Novel in vitro assay for the detection of pharmacologic inducers of fetal hemoglobin

Blood ◽  
2000 ◽  
Vol 96 (1) ◽  
pp. 321-326 ◽  
Author(s):  
Evangelia Skarpidi ◽  
George Vassilopoulos ◽  
Qiliang Li ◽  
George Stamatoyannopoulos
Keyword(s):  
Luciferase Activity ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Reporter Genes ◽  
Firefly Luciferase ◽  
Gene Activity ◽  
Renilla Luciferase ◽  
Luciferase Reporter ◽  
Vitro Assay ◽  
Highly Sensitive

Current techniques for identifying fetal hemoglobin (HbF) inducers are complex and time consuming. We developed a rapid and efficient method for detecting HbF inducers. Our system uses a recombinant DNA construct in which the coding sequences of 2 different luciferase reporter genes, firefly and renilla, are substituted for those of human γ and β globin genes, respectively. The activity of these genes can be distinguished by a simple, highly sensitive enzymatic assay in cell lysates. GM979 cells stably transfected with the construct are cultured in the presence of compounds, and their effects are determined by measuring the changes in activity of the 2 luciferase genes. Specific γ globin gene inducers are recognized by their ability to increase γ-firefly luciferase (γF) gene activity significantly more than β-renilla luciferase (βR) gene activity, identified by an increased ratio of γ-firefly luciferase activity over total luciferase activity. These results suggest that the use of the 2 luciferase reporter genes provides a simple, highly sensitive, and reproducible system for the detection of compounds that increase γ-globin gene expression. It can therefore be used for the screening of chemical agents that may have γ-globin gene inducibility.

Novel in vitro assay for the detection of pharmacologic inducers of fetal hemoglobin

Blood ◽  
2000 ◽  
Vol 96 (1) ◽  
pp. 321-326 ◽  
Author(s):  
Evangelia Skarpidi ◽  
George Vassilopoulos ◽  
Qiliang Li ◽  
George Stamatoyannopoulos
Keyword(s):  
Luciferase Activity ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Reporter Genes ◽  
Firefly Luciferase ◽  
Gene Activity ◽  
Renilla Luciferase ◽  
Luciferase Reporter ◽  
Vitro Assay ◽  
Highly Sensitive

Abstract Current techniques for identifying fetal hemoglobin (HbF) inducers are complex and time consuming. We developed a rapid and efficient method for detecting HbF inducers. Our system uses a recombinant DNA construct in which the coding sequences of 2 different luciferase reporter genes, firefly and renilla, are substituted for those of human γ and β globin genes, respectively. The activity of these genes can be distinguished by a simple, highly sensitive enzymatic assay in cell lysates. GM979 cells stably transfected with the construct are cultured in the presence of compounds, and their effects are determined by measuring the changes in activity of the 2 luciferase genes. Specific γ globin gene inducers are recognized by their ability to increase γ-firefly luciferase (γF) gene activity significantly more than β-renilla luciferase (βR) gene activity, identified by an increased ratio of γ-firefly luciferase activity over total luciferase activity. These results suggest that the use of the 2 luciferase reporter genes provides a simple, highly sensitive, and reproducible system for the detection of compounds that increase γ-globin gene expression. It can therefore be used for the screening of chemical agents that may have γ-globin gene inducibility.

scholarly journals In vivo analysis of the myosin heavy chain IIB promoter region

1998 ◽  
Vol 274 (3) ◽  
pp. C681-C687 ◽  
Author(s):  
Steven J. Swoap
Keyword(s):  
Reporter Gene ◽  
Luciferase Activity ◽  
Fiber Type ◽  
Firefly Luciferase ◽  
Renilla Luciferase ◽  
Luciferase Reporter ◽  
Base Pairs ◽  
Specific Expression ◽  
Mhc Iib

