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The additional sex combs-like (ASXL) family, a mammalian homolog of the additional sex combs (Asx) of Drosophila, has been implicated in transcriptional regulation via chromatin modifications. Abnormal expression of ASXL family genes leads to myelodysplastic syndromes and various types of leukemia. De novo mutation of these genes also causes developmental disorders. Genes in this family and their neighbor genes are evolutionary conserved in humans and mice. This review provides a comprehensive summary of epigenetic regulations associated with ASXL family genes. Their expression is commonly regulated by DNA methylation at CpG islands preceding transcription starting sites. Their proteins primarily engage in histone tail modifications through interactions with chromatin regulators (PRC2, TrxG, PR-DUB, SRC1, HP1α, and BET proteins) and with transcription factors, including nuclear hormone receptors (RAR, PPAR, ER, and LXR). Histone modifications associated with these factors include histone H3K9 acetylation and methylation, H3K4 methylation, H3K27 methylation, and H2AK119 deubiquitination. Recently, non-coding RNAs have been identified following mutations in the ASXL1 or ASXL3 gene, along with circular ASXLs and microRNAs that regulate ASXL1 expression. The diverse epigenetic regulations linked to ASXL family genes collectively contribute to tumor suppression and developmental processes. Our understanding of ASXL-regulated epigenetics may provide insights into the development of therapeutic epigenetic drugs. [ABSTRACT FROM AUTHOR]

Although tyrosine kinase inhibitor (TKI) therapies are highly effective in the treatment of chronic myeloid leukemia (CML), frequent recurrence limits their usage and demands new approaches for CML therapy. Stress-induced premature senescence (SIPS) is considered a potential anticancer treatment, but the underlying mechanism remains elusive. Here, we report that Sirtinol, a known SIRT1 inhibitor, induces premature senescence and growth arrest in K562 CML cells. Chromobox homolog 8 (CBX8) suppresses the Sirtinol-induced premature senescence, which is reversed by CBX8 knockdown. Upon Sirtinol treatment, the phosphorylation of AKT1, p27KIP1 and RB is severely downregulated. However, CBX8 overexpression enhances phosphorylation and, thereby, promotes the transcriptional activity of E2F1, both of which are impaired upon CBX depletion. These data suggest that CBX8 modulates SIPS through the RB-E2F1 pathway in CML cells and provide important insight into its application in CML treatment. [ABSTRACT FROM AUTHOR]

Additional sex comb-like (ASXL) family proteins are chromatin factors that function in transcriptional activation and repression. However, the underlying mechanisms and biological implications have not been well established. Here, we identified a LIM domain-containing protein, Wilms tumor 1-interacting protein (WTIP), as an ASXL1-binding partner. Biochemical assays confirmed an interaction between the murine homologs Asxl1 and Wtip. The suppressive role of Wtip in WT1 function and the expression of Wtip in kidney podocytes prompted us to investigate the role of Asxl1 in the kidney using Asxl1 -null mice. In homozygous Asxl1 −/− embryos, defects in kidney size and glomerular podocyte morphology were observed. Furthermore, up-regulation of Wt1/Wtip target genes was observed in the kidneys of Asxl1 -null embryos. Overall, these findings implicate Asxl1 in the maintenance of podocyte structure via its association with Wtip and in the regulation of WT1 signaling during early kidney development. [ABSTRACT FROM AUTHOR]

Highlights: [•] Adenovirus E1A interacts with a novel nucleolar protein, RaRF. [•] RaRF promotes the nucleolar translocation of E1A from the nucleoplasm. [•] E1A disrupts the interaction of RAR with RaRF, as well as RAR translocation to the nucleolus. [•] Nucleolar sequestration of E1A by RaRF could be a novel mechanism of RAR regulation. [ABSTRACT FROM AUTHOR]

Highlights: [•] CBX8 is a novel binding partner of SIRT1. [•] CBX8 represses Sirtinol-induced premature senescence and growth arrest. [•] CBX8 is required for SIRT1-mediated suppression of premature senescence. [•] CBX8 cooperates with SIRT1 for suppressing p53 acetylation and p21WAF1 expression. [ABSTRACT FROM AUTHOR]

Abstract: Retinoic acid (RA) plays pleiotropic roles in cellular differentiation and animal development. RA responses are mediated by transcriptional activation by the retinoic acid receptor (RAR) and retinoid X receptor (RXR) in cooperation with various types of coregulators at RA-responsive gene promoters. Here, we identified CDK2-associated cullin (CAC1) as a novel type of RARα coregulator that interacts with RARα and inhibits its transcriptional activity. The CoRNR box of CAC1 is required for the binding to and inactivation of RARα. In addition, CAC1 cooperates with histone deacetylases (HDACs) to suppress RARα, probably by associating with HDAC. Finally, depletion of CAC1 increases RA-induced neuronal differentiation of P19 cells, a response accompanied by significant upregulation of the neuronal marker nestin. From these results, we suggest that CAC1 is a novel corepressor of RARα that cooperates with HDACs and is involved in the regulation of RA-induced cellular differentiation. [Copyright &y& Elsevier]

Abstract: The regulation of estrogen-related receptor (ERR) transcriptional activity is poorly understood. To explore the underlying mechanism, we sought to isolate ERRα-binding protein(s). In a yeast two-hybrid screen, we identified a novel protein that has been characterized as a retinoic acid resistance factor (RaRF) (manuscript in-preparation). A specific interaction between RaRF and ERRα was confirmed in a GST pull-down assay in vitro and immunoprecipitation (IP) in mammalian cells. Further yeast two-hybrid assays and IP analyses indicated that the C-terminus of ERRα is required for RaRF binding. Consistent with our interaction data, transfection of RaRF significantly reduced the ability of ERRα, but not ERRγ, to transactivate an ERR-responsive luciferase reporter. In contrast, down-regulation of RaRF using shRNA increased ERRα activity without affecting that of ERRγ. RaRF was subsequently shown to repress the expression of the ERR target gene pS2. Further fluorescence microscopy revealed that ERRα or ERRγ is normally expressed in the nucleoplasm, with ERRα, but not ERRγ, translocating to the nucleolus when RaRF is expressed. Taken together, our data suggest that RaRF sequesters ERRα in the nucleolus through a specific interaction, thereby inhibiting its transcriptional activity. [Copyright &y& Elsevier]

Abstract: Thymine–DNA glycosylase (TDG), a DNA repair enzyme specific for G/T mismatches, plays a role in the regulation of gene expression through its physical interaction with transcription factors. Here, we show that TDG functionally associates with members of the p53 tumor suppressor family and directly modulates their activity. Yeast two-hybrid analysis indicated a physical interaction between a region including the oligomerization domain (OD) of p73α (residues 345–380) or p53 (residues 319–360) and residues 123–346 of TDG, which localizes in the G/T glycosylase catalytic domain (residues 123–372). This interaction was also detected in vitro and in vivo by GST pull-down and immunoprecipitation assays, respectively. TDG over-expression promoted the p73- and p53-mediated transcriptional activation of the p21Waf1 promoter in a dose-dependent manner. Further, TDG enhanced the p53 or p73α-induced growth repression. These observations suggest that TDG modulates the biological function of p53 and other members of the p53 family as a transcriptional coactivator. [Copyright &y& Elsevier]

