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At least 19 recordsLinked to original sources
PubMed2026

Screening of Estrogenic and Antiestrogenic Effects of Estradiol, Bisphenol A, and Fulvestrant Using 2D and 3D Breast Cancer Cell Systems With a Luciferase Reporter Gene Assay.

Endocrine-disrupting chemicals (EDCs) like bisphenol A (BPA) pose health risks by interfering with hormones. This study develops and utilizes in vitro 2D and 3D cell models to evaluate the estrogenic and antiestrogenic properties of compounds. Human breast cancer cell lines T47D and MCF7, stably transfected with a luciferase reporter gene (ERE-LUC), were first compared in 2D. Due to the significantly higher sensitivity and responsiveness observed in the T47D line during preliminary 2D screenings, this cell line was exclusively selected for the development of the 3D spheroid model. Cells were treated with 17β-estradiol (E2), BPA, and Fulvestrant (FUL) to assess cell viability and luciferase activity. In 2D models, T47D ERE-LUC cells showed higher responsiveness than MCF7 ERE-LUC, which failed to show significant luciferase induction with E2. In the 3D T47D model, cells exhibited significant and robust changes in luciferase activity in response to E2 and BPA, highlighting the enhanced fidelity of 3D cultures in replicating tissue conditions compared to their 2D counterparts. The study highlights the effectiveness of 3D models over 2D in evaluating estrogenic activity. Specifically, the 3D T47D ERE-LUC system serves as a superior, sensitive, and reliable platform for screening EDCs, offering benefits in cost, data speed, and reduced in vivo reliance.

Humans↗
PubMed2025

Reporter Gene Assays to Measure FOXO-Specific Transcriptional Activity.

The forkhead box O (FOXO) family of transcription factors translates environmental cues into precise gene expression patterns maintaining cellular equilibrium while influencing critical determinations of cell destiny and differentiation. FOXO proteins exert their effects through specific consensus binding to promoter sites within target genes. Notably, among the array of techniques available for assessing the transcriptional activity of FOXO factors, the utilization of luciferase-based reporters emerges as particularly distinctive. Luciferase, an enzyme sourced from bioluminescent organisms, instigates the oxidation of luciferin, culminating in the generation of oxyluciferin accompanied by discernible luminescence, a quantifiable event readily gauged using a luminometer. The adoption of luciferase activity as a measure in transcriptional assays is widespread due to its numerous advantages including simplicity, remarkable reproducibility, and high sensitivity. Moreover, the continuous advancements witnessed in luciferase-based vectors and measurement reagents bestow notable flexibility upon this methodology. Luciferase-based reporters offer a powerful tool for uncovering constituents within the signaling pathways governing FOXO factor function. Furthermore, these assays are also suitable for evaluating the efficacy of FOXO-targeting agents, whether they be inhibitors or activators. Here, we present a comprehensive, step-by-step elucidation of a commonly employed assay, adeptly quantifying the potential of small molecular compounds to amplify FOXO-specific transcriptional activity in U2OS cells.

Genes, Reporter↗
PubMed2025

Marburg Virus Minigenome Assays.

This chapter describes minigenome systems for Marburg virus (MARV), which reconstitute the viral polymerase complex functions of gene expression and genome replication. Procedures covered herein include passage and seeding of cells, transfection, sample collection, and reporter gene assays.

Marburgvirus↗
PubMed2026

The role of miRNA-32 in non-small cell lung cancer.