The myosin heavy chain (MHC) IIB gene is preferentially expressed in fast-twitch muscles of the hindlimb, such as the tibialis anterior (TA). The molecular mechanism(s) for this preferential expression are unknown. The goals of the current study were 1) to determine whether the cloned region of the MHC IIB promoter contains the necessary cis-acting element(s) to drive fiber-type-specific expression of this gene in vivo, 2) to determine which region within the promoter is responsible for fiber-type-specific expression, and 3) to determine whether transcription off of the cloned region of the MHC IIB promoter accurately mimics endogenous gene expression in a muscle undergoing a fiber-type transition. To accomplish these goals, a 2.6-kilobase fragment of the promoter-enhancer region of the MHC IIB gene was cloned upstream of the firefly luciferase reporter gene and coinjected with pRL-cytomegalovirus (CMV) (CMV promoter driving the renilla luciferase reporter) into the TA and the slow soleus muscle. Firefly luciferase activity relative to renilla luciferase activity within the TA was 35-fold greater than within the soleus. Deletional analysis demonstrated that only the proximal 295 base pairs (pGL3IIB0.3) were required to maintain this muscle-fiber-type specificity. Reporter gene expression of pGL3IIB0.3 construct was significantly upregulated twofold in unweighted soleus muscles compared with normal soleus muscles. Thus the region within the proximal 295 base pairs of the MHC IIB gene contains at least one element that can drive fiber-type-specific expression of a reporter gene.

scholarly journals An Intergenic SNP in the β–globin Gene Cluster Is Associated with Hyperuricemia and Influences Gene Transcription in Vitro

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1362-1362
Author(s):  
Chutima Kumkhaek ◽  
Daniel Shriner ◽  
Ayo P. Doumatey ◽  
Charles N. Rotimi ◽  
Griffin P. Rodgers
Keyword(s):  
Uric Acid ◽  
Serum Uric Acid ◽  
Globin Gene ◽  
K562 Cells ◽  
Firefly Luciferase ◽  
Renilla Luciferase ◽  
Luciferase Reporter ◽  
Genome Wide Association Studies ◽  
Chromosome 11 ◽  
Dna Fragment

Abstract Hemolysis as a suggested cause of hyperuricemia is based upon the fact that red blood cells contain uric acid. The development of hyperuricemia also generates from an increased synthesis of nucleic acids occurring as part of the erythropoietic response to hemolysis in hemoglobin disorders such as sickle cell anemia, α-thalassemia, and β-thalassemia. In addition, multiple genome-wide association studies (GWAS) have reported significant association between uric acid levels and specific genomic loci. However, the mechanism of hyperuricemia still remains controversial and it is also unknown whether African Americans have higher prevalence of hyperuricemia due to genetic vs environmental risk factors. Here, we used joint admixture mapping and association testing to identify genetic variants associated with serum uric acid levels in African American. Interestingly, we detected 6 SNPs (rs2855125, rs2855126, rs11036415, rs11036496, rs4320977, and rs4348933) in an intergenic region of the β–globin cluster on chromosome 11 are associated with high levels of serum uric acid in populations of African ancestry. Next, we explored the potential regulatory role of intergenic SNPs associated with hyperuricemia using luciferase reporter gene activity assays and electrophoretic mobility shift assays (EMSA). Each SNP-containing DNA fragment was amplified by PCR using human genomic DNA and inserted into a firefly luciferase reporter vector, pGL3-basic vector. 293T or K562 cells were co-transfected with these constructs and a Renilla luciferase vector to control for transfection efficiency. Expression of firefly luciferase driven by each SNP-containing DNA fragment was measured by a dual luciferase reporter assay and normalized by Renilla luciferase expression. SNPs rs2855126, rs11036496 and rs4348933 on chromosome 11 had significantly greater expression levels of firefly luciferase than pGL3-basic-transfected cells in both 293T and K562 cells. Of these, the SNP rs2855126 ancestral C allele (associated with higher serum uric acid levels) showed significantly higher luciferase activity than the derived G allele. Functionally, the luciferase activities from these constructs were determined to be very similar in both cell lines used. Alleles altering expression were further assessed for binding of nuclear extracted proteins by EMSA. We found specific gel shift bands for SNPs rs2855126, rs11036496, and rs4348933, suggesting these SNPs are situated in the binding site of potential transcription factors. These data provide new insights into the potential contribution of imbalanced β-globin gene expression to hyperuricemia. Disclosures No relevant conflicts of interest to declare.

scholarly journals Evaluation of Firefly and Renilla Luciferase Inhibition in Reporter-Gene Assays: A Case of Isoflavonoids

2021 ◽  
Vol 22 (13) ◽  
pp. 6927
Author(s):  
Maša Kenda ◽  
Jan Vegelj ◽  
Barbara Herlah ◽  
Andrej Perdih ◽  
Přemysl Mladěnka ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Firefly Luciferase ◽  
Qsar Model ◽  
In Vitro Assay ◽  
Biochanin A ◽  
Renilla Luciferase ◽  
Luciferase Reporter ◽  
Vitro Assay ◽  
Reporter Gene Assays