To synthesize glycyrrhetinic acid (GA) derivatives (3, 4, 5, 10, 13, 14, 15, and 16), we first removed the ketonic group in the C-11 position, and the carboxylic function at the C-30 position was kept intact, reduced to an alcohol, or transformed to an aldehyde corresponding derivatives 10 and 13. Glycyrrhetinic acid (GA) derivatives (3, 4, 5, 15, and 16) were coupled with 4-amino piperpyridine derivatives (12 and 14) and 4-fluorobenzyl bromide at C-30 carboxylic acid position of glycyrrhetinic acid. In subsequent tyrosinase assays, we found that GA derivatives 4, 5, and 16 were not active at early time points, but strongly inhibited tyrosinase activity at late time points. Of the GA derivatives examined, derivative 5 was most active, with an IC50 value of 50 μM after 2 h reaction. IC50 values of derivatives 4 and 16 were 120 and 170 μM, respectively. Further kinetic data indicated that these derivatives are slow-binding inhibitors of tyrosinase. The time-dependent inhibition was reversed when vitamin C or kojic acid was used, that is, both compounds showed active inhibition at early time points. These results suggest that GA derivatives are much more stable than vitamin C or kojic acid, although their intrinsic inhibitory potentials are relatively low. Higher stability and activity suggest that GA derivative 5 might be a useful candidate for skin whitening. [Copyright &y& Elsevier]

Arsenic trioxide (As2O3) has been implicated as a promising anticancer agent for treatment of many cancers including acute promylocytic leukemia. However, the molecular mechanisms are not yet fully defined in solid tumor cells, especially cervical cancer cells carrying human papillomavirus (HPV) genome. To analyze detailed mechanisms in vitro, we treated As2O3 to transformed HeLa cells, a well-studied cervical cancer cell line carrying HPV-18 sequence, and investigated its antiproliferative, antiviral and antimetastatic effects. As2O3 reduced survival and growth of HeLa cells in a dose- and time-dependent manner. Several indicatives of apoptosis were demonstrated by DNA fragmentation assay, DAPI nuclear staining and FACS analysis, respectively. Protein levels of p53 and cleavage of poly(ADP)-ribose polymerase were increased in a dose-dependent manner following treatment of As2O3. In parallel, semi-quantitative reverse transcription-polymerase chain reaction showed that the treatment inhibited HPV-18 E6/E7 viral gene expression in HeLa cells. Using transient transfection and CAT ELISA, we also found that AP-1 sites, located proximal to HPV-18 upstream regulatory region (URR) promoter, could be the major target sites for As2O3. Furthermore, As2O3-treated HeLa cells showed lesser capacity of invasion than those of untreated cells by in vitro invasion assay. Taken together, we proposed that antiviral effect, i.e. down-regulation of HPV E6/E7 oncogenes through targeting for AP-1 sites located in HPV URR might be associated with antiproliferative effect, i.e. induction of apoptosis as be resulted from the accumulation of p53, and that antimetastatic effect could be due to the targeted inactivation of AP-1, a transcription factor required for the expression of MMP-1 and -3. Therefore, our finding may provide a logical basis for the development of a new agent treating HPV-associated cervical neoplasia. [Copyright &y& Elsevier]

We have previously reported that human papillomavirus (HPV) E7 interacts with IRF-1, a key regulator of cellular immune response, and abrogates its transactivation function at the molecular level in vitro. To confirm our previous data, we investigated in vivo the E7-mediated down-regulation of IRF-1 using HPV E7-inducible cells and transgenic mice expressing HPV-18 E6/E7. When E7 was induced in the absence of tetracycline, the expression of target genes of IRF-1 (TAP-1, IFN-β, MCP-1 that are important for cellular immunity) was clearly reduced as determined by RT-PCR. In addition, the IRF-1 activity was down-regulated in E7-expressing cells into which IFN-β-CAT reporter plasmid was transfected. To further investigate the E7-mediated immune regulation in vivo, we constructed transgenic mice expressing E6 and E7 genes of HPV-18 under the control of HPV-18 promoter (URR). From several rounds of breeding, we obtained from a transgenic line that developed a cervical dysplasia and expressed E6/E7 as determined by histological examination and RT-PCR, respectively. Subsequent RT-PCR analysis indicated that TAP-1, IFN-β, and MCP-1 genes were less expressed in a cervical tissue derived from transgenic mouse, when compared with a cervix derived from normal mouse. From these results, we conclude that the E7 transgene expression inactivates the transactivation function of IRF-1 in vivo, which might be important for the elucidation of the E7-mediated immune evading mechanism that is frequently found in cervical cancer. [Copyright &y& Elsevier]

Studies the cryptic single-stranded-DNA binding activities of the phase lambda P and Escherichia coli DNAC replication initiation proteins facilitating the transfer of Escherichia coli DNAB helicase onto DNA. Reagents and enzymes; Gel-retardation assays; Oligonucleotides used in the study.

Hair loss caused by malfunction of the hair follicle stem cells (HFSCs) and physical damage to the skin is difficult to recover from naturally. To overcome these obstacles to hair follicle (HF) regeneration, it is essential to understand the three-dimensional (3D) microenvironment and interactions of various cells within the HFs. Therefore, 3D cell culture technology has been used in HF regeneration research; specifically, multicellular spheroids have been generally adapted to mimic the 3D volumetric structure of the HF. In this study, we culture HF-derived cells, which are mainly composed of HFSCs, in the form of 3D spheroids using a microwell array and discuss the effects of the 3D cellular environment on HF morphogenesis by expression measurements of Sonic hedgehog signaling and stem cell markers in the HF spheroids. Additionally, the influences of microwell depth on HF spheroid formation and biological conditions were investigated. The biomolecular diffusion and convective flow in the microwell were predicted using computational fluid dynamics, which allows analysis of the physical stimulations occurring on the spheroid at the micro-scale. Although a simple experimental method using the microwell array was adopted in this study, the results provide fundamental insights into the physiological phenomena of HFs in the 3D microenvironment, and the numerical analysis is expected to shed light on the investigation of the geometric parameters of the microwell system. [ABSTRACT FROM AUTHOR]

