A Split Renilla Luciferase Complementation Assay for the Evaluation of Hsp90/Aha1 Complex Disruptors and Their Activity at the Aha1 C-Terminal Domain.

Disruption of interactions between Hsp90 and the cochaperone protein, Aha1, has emerged as a therapeutic strategy to inhibit Aha1-driven cancer metastasis and tau aggregation in models of tauopathy. A combination of split Renilla luciferase assays was developed to screen and quantify the ability of small molecules to disrupt interactions between Hsp90 and both full length Aha1 protein (Aha1-FL) and the Aha1 C-terminal domain (Aha1-CTD). This luminescence-based approach was used to identify withaferin A and gedunin as disruptors of Hsp90/Aha1 interactions and provided insight into the binding regions for gambogic acid and gedunin on the Hsp90 homodimer. All compounds tested that disrupted Hsp90/Aha1-CTD interactions were found to disrupt interactions between Hsp90 and Aha1-FL, suggesting that interactions between Hsp90 and the Aha1-CTD play a key role in the stability of Hsp90/Aha1 complexes.

Nonviral DNA Delivery System with Supramolecular PEGylation Formed by Host-Guest Pseudo-Block Copolymers.

A cationic supramolecular system based on host-guest pseudoblock copolymers was developed for nonviral DNA delivery. In this system, the macromolecular host was a cationic star-shaped polymer composed of a ß-cyclodextrin (ß-CD) core and multiple poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) chains grafted on the core, while the macromolecular guest was a linear adamantyl-ended poly(ethylene glycol) (mPEG-Ad). Pseudoblock copolymers were self-assembled from the polymeric host-guest pairs (typically, 1:1 molar ratio) in aqueous media through the inclusion of an adamantyl group at the end of guest polymer into the ß-CD cavity of host polymers. Through such an approach, the resultant supramolecular system was integrated with not only a superior DNA condensing ability due to the host polymer but also an outstanding polyplex-stabilizing ability as well as biocompatibility due to the guest polymer. The cationic star-shaped host polymers alone were capable of condensing plasmid DNA efficiently into nanoparticles (70-100 nm) with positive surface charge. They showed obviously lower cytotoxicity than PEI 25K (commercial branched polyethylenimine with a molecular weight around 25 kDa) in cell lines of L929, MB231, and Hela under high dose. In serum-free or serum-containing culture conditions, these host polymers exhibited either higher or lower in vitro DNA transfection efficiency as compared with PEI 25K in the three cell lines under study, which was dependent on the N/P ratios and PDMAEMA arm length. Upon incorporation of the PEG block through host-guest complexation with mPEG-Ad (i.e., supramolecular PEGylation), the resulting host-guest supramolecular systems exhibited even lower cytotoxicity than the host polymers alone. The polyplexes between plasmid DNA (pDNA) and the host-guest systems showed significantly improved stability in BSA-PBS buffer solution (pH 7.4) and enhanced in vitro DNA transfection efficiency in the cases of higher N/P ratios or longer PDMAEMA arms in all tested cell lines under both serum-free and serum-containing culture conditions, as compared with the corresponding polyplexes without supramolecular PEGylation. Further, through forming pseudoblock copolymer, the DNA transfection ability of the supramolecular system can be easily modulated and optimized either by changing the ratio between the guest and host or by using different hosts with varied PDMAEMA arm lengths.

Improved dual luciferase reporter (DLR) assay to determine the protein stability.

We developed a binary vector co-expressing firefly luciferase (FF) and Renilla luciferase (REN) to detect protein stability in response to different stimuli, and verified the functionality of the vector. The StrigoQuant-like reporter expressing FF and REN in one transcript is a sensitive tool for detecting protein abundance in different genotypes. However, we found that significant differences in the relative FF/REN ratio of empty StrigoQuant vector in different genotypes. Therefore, to determine the actual protein abundance, the relative FF/REN ratio of the protein of interest should be normalized to that of the empty vector.

Creation of different bioluminescence resonance energy transfer based biosensors with high affinity to VEGF.