BACKGROUND: This study aimed to investigate whether miRNA-32 affects the proliferation and migration of non-small cell lung cancer (NSCLC) cells by regulating the expression of myocyte enhancer factor 2D (MEF2D). METHODS: Quantitative real-time polymerase chain reaction was utilized to evaluate the expression levels of miRNA-32 in clinical NSCLC tissue specimens and cell lines. Western blotting was employed to detect the protein expression levels of MEF2D, E-cadherin, N-cadherin, and CyclinD1, as well as to verify transfection efficiency. Cell proliferation and migration were assessed using Cell Counting Kit-8 and Transwell assays, respectively. Additionally, a dual-luciferase reporter gene assay was performed to validate the targeted regulatory relationship between miRNA-32 and MEF2D. RESULTS: miRNA-32 was significantly downregulated in lung cancer tissues and cell lines, whereas MEF2D exhibited significant upregulation. High miRNA-32 expression correlated with poor clinical outcomes in NSCLC across both our in-house cohort and the TCGA cohort. The stable overexpression of miRNA-32 in lung cancer cells markedly inhibited their proliferation and migratory capabilities. Mechanistically, miRNA-32 inhibited the translation of MEF2D by directly binding to its 3'UTR region. Crucially, the overexpression of MEF2D significantly reversed the inhibitory effects of miRNA-32 on lung cancer cell proliferation and migration. CONCLUSION: The miRNA-32/MEF2D signaling axis plays a pivotal role in the proliferation and metastasis of NSCLC, highlighting its potential as a diagnostic biomarker and prognostic indicator for the disease.

Humans↗
PubMed2026

Inflammatory Cytokines Impair Glucagon Expression and Secretion in Pancreatic α-Cells.

AIMS: Insulin resistance and obesity-associated inflammation are key drivers in the pathogenesis of Type 2 diabetes mellitus (T2DM). Whilst inflammatory cytokines are well known to impair β-cell function, their impact on pancreatic α-cells and glucagon (GCG) regulation remains poorly understood. In this study, we investigated the effects of the pro-inflammatory cytokines interleukin (IL)-1β, tumour necrosis factor (TNF)-α and interferon (IFN)-γ on GCG expression and secretion. MATERIALS AND METHODS: The viability and endocrine function of α-cell line αTC1 and isolated islets were investigated by WST-1 assay, LDH assay, qRT-PCR, Western blot analysis and ELISA. The transcriptional activity of the GCG promoter was analysed by reporter gene assays. The cellular composition of isolated islets was assessed by immunohistochemistry. RESULTS: We found that exposure of the α-cell line αTC1 to a mix of these cytokines activates cellular stress responses characterised by induction of the nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB) pathway and the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. Moreover, cytokine treatment markedly reduced GCG gene expression and secretion through repression of GCG promoter activity. Mechanistically, this was associated with a disrupted transcriptional network. These findings were confirmed in isolated mouse islets, where cytokine exposure significantly reduced GCG expression and secretion in islets of both male and female donors. CONCLUSIONS: Taken together, these findings indicate that inflammatory cytokines are potent modulators of α-cell function as well as GCG secretion and provide novel insights into inflammation-driven dysregulation of the endocrine function of pancreatic islets.

Glucagon-Secreting Cells↗
PubMed2026

LncRNA H19 promotes vascular remodeling by inhibiting MFN2.

Neointimal hyperplasia, featuring excessive proliferation and migration of vascular smooth muscle cells (VSMCs), is crucial in vascular remodeling diseases. Long non-coding RNA (lncRNA) H19 promotes vascular remodeling, yet underlying mechanisms remain incompletely elucidated. Here, we investigated whether H19 acts via its derivative miR-675-5p. Bioinformatics found a conserved H19/miR-675-5p/MFN2 (mitofusin 2) axis. Cell assays were performed to evaluate the effects of H19 and miR-675-5p on VSMC proliferation and migration. The dual-luciferase reporter gene assay was used to assess the interaction between miR-675-5p and Mfn2 mRNA. The mouse model of common carotid artery ligation was used to evaluate the role of H19 in neointimal hyperplasia. Our data suggested that knockdown of H19 inhibited VSMC proliferation and migration, as well as neointimal hyperplasia. Mechanistically, H19 regulated MFN2 through miR-675-5p, leading to ERK1/2 (extracellular signal-regulated kinase 1/2) activation. In conclusion, we suggest that targeting the H19/miR-675-5p/MFN2/ERK1/2 axis may help to treat vascular remodeling diseases.

Animals↗
PubMed1992

Transcriptional regulation of the human IgE receptor (Fc epsilon RII/CD23) by EBV. Identification of EBV-responsive regulatory elements in intron 1.

