Real-time monitoring of NF-kappaB activity in cultured cells and in animal models

Abstract

Nuclear factor kappa B (NFkB) is a transcription factor that plays a major role in many human disorders, including immune diseases and cancer. Monitoring of expression of this transcription factor should facilitate a better understanding of NFkB activation in pathological processes. We designed a reporter system based on NFkB responsive promoter elements driving expression of the secreted Gaussia luciferase (Gluc). We show that this bioluminescent reporter is a highly sensitive tool for non-invasive monitoring of the kinetics of NFkB activation and inhibition over time, both in conditioned medium of cultured cells, as well as in blood and urine of animals. NFkB activation was successfully monitored in real time in endothelial cells in response to tumor angiogenic signaling, as well as in monocytes in response to inflammation. Further, we demonstrated dual blood monitoring of both NFkB activation during tumor development as correlated to tumor formation using the NFkB Gluc reporter, as well as the secreted alkaline phosphatase reporter. This NFkB reporter system provides a powerful tool for monitoring NFkB activity in real time in vitro and in vivo.

INTRODUCTION

Over two decades ago, Baltimore and his colleagues described a nuclear factor that binds to DNA sequences in the promoter and enhancer regions of the mouse immunoglobulin (Ig) gene (osez1). This transcription activation mechanism originally thought to occur exclusively in B cells soon turned out to be a universal transcription process with important roles in many physiological disorders. This transcription factor is referred to as nuclear factor kappa B (NFkB). NFkB is a heterodimeric protein composed of different combinations of five members of the Rel family of transcription factors, including p65 (RelA), RelB, c-Rel, p50 and p52, containing a common Rel homology domain (RHD) within their N-terminal region (2). The RHD is responsible for DNA binding as well as homo- and heterodimerization. Only p65, RelB and c-Rel contain a transcription activation domain (TAD) allowing the induction of gene expression (3). The NFkB protein complex is usually present in the cytoplasm as homo- or heterodimers bound to the IkB family (mainly IkBα). Binding of IkB prevents NFkB translocation from the cytoplasm into the nucleus and subsequent binding of NFkB to the DNA (4). A wide variety of signals such as pro-inflammatory cytokines, growth factors, hormones, oxidative stress, viral infection, and DNA-damaging agents (5), can cause the activation of IkB kinase (IKK), which in turn phosphorylates the IkB causing the release of the NFkB dimers and their nuclear translocation. In the nucleus, the NFkB dimers bind to a kappaB site in the promoter or enhancer region of target genes thereby controlling gene expression. Activated NFkB can induce the transcription of many genes such as cytokines, growth factors, adhesion molecules and mitochondrial anti-apoptotic genes (2).

While the crucial role of NFkB in the immune response is well established (6), cumulative evidence has shown that it is a key mediator in inflammation as well as in tumor development, progression, and neovascularization (7). Constitutive NFkB activation has already been demonstrated in many cancer types (8-10). Activation of this signalling pathway can lead to the transcription of many anti-apoptotic genes and the inhibition of apoptosis, causing drug resistance (11). Further, NFkB has been shown to play an important role in tumor angiogenesis and invasiveness (12).

Bioluminescence imaging (BLI) has emerged as a powerful tool in biomedical research for monitoring of transgene expression, viral vector infection, tumor growth and metastasis, as well as inflammation and gene therapy (13). Recently, we have established a novel assay for ex vivo bioluminescence measurement of in vivo processes (14). By cloning the naturally secreted Gaussia luciferase (Gluc) under the control of the constitutively active cytomegalovirus (CMV) promoter, we were able to monitor different biological processes such as tumor growth and response to therapy, as well as tracking of circulating cells by assessing the Gluc activity in few microliters of blood, urine or serum in mice (14). Here, we constructed an NFkB reporter system comprising five tandem repeats of the NFkB transcription responsive elements (TRE; 12 bp each) and a TATA box driving the expression of the secreted Gluc. We showed that Gluc expression was induced up to 500-fold in response to NFkB activation in response to TNFα and was inhibited by 5-fold in response to sulfasalazine (SSZ) in a dose- and time-dependent manner in vitro. Further, we successfully monitored NFkB activation in real time in response to different stimuli including tumor angiogenesis, as well as in circulating monocytes upon induction of inflammatory responses. In addition, using two secreted enzyme reporters requiring different substrates (Gluc and the secreted alkaline phosphatase or SEAP), we established a dual-blood-assay system allowing the monitoring of both the NFkB activation during tumor development as correlated with cancer cell proliferation and tumor growth.