Firefly luciferase is susceptible to inhibition and stabilization by compounds under investigation for biological activity and toxicity. This can lead to false-positive results in in vitro cell-based assays. However, firefly luciferase remains one of the most commonly used reporter genes. Here, we evaluated isoflavonoids for inhibition of firefly luciferase. These natural compounds are often studied using luciferase reporter-gene assays. We used a quantitative structure–activity relationship (QSAR) model to compare the results of in silico predictions with a newly developed in vitro assay that enables concomitant detection of inhibition of firefly and Renilla luciferases. The QSAR model predicted a moderate to high likelihood of firefly luciferase inhibition for all of the 11 isoflavonoids investigated, and the in vitro assays confirmed this for seven of them: daidzein, genistein, glycitein, prunetin, biochanin A, calycosin, and formononetin. In contrast, none of the 11 isoflavonoids inhibited Renilla luciferase. Molecular docking calculations indicated that isoflavonoids interact favorably with the D-luciferin binding pocket of firefly luciferase. These data demonstrate the importance of reporter-enzyme inhibition when studying the effects of such compounds and suggest that this in vitro assay can be used to exclude false-positives due to firefly or Renilla luciferase inhibition, and to thus define the most appropriate reporter gene.

Aroyl-Pyrrolyl-Hydroxy-Amides (APHAs), a Novel Family of Synthetic Histone Deacetylases Inhibitors, Are Potent Inducers of Human g-Globin Gene Expression.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1216-1216
Author(s):  
Antonello Mai ◽  
Silvio Massa ◽  
Antonella Di Noia ◽  
Katija Jelicic ◽  
Elena Alfani ◽  
...  
Keyword(s):  
Gene Expression ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Histone Deacetylases ◽  
Globin Gene ◽  
Renilla Luciferase ◽  
In Vitro Assays ◽  
Luciferase Reporter ◽  
Globin Gene Expression

Abstract Post-natal pharmacological reactivation of HbF, by restoring the unbalanced α/non-α globin chain production in red cells of patients affected by β-thalassemia or sickle cell anemia, represents a potential cure for these diseases. Many classes of compounds have been identified capable to induce Hb F synthesis in vitro by acting at different levels of the globin gene expression regulatory machinery. One of these classes is represented by inhibitors of a family of enzymes, the histone deacetylases (HDACs), involved in chromatin remodelling and gene transcription regulation. HDACs act in multi-protein complexes that remove acetyl groups from lysine residues on several proteins, including histones and are divided into three distinct structural classes, depending on whether their catalytic activity is zinc (class I/II)- or NAD+ (class III)-dependent. The effects of the HDACs inhibitors identified so far on HbF synthesis is, however, modest and often associated with high toxicity. Therefore, the potential of their clinical use is unclear. We have recently described a new family of synthetic HDACs inhibitors, the Aroyl-pyrrolyl-hydroxy-amides (APHAs), that induce differentiation, growth arrest and/or apoptosis of transformed cell in culture [Mai A et al, J Med Chem2004;47:1098]. In this study, we investigate the capability of 10 different APHA compounds to induce Hb F in two in vitro assays. One assay is based on the ability of APHA compounds to activate either the human Aγ-driven Firefly (Aγ-F) or the β-promoter drives Renilla Luciferase (β-R) reporter in GM979 cells stably transfected with a Dual Luciferase Reporter construct. The second assay is represented by the induction of γ-globin expression (by quantitative RT-PCR) in primary adult erythroblasts obtained in HEMA cultures of mononuclear cells from normal donors. The majority of the compounds tested did not significantly increased the Aγ−F (Aγ−F+β−R) reporter ratio in GM979 cells. However, the compound MC1575 increased by 3-fold (from 0.09 to 0.30) the reporter ratio in GM979 cells at a concentration of 20 μM, with modest effects of the proliferation activity of GM979 cells over the three days of the assay. When MC1575 was added at a concentration of 2–10 μM in cultures of primary adult erythroblasts induced to differentiate in serum-free media for 4 days, it induced a three fold increase of the γ/(γ+β) globin ratio (from 0.04 to 0.12), with no apparent cellular toxicity. Among the HDAC inhibitors tested in this study, MC1575 was not the most potent inhibitor of total enzyme activity. However, it was the compound that most selectively inhibited the activity of the maize homologue of mammalian class IIa HDAC enzymes [Mai et al, J Med Chem2003;46:4826]. These results are consistent with the hypothesis that each class of histone deacetylases might have a specific biological function and indicate that those of class IIa might represent the enzymes most specifically involved in globin gene regulation. We suggest that, by targeting the chemical inhibitors toward the catalytic domain of this class of enzymes, it should be possible to identify more specific, more potent and less toxic compounds for pharmacological treatment of β-thalassemia or sickle cell anemia.