EBV infection of B cells induces the B cell activation Ag, CD23 (Fc epsilon RII). CD23 remains constitutively expressed at high levels in all EBV-immortalized B cells and likely plays an important role in the initiation and maintenance of immortalization by EBV. By utilizing an EBV-negative Burkitt's lymphoma line (BJAB) and EBV-positive sublines derived from it by in vitro infection, we have examined the molecular mechanisms involved in the regulation of CD23 by EBV. By nuclear runoff analysis, we have found that induction of CD23 is mediated by transcriptional activation that occurs in the presence of the transformation-competent B958 virus but not in the presence of the nontransforming P3HR-1 strain of EBV. To identify EBV-responsive transcriptional regulatory elements of CD23, we have performed reporter gene assays using plasmids containing fragments of the CD23 gene derived from its 5' terminus and adjacent flanking region transfected into EBV-positive and -negative BJAB lines. We have identified a 534-bp fragment of the gene which enhances transcription from a heterologous promoter (SV40) and reporter gene (chloramphenicol acetyltransferase) only in the presence of transformation-competent strains of EBV. Deletion of 144 bp of intron 1 from the 3' end of this fragment results in loss of EBV-responsive enhancer activity. The finding of an EBV-responsive enhancer element of CD23 is supported by mobility shift assays that demonstrated the formation of specific DNA-protein complexes between nuclear protein from transforming EBV-positive cells and the 144-bp intron sequence. These studies suggest that the transcriptional activation of CD23 by transforming strains of EBV involves regulatory elements that are located within the first intron of the gene.

Antigens, Differentiation, B-Lymphocyte↗
PubMed2025

A three-dimensional mouse liver organoid platform for assessing EDCs metabolites simulating liver metabolism.

Hepatic metabolism is an important process for evaluate the potential activity and toxicity of endocrine disrupting chemicals (EDCs) metabolites. Organization for Economic Co-operation and Development (OECD) has advocated the development of in vitro assays that mimic in vivo hepatic metabolism to eventually replace classical animal tests. In response to this need, we established a 3D mouse liver organoid (mLO) platform that mimics the animal model and is distinct from existing models. We evaluated the effects the activity of EDC metabolites generated through mLOs based on human cell-based reporter gene assays in addition to existing models. This study emphasizes the importance of hepatic ex-vivo and suggests the need a new metabolic model through a 3D mLOs platform. These results indicate that mLOs provides a novel biological method to screen for potential endocrine-disrupting activities of EDC metabolites.

Animals↗
PubMed2026

Cranberry juice potentiates sensitivity of uropathogenic Escherichia coli (UPEC) strains to fosfomycin and decreases occurrence of spontaneous resistance.

Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs). The growing prevalence of antimicrobial resistance underscores the need for alternative or complementary strategies to enhance antibiotic activity. Fosfomycin (FOS) remains a recommended first-line treatment for uncomplicated UTIs due to its broad activity and low resistance rates; however, spontaneous resistance frequently arises through mutations in bacterial transport systems. Cranberry juice is known for its anti-adhesive and anti-infective properties; however, its potential to modulate antibiotic activity remains poorly understood. Here, we show that cranberry juice markedly potentiates the antibacterial activity of FOS and limits the emergence of resistance in UPEC clinical isolates. In 72% of the 32 tested isolates, cranberry juice significantly increased FOS inhibition activities and reduced spontaneous FOS-resistant mutant frequencies by up to five orders of magnitude. Whole-genome sequencing revealed distinct mutational patterns: FOS-resistant mutants selected without cranberry juice primarily carried glpT mutations, whereas those obtained with juice harbored mutations in uhpT or associated regulatory genes. Reporter assays indicated that cranberry juice represses glpT expression while maintaining UhpT-mediated FOS uptake, thereby sustaining antibiotic entry and activity. These results demonstrate that cranberry juice alters bacterial carbohydrate transport regulation to potentiate FOS activity and suppress resistance emergence. This study provides novel evidence that a natural product can enhance FOS activity, highlighting its potential as an antibiotic adjuvant for UTI management.IMPORTANCEAntimicrobial resistance is a growing threat to public health, and new strategies are needed to preserve the activity of existing antibiotics. This study reveals that cranberry juice, a widely consumed natural product, enhances the antibacterial activity of fosfomycin against uropathogenic Escherichia coli by modulating bacterial sugar transport systems. By shifting fosfomycin uptake from GlpT- to UhpT-mediated pathways, cranberry juice both potentiates antibiotic activity and suppresses the emergence of resistant mutants. These findings provide new insight into how dietary components can influence antibiotic response, offering a promising basis for developing natural adjuvants that extend the lifespan of current antimicrobial agents.