MATERIALS AND METHODS

Vector construction

Five tandem repeats of NFkB transcription responsive elements (TREs; TGGGGACTTTCCGC) (15, 16), were designed and annealed using the complementary oligonucleotide sequences 5′-GATCTTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCA-3′ and 5′-CTAGTGCGGAAAGTCCCCAGCGGAAAGTCCCCAGCGGAAAGTCCCCAGCGGAAAGTCCCCAGCGGAAAGTCCCCAA-3′. Additional NFkB reporters with multiple tandem repeats of 5, 10 and 15 NFkB TRE, were constructed with 0, 4, 8, 12 or 30 random base-pairs (bp) acting as spacers separating each of the tandem copies (Fig. 1a). All of these promoter elements were designed to generate sticky ends compatible with BglII and NheI restriction sites at the 5′ and 3′ ends, respectively. The humanized Gaussia luciferase cDNA (Nanolight, Pinetop, Az) (17) was amplified by PCR adding a TATA box from the human immunodeficiency virus type 1 subtype E promoter (ATATA) 30 bp upstream of the Gluc cDNA. The TATA-Gluc fragment was purified from a 1% agarose gel and ligated into the mammalian expression plasmid pHGCx (from Dr. Yoshimura Saeki, MGH, Boston MA) (18) by replacing the CMV promoter with NFkB elements generating pHGNF-Gluc. Similarly, the CMV promoter in pHGCX was replaced with the SV40 promoter (amplified from pSEAP2-control plasmid, Clontech, Palo Alto, CA) generating pHGSV40-Gluc. The NF-Gluc expression cassette was subsequently subcloned into CSCW, a self-inactivating lentivirus vector (19), at the BamHI and XhoI sites producing pCSNF-Gluc. A DNA fragment spanning two copies of the 1.2 Kb chicken β-globin (HS4) insulator elements, amplified from the pJC13-1 plasmid (20) (a kind gift of Dr. Adam West, National Institutes of Health, Bethesda, MD), was inserted into the U3 region of the pCSNF-Gluc plasmid using the PmeI and KpnI restriction sites creating pCSHSNF-Gluc (designated lenti-NF-Gluc throughout the text). Using the pCSCW-IG self-inactivating vector (19), we cloned the mCherry fluorescent protein (kindly provided by Dr. Roger Tsien, UCSD, CA) cDNAs under the control of the CMV promoter, thereby generating LV-mCherry. SEAP and mCherry separated by an internal ribosome entry site (IRES) were cloned in a similar vector by amplifying the SEAP from the pSEAP2-basic vector (Clontech, Palo Alto, CA) (14). The integrity of all constructs was verified by sequencing. All lentivirus vectors were produced and titered as transducing units (TU)/ml as previously described (19).

Figure 1.

The NFkB-Gluc reporter system. (a) Schematic overview of the different NF-Gluc expression cassettes with the corresponding Gluc induction levels. 293T cells were transfected with plasmids carrying the expression cassette of Gluc under the control of different NFkB responsive elements. Cells were either not treated or irradiated with 5 Gy of IR and 24 h later, Gluc activity was measured in an aliquot of conditioned medium. (b) 293T cells were transduced with lentivirus vector encoding Gluc under NFkB-0 TREs with or without a TATA box. Cells were treated with TNFα (10 ng/ml) and Gluc activity was measured in an aliquot of the conditioned medium 24 h later. (c) 293T cells were transduced with lentivirus vector expressing 5NFkB-0-Gluc with or without HS4 insulator elements. Forty-eight h later, Gluc was measured in an aliquot of the conditioned medium. Data in (b-d) is presented as the average fold increase in Gluc activity ± S.D (n=4). **p≤0.01, student t-test.

Cell culture and reagents

293T human kidney fibroblasts cells (from Dr. Maria Calos, Stanford University, Stanford, CA), U87 human glioma (ATCC, U87 MG), Gli36 human glioma (from Dr. Anthony Capanogni, UCLA, Los Angeles) (21), A549 human lung carcinoma (ATCC) and U937 monocytic/histiocytic lymphoma (ATCC) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Sigma, St. Louis, MO), 100 U/ml penicillin, and 0.1 mg/ml streptomycin (Sigma). HEI193 human schwannoma cells (22) were maintained in DMEM supplemented with 10% FBS, 2 μM forskolin (Calbiochem Corp., LaJolla, CA), 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 50 mg/ml gentamycin (Invitrogen, Carlsbad, CA). U87-CFP cells were produced by stably transducing U87 cells with a CMV-controlled Cerulean expression cassette using a lentivirus vector (23). Human brain microvascular endothelial cells (HBMVECs; Cell Systems ACBRI-376) were cultured in EGM medium (Cambrex) for no more than 10 passages. HBMVEC-mCherry cells were produced by stably transducing HBMVECs (passage 5) with lentivirus vector expressing mCherry. After 3 passages the cells were discarded. All cells were grown at 37°C in a 5% CO₂ humidified incubator. Sulfasalazine (SSZ), parthenolide (PTL), etoposide and doxorubicin were purchased from Sigma. Recombinant human tumor necrosis factor alpha (TNFα) was obtained from R&D systems, Minneapolis, MN. Ionizing radiation treatments were performed at 3-10 gray (Gy) using a ¹³⁷Cs source.