In age-related macular degeneration (AMD) or diabetic retinopathy (DR), hypoxia and inflammatory processes lead to an upregulation of the vascular endothelial growth factor (VEGF) expression and thereby to pathological neovascularisation with incorrectly formed vessels prone to damage, thus increasing the vascular permeability and the risk of bleeding and oedema in the retina. State of the art treatment is the repeated intraocular injection of anti-VEGF molecules. For developing improved individualized treatment approaches, a minimally invasive, repeatable method for in vivo quantification of VEGF in the eye is necessary. Therefore, we designed single molecule eBRET2 VEGF biosensors by directly fusing a Renilla luciferase mutant (Rluc8) N-terminal and a green fluorescent protein (GFP2) C-terminal to a VEGF binding domain. In total, 10 different VEGF biosensors (Re01- Re10) were generated based on either single domains or full length of VEGF receptor 1 or 2 extracellular regions as VEGF binding domains. Full length expression of the biosensors in HEK293-T cells was verified via Western Blot employing an anti-Rluc8-IgG. Expression of alternative splice variants was eliminated through the deletion of the donor splice site by introduction of a silent point mutation. In all ten biosensors the energy transfer from the Rluc8 to the GFP2 occurs and generates a measurable eBRET2 ratio. Four biosensors show a relevant change of the BRET ratio (ΔBR) after VEGF binding. Furthermore, each biosensor shows a unique detection range for VEGF quantification and especially Re06 and Re07 have a high sensitivity in the range of in vivo VEGF concentrations in the eye, previously measured by invasive methods. In conclusion, we generated several eBRET2 biosensors that are suitable for VEGF quantification in vitro and could identify two eBRET2 biosensors, which may be suitable for non-invasive in vivo VEGF quantification with an implantable device.

Engineering of galectin-3 for glycan-binding optical imaging.

Galectin-3 (Gal-3) is a multifunctional glycan-binding protein that participates in many pathophysiological events and has been described as a biomarker and potential therapeutic target for severe disorders, such as cancer. Several probes for Gal-3 or its ligands have been developed, however both the pathophysiological mechanisms and potential biomedical applications of Gal-3 remain not fully assessed. Molecular imaging using bioluminescent probes provides great sensitivity for in vivo and in vitro analysis for both cellular and whole multicellular organism tracking and target detection. Here, we engineered a chimeric molecule consisting of Renilla luciferase fused with mouse Gal-3 (RLuc-mGal-3). RLuc-mGal-3 preparation was highly homogenous, soluble, active, and has molecular mass of 65,870.95 Da. This molecule was able to bind to MKN45 cell surface, property which was inhibited by the reduction of Gal-3 ligands on the cell surface by the overexpression of ST6GalNAc-I. In order to obtain an efficient and stable delivery system, RLuc-mGal-3 was adsorbed to poly-lactic acid nanoparticles, which increased binding to MKN45 cells in vitro. Furthermore, bioluminescence imaging showed that RLuc-mGal-3 was able to indicate the presence of implanted tumor in mice, event drastically inhibited by the presence of lactose. This novel bioluminescent chimeric molecule offers a safe and highly sensitive alternative to fluorescent and radiolabeled probes with potential application in biomedical research for a better understanding of the distribution and fate of Gal-3 and its ligands in vitro and in vivo.

Bioluminescence Resonance Energy Transfer (BRET) Coupled Near-Infrared Imaging of Apoptotic Cells.

Detection of apoptotic cells is crucial for understanding the mechanism of diseases and for therapy development. So far, visible-emitting fluorescent probes such as FITC-labeled Annexin V has been widely used for the detection of apoptotic cells. However, such probes cannot be applied to noninvasive imaging in the near-infrared (NIR) region. Compared with visible light, NIR light is highly permeable in turbid biological samples and tissues. In addition, NIR optical imaging has several advantages such as lower autofluorescence and scattering from biological samples, leading to clearer images with high signal to background ratios. Here, we describe the synthesis and application of bioluminescence resonance energy transfer (BRET)-coupled quantum dots (QDs) for the NIR optical imaging of apoptotic cells.

