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Activation of alpha- and beta-estrogen receptors by persistent pesticides in reporter cell lines.

Many persistent pesticides have been implicated in reproductive and developmental adverse effects, in man and wildlife. It has been hypothesized that these so-called xeno-hormones could upset the endocrine system function by binding to human estrogen receptor alpha and beta (ERalpha, beta) and thus be responsible for the higher incidence of breast and cervical cancer, infertility and endometriosis. In this report, forty-nine pesticides were tested for ERalpha and beta activation or inhibition in stable reporter cell lines, HELN ERalpha and ERbeta. Stable transfection of the ERalpha and ERbeta constructs together with an estrogen reporter luciferase vector into the HeLa cell line resulted in two estradiol-sensitive cell lines. In our model, fifteen of the tested pesticides were found to agonize the ERalpha-mediated transcription in a dose-dependent manner and DDT, trans-nonachlor, chlordane, fenvalerate and toxaphene were also capable to activate ERbeta. Antagonistic activities toward hERalpha and hERbeta were shown in three (carbaryl, pentachlorophenol and 2,4,5-trichlorophenoxyacetic acid) and seven (chlordecone, methoxychlor, carbaryl, endosulfan, endrin, dieldrin, aldrin) pesticides, respectively. Remarkably chlordecone and methoxychlor which were the most effective antagonist compounds for hERbeta, were agonists for hERalpha. Although the ERalpha activation potential of the pesticides was lower than that of estradiol, the overall body scale response might be amplified by the ability of pesticides to act via several mechanisms and by frequent and prolonged exposure to different pesticides, even at low concentrations.

Animals↗

Estrogenic activity of cosmetic components in reporter cell lines: parabens, UV screens, and musks.

In this work, the estrogenic effects of three classes of substances included in cosmetic formulations-parabens, ultraviolet (UV) screens, and musk fragrances-were studied. Their estrogenic activity was measured with the use of three reporter cell lines: HELN, HELN ERalpha, and HELN ERbeta. These three cell lines allowed for the measurement of estrogenic activity toward estrogen receptors alpha and beta (ERalpha and ERbeta, while taking nonspecific interactions into account. Eight of the 15 substances tested showed specific estrogenic activity with the following degree of potency on ERalpha butylparaben > propylparaben > homosalate = octyl-dimethyl-PABA = 4-methyl-benzylidenecamphor = octyl-methoxycinnamate > ethylparaben = galaxolide. Among these active substances, parabens activated ERalpha and ERbeta similarly, UV screens activated ERalpha moderately and had almost no effect on ERbeta, and fragrances did not activate ERbeta. Methylparaben, ethylparaben, musk moskene, celestolide, and cashmeran did not activate estrogenic responses up to 10(-5) M. Musk ketone and benzophenone-3 were not considered estrogenic at 10(-5) M.

Cell Line↗

Gene-trapping to identify and analyze genes expressed in the mouse hippocampus.

Mice harboring random gene-trap insertions of a lacZ (beta-galactosidase)-neomycin resistance fusion cassette (beta-geo) were analyzed for expression in the hippocampus. In 4 of 15 lines reporter gene activity was observed in the hippocampal formation. In the obn line, enzyme activity was detected in the CA1-3 hippocampal subfields, in hpk expression was restricted to CA1, but in both lines reporter activity was also present in other brain regions. In the third line, kin, reporter activity was robustly expressed throughout the stratum pyrimidale of CA1-3, with only low-level expression elsewhere. The final line (glnC) displayed ubiquitous expression of the reporter and was not analyzed further. Fusion transcripts for the first three lines were characterized; all encode polypeptides with features of membrane-associated signalling proteins. The obn fusion identified a human cDNA (B2-1) encoding a pleckstrin homology (PH) domain, while hpk sequences matched the Epstein-Barr Virus (EBV) inducible G-protein coupled receptor, EBI-1. kin identified an alternative form of the abl-related nonreceptor tyrosine kinase c-arg. Electrophysiological studies on mice homozygous for the insertions revealed normal synaptic transmission, paired pulse facilitation and paired-pulse depression at Schaffer collateral-commissural CA1 synapses, and normal long-term potentiation (LTP) in obn and kin. hpk mice displayed an increase in hippocampal CA1 long-term potentiation (LTP), suggesting a role for this receptor in synaptic plasticity.