Transfection and lentivirus transduction

293T human fibroblast cells were plated in a 6-well plate (1.5×10⁴ cells/well) and transfected with plasmids expressing Gluc under either NFkB, CMV or SV40 promoters, using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s guidelines. To achieve stable gene expression in most cells (>90%), different cell types were transduced with lentivirus vectors at 100 transduction units (TU)/cell.

In vitro Gaussia luciferase activity

For Gluc assays, 15 μl aliquots of the cell-free conditioned medium were collected at different time points. Gluc activity was assayed by adding 20 μM coelenterazine, the Gluc substrate (Nanolight, Pinetop, AZ) to the supernatant and measuring photon counts in a 96-well plate luminometer (Dynex, Richfield, MN) over 10 sec.

In vitro angiogenesis assay

Low passage HBMVEC-mCherry cells transduced with lentivirus vector to express NF-Gluc (passage <7) were cultured on Matrigel (Beckton Dickinson, San Jose, CA) in EBM basal medium (Cambrex, San Diego, CA) in the presence or absence of U87-CFP cells, or EGM cocktail (Cambrex). Twenty-four h later, aliquots of conditioned medium were analyzed for Gluc activity and the cultures were analyzed by a combination of light and fluorescence microscopy.

In vivo experiments

All animal experiments were approved by the Massachusetts General Hospital Subcommittee on Research Animal Care. One million HEI193, 293T or Gli36 expressing NF-Gluc and/or SEAP were injected subcutaneously (s.c.) into nude mice. TNFα (16 μg/kg body weight) and SSZ (15 mg/kg body weight) were injected intravenously (i.v.) unless otherwise specified. For circulating tumor cells, 0.5 million cells expressing NF-Gluc and SEAP were injected intra-ocularly.

Monocyte model

U937 human leukemic monocyte lymphoma cells were transduced with lenti-NF-Gluc at 100 TU/cell. Cells were plated in wells of 96-well plates and treated with different concentrations of human TNFα (R&D Systems). At different time points, Gluc activity was assayed in the medium as above. For the in vivo studies, 1 million U937 cells expressing NF-Gluc were injected intraperitoneally (i.p.) into 8 mice and 1 h later, 4 mice were injected with PBS (control) and the other four mice with 2 μg of TNFα (in 100 μl) via the same route. Before PBS/TNFα injection and at different time points after injection, 5 μl blood was withdrawn and assayed for Gluc activity (see below).

Gluc/SEAP blood assay

For the Gluc assay, 5 μl of blood was collected by making a small nick in the mouse tail and mixed immediately with 1 μl 20 mM EDTA. Gluc activity was measured using the luminometer after injecting 100 μl 100 μM coelenterazine and acquiring photon counts over 10 sec. The SEAP chemiluminescence activity was measured in 5 μl serum using the Great EscAPe SEAP assay kit (Clontech) as per manufacturer’s instructions using the luminometer.

In vivo bioluminescence imaging

Mice were anesthetized by i.p. injections of ketamine (100 mg/kg) and xylazine (5 mg/kg) and then received an intra-ocular injection of coelenterazine (4 mg/kg body weight diluted in PBS). Photon counts were acquired for 5 minutes using a cooled CCD camera system. White-light surface images were also taken before each Gluc imaging session providing an anatomical view of the animal. Image processing and signal intensity quantification and analysis were performed using CMIR-Image, software developed by the Center for Molecular Imaging Research at Massachusetts General Hospital using image display and analysis suite developed by IDL (Research Systems Inc., Boulder, CO). Images were represented in a pseudo-color photon count manner, superimposed on a gray-scale anatomic white-light image, exposing the bioluminescence intensity as well as the animal anatomy. Bioluminescence signals were defined by an automatic intensity highlight procedure after subtracting background noise. The sum of the photon counts was calculated as a measure of Gluc reporter activity.

