal DNA maintenance factor encoding gene expression.

Structural insights into the active site of poly(ADP-ribose) glycohydrolase using docking modes of 6-hydroxy-3H-xanthen-3-one derivative inhibitors.

Poly(ADP-ribose) glycohydrolase (PARG) plays an essential role in poly(ADP-ribose) (PAR) turnover, and thereby regulating DNA transactions, such as DNA repair, replication, transcription and recombination. Here, we examined the inhibitory activities of 6-hydroxy-3H-xanthene-3-one (HXO) derivatives and analyzed their binding modes in the active site of PARG by in silico docking study. Among the derivatives, Rose Bengal was found to be the most potent inhibitor of PARG and its halogen groups were revealed to cooperatively potentiate the inhibitory activity. Importantly, the binding mode of Rose Bengal occupied the active site of PARG revealed the presence of unique "Sandwich" residues of Asn869 and Tyr792, which enable the inhibitor to bind tightly with the active pocket. This sandwich interaction could stabilize the π-π interactions of HXO scaffold with Phe902 and Tyr795. In addition, to increase the binding affinity, the iodine and chlorine atoms of this inhibitor could contribute to the inducing of favorable disorders, which promote an entropy boost on the active site of PARG for structural plasticity, and making the stable configuration of HXO scaffold in the active site, respectively, as judged by the analysis of binding free energy. These results provide new insights into the active site of PARG and an additional opportunity for designing selective PARG inhibitors.

Association of ABC Transporter With Resistance to FK866, a NAMPT Inhibitor, in Human Colorectal Cancer Cells.

BACKGROUND/AIM: Nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme in the NAD+ biosynthetic pathway, is a drug target of potent anticancer candidates, including FK866 and other reported NAMPT inhibitors. However, it is known that NAMPT point-mutations render resistance to specific NAMPT inhibitors in several cancer cells. We investigated the resistance mechanisms of NAMPT inhibitor FK866 in human colorectal cancer (CRC) cells. MATERIALS AND METHODS: We used CRC human cell line HCT116 to determine the expression profiles of FK866-sensitive parental HCT116 cells and FK866-resistant HCT116 (HCT116RFK866) cells by DNA microarray analysis. The levels of multidrug resistance protein 1 (MDR1) were assessed via western blot. In addition, we analyzed the sensitivity of FK866 in parental HCT116 cells and HCT116RFK866 cells by co-treatment with MDR1 inhibitor verapamil. RESULTS: Our results revealed an association between ATP-binding cassette (ABC) transporter gene ABCB1 and resistance to NAMPT inhibitor FK866 in both HCT116RFK866 cells and parental HCT116 cells. The expression of ABCB1, which encodes MDR1, was lower in HCT116RFK866 cells than in parental HCT116 cells. Furthermore, the protein level of MDR1/ATP-binding cassette sub-family B member 1 (ABCB1) was 0.5-fold lower in HCT116RFK866 cells than in parental HCT116 cells. Additionally, HCT116RFK866 cells showed improved sensitivity to FK866 when co-treated with verapamil, an ABCB1 inhibitor. Interestingly, the efficacy of FK866 in parental HCT116 cells was the same for the treatment with FK866 alone or in combination with verapamil. CONCLUSION: The change in expression of ABCB1 plays a key role in CRC drug resistance to NAMPT inhibitor FK866. This suggests that the MDR1/ABCB1 mechanism may regulate the resistance of anticancer NAMPT inhibitor FK866.

Characterization of the human zinc finger nfx­1­type containing 1 encoding ZNFX1 gene and its response to 12­O­tetradecanoyl­13­acetate in HL­60 cells.