Fosfomycin↗
PubMed1992

Liver-specific expression of the gene coding for human factor X, a blood coagulation factor.

Factor X is a vitamin K-dependent glycoprotein that plays an essential role in both the intrinsic and extrinsic pathways of blood coagulation. Studies on a recombinant lambda phage containing the 5'-flanking region of the human factor X gene showed that the factor X gene was linked to and was located at the 3' end of the factor VII gene: the initiation codon of the factor X gene was 2823 base pairs (bp) downstream from the polyadenylation site of the factor VII gene. This 2.8-kilobase intergenic region, and progressively deleted fragments of it, was fused to the chloramphenicol acetyltransferase gene, and transient expressions in HepG2 cells, human fibroblasts, and Chinese hamster ovary cells were measured. A liver-specific promoter element, FXP1-binding site, essential for hepatocyte-specific transcription was identified. This promoter sequence, further localized to -63 to -42 bp in DNase I footprint studies, was homologous to LF-A1 or hepatic nuclear factor-4 recognition sequence and was equally functional in the normal and inverse orientations. FXP1 site bound to nuclear protein(s) from HepG2 cells and complex formation was partially abolished by the presence of duplex oligonucleotides containing liver factor-A1 or hepatic nuclear factor-4-binding sequences. Two additional positive elements located upstream of the promoter region, spanning from -215 to -149 bp (FXP2 site), and -457 to -351 bp (FXP3 site), were also established by reporter gene assays.

Amino Acid Sequence↗
PubMed1992

Novel DNA binding proteins participate in the regulation of human neurofilament H gene expression.

By a combination of DNase I footprinting, methylation interference, and gel shift analyses we have identified multiple binding sites for nuclear proteins within the promoter region of the human neurofilament H gene. Two sites likely bind the transcription factor Sp1 while two others may be targets for previously unrecognized DNA binding proteins. One site, PAL, occurs within the 10 bp sequence GGGGAGGAGG. Two copies of the PAL sequence form an interrupted palindrome around one of the Sp1 sites. A second site, PROX, is found within the sequence GGTTGGACC. Nuclear extracts prepared from both neural and non-neural cell lines, mouse brain, and mouse liver contain proteins that recognize and bind to the PROX and PAL sequences indicating that proteins which bind to these target sequences are widespread. The appearance of these target sequences in the 5' upstream region of several neuron specific genes suggests that they play key roles in the transcription of neuron specific genes. The functional activity of these target DNA sequences was demonstrated by transfection assays using a reporter gene fused to nested deletions of the NF(H) promoter region. Interestingly, these assays revealed that maximal transient expression was obtained with DNA fusion genes containing the PAL, PROX and TATA sequences. Inclusion of the Sp1 sites into the fusion genes failed to enhance the expression of the reporter gene. To determine if the NF(H) promoter can be activated in a tissue specific manner during development transgenic mice containing the promoter region linked to a beta-galactosidase reporter gene were generated. In one line sporadic expression of the transgene occurred in the CNS and testis while in four other lines no expression occurred. Collectively these results suggest that the NF(H) gene promoter is active in a tissue specific manner only by interactions with regulatory elements that lie further upstream or downstream of the start site of initiation.

Animals↗
PubMed1992

The Epstein-Barr virus nuclear protein 1 promoter active in type I latency is autoregulated.