Amino Acid Sequence↗

Construction of a lipopolysaccharide reporter cell line and its use in identifying mutants defective in endotoxin, but not TNF-alpha, signal transduction.

Gram-negative bacterial LPS is a potent activator of inflammatory responses. The binding of LPS to CD14 initiates signal transduction; however, the molecular processes immediately following this event remain unclear. We engineered an LPS-inducible fibroblast reporter cell line to facilitate the use of molecular genetic techniques to study the LPS signaling pathway. A plasmid containing the human Tac Ag cDNA under transcriptional control of the human E selectin promoter was cotransfected into Chinese hamster ovary (CHO)-K1 cells together with a CD14 expression plasmid. A cell line was obtained, 3E10, which upregulated expression of Tac following stimulation with LPS. Pools of mutagenized cells were exposed to LPS and then labeled with anti-Tac mAb. Cells that failed to up-regulate Tac expression were enriched by flow cytometry. Thirty clonal mutant cell lines were identified that continued to express CD14 and bind LPS, but failed to express Tac or translocate nuclear factor-kappaB (NF-kappaB) following LPS exposure. TNF-alpha-treated mutant cells continued to express Tac and translocate NF-kappaB. An analysis of LPS-induced NF-kappaB activity in heterokaryons derived from polyethylene glycol-fused cell lines indicated that recessive mutations in genes encoding components of the LPS signaling pathway accounted for the signaling defects. To date, two complementation groups have been identified from 11 cell lines analyzed. These data demonstrate that the TNF-alpha signaling pathway diverges from the LPS pathway early in the signal-transduction cascade despite similarities in LPS- and TNF-alpha-induced responses. Identification of the genes affected in these mutant reporter cells should identify heretofore-elusive components of the LPS signaling cascade.

Acute-Phase Proteins↗

Evaluation of ligand selectivity using reporter cell lines stably expressing estrogen receptor alpha or beta.

Estrogens control transcriptional responses through binding to two different nuclear receptors, estrogen receptor alpha (ERalpha) and beta (ERbeta). Since these two ER subtypes are thought to mediate different biological effects, there is intense interest in designing subtype-selective ER ligands. In this study, we evaluated the ERalpha and ERbeta selectivity of 19 known estrogens and antiestrogens using reporter cell lines previously developed in our laboratory. The HELN-ERalpha and HELN-ERbeta cells stably express full-length ERalpha and ERbeta, respectively, and are derived from HELN cells (HeLa cells stably transfected with an ERE-driven luciferase plasmid). We report that 16alpha-LE2, PPT and 3beta,5alpha-GSD have a high ERalpha-selective agonist potency while 8beta-VE2, DPN, genistein and biochanin A show ERbeta selectivity with 8beta-VE2 being the most potent and selective ERbeta agonist. We also tested ER antagonists and we showed that raloxifene and RU486 are ERalpha and ERbeta-selective antiestrogens, respectively. In all cases, selectivity is due to differences in binding affinities as indicated by whole-cell ligand-binding assays. Very interestingly, we demonstrate that a combination of genistein and raloxifene produces a full-ERbeta specific response. Together these results demonstrate the usefulness of our stably transfected cell lines to characterize ER ligands and indicate that treatments combining agonist/antagonist ligands produce full-ERbeta selectivity.

Binding Sites↗

Product-line performance reporting: a key to cost management.

For many hospitals, performance reporting is the most important and valuable function of a cost management system. Performance reporting provides hospitals--or any organization, for that matter--with an enhanced ability to reduce and control costs and monitor profitability. By periodically comparing strategic and operational plans with actual results, managers are provided with measurements of their performance for the aspects of the organization they control. The following is an excerpt from the book, Cost Management for Hospitals. This section deals with product-line performance reporting and the variances that measure the effects different factors have on performance.