Human promyelocytic HL­60 cells can be differentiated into macrophage­like cells by treatment with 12­O­tetra decanoylphorbol­13­acetate (TPA). Certain 5' upstream regions of the zinc finger protein (ZNF)­encoding genes contain duplicated GGAA motifs, which are frequently found in the TPA­responding gene promoter regions. To examine transcriptional responses to TPA, 5'flanking regions of human zinc finger CCCH­type containing, antiviral, ZNF252, ZNF343, ZNF555, ZNF782 and zinc finger nfx­1­type containing 1 (ZNFX1) genes were isolated by polymerase chain reaction (PCR) and ligated into a multiple­cloning site of the pGL4.10[luc2] vector. Transient transfection and a luciferase assay revealed that the ZNFX1 promoter most prominently responded to the TPA treatment. Deletion and point mutation experiments indicated that the duplicated GGAA motif in the 100­bp region positively responded to TPA. In addition, reverse transcription­quantitative PCR and western blotting showed that the mRNA and protein of ZNFX1 accumulate during the differentiation of HL­60 cells. These results indicated that expression of the TPA­inducible ZNFX1 gene, which belongs to the group of interferon­responsive genes, is regulated by the cis­action of the duplicated GGAA motif.

Targeting poly(ADP-ribose) glycohydrolase to draw apoptosis codes in cancer.

Poly(ADP-ribosyl)ation is a unique post-translational modification of proteins. The metabolism of poly(ADP-ribose) (PAR) is tightly regulated mainly by poly(ADP-ribose) polymerases (PARP) and poly(ADP-ribose) glycohydrolase (PARG). Accumulating evidence has suggested the biological functions of PAR metabolism in control of many cellular processes, such as cell proliferation, differentiation and death by remodeling chromatin structure and regulation of DNA transaction, including DNA repair, replication, recombination and transcription. However, the physiological roles of the catabolism of PAR catalyzed by PARG remain less understood than those of PAR synthesis by PARP. Noteworthy biochemical studies have revealed the importance of PAR catabolic pathway generating nuclear ATP via the coordinated actions of PARG and ADP-ribose pyrophosphorylase (ADPRPPL) for the driving of DNA repair and the maintenance of DNA replication apparatus while repairing DNA damage. Furthermore, genetic studies have shown the value of PARG as a therapeutic molecular target for PAR-mediated diseases, such as cancer, inflammation and many pathological conditions. In this review, we present the current knowledge of de-poly(ADP-ribosyl)ation catalyzed by PARG focusing on its role in DNA repair, replication and apoptosis. Furthermore, the induction of apoptosis code of DNA replication catastrophe by synthetic lethality of PARG inhibition and the recent progresses regarding the development of small molecule PARG inhibitors and their therapeutic potentials in cancer chemotherapy are highlighted in this review.

Characterization of the human E2F4 promoter region and its response to 12-O-tetradecanoylphorbol-13-acetate.

The E2F transcription factors (TFs), which control the progression of the cell cycle in response to DNA-damage and various stresses, are known to interact with a tumour suppressor, Retinoblastoma 1 (RB1). We previously showed that the response of the human RB1 promoter to a 12-O-tetradecanoylphorbol-13-acetate (TPA) in HL-60 cells is mediated by a duplicated GGAA motif, which is also present in the 5'-upstream of the E2F family genes. The motifs are especially rich in the 5'-upstream of the E2F4 gene. In the present study, we constructed luciferase (Luc) expression vectors containing a 466 bp of the 5'-upstream of the human E2F4 gene. The transfection of this plasmid and deletion/mutation-introduced derivatives into HL-60 cells and a Luc reporter assay showed that duplicated and triplicated GGAA (TTCC) motifs in the E2F4 promoter respond to TPA. As expected, electrophoretic mobility shift assay indicated that SPI1 (PU.1) binds to the GGAA motif-containing element. A quantitative RT-PCR and western blotting showed that the E2F4 transcripts and its encoding proteins accumulate during the differentiation of HL-60 into macrophage-like cells. In contrast, the expression of the E2F1 gene and the protein, which possibly acts as a cell cycle accelerator, was greatly diminished.

Characterization of a novel microRNA, miR-188, elevated in serum of muscular dystrophy dog model.