RESULTS

Construction of the NF-Gluc reporter

Initially, we designed and compared various NFkB responsive promoters by cloning multiple tandem repeats of the NFkB TRE with different linker between each of the repeats (to reduce potential steric hindrance) to drive the expression of the Gluc reporter (Fig. 1a). 293T cells were transfected with each of these reporters and 24 h later, NFkB was activated either by ionizing radiation (IR) (24), or TNFα {Osborn, 1989 #76}. Gluc induction levels in response to NFkB activation were assessed in aliquots of conditioned medium 24 h post-treatment. A significant increase in Gluc expression was detected after stimulation of these cells with IR for all six different constructs, with 5NFkB-0 and 2*5NFkB giving 6-fold induction (Fig. 1a). Adding >5 tandem repeats of the TRE or different linkers between each repeat did not enhance the Gluc induction level and therefore we decided to proceed with the construct containing 5 TREs of NFkB with no spacers (5NFkB-0] in all subsequent studies. In order to enhance the transcriptional activity of the reporter, a TATA box was inserted downstream of the NFkB TRE. As expected, a >28-fold increase in Gluc induction was observed in response to NFkB activation upon TNFα treatment as compared to the reporter without the TATA box (Fig.1b). When we packaged the NF-Gluc into a lentivirus vector and transduced 293T cells, we noticed a high basal level of Gluc expression as compared to transient transfection. In order to reduce any possible NFkB-independent transactivation of Gluc expression, we inserted chicken β-globin insulators (20) in the U3 region of the lentivirus vector, thereby flanking the Gluc expression cassette upon expression. This vector was designated as NF-Gluc. The addition of the insulators showed much tighter system with a significant reduction of the Gluc basal level in the absence of NFkB activating stimuli (Fig. 1c; **p≤0.01).

In vitro monitoring of NFkB activation and inhibition

In order to confirm that the increase in Gluc expression is due to NFkB activation, 293T cells were transfected with plasmids expressing Gluc under either NFkB TRE, SV40 or CMV promoters and treated with TNFα (a renowned inducer of NFkB activity (25)) or PBS as a control. Twenty-four h later, 20 μl aliquots of conditioned medium were transferred into a 96-well plate and assayed for Gluc activity using the luminometer (Fig. 2a). The plate was also imaged using the CCD camera (Fig. 2b). As expected, cells expressing Gluc under the control of NFkB TREs showed a >100-fold increase in Gluc expression in response to TNFα, indicating NFkB-mediated transcriptional activation of the NF-Gluc reporter (Fig. 2a & b). On the other hand, the SV40 promoter did not show any increase in Gluc activity in response to TNFα, while surprisingly the CMV promoter showed around a 2-fold increase in Gluc expression in response to TNFα. These results support previous reports that the CMV promoter can be induced by different drugs and that TNFα can further stimulate the activation of the CMV promoter via induction of NFkB (26, 27).

Figure 2.

Monitoring of NFkB activation in culture. (a & b) 293T cells were transfected with a plasmid carrying the expression cassette for Gluc under control of the NFkB, CMV or SV40 promoter and treated with TNFα (10 ng/ml). 24 h later, aliquots of the conditioned medium were assayed for Gluc activity either using the luminometer (a) or the CCD camera (b) after the addition of coelenterazine. (c) 293T cells stably expressing NF-Gluc were subjected to different treatments: TNFα (20 ng/ml), etoposide (2 μM), doxorubicin (0.125 μM), bleomycin sulfate (50 μg/ml) or IR (5 Gy). (d) Different cells lines, Gli36, A549, HEI193 and 293T cells were transduced with lenti-NF-Gluc and treated with TNFα (20 ng/ml). (e) 293T cells expressing NF-Gluc were treated with different concentrations of TNFα. (f) Time response curve for TNFα induction. 293T cells-expressing NF-Gluc were treated with TNFα (2.5 ng/ml). In (c-f) 15 μl aliquots of the conditioned medium were assayed for Gluc activity 24 h post-treatment (c-e) or at different time points (f). Data are presented as average of fold increase in Gluc activity in treated cells as compared to untreated ± S.D (n=4). dox = doxorubicin, etop = etoposide, BS = bleomycin sulfate, IR = ionizing radiation.