Medium-Throughput Detection of Hsp90/Cdc37 Protein-Protein Interaction Inhibitors Using a Split Renilla Luciferase-Based Assay.

The protein-folding chaperone Hsp90 enables the maturation and stability of various oncogenic signaling proteins and is thus pursued as a cancer drug target. Folding in particular of protein kinases is assisted by the co-chaperone Cdc37. Several inhibitors against the Hsp90 ATP-binding site have been developed. However, they displayed significant toxicity in clinical trials. By contrast, the natural product conglobatin A has an exceptionally low toxicity in mice. It targets the protein-protein interface (PPI) of Hsp90 and Cdc37, suggesting that interface inhibitors have an interesting drug development potential. In order to identify inhibitors of the Hsp90/Cdc37 PPI, we have established a mammalian cell lysate-based, medium-throughput amenable split Renilla luciferase assay. This assay employs N-terminal and C-terminal fragments of Renilla luciferase fused to full-length human Hsp90 and Cdc37, respectively. We expect that our assay will allow for the identification of novel Hsp90/Cdc37 interaction inhibitors. Such tool compounds will help to evaluate whether the toxicity profile of Hsp90/Cdc37 PPI inhibitors is in general more favorable than that of ATP-competitive Hsp90 inhibitors. Further development of such tool compounds may lead to new classes of Hsp90 inhibitors with applications in cancer and other diseases.

Development of a quantitative method to measure EV uptake.

The outstanding potential of Extracellular Vesicles (EVs) in medicine, deserves a detailed study of the molecular aspects regulating their incorporation into target cells. However, because EV size lies below the limit of resolution of optical techniques, quantification together with discrimination between EV binding to the target cell and uptake is usually not completely achieved with current techniques. Human tetraspanins CD9 and CD63 were fused to a dual EGFP-Renilla-split tag. Subcellular localization and incorporation of these fusion proteins into EVs was assessed by western-blot and fluorescence microscopy. EV binding and uptake was measured using either a classical Renilla substrate or a cytopermeable one. Incubation of target cells expressing DSP2 with EVs containing the complementary DSP1 portion could not recover fluorescence or luciferase activity. However, using EVs carrying the fully reconstituted Dual-EGFP-Renilla protein and the cytopermeable Renilla luciferase substrate, we could distinguish EV binding from uptake. We provide proof of concept of the system by analysing the effect of different chemical inhibitors, demonstrating that this method is highly sensitive and quantitative, allowing a dynamic follow-up in a high-throughput scheme to unravel the molecular mechanisms of EV uptake in different biological systems.

Cell-specific CRISPR-Cas9 activation by microRNA-dependent expression of anti-CRISPR proteins.

The rapid development of CRISPR-Cas technologies brought a personalized and targeted treatment of genetic disorders into closer reach. To render CRISPR-based therapies precise and safe, strategies to confine the activity of Cas(9) to selected cells and tissues are highly desired. Here, we developed a cell type-specific Cas-ON switch based on miRNA-regulated expression of anti-CRISPR (Acr) proteins. We inserted target sites for miR-122 or miR-1, which are abundant specifically in liver and cardiac muscle cells, respectively, into the 3'UTR of Acr transgenes. Co-expressing these with Cas9 and sgRNAs resulted in Acr knockdown and released Cas9 activity solely in hepatocytes or cardiomyocytes, while Cas9 was efficiently inhibited in off-target cells. We demonstrate control of genome editing and gene activation using a miR-dependent AcrIIA4 in combination with different Streptococcus pyogenes (Spy)Cas9 variants (full-length Cas9, split-Cas9, dCas9-VP64). Finally, to showcase its modularity, we adapted our Cas-ON system to the smaller and more target-specific Neisseria meningitidis (Nme)Cas9 orthologue and its cognate inhibitors AcrIIC1 and AcrIIC3. Our Cas-ON switch should facilitate cell-specific activity of any CRISPR-Cas orthologue, for which a potent anti-CRISPR protein is known.

A potential gene delivery strategy using BK virus.