MicroRNAs (miRNAs) are non-coding small RNAs that regulate gene expression at the post-transcriptional level. Several miRNAs are exclusively expressed in skeletal muscle and participate in the regulation of muscle differentiation by interacting with myogenic factors. These miRNAs can be found at high levels in the serum of patients and animal models for Duchenne muscular dystrophy, which is expected to be useful as biomarkers for their clinical conditions. By miRNA microarray analysis, we identified miR-188 as a novel miRNA that is elevated in the serum of the muscular dystrophy dog model, CXMDJ. miR-188 was not muscle-specific miRNA, but its expression was up-regulated in skeletal muscles associated with muscle regeneration induced by cardiotoxin-injection in normal dogs and mice. Manipulation of miR-188 expression using antisense oligo and mimic oligo RNAs alters the mRNA expression of the myogenic regulatory factors, MRF4 and MEF2C. Our results suggest that miR-188 is a new player that participates in the gene regulation process of muscle differentiation and that it may serve as a serum biomarker reflecting skeletal muscle regeneration.

Genomic and tumor biological aspects of the anticancer nicotinamide phosphoribosyltransferase inhibitor FK866 in resistant human colorectal cancer cells.

Cancer cells' resistance to drugs remains an important problem affecting cancer treatment strategies. We previously studied the nicotinamide phosphoribosyltransferase (NAMPT) inhibitor FK866's resistance mechanisms in the human colorectal cancer HCT116 cells. We established an acquired FK866-resistant cell line, HCT116RFK866. In this study, we investigated gene mutations in parental HCT116 and HCT116RFK866 cells using exome sequencing technology. The results indicated cluster genes related to NAD+ biosynthesis (including NAMPT), DNA repair, and ATP-binding cassette transporters were differentially altered in these cells. Interestingly, HCT116RFK866 cells, which are resistant to other class NAMPT inhibitors, were more sensitive to the anticancer 5-fluorouracil and cisplatin and γ-ray irradiation compared to parental HCT116 cells. This higher sensitivity appears to cause a genetic change in the identified gene clusters by resistance to the NAMPT inhibitor FK866. Collectively, these novel findings provide a better understanding of anticancer candidate NAMPT inhibitors with regard to resistance mechanisms and cancer chemotherapy strategies.

Cross resistance to diverse anticancer nicotinamide phosphoribosyltransferase inhibitors induced by FK866 treatment.

Cross-resistance to drugs remains an unsolved problem in cancer chemotherapy. This study elucidates a molecular mechanism of cross-resistance to diverse inhibitors of nicotinamide phosphoribosyltransferase (NAMPT) with anticancer activity. We generated a variant of the human colon cancer cell line HCT116, HCT116RFK866, which exhibited primary resistance to the potent NAMPT inhibitor FK866, and was approximately 1,000-fold less sensitive to the drug than the parental HCT116. HCT116RFK866 was found to be cross-resistant to diverse NAMPT inhibitors, including CHS-828, GNE-617, and STF-118804. Whole-exon sequencing revealed two point mutations (H191R and K342R) in NAMPT in HCT116RFK866, only one of which (K342R) was present in the parental HCT116. Importantly, the protein level, NAMPT enzyme activity, and intracellular NAD+ level were similar between HCT116RFK866 and HCT116. Hence, we investigated NAMPT-binding partners in both cell lines by focused proteomic analyses. The amount of NAMPT precipitated with anti-NAMPT monoclonal antibody was much higher in HCT116RFK866 than in the parental. Furthermore, in HCT116, but not in HCT116RFK866, NAMPT was revealed to interact with POTE ankyrin domain family member E and beta-actin. Thus, these results suggest that NAMPT usually interacts with the two partner proteins, and the H191R mutation may prevent the interactions, resulting in resistance to diverse NAMPT inhibitors.

Identification of Minimal p53 Promoter Region Regulated by MALAT1 in Human Lung Adenocarcinoma Cells.

The MALAT1 long noncoding RNA is strongly linked to cancer progression. Here we report a MALAT1 function in repressing the promoter of p53 (TP53) tumor suppressor gene. p21 and FAS, well-known p53 targets, were upregulated by MALAT1 knockdown in A549 human lung adenocarcinoma cells. We found that these upregulations were mediated by transcriptional activation of p53 through MALAT1 depletion. In addition, we identified a minimal MALAT1-responsive region in the P1 promoter of p53 gene. Flow cytometry analysis revealed that MALAT1-depleted cells exhibited G1 cell cycle arrest. These results suggest that MALAT1 affects the expression of p53 target genes through repressing p53 promoter activity, leading to influence the cell cycle progression.