Role of Cjun in Gγ-Globin Gene Trans-Activation.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1870-1870
Author(s):  
Sirisha Kodeboyina ◽  
Sima Zein ◽  
Moosueng Lee ◽  
Parimaladevi Balamurugan ◽  
Xiao Yao ◽  
...  
Keyword(s):  
Electrophoretic Mobility ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
K562 Cells ◽  
Luciferase Reporter ◽  
Complete Analysis ◽  
Dependent Manner ◽  
Mobility Shift ◽  
Electrophoretic Mobility Shift Assays

Abstract Previous studies from our laboratory demonstrated the role of the G-CRE (Gγ-globin cAMP response element) in drug-mediated fetal hemoglobin induction. The G-CRE located at −1222 to −1229 in the promoter of Gγ-globin gene, contains binding site for trans-factors CREB1, ATF-2 and cJun. We previously demonstrated binding of phosphorylated CREB1 and ATF-2 to this element via p38 MAPK signaling triggered by sodium butyrate (NaB) and trichostatin A (TSA). Electrophoretic mobility shift assays with a probe containing the AC → TG mutation in the G-CRE (TGTGGTCA, m2) abolished trans-factor binding to the G-CRE. Furthermore, Gγ promoter activity was abolished in the PGL3 luciferase reporter vector driven by the Gγ promoter (−1500 to +36) carrying the m2 mutation. (Sangerman et al. Blood108:3590–9, 2006). Subsequent studies in our laboratory were aimed at understanding the role of trans-factor cJun, an AP-1 family member, as a regulator of Gγ-globin expression via the G-CRE site. In K562 cells treated with 2mM NaB or 0.3μM TSA for 48 hrs, cJun phosphorylation increased 2.8-fold and 6.4-fold respectively by western blot analysis. Chromatin immunoprecipitation studies showed 16-fold chromatin enrichment in the −1225 Gγ-globin region compared to IgG control studies indicative of significant cJun binding in vivo at steady state. Electrophoretic mobility shift assays using cJun monoclonal antibody demonstrated a supershifted DNA-protein complex confirming binding of cJun to the G-CRE probe. To gain evidence for a functional role of cJun, we performed enforced expression studies using the pLen-cJun vector. In a concentration dependent manner, over-expression of cJun increased luciferase activity up to 350-fold in the luciferase reporter plasmid controlled by the Gγ-promoter (−1500 to +36). As predicted from binding studies, the m2 mutation in this promoter abolished the cJunmediated trans-activation confirming that the G-CRE is required to mediate effects of cJun. We are currently investigating the ability of cJun to trans-activate the endogenous Gγ-globin gene in K562 cells. To achieve this goal, K562 stable lines were established with the expression vectors pLen-cJun and empty vector. A complete analysis of the stable lines is in progress. Future investigations to identify other components of the functional CREB1/ATF2/cJun enhanceosome complex bound to the G-CRE will be performed using affinity chromatography and mass spectrometry. This information will be used to develop strategies for fetal hemoglobin induction.

scholarly journals Use of Leishmania donovani Field Isolates Expressing the Luciferase Reporter Gene in In Vitro Drug Screening

2005 ◽  
Vol 49 (9) ◽  
pp. 3776-3783 ◽  
Author(s):  
Ashutosh ◽  
Suman Gupta ◽  
Ramesh ◽  
Shyam Sundar ◽  
Neena Goyal
Keyword(s):  
Reporter Gene ◽  
High Throughput ◽  
High Throughput Screening ◽  
Leishmania Donovani ◽  
Luciferase Activity ◽  
Firefly Luciferase ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Field Isolates

ABSTRACT Currently available primary screens for the selection of candidate antileishmanial compounds are not ideal. These techniques are time-consuming, laborious, and difficult to scale and require macrophages, which limit their use for high-throughput screening. We have developed Leishmania donovani field isolates that constitutively express the firefly luciferase reporter gene (luc) as a part of an episomal vector. An excellent correlation between parasite number and luciferase activity was observed. luc expression was stable, even in the absence of drug selection, for 4 weeks. The transfectants were infective to macrophages, and intracellular amastigotes exhibited luciferase activity. The suitability of these recombinant field isolates for in vitro screening of antileishmanial drugs was established. The luciferase-expressing sodium stibogluconate-resistant cell lines offer a model for the screening of compounds for resistance. The system is in routine use at the Central Drug Research Institute, Lucknow, India, for high-throughput screening of newly synthesized compounds.