Recombinant BK virus (rBKV) is able to express polypeptides under control of its native BKV late promoter. This ability helps to use this construct as a good reporter since it can infect human cells. In this study, we generate a BKV construct containing Renilla luciferase (Rluc) sequences under control of the BKV late promoter. The activity of Rluc was strongly detected in Vero-76 and Cos-1 cells transfected with rBKV-Rluc-myc-2A-VP2 construct, indicating the production of a functional enzyme driven by the native late promoter. Furthermore, a construct made of rBKV-IL2SP-Rluc-myc-2A-VP2 by introducing human IL2 secretion peptide (IL2 SP) caused secretion of IL2SP-Rluc-myc into the culture medium. As a concluding remark, a potential infectious rBKV that can express foreign antigens such as Rluc was generated successfully. The proposed strategy would be useful to engineer recombinant forms of rBKV with many potential applications including development of antiviral assay for new drugs, human vaccines and gene delivery systems for immunotherapeutic or cell transduction.

New Optical Imaging Reporter-labeled Anaplastic Thyroid Cancer-Derived Extracellular Vesicles as a Platform for In Vivo Tumor Targeting in a Mouse Model.

Extracellular vesicles (EVs), originating from multivesicular bodies by invagination of the endosomal membrane, are communication channels between distant cells. They are natural carriers of exogeneous cellular materials and have been exploited as drug delivery carriers in various diseases. Here, we found that tumor cell-derived EVs can be used as efficient targets in tumors by monitoring with an optical reporter system. Anaplastic thyroid cancer (CAL62) cell-derived EVs with Renilla luciferase (Rluc) were used to target CAL62 tumors in a mouse model. Optical imaging revealed that cancer cell-derived EVs (EV-CAL62/Rluc) targeted the original tumor (CAL62) in mice within 30 min after systemic injection. Furthermore, fluorescence imaging revealed that EV-CAL62/Rluc were internalized into CAL62 tumors in the mice. Ex vivo Optical imaging further confirmed the in vivo finding. Here, we successfully monitored the tumor targeting ability of tumor cell-derived EVs by optical imaging. Based on these results, tumor cell-derived EVs are highly effective natural carriers for drug delivery for cancer therapies.

Molecular Imaging of Tumor Angiogenesis and Therapeutic Effects with Dual Bioluminescence.

BACKGROUND: Angiogenesis is critical for the growth of tumor by supplying nutrients and oxygen that exacerbates the metastasis and progression of cancer. Noninvasive imaging of angiogenesis during the tumor therapeutic processes may provide novel opportunities for image-guided tumor management. OBJECTIVE: Here, we want to develop a mouse animal model for assessing cancer progression and angiogenesis in the same individuals by molecular imaging. METHODS: Breast cancer model was developed with mouse breast cancer cell line 4T1 carrying a reporter system encoding a triple fusion (TF) reporter gene consisting of renilla luciferase (Rluc), red fluorescent protein (RFP) and herpes simplex virus truncated thymidine kinase (HSV-ttk) in transgenic mice, which expressed firefly luciferase (Fluc) under the promoter of vascular endothelial growth factor receptor 2 (Vegfr2-luc). The mice were subsequently treated with ganciclovir (GCV) and the tumor angiogenesis was tracked by Fluc imaging and the growth status of tumor was monitored by imaging of Rluc simultaneously. CONCLUSION: Overall, this traceable breast cancer model can simultaneously image the tumor growth and angiogenesis in single individual, which may facilitate a better understanding the mechanisms of angiogenesis in the progression and regression of tumor.

A Luciferase Functional Quantitative Assay for Measuring NF-ĸB Promoter Transactivation Mediated by HTLV-1 and HTLV-2 Tax Proteins.

HTLV-1 and HTLV-2 viruses express Tax transactivator proteins required for viral genome transcription and capable of transforming cells in vivo and in vitro. Although Tax oncogenic potential needs to be further elucidated, it is well established that Tax proteins activate, among others, transcription factors of the NF-ĸB family, which are involved in immune and inflammatory responses, cell growth, apoptosis, stress responses and oncogenesis. Here, we describe a reporter gene assay applied for quantitative analysis of Tax-dependent NF-ĸB activation. The procedure is based on co-transfection of two individual vectors containing the cDNA for firefly and Renilla luciferase enzymes and vectors expressing Tax proteins. The luciferase expression is driven by cis-NF-ĸB promoter regulatory elements responsive to Tax transactivating factor. This assay is particularly useful to investigate Tax influence on NF-ĸB activation mediated by viral or host factors.