The androgen receptor (AR) plays a critical role in the initiation and progression of prostate cancer (PCa), and thus its regulation is an important tool in PCa therapy. Here, we report that CDK2-associated cullin 1 (CACUL1) directly associates with AR and suppresses AR transcriptional activity. In addition, CACUL1 represses histone demethylase LSD1-mediated AR transactivation by competing with LSD1 for AR binding. Depletion of CACUL1 enhances the LSD1 occupancy of the AR-target promoter, accompanied by decreased accumulation of H3K9me2, a repressive transcriptional marker. CACUL1 and LSD1 oppositely regulate CDX-induced cell death in AR-positive LNCaP and metastatic castrate-resistant LNCaP-LN3 cells. These data suggest that CACUL1 impairs LSD1-mediated activation of AR, thereby implicating it as a potential antitumor target in PCa. [ABSTRACT FROM AUTHOR]

We previously demonstrated that kaempferol, a flavonoid present in various herbs, inhibits adipogenesis by repressing peroxisome proliferator-activated receptor γ (PPARγ) activity. Here, we focused on elucidation of the underlying mechanism using genome-wide tools. First, RNA sequencing (RNA-seq) analysis showed downregulation of genes involved in adipogenesis in response to kaempferol. Subsequent ChIP assays revealed that kaempferol regulates the expression of adipogenic (Adipoq , Fabp4 , Lpl) genes by modulating enrichment of active H3K4me3 and repressive H3K27me3 histone codes on target promoters. Second, we performed ChIP sequencing analysis of active H3K4me3, and co-analysis with RNA-seq identified PPARγ responsive sites in genes downregulated by kaempferol, in terms of expression and H3K4me3 deposition. Third, direct kaempferol binding to PPARγ, for which the K D value was 44.54 μM, was determined by microscale thermophoresis. Further RT-qPCR and GST pull-down assays demonstrated that kaempferol antagonizes rosiglitazone-induced PPARγ activation and impairs the rosiglitazone-dependent interaction between PPARγ and its coactivator CBP. Overall, our data suggest that kaempferol, as a PPARγ antagonist, mediates epigenetic repression of lipid accumulation by regulating histone methylation, and could serve as a candidate epigenetic drug to treat obesity-related diseases. [Display omitted] • Kaempferol downregulates adipogenic PPARγ target genes by reducing the enrichment of H3K4me3, an active histone code. • ChIP-sequencing identified genes with attenuated H3K4me3 levels by kaempferol in 3T3-L1 cells. • Inhibitory effect of kaempferol on H3K4me3 accumulation was validated using newly identified PPARγ target genes. • Kaempferol binds to the ligand-binding domain of PPARγ and acts as an antagonist. [ABSTRACT FROM AUTHOR]

Abstract: Retinoids including all-trans retinoic acid (RA) have been widely used for cancer therapy. However, the major obstacle for RA therapy is the acquired resistance of which mechanism remained obscure thus far. Here, we first identified Zyxin that cooperates with PTOV1 for the negative regulation of RA signaling. Our studies on the underlying mechanism indicated that Zyxin, translocating to the nucleus in response to RA, mediates RAR repression by forming a ternary complex with PTOV1 and the RAR coactivator CBP, thereby promoting dissociation of CBP from RAR at the RA-responsive promoter. Consistently, RA-induced cancer cell cytotoxicity was significantly impaired by Zyxin or PTOV1. Overall, our findings suggest that Zyxin and PTOV1 should be considered as critical determinants in cancer therapy with retinoids. [Copyright &y& Elsevier]

Background & Aims: Oleuropein, a secoiridoid derived from olives and olive oil, has been known to possess antimicrobial, antioxidative, and anticancer activities. The purpose of the present study was to determine whether oleuropein has a protective effect against hepatic steatosis induced by a high fat diet (HFD) and to elucidate its underlying molecular mechanisms in mice. Methods: Male C57BL/6N mice were fed a normal diet (ND), HFD, or an oleuropein-supplemented diet (OSD) for 10weeks. The plasma and hepatic lipid levels were determined, and the hepatic gene and protein expression levels were analysed via RT-PCR and Western blotting, respectively. Results: The supplementation of HFD with oleuropein reversed the HFD-induced increases in liver weight along with plasma and hepatic lipid levels in mice. The expression of Wnt10b inhibitor genes, such as secreted firizzed-related sequence protein 5 and dickkopf homolog 2, was downregulated, whereas the β-catenin protein expression was upregulated in the liver of OSD-fed mice compared to HFD-fed mice. Fibroblast growth factor receptor 1 (FGFR1), phosphoextracellular-signal-regulated kinase 1/2, cyclin D, and E2F transcription factor 1, along with several key transcription factors and their target genes involved in adipogenesis, were downregulated by oleuropein. OSD-fed mice exhibited decreased expression of the toll-like-receptor-(TLR)-mediated signaling molecules (TLR2, TLR4, and myeloid differentiation primary-response gene 88) and proinflammatory cytokines, in their livers, as compared to HFD mice. Conclusions: These results suggest that the protective effects of oleuropein against HFD-induced hepatic steatosis in mice appear to be associated with the Wnt10b- and FGFR1-mediated signaling cascades involved in hepatic lipogenesis, along with the TLR2- and TLR4-mediated signaling implicated in hepatic steatosis. [Copyright &y& Elsevier]

Abstract: To characterize the regulatory mechanism of the interferon regulatory factor (IRF) family, we performed yeast two-hybrid screening with IRF-2 and isolated the small ubiquitin-related modifier (SUMO)-conjugating enzyme Ubc9, which also interacts with other IRF family members IRF-1 and ICSBP. Subsequent assays indicated that among the IRF family members, only IRF-1 interacts with SUMO-1 through its transcriptional activation domain. The interaction between IRF-1 and SUMO-1 was confirmed in vitro by GST pull-down assays and in vivo by co-localization assays. Furthermore, this interaction led to the Ubc9-mediated sumoylation of IRF-1 in vitro and in vivo. Transient transfection assays revealed that Ubc9 or SUMO inhibits the transcriptional activity of IRF-1 in a dose-dependent manner. Finally, Ubc9 and SUMO cooperate in the transcriptional repression of IRF-1. Taken together, these observations suggest that Ubc9 functions as a transcriptional repressor of IRF-1 by inducing sumoylation, and that this effect may be required for the physiological activity of IRF-1. [Copyright &y& Elsevier]

Abstract: The transcription regulator p73α is structurally different from p53 in that it possesses a unique C-terminal domain, which has been implicated in transcriptional repression. To dissect the mechanism of repression by this domain, we performed a yeast two-hybrid screen of a HeLa cDNA library using residues 487–636 of p73α as bait and isolated a cDNA clone encoding the C-terminal portion (residues 2210–2647) of filamin A, a 280-kDa actin-binding protein. Additional yeast two-hybrid assays indicated that filamin A specifically interacts with the p73α C-terminus, which is lacking in p53 and p73β. The interaction was confirmed by GST pull-down assays in vitro and by immunoprecipitation analysis in vivo. Immunofluorescence microscopy revealed that p73α remained in the cytoplasm in A7 melanoma cells stably expressing filamin A, whereas it was localized in the nucleus of filamin A-deficient M2 cells. Deletion of the C-terminus of p73α (residues 487–636) resulted in nuclear localization in both cell types. Consistent with our interaction data, transient co-expression of filamin A resulted in the down-regulation of p73α, but not of p53, transcriptional activity on various p53-responsive promoters. Taken together, our data suggest that p73α is sequestered in the cytoplasm by filamin A, thereby inhibiting its transcriptional activity. [Copyright &y& Elsevier]

