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

Abstract

Nuclear factor kappaB (NF-kappaB) is a transcription factor that plays a major role in many human disorders, including immune diseases and cancer. We designed a reporter system based on NF-kappaB responsive promoter elements driving expression of the secreted Gaussia princeps luciferase (Gluc). We show that this bioluminescent reporter is a highly sensitive tool for noninvasive monitoring of the kinetics of NF-kappaB activation and inhibition over time, both in conditioned medium of cultured cells and in the blood and urine of animals. NF-kappaB 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 NF-kappaB activation during tumor development as correlated to tumor formation using the NF-kappaB Gluc reporter, as well as the secreted alkaline phosphatase reporter. This NF-kappaB reporter system provides a powerful tool for monitoring NF-kappaB activity in real time in vitro and in vivo.

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.

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.

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.

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.

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.

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).

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).