The only member of the Epstein-Barr virus family of nuclear proteins (EBNAs) expressed during type I and type II latent infections is EBNA-1. This is in contrast to type III latency, during which all six nuclear proteins are expressed from a common transcription unit. The exclusive expression of EBNA-1 during type I and II latency is mediated through a recently identified promoter, Fp. The objective of this study was to characterize Fp in the Burkitt lymphoma cell background, where it is known to be differentially utilized. Using a short-term transfection assay and reporter gene plasmids containing Fp linked to the human growth hormone, we examined Fp activity in type I and type III latently infected and virus-negative Burkitt lymphoma cells. The data suggested that Fp is predominantly regulated through two distinct elements located between +24 and +270 relative to the transcription start site. One element positively mediates Fp activity, probably at the level of transcription, and acts in a virus-independent manner. The second element contains the EBNA-1 DNA binding domain III and negatively regulates Fp-directed gene expression in trans with EBNA-1 in type III as well as type I latency. Thus, we have identified a third function of EBNA-1, i.e., that of a repressor of gene expression, in addition to its known role in viral DNA replication and its ability to trans-activate gene expression. The overall activity of Fp in type I latently infected Burkitt cells was approximately sixfold lower than in virus-negative Burkitt cells, in which there is no autoregulation, suggesting that there is a fine balance between these two opposing regulatory elements during type I latency.

Antigens, Viral↗
PubMed1992

Behavior of genes directly injected into the rat heart in vivo.

Gene transfer can be achieved in the adult rat heart in vivo by direct injection of plasmid DNA. In this report we define the spatial and temporal limits of reporter gene expression after a single intracardiac injection. pRSVCAT (100 micrograms), in which the Rous sarcoma virus long terminal repeat is fused to the chloramphenicol acetyltransferase reporter gene, and p alpha MHCluc (100 micrograms), in which the alpha-cardiac myosin heavy chain promoter is fused to the firefly luciferase gene, were injected into hearts, and reporter gene activities were assayed at various times. Both chloramphenicol acetyltransferase and luciferase were detectable in 100% of the rats from 1 to 7 days, in 60% of the rats from 17 to 23 days, and in 30% of the rats from 38 to 60 days after injection. Reporter gene activity was largely limited to a 1-2-mm region of the ventricle surrounding the injection site. Closed circular DNA was far more effective than linear DNA in transfecting cells in vivo. The relative strengths of three different promoters, Rous sarcoma virus long terminal repeat, alpha-myosin heavy chain, and alpha 1-antitrypsin, all fused to the luciferase reporter gene were determined. The constitutive viral promoter was approximately 20-fold more active than the cardiac-specific cellular promoter, and the liver-specific cellular promoter was not active at all in the cardiac environment. Thus, direct injection of genes into the heart offers a simple and powerful tool with which to assess the behavior of genes in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Interaction with the nuclear matrix of a chimeric construct containing a replication origin and a transcription unit.

We have studied the interaction of a chimeric construct containing an origin of replication (from bovine papilloma virus) and a hormonally regulated transcription unit (long terminal repeat from the mouse mammary tumor virus, driving the v-Ha-ras gene) with the nuclear scaffold and matrix from mouse fibroblasts. We used two experimental approaches because the nuclear matrix protein composition depends largely on the isolation conditions, making its definition mostly operational. In situ studies and in vitro experiments performed in 1361.5 cells, a cell line in which multiple copies of the construct have been established, indicate that two interesting regions of the construct interact with the nuclear matrix. The first region is located in the v-Ha-ras gene 5'-flanking sequences. These sequences come from the Harvey virus and contain a piece of the virus like 30S (VL30) sequences in which the v-Ha-ras gene is embedded. This DNA fragment was coupled to the thymidine kinase (TK) promoter driving the reporter luciferase gene and assayed in transient transfection experiments. Its insertion, in the sense orientation, upstream of the TK promoter resulted in a moderate enhancement (2-3-fold) of the luciferase activity. The second region is the most interesting from a physiological point of view. It contains the plasmid maintenance sequence 1 (PMS-1) and the core origin of replication of the bovine papilloma virus. Differences in the results from in situ (nuclear scaffold) and in vitro (nuclear matrix) experiments suggest that the components involved in the interaction with PMS-1 and the viral origin of replication are different. This may be of importance in the context of the recently proposed view that PMS-1 could be part of a composite origin of replication and provide information at a distance.