To test whether the NF-Gluc reporter system could be used to monitor NFkB activation in response to different stimuli, 293T human embryonic kidney cells were transduced with a lentivirus vector expressing NF-Gluc (lenti-NF-Gluc) to stably express Gluc under the control of NFkB. These cells were treated with different drugs previously described to induce NFkB activation, the inflammatory cytokine TNFα {Osborn, 1989 #76}, the chemotherapeutic drugs etoposide and doxorubicin (28), as well the radiomimetic drug, bleomycin sulfate (29). Alternatively, cells received a single dose of 5 Gy of IR (24). The Gluc expression was assayed 24 h post-treatment. The increase in Gluc expression as a measure of NFkB activation varied among the different treatments, with TNFα resulting in >500-fold induction (Fig. 2c). To investigate the activation of this reporter in different cell types, we transduced different human tumor cell lines, Gli36 glioma cells, HEI193 schwannoma cells and A549 lung adenocarcinoma cells, as well as 293T human fibroblast cells with lenti-NF-Gluc. These cells have >95% transduction efficiency with lentivirus vector under the conditions used (30, 31). These cells were treated with TNFα and the Gluc activity was measured in an aliquot of conditioned medium 24 h later. As expected, HEI193 human schwannoma cells [a benign tumor; (32-35)] and 293T fibroblast cells showed the lowest basal levels of NFkB activity (around 500 RLU) and consequently the highest Gluc induction levels (>100 fold; Fig. 2d). On the other hand, Gli36 human glioma and A549 human lung carcinoma cells [both malignant and invasive tumor types; (36, 37)] presented with high basal NFkB activity (around 120,000 RLU, data not shown) and a moderate additional increase in Gluc expression (3-5 fold) upon TNFα treatment (Fig. 2d).

To determine the usefulness of the NF-Gluc reporter in monitoring NFkB activation in a dose- and time-dependent manner and in real-time, 293T cells stably expressing NF-Gluc were treated with different concentrations of TNFα. The Gluc signal increased upon increasing the TNFα dose (Fig. 2e). Notably, even the lowest concentration (75 pg/ml) resulted in >50-fold induction. When conditioned medium was collected at different time points and assayed for Gluc activity, an increase was observed as early as 2 h after TNFα treatment, reaching a maximum at 48 h (Fig. 2f).

Sulfasalazine (SSZ), an anti-inflammatory and immunosuppressive agent, inhibits IKKα and IKKβ thereby blocking NFkB activation (38, 39). To test whether NFkB inhibition could be monitored using our reporter system, cells expressing NF-Gluc were treated with 500 μM SSZ, a concentration reported to cause a 50% decrease in the binding of NFkB to TRE elements (39). As expected, we observed a 2-fold decrease in Gluc basal level in response to SSZ treatment (Fig. 3a). SSZ also produced a decrease in NFkB induction caused by TNFα by 4-fold (Fig. 3b). A similar decrease in NFkB induction caused by doxorubicin was also observed (data not shown). We further validated the inhibition of the TNFα-mediated NFkB activation by testing another NFkB inhibitor, parthenolide (PTL), which blocks the IKK complex (40). PTL almost abolished the activation of NFkB by TNFα, yielding a modest 3-fold increase in Gluc activity when combined with TNFα (Fig. 3b). In order to determine the kinetics of the SSZ-mediated inhibition, we treated cells with either SSZ, TNFα alone or a combination of both TNFα and SSZ. Aliquots of the cell-free conditioned medium were collected over time and assayed for Gluc activity. The inhibitory effect of SSZ on NFkB was sustained up to 48 h (Fig. 3c). Further, a dose increase of SSZ in TNFα- treated cells correlated with a decrease in Gluc activity (Fig. 3d). In conclusion, the NF-Gluc reporter system proved to be a useful tool in monitoring both activation and inhibition of NFkB in real-time.

Figure 3.

Monitoring of NFkB inhibition. 293T cells-expressing NF-Gluc were plated in 96-well plate and Gluc activity was assayed in an aliquot of conditioned medium 24 h post-treatment with different drugs. (a) Cells were treated with either PBS (control) or SSZ (500 μM). Data is presented as RLU/sec ± SD (n=4). (b) Cells were treated with TNFα (5 ng/ml) in the presence or absence of SSZ (500 μM) or PTL (2 μM). (c) Time course for NFkB induction and inhibition after TNFα (20 ng/ml) and/or SSZ (500 μM) treatment. Aliquots from the conditioned medium were assayed for Gluc at different time points post-treatment. (d) Cells were treated with TNFα (20 ng/ml) and different doses of SSZ. In (b-d) data presented as fold change in Gluc activity, as compared to untreated samples ± SD (n=4). *p≤0.05, student t-test.