New Insights into the Roles of NAD+-Poly(ADP-ribose) Metabolism and Poly(ADP-ribose) Glycohydrolase.

Accumulating evidence has suggested the fundamental functions of NAD+-poly(ADP-ribose) metabolism in cellular and physiological processes, including energy homeostasis, signal transduction, DNA transaction, genomic stability and cell death or survival. The NAD+ biosynthesis and poly(ADP-ribose) [(ADP-R)n] turnover are tightly controlled by several key enzymes, such as nicotinamide phosphoribosyltransferase (NmPRT), nicotinamide mononucleotide adenylyltransferases (NMNATs), poly(ADP-ribose) polymerase (PARP), poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribose pyrophosphorylase (ADPRPPL). Many researches investigating the roles of these enzymes in cells have revealed the physiological and pathological importance, and thereby the therapeutical values. Among these enzymes, the polymer degrading enzyme PARG has not yet been intensively studied, because of the low cellular content, lack of cell-available PARG chemical inhibitors and PARG genetic models. So, the biological roles of (ADP-R)n catabolism by PARG are still being elucidated as compared to those of synthesis by PARP. However, recent studies delineate that PARG-dependent (ADP-R)n degradation is critical for many pathological conditions, and thus PARG is an important target for chemical therapeutics for several diseases. This review will present the recent progresses about the roles of NAD+-(ADP-R)n metabolism and the structures and functions of PARG, with a focus on its role in DNA repair and cell death by apoptosis in relation to central regulatory network, and the therapeutic potentials of PARG inhibitors in cancer chemotherapy.

Characterization of the 5'-flanking region of the human DNA helicase B (HELB) gene and its response to trans-Resveratrol.

Human DNA helicase B (HELB/HDHB) regulates DNA replication through association with human DNA polymerase α-primase. In the present study, an 866-base pair (bp) of the 5'-flanking region of the human HELB gene-containing Luciferase (Luc) reporter plasmid, pHDHB-Luc was transfected into various cell lines and Luc activity was analyzed. Deletion analyses revealed that a 121-bp containing the major transcription start site (TSS) was essential for the basal promoter activity in all tested cells. TF-SEARCH analysis indicated that GC-box/Sp1 and duplicated GGAA-motifs containing putative STAT-x and c-ETS binding sites are located close to the TSS. Furthermore, chromatin immunoprecipitation (ChIP) analysis showed that PU.1 and Sp1 bind to the 121-bp region. Reverse transcriptase-polymerase chain reaction (RT-PCR) and western blot analyses showed the HELB gene and protein expression was up-regulated by trans-Resveratrol (Rsv) treatment in HeLa S3 cells. Moreover, transfection experiment indicated that mutations on the GC-boxes and the duplicated GGAA-motif greatly reduced promoter activity and the response to Rsv in HeLa S3 cells. These results suggest that Rsv, which is a natural compound that has been found to elongate the lifespan of various organisms, regulates HELB promoter activity through co-operation of the GC-boxes and the duplicated GGAA-motif in the 121-bp.

Characterization of the 5'-flanking region of the human TP53 gene and its response to the natural compound, Resveratrol.

Tumour suppressor p53, which is encoded by theTP53gene, is widely known to play an important role in response to DNA damage and various stresses. It has recently been reported that p53 regulates glucose metabolism and that an increase in p53 protein level is induced after serum deprivation or treatments with a natural compound,trans-Resveratrol (Rsv). In this study, we constructed a Luciferase expression vector, pGL4-TP53-551, containing 551 bp of the 5'-upstream region of the humanTP53gene, which was then transfected into HeLa S3 cells. A Luciferase assay showed that Rsv treatment increased the promoter activity of theTP53gene in comparison to that ofPIF1 Detailed deletion and mutation analyses revealed that Nkx-2.5 and E2F-binding elements are required in addition to duplicated GGAA (TTCC), for the regulation ofTP53promoter activity. In this study, it is suggested that the transient induction ofTP53gene expression by Rsv treatment might be partly involved in its anti-aging effect through maintenance of chromosomal DNAs.