Accounting↗

Use of reporter cell lines for detection of endocrine-disrupter activity.

We have studied stable transformed human mammary cell lines with highly inducible steroid receptor-mediated luciferase reporter gene expression. Cells responding specifically to glucocorticoids, progestagens, androgens, or estrogens are described and characterized. The use of this high-throughput, cell-based assay for analysis of steroid (ant)agonists is reported. Systematic characterization of endocrine-disrupting activity on human receptors and in a human-cell system is interpreted for a selection of xenobiotics. We show that the phytoestrogens apigenin and genistin have progestagenic and androgenic activity, respectively. Finally, application of cell-based assays to the analysis of environmental samples is discussed.

Cell Line, Tumor↗

Generation of an NKX2-1-EGFP reporter iPSC line with inducible Cas9 for lung progenitor cell tracing.

NK2 homeobox 1 (NKX2-1), a master regulator robustly expressed in lung, thyroid, and forebrain, is indispensable for specifying lung epithelial fate and serves as a definitive marker of lung progenitors. Here, we generated a human induced Pluripotent Stem Cell (iPSC) line harboring a doxycycline (dox)-inducible Cas9 and an NKX2-1-EGFP-puro reporter via CRISPR/Cas9-mediated homology-directed repair. This dual-function line combines inducible genome editing with real-time tracing of early lung progenitors, enabling their prospective isolation and screening for stage-specific maturation regulators. Therefore, this engineered iCas9-NKX2-1 EGFP line is a key resource for dissecting human lung development, modeling pulmonary disease, and advancing regenerative therapies.

Humans↗

Use of a novel GFP reporter cell line to examine replication capacity of CXCR4- and CCR5-tropic HIV-1 by flow cytometry.

The rate of HIV-1 disease progression correlates strongly with plasma viral load and is likely to be influenced by both host and viral determinants. Though interest in the impact of viral replication capacity during HIV-1 infection has been increasing, especially with respect to drug resistance mutations, its influence on disease course remains poorly understood. This is due in part to significant drawbacks in conventional means of measuring HIV-1 growth in vitro (i.e. expense, inconvenience, and experimental variability). A FACS-based method is described here to measure HIV-1 replication sensitively and a modification of this method can be used to determine viral titer accurately. Importantly, the target cells used are permissive to CXCR4- and CCR5-tropic HIV-1 strains. In pilot experiments, the growth kinetics of laboratory-adapted strains NL4-3 and IIIB were examined carefully. Using this method, differences were observed in growth kinetics between three laboratory strains and seven primary isolates, indicating the potential for a broad range of in vitro replication capacities among individual isolates. In conclusion, this FACS-based method provides a sensitive approach to measure the replication capacity of HIV-1 and may prove useful in studies examining the impact of viral fitness on disease progression.

Cell Line↗

BAC constructs in transgenic reporter mouse lines control efficient and specific LacZ expression in hypertrophic chondrocytes under the complete Col10a1 promoter.

During endochondral ossification hypertrophic chondrocytes in the growth plate of fetal long bones, ribs and vertebrae play a key role in preparing growth plate cartilage for replacement by bone. In order to establish a reporter gene mouse to facilitate functional analysis of genes expressed in hypertrophic chondrocytes in this process, Col10a1- BAC reporter gene mouse lines were established expressing LacZ specifically in hypertrophic cartilage under the control of the complete Col10a1 gene. For this purpose, a bacterial artificial chromosome (BAC RP23-192A7) containing the entire murine Col10a1 gene together with 200 kb flanking sequences was modified by inserting a LacZ-Neo cassette into the second exon of Col10a1 by homologous recombination in E. coli. Transgenic mice containing between one and seven transgene copies were generated by injection of the purified BAC-Col10a1- lLacZ DNA. X-gal staining of newborns and embryos revealed strong and robust LacZ activity exclusively in hypertrophic cartilage of the fetal and neonatal skeleton of the transgenic offspring. This indicates that expression of the reporter gene in its proper genomic context in the BAC Col10a1 environment is independent of the integration site and reflects authentic Col10a1 expression in vivo. The Col10a1 specific BAC recombination vector described here will enable the specific analysis of effector gene functions in hypertrophic cartilage during skeletal development, endochondral ossification, and fracture callus healing.