Abstract: To elucidate the regulatory mechanism of p73 gene expression, we analyzed the human p73 promoter and found three putative Egr-1-binding sites located upstream of exon 1 (−1728, −321, and −38). The Egr-1 responsiveness of these sites was analyzed by transient transfection assays using 5′- and 3′-serial truncations of the p73 promoter, subcloned in a CAT reporter vector. The functional significance of the region was further confirmed by an electrophoretic mobility shift assay using the Egr-1 protein synthesized in vitro and a [32P]-labeled middle site sequence, followed by competition with unlabeled wild-type or mutant oligonucleotides and supershift assays using an anti-Egr-1 antibody. When induced by either the nitric oxide donor NOC-18 or the PPARγ agonist troglitazone, Egr-1 bound to the p73 promoter, as assessed by chromatin immunoprecipitation assays, accompanied by increased expression of p73. MTT assays revealed that cell growth was significantly inhibited on treating the cells with troglitazone. Overall, our results provide direct evidence that Egr-1 positively regulated p73 expression by binding to its promoter in vivo, consistent with Egr-1 and p73 being involved in p53-independent tumor suppression. [Copyright &y& Elsevier]

Abstract: To understand the molecular mechanism underlying HIPK2 regulation of the transcriptional activation by p53, we sought to identify the protein that interacts with HIPK2. From our yeast two-hybrid screen, we found that four and a half LIM domains 2 (FHL2) could bind to the C-terminal half of HIPK2. Further assays in yeast mapped the minimal interaction domain to amino acids 812–907 in HIPK2. The interaction was confirmed using a GST pull-down assay in vitro, and an immunoprecipitation (IP) assay and fluorescence microscopy in vivo. FHL2 alone spread throughout both the cytoplasm and nucleus but was redistributed to dot-like structures in the nucleus when HIPK2 was coexpressed in HEK293 cells. When tethered to the Gal4-responsive promoter through the Gal4 DBD fusion, FHL2 showed autonomous transcriptional activity that was enhanced by wild-type HIPK2, but not by the kinase-defective mutant. In addition, FHL2 increased the p53-dependent transcriptional activation and had an additive effect on the activation when coexpressed with HIPK2, which was again not observed with the kinase-defective mutant of HIPK2. Finally, we found a ternary complex of p53, HIPK2, and FHL2 using IP, and their recruitment to the p53-responsive p21Waf1 promoter in chromatin IP assays. Overall, our findings indicate that FHL2 can also regulate p53 via a direct association with HIPK2. [Copyright &y& Elsevier]

Abstract Additional sex comb-like1 (Asxl1) is known as a chromatin modulator that plays dual functions in transcriptional regulation depending on the cell type. Recent studies using Asxl1 knockout mice revealed that Asxl1 is important for the proliferation and differentiation of hematopoietic progenitor cells, and the development of organs. Although we previously reported Asxl1 as a Sox2 target gene, its function in embryonic stem cells (ESCs) remains largely unknown. For this purpose, we isolated ESCs from the blastocyst inner cell mass of Asxl1 −/− mice. Asxl1 deficiency in ESCs exhibited no effect on cell proliferation, expression of core pluripotent transcription factors, or alkaline phosphatase activity, suggesting dispensability of Asxl1 for self-renewal of ESCs. By contrast, the differentiation of Asxl1 −/− ESCs was significantly affected as shown by size reductions of embryoid bodies accompanied with apoptosis, aberrant expression of differentiation genes, downregulation of bivalent neurogenesis genes, and abnormal axon formation in neurons. Overall, our findings indicated that Asxl1 played a critical role in regulating genes associated with neural differentiation without affecting self-renewal of mouse ESCs. Highlights • Asxl1 is dispensable for the maintenance of ESC self-renewal and pluripotency. • Asxl1 deficiency leads to defective EB formation. • Differentiation-associated genes are dysregulated in Asxl1 -deleted EBs. • Asxl1 is required for the proper morphology of neuronal axons. [ABSTRACT FROM AUTHOR]

Summary: Alzheimer's disease (AD) is the leading cause of dementia in the elderly. Despite decades of study, effective treatments for AD are lacking. Mitochondrial dysfunction has been closely linked to the pathogenesis of AD, but the relationship between mitochondrial pathology and neuronal damage is poorly understood. Sirtuins (SIRT, silent mating type information regulation 2 homolog in yeast) are NAD‐dependent histone deacetylases involved in aging and longevity. The objective of this study was to investigate the relationship between SIRT3 and mitochondrial function and neuronal activity in AD. SIRT3 mRNA and protein levels were significantly decreased in AD cerebral cortex, and Ac‐p53 K320 was significantly increased in AD mitochondria. SIRT3 prevented p53‐induced mitochondrial dysfunction and neuronal damage in a deacetylase activity‐dependent manner. Notably, mitochondrially targeted p53 (mito‐p53) directly reduced mitochondria DNA‐encoded ND2 and ND4 gene expression resulting in increased reactive oxygen species (ROS) and reduced mitochondrial oxygen consumption. ND2 and ND4 gene expressions were significantly decreased in patients with AD. p53‐ChIP analysis verified the presence of p53‐binding elements in the human mitochondrial genome and increased p53 occupancy of mitochondrial DNA in AD. SIRT3 overexpression restored the expression of ND2 and ND4 and improved mitochondrial oxygen consumption by repressing mito‐p53 activity. Our results indicate that SIRT3 dysfunction leads to p53‐mediated mitochondrial and neuronal damage in AD. Therapeutic modulation of SIRT3 activity may ameliorate mitochondrial pathology and neurodegeneration in AD. [ABSTRACT FROM AUTHOR]

BRCA1-associated protein 1 (BAP1) has been implicated in diverse biological functions, including tumor suppression. However, its regulation via glycosylation and its role in embryonic stem (ES) cells are poorly defined. BAP1 was recently reported to interact with O-linked N -acetylglucosamine ( O -GlcNAc) transferase (OGT). Here, we confirmed the physical interaction and investigated its functional significance. The O -GlcNAcylation of BAP1, which requires OGT, was examined in vivo and in vitro , and was proven using alloxan, an OGT inhibitor. OGT promoted the BAP1-induced repression of retinoic acid (RA)-induced RA receptor (RAR) activation. The repressive activity of BAP1 was relieved by alloxan but exacerbated by PUGNAc, an O -GlcNAcase (OGA) inhibitor. Finally, we addressed the role of O -GlcNAcylation in the RA-induced differentiation of murine ES cells. Alkaline phosphatase staining revealed the cooperation of RA and alloxan for impairing the pluripotency of ES cells. This cooperation was also observed by measuring the size of embryonic bodies and the expression of Sox2, a pluripotency marker. Overall, our data suggest that OGT-mediated O -GlcNAcylation of BAP1 prefers the maintenance of pluripotency, whereas its inhibition facilitates RA-induced differentiation in ES cells. [ABSTRACT FROM AUTHOR]