Next, we determined whether we could use the NF-Gluc reporter system to monitor the induction of NFkB in a biologically relevant system. We decided to determine the induction of NFkB in angiogenic endothelial cells, a well studied process (41). Low passage HBMVEC-mCherry cells were transduced to express NF-Gluc. These cells formed a confluent monolayer and were viable for at least 3 passages as determined by microscopic monitoring (Fig. 4a). Upon co-culturing of these cells on a Matrigel substratum with U87-Cerulean cells, or in EBM containing an angiogenic cocktail (EGM), a tubule network was visualized using fluorescence microscopy (42) (Fig. 4a). The induction of endothelial tubules in vitro has been described to be partly mediated by NFkB (41). In parallel to the fluorescence microscopic analysis, we analyzed the conditioned medium from these cells for Gluc activity. A clear induction of the NFkB-controlled Gluc signal was measured after stimulation of the HBMVECs with U87 glioma cells or the EGM medium containing a cocktail of angiogenic factors (Fig. 4b), confirming a role for NFkB in glioma angiogenesis in vitro.

Figure 4.

Analysis of NFkB activation during tumor angiogenesis in vitro. (a) HBMVEC-mCherry cells infected with lenti-NF-Gluc were cultured under different conditions, as a monolayer on plastic (top left), or on Matrigel-coated plates in basal medium only (EBM) (top right), or basal medium supplemented with a cocktail of angiogenic factors (EGM) (bottom right), or with U87-CFP glioma cells (bottom left) [size bar, 300 μm]. Images are obtained 24 h post-culture (b) Twenty-four h after culturing, Gluc activity was assayed in the conditioned medium. Data is presented as average of fold increase in Gluc activity as compared to control monolayer culture ± S.D (n=4). *p≤0.05, student t-test.

In vivo blood monitoring of NFkB activity

Recently, we have shown that the level of Gluc in the blood of experimental animals bearing cells expressing this reporter under a constitutively active promoter can be used as a marker for cell viability and proliferation (14). We therefore hypothesized that under the NFkB TREs, the Gluc expression should reflect the physiological state of NFkB activation. Hence, we used our reporter construct for in vivo assessment of NFkB activity by monitoring Gluc activity in the blood. HEI193 human schwannoma cells expressing either NF-Gluc or Fluc-mCherry as a control were implanted subcutaneously (s.c.) in nude mice. One week later, 10 μl aliquots of blood were collected and assayed for Gluc activity. In parallel, the Gluc activity in tumors was quantified by in vivo bioluminescence imaging using the CCD camera after intravenous (i.v.) injection of coelenterazine. Mice were then i.v. injected with 100 μl of TNFα (16 μg/kg body weight) or PBS control and blood was collected at different time points. An increase in Gluc activity in the blood was observed starting at 4 h after TNFα injection and reaching a maximum at 16 h followed by a gradual decrease to the basal level 36 h post-treatment (Fig. 5a). In parallel, the mice were also imaged with the CCD camera for Gluc expression 12 h and 48 h after TNFα injection. An increase in Gluc expression was also detected by in vivo bioluminescence imaging 12 h post-treatment followed by a decrease to the basal level 48 h later, which correlated with the Gluc-blood level (Fig. 5b>). To investigate whether the NF-Gluc reporter could be used to monitor NFkB inhibition by SSZ in vivo, similar mice bearing xenografts of HEI193-NF-Gluc cells were treated with either SSZ (15 mg/kg body weight), or TNFα and SSZ. A 2.5-fold decrease in NFkB basal level in blood was detected upon SSZ treatment. Moreover, SSZ inhibited the NFkB induction in HEI193 tumors after TNFα treatment (Fig. 5a). To confirm these results in another cell type, we implanted subcutaneously 293T cells expressing either NF-Gluc or Fluc-mCherry as a control, and treated mice with similar doses of TNFα, SSZ alone, or a combination of both. Similar induction of Gluc expression in response to TNFα-mediated NFkB activation and a similar decrease in Gluc activity upon treatment with either SSZ alone or TNFα and SSZ was observed (data not shown).

Figure 5.

In vivo monitoring of NFkB activation and inhibition in real-time. (a) One million HEI193 cells were implanted s.c. in nude mice. One week later, mice were injected i.v. with either TNFα (16 μg/kg of body weight), TNF + SSZ (15 mg/kg of body weight), SSZ only or PBS (control). At different time points, 5 μl of blood was withdrawn and assayed for Gluc activity. (b) Bioluminescence obtained from HEI193-NF-Gluc cells using a CCD camera at time 0, 12 and 48 h post-treatment. Data presented as RLU/sec ± SD (n=5) with CCD image of one representative mouse of each group.