Animals↗

Activities of MAP-kinase pathways in normal uroepithelial cells and urothelial carcinoma cell lines.

It is often assumed that MAPK pathways drive proliferation of normal uroepithelial (UEC) and urothelial carcinoma (TCC) cells. To check this assumption, activities and inducibilities of promoters containing serum-response elements (SRE) or AP-1 binding sites were investigated in cultured UEC and seven TCC lines. Reporter plasmids dependent on SRE or AP-1 sites were highly active in UEC, but significantly less so in TCC lines. Reporter activity in TCC lines could be induced by constitutively active MEKK4 or TPA. Accordingly, phosphorylation of the MAPK pathway components MEK, ERK, and ELK1 was most pronounced in UEC and lower in TCC lines. MAPK-dependent promoter activities and bromodeoxyuridine incorporation decreased in UEC upon withdrawal of growth factors, but less so in TCC lines, in which serum diminution increased apoptosis. Likewise, E2F-dependent promoters responded to growth factors in UEC, but were more serum-independent in the TCC lines, which lack either RB1 or p16(INK4A). MEK inhibitors inhibited BrdU incorporation in UEC more strongly than in TCC lines. Thus, proliferation of normal uroepithelial cells is indeed associated with activation of MAPK pathways. However, autonomous proliferation of TCC lines--unexpectedly--appears much less dependent on MAPK activation and may rather be promoted by defects in cell cycle regulation.

Binding Sites↗

Beau's lines. A case report.

Beau's lines are a depression of the nail surface associated with a severe disturbance which temporarily interferes with the rate of nail growth. They appear several weeks or more after incurrence of the damage and continue to extend during nail growth. These lines were observed soon after birth in all twenty fingernails of an infant girl. The depression extended through the whole thickness of the nails. This would appear to be the first reported case in which Beau's lines have developed so early in life, probably as a result of intrauterine distress.

Female↗

The development of Xenopus tropicalis transgenic lines and their use in studying lens developmental timing in living embryos.

The generation of reporter lines for observing lens differentiation in vivo demonstrates a new strategy for embryological manipulation and allows us to address a long-standing question concerning the timing of the onset of differentiation. Xenopus tropicalis was used to make GFP reporter lines with (gamma)1-crystallin promoter elements directing GFP expression within the early lens. X. tropicalis is a close relative of X. laevis that shares the same ease of tissue manipulation with the added benefits of a diploid genome and faster life cycle. The efficiency of the Xenopus transgenic technique was improved in order to generate greater numbers of normal, adult transgenic animals and to facilitate in vivo analysis of the crystallin promoter. This transgene is transmitted through the germline, providing an accurate and consistent way to monitor lens differentiation. This line permitted us to distinguish models for how the onset of differentiation is controlled: by a process intrinsic to differentiating tissue or one dependent on external cues. This experiment would not have been feasible without the sensitivity and accuracy provided by the in vivo reporter. We find that, in specified lens ectoderm transplanted from neural tube stage donors to younger neural-plate-stage hosts, the onset of differentiation, as measured by expression of the crystallin/GFP transgene, is delayed by an average of 4.4 hours. When specified lens ectoderm is explanted into culture, the delay was an average of 16.3 hours relative to control embryos. These data suggest that the onset of differentiation in specified ectoderm can be altered by the environment and imply that this onset is normally controlled by external cues rather than by an intrinsic mechanism.

Animals↗

Endogenous tagging of the TBXT (Brachyury) gene with 2A-EGFP in BJNhem19 human embryonic stem cells using CRISPR-Cas9 genome editing.