Elevated expression of preferentially expressed antigen in melanoma (PRAME) has been implicated in disease progression in a variety of cancers. However, the mechanisms underlying the transcriptional regulation of PRAME remain largely unexplored. Initially, we observed that PRAME was elevated in proportion to the malignant potential of melanoma cells. From the in silico prediction of PRAME gene structure, we identified the putative myeloid zinc finger 1 (MZF1) binding sites, which overlap with a CpG-rich region located in the first intron. The transcription factor MZF1 increased PRAME expression via its direct binding to the intron DNA. Upon treatment with a DNA methylation inhibitor, 5-aza-2'-deoxycitidine (5-azaC), together with ectopic expression of MZF1, PRAME expression was significantly enhanced at both the protein and mRNA levels. More pronounced MZF1 binding to the PRAME DNA was observed in the presence of 5-azaC. DNA methylation was inversely correlated with PRAME expression in melanoma cells. Finally, we observed that MZF1, like PRAME, promotes the colony-forming ability in melanoma cells. Overall, our findings suggest that MZF1, via stimulation of PRAME expression, may be a potential prognostic and therapeutic target in melanoma. [ABSTRACT FROM AUTHOR]

Mechanisms associated with the progression of non-alcoholic fatty liver disease (NAFLD) remain unclear. We attempted to identify the pattern of altered gene expression at different time points in a high fat diet (HFD)-induced NAFLD mouse model. The early up-regulated genes are mainly involved in the innate immune responses, while the late up-regulated genes represent the inflammation processes. Although recent studies have shown that microRNAs play important roles in hepatic metabolic functions, the pivotal role of microRNAs in the progression of NAFLD is not fully understood. We investigated the functions of miR-451, which was identified as a target gene in the inflammatory process in NAFLD. miR-451 expression was significantly decreased in the palmitate (PA)-exposed HepG2 cells and in liver tissues of HFD-induced non-alcoholic steatohepatitis (NASH) mice. Its decreased expressions were also observed in liver specimens of NASH patients. In vitro analysis of the effect of miR-451 on proinflammatory cytokine provided evidence for negative regulation of PA-induced interleukin (IL)-8 and tumor necrosis factor-alpha (TNF-α) production. Furthermore, miR-451 over-expression inhibited translocation of the PA-induced NF-κB p65 subunit into the nucleus. Our result showed that Cab39 is a direct target of miRNA-451 in steatotic cells. Further study showed that AMPK activated through Cab39 inhibits NF-κB transactivation induced in steatotic HepG2 cells. miR-451 over-expression in steatotic cells significantly suppressed PA-induced inflammatory cytokine. These results provide new insights into the negative regulation of miR-451 in fatty acid-induced inflammation via the AMPK/AKT pathway and demonstrate potential therapeutic applications for miR-451 in preventing the progression from simple steatosis to severely advanced liver disease. [ABSTRACT FROM AUTHOR]

Among the members of the additional sex comb-like (ASXL) family, ASXL3 remains unexplored. Here, we showed that ASXL3 interacts with HP1α and LSD1, leading to transcriptional repression. We determined that ASXL3 depletion augments the ligand-induced transcriptional activities of LXRα and TRβ, which were repressed by ASXL3 overexpression. The ligand-dependent interactions of ASXL3 with LXRα and TRβ were demonstrated by the GST pull-down and immunoprecipitation analyses. We confirmed that ASXL3 suppresses the expression of LXRα target genes through its recruitment to the LXR-response elements. Finally, we observed that lipid accumulation in Hep3B cells is downregulated upon ASXL3 overexpression but upregulated upon ASXL3 depletion. Overall, our data suggest that ASXL3 is another corepressor of LXRα, promoting to the regulation of lipid homeostasis. [ABSTRACT FROM AUTHOR]

Highlights: [•] ASXL1 and ASXL2 directly interact with ligand-bound LXRα. [•] Ligand-induced LXRα activity is repressed by ASXL1 and activated by ASXL2. [•] ASXL1 and ASXL2 bind to the LXRE of the LXRα target promoter. [•] ASXL1 and ASXL2 reciprocally regulate lipogenesis in liver cells. [Copyright &y& Elsevier]

Summary: Despite the importance of retinoic acid (RA) signaling and histone monoubiquitination in determining cell fate, the underlying mechanism linking the two processes is poorly explored. We describe that additional sex comb-like 1 (ASXL1) represses RA receptor activity by cooperating with BRCA1-associated protein 1 (BAP1), which contains the ubiquitin C-terminal hydrolase (UCH) domain. Both the UCH- and ASXL1-binding domains of BAP1 were required for cooperation. In contrast to Drosophila BAP1, mammalian BAP1 cleaved ubiquitin from histone H2B. As supported by BAP1 mutants, ASXL1 was critical for BAP1 recruitment to chromatin and its activation therein. ASXL1 requirement was supported using Asxl1 null mice embryonic fibroblasts. Both ASXL1 and BAP1 were downregulated during RA-induced P19 cell differentiation with concomitant increase of ubiquitinated H2B, leading to activation of Hox genes. Our data demonstrate the critical role of ASXL1 cooperation with BAP1 in cell differentiation through the regulation of RA signaling associated with H2B ubiquitination. [Copyright &y& Elsevier]

Abstract: To investigate the underlying mechanism of targets of cyanidin, a flavonoid, which exhibits potent anti-atherogenic activities in vitro and in vivo, a natural chemical library that identified potent agonistic activity between cyanidin and peroxisome proliferator-activated receptors (PPAR) was performed. Cyanidin induced transactivation activity in all three PPAR subtypes in a reporter gene assay and time-resolved fluorescence energy transfer analyses. Cyanidin also bound directly to all three subtypes, as assessed by surface plasmon resonance experiments, and showed the greatest affinity to PPARα. These effects were confirmed by measuring the expression of unique genes of each PPAR subtype. Cyanidin significantly reduced cellular lipid concentrations in lipid-loaded steatotic hepatocytes. In addition, transcriptome profiling in lipid-loaded primary hepatocytes revealed that the net effects of stimulation with cyanidin on lipid metabolic pathways were similar to those elicited by hypolipidemic drugs. Cyanidin likely acts as a physiological PPARα agonist and potentially for PPARβ/δ and γ, and reduces hepatic lipid concentrations by rewiring the expression of genes involved in lipid metabolic pathways. [Copyright &y& Elsevier]