Monitoring of NFkB activation in monocytes

NFkB plays a critical role in regulated expression of a large variety of genes involved in immune and inflammatory responses, such as cell adhesion molecules, chemokines and cytokines (43). Some cytokines, such as TNFα directly activate NFkB in order to amplify the primary inflammatory response. Monitoring of NFkB activation in vivo may contribute to a better understanding of the innate immune response activation. We transduced U937 human leukemic monocyte lymphoma cells with lenti-NF-Gluc. Initially, these cells were exposed to different amounts of TNFα in culture. At different time points, aliquots of the conditioned medium were assayed for the Gluc activity. A dose-dependent increase in Gluc expression in the treated U937 cells compared to non-treated cells indicated that NFkB was activated by up to 8-fold (24 h post-treatment) in response to TNFα (Fig. 6a). To corroborate these findings in an in vivo model, U937 cells expressing NF-Gluc were injected i.p. and 1 h later mice were injected with either PBS or 80 μg/kg body weight of TNFα. in a similar route. Before TNFα injection and at different time points after injection, 5 μl aliquots of blood was withdrawn and assayed for Gluc activity. The Gluc blood level measured in response to NFkB activation showed a maximum at 24 h post-TNFα injection (8-fold), after which the signal decreased to the basal level (48 h later), suggesting a transient induction of NFkB activation. At no time point did the PBS control group show any change in Gluc blood activity (Fig. 6b). Also, prior to TNFα injection and 24 h post-injection, mice were imaged with the CCD camera after i.v. injection of coelenterazine. At neither time points there was any positive signal with the CCD camera supporting the high sensitivity of the Gluc blood assay as compared to in vivo bioluminescence imaging in monitoring NFkB activation in dispersed monocytes. The NF-Gluc reporter together with the Gluc blood assay provide a means to monitor NFkB activation in immune cells, which can serve as a tool to study the dynamics of immune activation in subsets of immune cells in small animals.

Figure 6.

Monitoring of NFkB activation in monocyes. (a) U937 cells expressing NF-Gluc were treated with different concentrations of TNFα. At different time points, the Gluc activity was assayed in 10 μl of conditioned medium. (b) U937-NF-Gluc cells were injected i.p. and 1 h later, mice were injected with either PBS (control) or TNFα (80 μg/kg of body weight) in the same route before and at different time points after treatment, Gluc activity was monitored in 20 μl blood. (b) CCD camera images obtained before and 24 h after TNFα injection. Data shown are average RLU/sec ± SD (n=6).

Dual blood monitoring of NFkB activity and cell growth during tumor development

Since NFkB is involved in tumor progression (12), we sought to look for NFkB activation during tumor development. Gli36 human glioma cells were co-transduced with lenti-NF-Gluc, lenti-Fluc-mCherry and lenti-SEAP and implanted subcutaneously in mice. Before and at different time points post-implantation, serum was collected and assayed for Gluc or SEAP activity as an index for NFkB activation and cell growth, respectively. An increase in Gluc signal and therefore NFkB activation was observed over time, which correlated with an increase in SEAP signal, a marker for tumor cells proliferation (Fig 7a). In parallel, 5 μl of urine was collected and also assayed for Gluc activity which showed similar increase in Gluc expression proving that the Gluc level in urine can also be used as an index of NFkB activation (Fig. 7b). In another experiment, mice were implanted with different amount of these cells. One week later, tumor volume was monitored using in vivo Fluc bioluminescence imaging after i.p. injection of d-luciferin and serum were assayed for Gluc or SEAP activity. The Gluc level in serum as a marker for NFkB activation correlated with serum SEAP level, which in turn correlated with tumor volume as assessed by in vivo bioluminescence imaging (Fig. 7c). On the other hand, when these tumor cells were injected i.v., an increase in Gluc value (~7-10-fold) was observed in serum 2 h post-injection indicating an immediate activation of NFkB in these cells which than dropped back to basal level (near SEAP signals) 18 h later (Fig.7d).

Figure 7. Dual-monitoring of tumor formation and NFkB activation.

One million Gli36 human glioma cells expressing Fluc, NF-Gluc and SEAP were implanted subcutaneously in nude mice. (a-b) At different time points, blood or urine were withdrawn and serum was assayed for either Gluc or SEAP activity (a) and urine for Gluc activity (b). (c) one week post-implantation, tumor volume was imaged with in vivo Fluc bioluminescence imaging using the CCD camera and Gluc and SEAP serum levels were assayed as in (a). (d) Gli36 cells expressing NF-Gluc and SEAP were injected i.v. and the serum Gluc and SEAP activity was monitored at different time points as in (a).