3T3 Cells↗
PubMed1992

Identification and sequence analysis of the promoter for the leukocyte integrin beta-subunit (CD18): a retinoic acid-inducible gene.

Leukocyte adhesion receptors (LFA-1; Mac-1; p150,95) are a family of heterodimeric cell-surface adhesion molecules expressed exclusively in granulocytes, lymphocytes, and macrophages. Expression of these proteins is under complex regulatory control, but to date promoters for these genes have not been identified. The CD18 gene codes for the common beta-subunit of the leukocyte adhesion receptors. Transcription of CD18 is highly tissue-specific, hormonally inducible (by retinoic acid [RA]), and coordinately regulated with leukocyte integrin alpha-chains. To identify the CD18 promoter, we screened a human genomic phage library with a human CD18 cDNA probe and obtained a clone that contains an exon coding for the 5' untranslated region (UTR). Using rapid amplification of cDNA ends (RACE), RNAse protection, S1 nuclease, and primer extension assays, we demonstrated the existence of multiple transcription start sites clustered in a 45-nt region. We investigated the transcription-promoting activity of the genomic sequences 5' to the CD18 gene by performing transient expression assays with a growth hormone reporter gene in various hematopoietic cell lines. The CD18 promoter was active in Jurkat cells, a lineage that normally expresses CD18 but was considerably less active in K562, an early erythroid line that does not normally express CD18. The genomic sequences upstream of the start site cluster lack CAAT and TATA boxes, but have two Sp1 binding sites and 10 T(G/C)AC(C/A) boxes, which may represent binding sites for RA receptors (RAR). These features distinguish the CD18 promoter from the promoters of other tissue-specific, hormone-inducible genes, and may be representative of leukocyte integrin promoters in general.

Antigens, CD↗
PubMed1992

Detection of the neor gene expression in animal cells after genetic transformation.

A reliable assay is reported for the detection of the marker gene aminoglycoside phosphotransferase activity in cells that express this enzyme transiently or as a result of stable genetic transformation. This method combines the simplicity of the dot assays with the reliability of the more elaborate and time consuming electrophoretic or chromatographic methods. Inhibition of phosphatases and protein kinases during the reaction reduces labeled ATP consumption by these enzymes. As a result, this assay allows the detection of approx 10 times lower levels of the enzyme than currently used methods. To detect the expression of reporter genes in transformed cells aminoglycoside phosphotransferase can be used as well as the widely used chloramphenicol acetyltransferase enzyme.

Animals↗
PubMed1992

Transgenic indicator mice for studying activated retinoic acid receptors during development.

Retinoic acid (RA) receptors (RARs) are ligand-inducible transcription factors that bind to specific DNA sequences associated with the regulatory regions of RA-regulatable genes. Since RA has been implicated as an important factor both in normal development and in teratological studies, one would like to have a model system that detects the presence of activated receptors during development. We have constructed a recombinant reporter gene that has three copies of the RA response element (RARE) from the RAR beta-2 promoter 5' to the herpes simplex virus thymidine kinase promoter; this regulatory region is coupled to the bacterial beta-galactosidase reporter gene. This construct was RA inducible in transient transfection assays in F9 embryonal carcinoma cells. Transgenic embryos with this reporter gene construct exhibited restricted and reproducible patterns of beta-galactosidase activity during embryogenesis, beginning between gestational ages day 7.5 and 8.5. At day 8.5, beta-galactosidase activity was detected in the closed neurotube and somites. Day 8.5 embryos, from pregnant females fed RA 14 hr earlier, exhibited a greater intensity and distribution of beta-galactosidase activity. Similarly, at later stages of gestation, maternal RA exposure resulted in enhanced embryonic beta-galactosidase expression. This type of transgenic indicator mouse should be useful in detailing the role of activated RARs during embryonic development.

Animals↗