False responses of Renilla luciferase reporter control to nuclear receptor TR4.

Renilla luciferase reporter is a widely used internal control in dual luciferase reporter assay system, where its transcription is driven by a constitutively active promoter. However, the authenticity of the Renilla luciferase response in some experimental settings has recently been questioned. Testicular receptor 4 (TR4, also known as NR2C2) belongs to the subfamily 2 of nuclear receptors. TR4 binds to a direct repeat regulatory element in the promoter of a variety of target genes and plays a key role in tumorigenesis, lipoprotein regulation, and central nervous system development. In our experimental system using murine pituitary corticotroph tumor AtT20 cells to investigate TR4 actions on POMC transcription, we found that overexpression of TR4 resulted in reduced Renilla luciferase expression whereas knockdown TR4 increased Renilla luciferase expression. The TR4 inhibitory effect was mediated by the TR4 DNA-binding domain and behaved similarly to the GR and its agonist, Dexamethasone. We further demonstrated that the chimeric intron, commonly present in various Renilla plasmid backbones such as pRL-Null, pRL-SV40, and pRL-TK, was responsible for TR4's inhibitory effect. The results suggest that an intron-free Renilla luciferase reporter may provide a satisfactory internal control for TR4 at certain dose range. Our findings advocate caution on the use of Renilla luciferase as an internal control in TR4-directed studies to avoid misleading data interpretation.

Identification of several high-risk HPV inhibitors and drug targets with a novel high-throughput screening assay.

Human papillomaviruses (HPVs) are oncogenic viruses that cause numerous different cancers as well as benign lesions in the epithelia. To date, there is no effective cure for an ongoing HPV infection. Here, we describe the generation process of a platform for the development of anti-HPV drugs. This system consists of engineered full-length HPV genomes that express reporter genes for evaluation of the viral copy number in all three HPV replication stages. We demonstrate the usefulness of this system by conducting high-throughput screens to identify novel high-risk HPV-specific inhibitors. At least five of the inhibitors block the function of Tdp1 and PARP1, which have been identified as essential cellular proteins for HPV replication and promising candidates for the development of antivirals against HPV and possibly against HPV-related cancers.

An alternative approach in regulation of expression of a transgene by endogenous miR-145 in carcinoma and normal breast cell lines.

MicroRNAs are small noncoding RNAs that regulate gene expression by repressing translation of target cellular transcripts. Increasing evidences indicate that miRNAs have different expression profiles and play crucial roles in numerous cellular processes. Delivery and expression of transgenes for cancer therapy must be specific for tumors to avoid killing of healthy tissues. Many investigators have shown that transgene expression can be suppressed in normal cells using vectors that are responsive to microRNA regulation. To overcome this problem, miR-145 that exhibits downregulation in many types of cancer cells was chosen for posttranscriptional regulatory systems mediated by microRNAs. In this study, a psiCHECK-145T vector carrying four tandem copies of target sequences of miR-145 into 3'-UTR of the Renilla luciferase gene was constructed. Renilla luciferase activity from the psiCHECK-145T vector was 57% lower in MCF10A cells with high miR-145 expression as compared to a control condition. Additionally, overexpression of miR-145 in MCF-7 cells with low expression level of miR-145 showed more than 76% reduction in the Renilla luciferase activity from the psiCHECK-145T vector. Inclusion of miR-145 target sequences into the 3'-UTR of the Renilla luciferase gene is a feasible strategy for restricting transgene expression in a breast cancer cell line while sparing a breast normal cell line.

In Vivo Hepatic Enhancer Elements in the Human ABCG2 Locus.