DISCUSSION

We have developed a novel NFkB reporter system based on the naturally secreted Gaussia luciferase (14, 17). The NF-Gluc reporter proved to be a useful tool for sensing both NFkB activation and inhibition in different models including tumors, angiogenesis, and inflammation. Moreover, the Gluc reporter allowed monitoring of NFkB activity by measuring its level in an aliquot of the conditioned medium of cultured cells or in blood or urine of animals at sequential time-points. Further, in the context of concentrated local NF-Gluc reporter in vivo, the Gluc signal can be confirmed and localized using in vivo bioluminescence imaging.

NFkB is a ubiquitous transcription factor that plays a critical role in regulating expression of a large variety of genes involved in immune and inflammatory responses, such as cell adhesion molecules, chemokines and cytokines, but also genes controlling other biological processes such as cell survival, apoptosis and differentiation. Activation of this transcription factor is associated with several physiological disorders, notably inflammation and cancer. Monitoring of NFkB activation may contribute to a better understanding of such biological processes and pathological conditions. Several reporter systems have been used to study the expression and activation of NFkB. Previous studies have used NFkB responsive elements driving the expression of LacZ (44, 45) Fluc (46), eGFP (47) or SEAP (48) reporter genes. Although these systems proved to be useful in detecting NFkB activation, they may each have several disadvantages in the context of in vivo analysis and sequential monitoring of NFkB activity as compared to the NF-Gluc reporter developed here. Fluc requires d-luciferin injection before imaging, thus a total clearance of the substrate is essential before the next imaging session can be started, complicating the monitoring of NFkB activation kinetics, unless using a pump system which continuously delivers the substrate to the animal (49). In addition, anesthetizing the animals can be a major limitation during kinetic studies, requiring multiple imaging sessions over a short period of time or during studies in which the animal needs to stay awake. The eGFP reporter gene is partly compromised by its relatively low sensitivity, and in most cases requires animal sacrifice before fluorescent analysis, or alternatively, the use of more elaborate techniques such as epi-fluorescence microscopy. A high signal to noise ratio due to auto-fluorescence in many organs decreases dramatically the sensitivity of in vivo fluorescence imaging. LacZ staining requires animal sacrifice and tissue sectioning before analysis. SEAP overcomes most of these problems, however we recently showed that this reporter is 20,000-fold less sensitive than Gluc in cultured cells with a linear range with respect to cell number covering <3 orders of magnitudes (50). Further, the SEAP assay requires several different incubation steps before analysis making this assay more laborious and time consuming. On the other hand, the Gluc blood/urine assay is simple and short requiring only the addition of its substrate coelenterazine and luminometer analysis, and has a linear range of over 5 orders of magnitude with respect to cell number (14).

The NFkB transcription factor is known to be activated in many cancer types including lung, ovarian, astrocytomas, melanoma, prostate adenocarcinoma, and glioblastoma, and was shown to correlate with disease progression (6, 7, 32-34, 36, 37, 51). Therefore, this reporter could be used to identify and monitor potential chemotherapeutics based on interference or potentiation of NFkB activity in vitro and in vivo. Since NFkB has been shown to be involved in tumor angiogenesis and invasiveness (12), the NF-Gluc reporter can also be used as a sensor for tumor angiogenesis. The neovascularization process occurs through a series of organized steps, which is initiated by remodeling of the extracellular matrix. NFkB plays a major role in this remodeling process by inducing expression of various genes such as VEGF, plasminogen activator inhibitors and matrix metalloproteinases in endothelial cells (52-54). Further, NFkB plays a major role in migration and invasion of endothelial cells (55, 56), therefore the NF-Gluc reporter could be used to determine the kinetics of NFkB activation during the various steps of angiogenesis.

Besides cancer, identifying and testing of novel drugs that interfere with NFkB activity can be important for the treatment of a number of disorders, including neurological disorders (57), rheumatoid arthritis (58), inflammatory bowel disease (59) and asthma (60). The NF-Gluc reporter may also prove useful for high-throughput drug screening for NFkB activators and inhibitors, which subsequently can be validated in vivo using the NF-Gluc-blood assay. Not many systems are available such as NF-Gluc, allowing both in vitro screening and kinetic analysis as well as in vivo validation of a relatively large number of drugs in a short period of time. Moreover, this reporter system is easy to implement, is cost and time-effective, and can be extended to study other physiologically regulated transcription elements, such as cAMP, p53, ISRE, TARE, and SRF, making it a versatile tool for studying transcriptional activation in vitro and in vivo in a non-invasive and quantitative manner.