Abstract: ERα, a critical transcriptional factor for breast cancer proliferation, is regulated by a complex binding repertoire that includes coactivators and corepressors. Here, we identified a novel class of ERα coregulator called CAC1. The CoRNR box of CAC1 was required for the binding to and inactivation of ERα. CAC1 also associated with histone demethylase LSD1 and suppressed LSD1-enhanced ERα activity. CAC1 impaired recruitment of ERα and LSD1 to the ERα-responsive promoter, leading to greater H3K9me3 accumulation. This effect was reversed by CAC1 depletion. Finally, CAC1 increased paclitaxel-induced cell death in ERα-positive MCF7 cells, which are paclitaxel-resistant. Overall, our study provides the first evidence that CAC1, associated with LSD1, functions as an ERα corepressor, implicating a potential antitumor target in ERα-positive breast cancer. Structured summary of protein interactions: ER-alpha physically interacts with CAC1 by anti tag coimmunoprecipitation (View Interaction: 1, 2, 3) LSD1 physically interacts with CAC1 by anti tag coimmunoprecipitation (View interaction) CAC1 binds to ER-alpha by pull down (View interaction) CAC1 and ER-alpha colocalize by fluorescence microscopy (View interaction) [Copyright &y& Elsevier]

Abstract: This study investigated the role of piperine in the transcriptional regulation of liver X receptor α (LXRα) and the effects of dietary piperine on high-fat diet (HFD)-induced hepatic steatosis and insulin resistance in mice. Furthermore, we explored the potential molecular mechanisms through which the protective effects of piperine may work. In the present study, piperine significantly reduced ligand-induced LXRα activity in a dose-dependent manner and gradually disrupted the interaction between ligand-bound LXRα and GST–CBP. In mice, an HFD supplemented with 0.05% piperine (PSD) significantly decreased body and liver weight as well as plasma and hepatic lipid levels. In agreement with our in vitro study, in mice fed an HFD, dietary piperine markedly decreased LXRα mRNA expression and its lipogenic target genes (i.e., SREBP1c, ChREBPα, FAS, and CD36). Piperine also significantly decreased plasma insulin and glucose concentrations, while increasing insulin sensitivity in mice fed an HFD. In addition, piperine downregulated the expression of genes involved in ER stress, including GRP78, activating transcription factor 6, and eukaryotic translation initiation factor 2α, and upregulated GLUT2 translocation from the cytosol to the plasma membrane in the livers of PSD mice. Piperine antagonized LXRα transcriptional activity by abolishing the interaction of ligand-bound LXRα with the co-activator CBP. The effects of piperine on hepatic lipid accumulation were likely regulated via alterations in LXRα-mediated lipogenesis in mice fed an HFD. Dietary piperine also led to reduced ER stress and increased insulin sensitivity and prevented hepatic insulin resistance in mice fed the HFD. [Copyright &y& Elsevier]

Abstract: Oleuropein, a bitter glucoside found in green olive leaves, and its metabolite hydroxytyrosol display powerful antioxidant activity both in vivo and in vitro. In this study, we hypothesized that the antioxidant activity of oleuropein could attenuate hepatic steatosis. To test this hypothesis, we established steatotic hepatocytes using HepG2 and FL83B cells treated with free fatty acids (FFAs) (oleate:palmitate, 2:1). To confirm hepatic steatosis, the intracellular lipid levels were quantitatively measured by Nile Red staining, and the sizes and distributions of lipid droplets were visualized by transmission electron microscopy. The expression of PAT family proteins as well as of adipose differentiation-related protein and tail interacting protein (TIP47) was evaluated by reverse transcriptase polymerase chain reaction and immunoblotting. To examine the cellular and molecular events associated with oleuropein, annexin V/propidium iodide staining and immunoblotting were performed. Oleuropein decreased the number and size of lipid droplets in FFA-treated cells and reduced intracellular triglyceride accumulation. However, it did not affect the expression of lipid droplets-associated PAT family proteins, including adipose differentiation-related protein and TIP47. In addition, oleuropein reduced FFA-induced extracellular signal-regulated kinase activation but had no effect on c-Jun N-terminal kinase or AKT activation. Given its protective effects against FFA-induced hepatocellular steatosis, oleuropein may be a lipid-lowering agent. [Copyright &y& Elsevier]

Abstract: Additional sex comb-like 1 (ASXL1) has been suggested to be an enhancer of trithorax and polycomb proteins, and functions as a dual co-regulator of retinoid acid (RA) signaling. However, the mechanism by which ASXL1 gene expression is regulated remains unresolved. Concomitant downregulation of both SOX2 and ASXL1 during the RA-induced differentiation of P19 cells prompted us to investigate the role of SOX2 in the regulation of ASXL1. Knockdown of SOX2 in SOX2-rich NT2 cells resulted in the reduction of ASXL1 expression, whereas SOX2 overexpression in SOX2-deficient H1299 cells increased ASXL1 expression. Using a cloned ASXL1–luciferase reporter, we demonstrated that SOX2 directly transactivates the ASXL1 promoter. Serial deletion and mutation studies mapped the SOX2 response element region in the ASXL1 promoter to −1600 to −1400bp. We showed by chromatin immunoprecipitation assay that SOX2 directly binds to the ASXL1 promoter region. Finally, formation of embryonic bodies by ASXL1-depleted murine E14TG2a embryonic stem cells was significantly impaired, similar to SOX2-knockdown cells. From these results, we suggest that ASXL1 may be a direct target of SOX2 and may play a role in maintaining the pluripotency of stem cells. [Copyright &y& Elsevier]

Abstract: Retinoic acid (RA) plays a role in cancer therapy. However, its long-term treatment is hindered by the acquired resistance which is not fully understood. Our previous study indicated that the transcriptional activity of RA receptor (RAR) is enhanced by association of MED25 with CREB-binding protein (CBP) through the PTOV domain, which is also present in prostate tumor over-expressed protein 1 (PTOV1). Here, we show that MED25 and PTOV1 reciprocally regulate RAR transcriptional activity through competitive bindings to CBP and opposite regulation of CBP recruitment to the RA-responsive gene promoter. Finally, we demonstrate that MED25 and PTOV1 differentially modulate RA sensitivity in cancer cells depending on their expression levels, suggesting a potential molecular mechanism underlying RA resistance which frequently emerges during cancer treatments. [ABSTRACT FROM AUTHOR]