ABCG2 encodes the mitoxantrone resistance protein (MXR; breast cancer resistance protein), an ATP-binding cassette (ABC) efflux membrane transporter. Computational analysis of the ∼300 kb region of DNA surrounding ABCG2 (chr4:88911376-89220011, hg19) identified 30 regions with potential cis-regulatory capabilities. These putative regulatory regions were tested for their enhancer and suppressor activity in a human liver cell line using luciferase reporter assays. The in vitro enhancer and suppressor assays identified four regions that decreased gene expression and five regions that increased expression >1.6-fold. Four of five human hepatic in vitro enhancers were confirmed as in vivo liver enhancers using the mouse hydrodynamic tail vein injection assay. Two of the in vivo liver enhancers (ABCG2RE1 and ABCG2RE9) responded to 17ß-estradiol or rifampin in human cell lines, and ABCG2RE9 had ChIP-seq evidence to support the binding of several transcription factors and the transcriptional coactivator p300 in human hepatocytes. This study identified genomic regions surrounding human ABCG2 that can function as regulatory elements, some with the capacity to alter gene expression upon environmental stimulus. The results from this research will drive future investigations of interindividual variation in ABCG2 expression and function that contribute to differences in drug response.

The isoflavone fraction from soybean presents mRNA maturation inhibition activity.

Recent findings indicate that mRNA splicing inhibitors can be potential anticancer candidates. We have previously established a screening system which monitors mRNA processing in order to identify mRNA processing inhibitors. Among a number of dietary resources, isoflavone fractions showed an inhibitory effect of mRNA processing. These findings demonstrate that a variety of dietary sources have an impact on mRNA biogenesis.

Ligand Activity of Group 15 Compounds Possessing Triphenyl Substituent for the RXR and PPARγ Nuclear Receptors.

We investigated the ability of group 15 compounds with a triphenyl substituent to bind to and activate human retinoic X receptor (RXR) and peroxisome proliferator-activated receptor (PPAR) γ and their ability to activate the receptor. Triphenylphosphine oxide (TPPO) transcriptionally activated both RXR and PPARγ. Triphenylbismuth (TPBi) transcriptionally activated PPARγ but not RXR. However, TPBi significantly inhibited RXR transcriptional activity induced by 9-cis retinoic acid (9cRA) and PPARγ transcriptional activity induced by rosiglitazone (Rosi). Triphenylarsine (TPAs) also significantly inhibited the 9cRA- and Rosi-induced transcriptional activity of both receptors, whereas TPAs alone had no effect on the transcriptional activity of RXR and PPARγ. Consistent with these results, TPAs and TPBi blocked the binding of [3H]9cRA to RXR and of [3H]Rosi to PPARγ in a competitive manner. However, contrary to the results of the reporter gene assay, TPPO did not compete with [3H]9cRA and [3H]Rosi for binding to RXR and PPARγ, respectively. Our findings indicate that 1) TPPO is a transcriptional activator-but not a ligand-of RXR and PPARγ; 2) TPBi is an antagonist of RXR and a partial agonist of PPARγ; and 3) TPAs is a dual antagonist of RXR and PPARγ. These results suggest that TPPO, TPAs, and TPBi are potential endocrine disrupters of the PPARγ-RXR signaling pathway.

Cell-based high-throughput compound screening reveals functional interaction between oncofetal HMGA2 and topoisomerase I.

HMGA2 is an important chromatin factor that interacts with DNA via three AT-hook domains, thereby regulating chromatin architecture and transcription during embryonic and fetal development. The protein is absent from differentiated somatic cells, but aberrantly re-expressed in most aggressive human neoplasias where it is causally linked to cell transformation and metastasis. DNA-binding also enables HMGA2 to protect cancer cells from DNA-damaging agents. HMGA2 therefore is considered to be a prime drug target for many aggressive malignancies. Here, we have developed a broadly applicable cell-based reporter system which can identify HMGA2 antagonists targeting functionally important protein domains, as validated with the known AT-hook competitor netropsin. In addition, high-throughput screening can uncover functional links between HMGA2 and cellular factors important for cell transformation. This is demonstrated with the discovery that HMGA2 potentiates the clinically important topoisomerase I inhibitor irinotecan/SN-38 in trapping the enzyme in covalent DNA-complexes, thereby attenuating transcription.