Brachyury (TBXT) is a T-box transcription factor essential for directing human embryonic stem cells (hESCs) toward mesodermal lineage specification. Here, we report the generation of a Brachyury/TBXT-2A-EGFP knock-in reporter line in the BJNhem19 hESC line using CRISPR/Cas9-mediated homology-directed repair. Accurate integration of the reporter cassette was confirmed by DNA sequencing and validated by the comparison of EGFP expression to endogenous TBXT gene expression following directed mesodermal differentiation. This reporter line provides a tractable fluorescence-based platform for real-time monitoring of mesodermal commitment, which will help in high-content screening of differentiation protocols.

Journal Article↗

LuSIV cells: a reporter cell line for the detection and quantitation of a single cycle of HIV and SIV replication.

A single cycle of viral replication is the time required for a virus to enter the host cell, replicate its genome, and produce infectious progeny virions. The primate lentiviruses, human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), require on average 24 h to complete one cycle of replication. We have now developed and characterized a reporter assay system in CEMx174 cells for the quantitative measurement of HIV/SIV infection within a single replication cycle. The SIV(mac)239 LTR (-225 --> +149) was cloned upstream of the firefly luciferase reporter gene and this reporter plasmid is maintained in CEMx174 cells under stable selection. This cell line, designated LuSIV, is highly sensitive to infection by primary and laboratory strains of HIV/SIV, resulting in Tat-mediated expression of luciferase, which correlates with viral infectivity. Furthermore, manipulation of LuSIV cells for the detection of luciferase activity is easy to perform and requires a minimal amount of time as compared to current HIV/SIV detection systems. The LuSIV system is a powerful tool for the analysis of HIV/SIV infection that provides a unique assay system that can detect virus replication prior to 24 h and does not require virus to spread from cell to cell. Thus these cells can be used for the study of replication-deficient viruses and the high throughput screening of antivirals, or other inhibitors of infection.

Cell Line↗

Reporter cell lines to study the estrogenic effects of xenoestrogens.

In order to characterize the estrogenic activity of chemicals, we established complementary in vitro recombinant receptor-reporter gene assays in stably transfected MCF-7 and HeLa cells. MCF-7 cells which express the endogenous estrogen receptor alpha (ER alpha) were stably transfected with only an estrogen-regulated luciferase gene. These cells enable the detection of compounds which bind to ER alpha or interfere with the induction of ER alpha mediated gene expression. Furthermore, HeLa cells, which do not express endogenous ERs, were transfected with an ER alpha or an ER beta construct together with an estrogen-regulated luciferase gene, or a chimeric GAL4-ER alpha receptor and the corresponding luciferase reporter gene. Finally, we tested these four cellular models as tools to check the estrogenic activities of several potential xenoestrogens and to detect estrogenic activity in wastewater sewage treatment effluents. In all of the models, nonylphenol mixture (NPm), 4n-nonylphenol (4nNP), 2,4'-DDE, 4,4'-DDE and wastewater sewage treatment effluent were active, while PCB mixture (Aroclor 1254), PCB 77, atrazine and lindane (gamma hexachlorocyclohexane) were inactive. Dioxin partially activates the estrogen receptor in MCF-7 cells while in HeLa-derived cell lines, it decreased the estrogenic-induced expression of luciferase.

Cell Line↗

GFAP-positive progenitor cells produce neurons and oligodendrocytes throughout the CNS.

Once thought to merely act as scaffolds in neuronal migration, recent evidence suggests that radial glia may serve as progenitors for the majority of neurons in the CNS. Cre/loxP fate-mapping experiments were carried out using a fragment of a glial-specific promoter (glial fibrillary acidic protein; GFAP) to drive expression of Cre recombinase. We show that GFAP+ progenitor cells give rise to neurons and oligodendrocytes throughout the CNS. We find very little regional heterogeneity in the neurogenic potential of radial glia between dorsal and ventral telencephalon. Additionally, radial glia serve as precursors for subpopulations of interneurons in the ventral telencephalon. Interestingly, the human GFAP promoter but not the mouse GFAP promoter is active in oligodendrocyte progenitor cells. We also demonstrate that the most commonly used Cre reporter lines are very inefficient in detecting Cre-dependent recombination in astrocytes and describe a new Cre reporter line for assessing recombination in astrocytes.

Animals↗