Identification of New and Diverse Inducers of Fetal Hemoglobin with High Throughput Screening (HTS)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4277-4277 ◽  
Author(s):  
Jose I Sangerman ◽  
Michael S Boosalis ◽  
Ling Shen ◽  
Sarah Haigh ◽  
Ada Kane ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Globin Gene ◽  
Clinical Stage ◽  
Gene Promoter ◽  
Fetal Hemoglobin ◽  
Luciferase Reporter ◽  
Significant Activity ◽  
Globin Mrna ◽  
Globin Promoter

Abstract Abstract 4277 Pharmacologic augmentation of fetal hemoglobin (HbF, γ-globin) production, to replace diminished β-globin chains in the β-thalassemias and to inhibit HbS polymerization in sickle cell disease, is a definitive therapeutic modality. Despite long-term efforts, regulatory approval has been obtained for only one chemotherapeutic agent. Pharmacologic reactivation of high-level HbF expression with non-cytotoxic, tolerable therapeutics is still an unmet medical need for this global health burden. To investigate potential therapeutic libraries for unrecognized HbF inducers, we developed a high-throughput screening (HTS) program to interrogate diverse chemical libraries, including a library of FDA-approved and clinical stage drugs. This program has identified unexpected new and highly potent HbF-inducing drugs, some of which are already in clinical use for other medical indications and have established safety profiles. A human cell-based assay which was previously used in low throughput assays, utilizing a 1.4-kilobase (kb) KpnI-BglII fragment of the HS2 of the locus control region (LCR) linked to the γ-globin gene promoter and the enhanced green fluorescent protein (EGFP) reporter gene, was adapted for high throughput screening and employed as the primary screen. Cytotoxic activity was assayed in a simultaneous counter screen. A number of hits were identified as being more potent than positive controls (such as butyrate). Several hits were immediately eliminated from further development as potential hemoglobinopathy therapeutics because of cytotoxicity (e.g., Idarubicin) or undesirable off-target effects, but nonetheless validated the HTS itself and were validated in secondary confirmatory assays as highly-potent HbF-inducers. The HTS assay identified 8 FDA-approved drugs as potent inducers of γ-globin gene expression, with activity at 1–2 logs lower concentrations (1000-fold higher potency) than prior generation therapeutic candidates. The γ-globin-specificity of hits was determined in a secondary assay employing a stably-transfected dual-luciferase reporter construct containing the LCR and the β-globin promoter linked to renilla luciferase and the Aγ-globin promoter linked to firefly luciferase (μLCRβprRlucAγprFluc cassette). Clinical-stage or clinically-approved agents, including Ambroxol at 1 μM, Desloratadine at 1 μM, Resveratrol at 10 μM, Benserazide at 5 μM, the HDAC inhibitor MS-275 at 5 μM, and an established bioactive, NSC-95397, at 1 μM were all significantly more active in this assay than Butyrate at 2000 μM, with MS-275 and Resveratrol being the most active. These drugs were then assayed for their ability to induce γ-globin mRNA expression in cultured primary human erythroid progenitors, at concentrations which are pharmacologically achievable in humans. Drugs significantly more active in γ -globin mRNA induction than the positive control (2-fold induction) in this system included Ambroxol (3-fold), Desloratadine (up to 6-fold), Resveratrol (up to 3-fold), Benserazide (up to 5-fold), and MS-275 (up to 3.7-fold). Two agents were subsequently studied in anemic baboons, and demonstrated in vivo induction of γ-globin mRNA, HbF, and F-reticulocytes. Unexpectedly, rises in total hemoglobin (>1 gm/dL) also occurred with 2 agents. Thus, a panel of structurally- and functionally-unrelated compounds demonstrate greater HbF-inducing activity, with up to 1000-fold higher potency, than current HbF-inducers which have significant activity in clinical trials. Some of the drugs identified by HTS have entirely benign safety profiles. These candidates could be clinically evaluated rapidly and at significantly less cost than new chemical entities, which require extensive toxicology, manufacturing, and clinical evaluation. These findings demonstrate the utility of a high-throughput screening program based on γ-globin gene promoter induction. Disclosures: No relevant conflicts of interest to declare.

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