Abstract: The retinoic acid receptor (RAR), as one of the retinoic acid (RA)-responsive transcription activators, mediates various biological processes by regulating RA target gene expression. In studying how RAR activity is regulated, we isolated thioredoxin glutathione reductase (TGR), a member of the thioredoxin reductase family. Systematic yeast two-hybrid assays showed that in the presence of RA, TGR interacts with RAR via the LxxLL motif (NR box) located between the Grx and TrxR domains of TGR. This interaction was confirmed by GST pull-down and immunoprecipitation assays. The stable over-expression or knockdown of TGR in TGR-deficient NIH3T3 or TGR-abundant TM4 Sertoli cells, respectively, revealed that TGR enhances the transcriptional activity of RAR by increasing its DNA-binding capacity and restores RAR activity after impairment by reactive oxygen species (ROS). Furthermore, we demonstrated that the transactivation potential and DNA-binding activity of RAR in response to ROS depends on the cellular level of TGR. Overall, our data suggest that the redox regulation function of TGR protects the DNA-binding activity of RAR against cellular ROS damage. [Copyright &y& Elsevier]

Summary: Human SIRT1 is an NAD+-dependent deacetylase protein that plays a role in cell death/survival, senescence, and endocrine signaling. While its substrates, including p53, have been well characterized, no direct regulators are known. We describe here a nuclear protein, active regulator of SIRT1 (AROS), which directly regulates SIRT1 function. AROS enhanced SIRT1-mediated deacetylation of p53 both in vitro and in vivo, and it inhibited p53-mediated transcriptional activity. AROS activity was abrogated by the SIRT1 inhibitors splitomicin and nicotinamide and by SIRT1 small interfering RNA (siRNA). In addition, AROS was unable to cooperate in p53 inactivation in an AROS-binding-defective SIRT1 mutant. Finally, knockdown of endogenous AROS using an antisense expression vector enhanced p21WAF1 expression and increased both the G0/G1 population and apoptosis in response to DNA damage, while AROS overexpression improved cell survival. To our knowledge, AROS is the first direct SIRT1 regulator to be identified that modulates p53-mediated growth regulation. [Copyright &y& Elsevier]

Abstract: Proteomic analyses can efficiently detect the variation of protein in high throughput screening. Using 2DE/MALDI-TOF-MS and SELDI-TOF-MS, we tried to search several cellular proteins that are responsive to ursolic acid (UA) in HeLa cervical carcinoma cells. Compared to control, UA-treated HeLa cells unfolded 25 proteins in significant changes by 2DE/MALDI-TOF-MS, most of which were involved in apoptosis. SELDI-TOF-MS with two types of protein chips profiled and analyzed proteomic features after administration of UA. Interestingly, eight polypeptide peaks can be detected. Further identification and characterization of these proteins may build the molecular basis of UA-induced apoptosis and provide insight into the anti-proliferative mechanism in cervical carcinoma cells. [Copyright &y& Elsevier]

Abstract: Objectives. : Paclitaxel (Taxol®), a potent drug of natural origin isolated from the bark of the Pacific yew, is widely used in the treatment of ovarian, lung and breast cancer. At present, there is little information regarding the anti-cancer mechanism of paclitaxel against cervical carcinoma cells. We thus tried to show the anti-cancer effect of paclitaxel on cervical carcinoma cell line carrying HPV by using a proteomic analysis and to investigate the mechanism of actions. Methods. : We treated paclitaxel to cervical carcinoma cells and then carried out MTT assay to observe the anti-proliferate activity. Using proteomics analysis including two-dimensional (2-DE) gel electrophoresis and MALDI-TOF-MS, we tried to find the anti-proliferate activity-related proteins. Among them, paclitaxel treatment suppressed the expression of the mitotic checkpoint protein BUB3. Functional proteomic analysis by small interfering RNA (siRNA) targeting was tried to illuminate a role of mitotic checkpoint protein BUB3 in cell cycle progression. Results. : The cytotoxicity effects of paclitaxel were determined in HPV-16 positive CaSki, HPV-18 positive HeLa and HPV-negative C33A cervical carcinoma cell lines. Using efficient proteomics methods including 2-DE/MALDI-TOF-MS, we identified several cellular proteins that are responsive to paclitaxel treatment in HeLa cells. Paclitaxel treatment elevated mainly apoptosis-related, immune response-related and cell cycle check point-related proteins. On the other hand, paclitaxel treatment diminished growth factor/oncogene-related proteins and transcription regulation-related proteins. Paclitaxel showed anti-proliferate activity through the membrane death receptor (DR)-mediated apoptotic pathway involving activation of caspase-8 with a TRAIL-dependent fashion as well as the mitochondrial-mediated pathway involving down-regulation of bcl-2 by cytochrome c release. Furthermore, we found siRNA-induced BUB3 knock down on cell cycle progression blocked by cell cycle arrest after paclitaxel treatment. Conclusions. : The proteome profiling technique provided a broad-base and effective approach for the identification of protein changes induced by paclitaxel and showed anti-proliferate activity through the membrane death receptor-mediated apoptotic pathway, the mitochondrial-mediated pathway. This study shows the power of proteomic profiling with functional analysis using RNAi technology for the discovery of novel molecular targets and a better understanding of the actions of paclitaxel at the molecular level in cervical carcinoma cells. [Copyright &y& Elsevier]

cDNA microarray and proteomics studies were performed to analyze the genomic and proteomic expression patterns in HPV-16 E6 gene transfected stable human carcinoma cell lines. Among 1024 known genes and ESTs tested by cDNA microarray, we found 50 upregulated and 35 downregulated genes in RC10.1 HPV-16 E6 transfected human colon adenocarcinoma cells compared to RKO cells, and 27 upregulated and 43 downregulated genes in A549E6 HPV-16 E6 transfected human lung adenocarcinoma cells compared to A549 cells. Employing two dimensional gel electrophoresis and MALDI-TOF-MS, the global pattern of protein expressions in RC10.1 human colon adenocarcinoma and A549E6 human lung adenocarcinoma cell lines stably expressing the HPV 16-E6 gene were compared with those of RKO and A549 cell lines to generate a differential protein expression catalog. We found 13 upregulated and 13 downregulated proteins in RC10.1 (E6-expressing RKO) cells compared to RKO cells and 12 upregulated and 14 downregulated proteins in A549E6 (E6-expressing A549) cells compared to A549 cells. The identified genes and proteins were classified into several groups according to the subcellular function. Expressing pattern of three genes and proteins (CDK5, Bak, and I-TRAF) were matched in both analyses of cDNA microarray and proteomics. These powerful approaches using cDNA microarray and proteomics could provide in-depth information on the impact of HPV-16 E6-related genes and proteins. Differential gene and protein expression patterns by transfection of HPV-16 E6 will provide the nucleus of valuable resource for investigation of the biochemical basis of cervical carcinogenesis. Further understanding of this data base may provide valuable resources for developing novel diagnostic markers and therapeutic targets of cervical cancer. [ABSTRACT FROM AUTHOR]

The global effects of 5-fluorouracil (FU) on cervical carcinoma cells were analyzed using an efficient proteomic method. More than 50 proteins showed a significant change in 5-FU–treated cervical carcinoma cells compared to control cells. Among them, 34 proteins have been identified by employing two-dimensional gel electrophoresis and MALDI-TOF-MS using peptide mass fingerprinting. In results, 22 proteins were upregulated (CIDE-B [cell death-inducing DFFA-like effector B], caspase-3, caspase-8, Apo-1/CD95 (Fas), etc.) and 12 proteins were downregulated (mitotic checkpoint protein BUB3, myc proto-oncogene protein [c-myc], src substrate cortactin, transforming protein p21A, etc.) by 5-FU treatment in HeLa cervical carcinoma cells as determined by spot volume (P < 0.05). Our experiments showed that 5-FU engaged the mitochondrial apoptotic pathway involving cytosolic cytochrome c release and subsequent activation of caspase-9 and caspase-3 as well as the membrane death receptor (DR)-mediated apoptotic pathway involving activation of caspase-8 with an Apo-1/CD95 (Fas)-dependent fashion. In addition, we could observe reduction of HPV-18 E6/E7 gene expression and activation of p53, pRb, and p21waf1 proteins by 5-FU treatment in HeLa cervical carcinoma cells. In conclusion, we suggest that 5-FU suppresses the growth of cervical cancer cells not only by antiproliferative effect but also antiviral regulation. Our findings may offer new insights into the mechanism of anticancer effect affected by 5-FU treatment in cervical cancer cells and its mode of action. [ABSTRACT FROM AUTHOR]

Retinol (vitamin A) is used as an antiwrinkle agent in the cosmetics industry. However, its photo-instability makes it unsuitable for use in general cosmetic formulations. To improve the photo-stability of retinol, three derivatives (3, 4, and 5) were synthesized and their biological activities were analyzed. 1H NMR and HPLC analysis indicated that derivatives 3 and 5 were much more stable than retinol under our sunlight exposure conditions. When human adult fibroblasts were treated, the IC50 of derivative 3 was 96 μM, which is similar to that of retinol, as determined by the MTT assay. Derivatives 4 and 5 were 2.5 and 8 times more toxic than retinol, respectively. At 1 μM treatment, like retinol, derivatives 3 and 4 were specifically active for RARα out of six retinoid receptors (RAR/RXRα, β, γ). Dose-dependent analysis confirmed that derivative 4 was as active as retinol and the other two derivatives were less active for RARα. The effect of our derivatives on the expression of collagenase, an indicator of wrinkle formation, was measured using the transient co-expression of c-Jun and RT-PCR in HaCaT cells. Collagenase promoter activity, which is increased by c-Jun expression, was reduced 42% by retinol treatment. The other derivatives inhibited collagenase promoter activity similarly. These results were further confirmed by RT-PCR analysis of the collagenase gene. Taken together, our results suggest that retinol derivative 3 is a promising antiwrinkle agent based on its higher photo-stability, lower RARα activity (possibly indicating reduced side effects), and similar effect on collagenase expression. [Copyright &y& Elsevier]

: ObjectiveThe goal of this study was to study whether estrogen could induce progression of cervical neoplasia by the influence of direct hormonal transactivation of the viral genes.: MethodsWe examined the in vivo effect of estrogen on HPV-18 URR E6/E7 transgenic mice. We analyzed the growth stimulation of epithelial cells at squamo-columnar junction and vagina and the expression of HPV E6/E7 in transgenic mice. The promoter activity of HPV-18 URR after treatment of estrogen was also evaluated by in vitro transient transfection assay.: ResultsThe dysplastic lesions of lower genital tract were more frequently seen in the HPV-18 URR E6/E7 transgenic mice and estrogen-treated mice, when compared to those of control group (P < 0.05). The majority of cells in whole epithelial layer of cervix and vagina were proliferating cell nuclear antigen-positive (PCNA) by immunohistochemical staining in the estrogen-treated transgenic mice. Hyperplastic glandular lesions were also identified in estrogen-treated HPV-18 URR E6/E7 transgenic mice (5 of 21) and estrogen-treated nontransgenic mice (2 of 10). The level of E6/E7 transcripts in transgenic mouse was increased in the presence of estradiol. Treatment with 0.5 × 10−6 M estradiol in the presence of cotransfection with the estrogen receptor expression vector and URR-CAT showed an additive effect of CAT activity (4.8-fold increase).: ConclusionThe HPV E6 and E7 oncogenes implicated in cervical cancer are indeed capable of potentiating tumor formation in animal model. Continual estrogen-induced proliferation might be viewed by in vivo and in vitro mechanisms by which squamous cells as well as glandular cells in cervix and vagina became permissive for neoplastic progression in HPV-18 URR E6/E7 transgenic mice and their molecular systems. [Copyright &y& Elsevier]

Interferon regulatory factor-1 (IRF-1), a putative tumor suppressor protein, is a transcriptional mediator of interferon-responsive signaling pathways that are involved in antiviral defense, apoptosis, immune response, and cell growth regulation. To delineate the IRF-1 domain responsible for transactivation, we performed a detailed deletion analysis of IRF-1. We found that the amino acid segment 217–260 was necessary and sufficient for transactivation. The structure of this region was predicted to be loop–helix–loop–sheet using the program PHD. Further studies indicated that casein kinase II and protein kinase C sites on each of the two loops are not important for transactivation, and a region containing amino acids 233–255 comprises the core activation domain. To verify the physiological role of segment 233–255, we constructed an IRF-1 deletion mutant lacking these amino acids and examined it using IRF-1 transactivation assay, fluorescence-activated cell sorting, and in situ β-galactosidase staining techniques. From the results of these studies, we conclude that the amino acid segment 233–255 of IRF-1 comprises the core activation domain required for the physiological functions of IRF-1. [Copyright &y& Elsevier]

The E6 and E7 oncoproteins of human papillomavirus (HPV) play a major role in the development of cervical carcinoma. In this study, a recombinant adenovirus that expresses the bovine papillomavirus (BPV) E2, which has been shown to inhibit HPV early gene expression, was delivered to two HPV-immortalized cell lines as well as CaSki, a cervical carcinoma cell line. We tested whether the carcinoma and the immortal cells were equally affected by the expression of BPV E2. In all cell lines, BPV E2-mediated inhibition of HPV E6/E7 expression caused a dramatic suppression of cell growth, being preceded by the activation of the p53–Rb growth-inhibitory pathway, and a decrease in hTERT mRNA expression and telomerase activity. This suggests that the HPV E6 and E7 proteins are required not only for induction of the proliferative phenotype and telomerase activity, but also for their maintenance. In both the carcinoma and the immortal lines, the number of cells with enlarged cytoplasm and senescence-associated β-galactosidase activity, which are markers for cellular senescence, was significantly increased. These results suggest that a senescence program exists in cells immortalized by HPV DNA as well as in cervical carcinoma cells. [Copyright &y& Elsevier]