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. 2009 Aug 4;17(10):1703–1711. doi: 10.1038/mt.2009.171

A Novel Estrogen Receptor Intramolecular Folding–based Titratable Transgene Expression System

Ramasamy Paulmurugan 1,2, Parasuraman Padmanabhan 1,2, Byeong-Cheol Ahn 1,2,3, Sunetra Ray 1,2, Juergen K Willmann 1,2, Tarik F Massoud 1,2,4, Sandip Biswal 1,2, Sanjiv S Gambhir 1,2,5,6
PMCID: PMC2835012  PMID: 19654568

Abstract

The use of regulated gene expression systems is important for successful gene therapy applications. In this study, ligand-induced structural change in the estrogen receptor (ER) was used to develop a novel ER intramolecular folding–based transcriptional activation system. The system was studied using ER-variants of different lengths, flanked on either side by the GAL4-DNA-binding domain and the VP16-transactivation domain (GAL4DBD-ER-VP16). The ER ligands of different types showed efficient ligand-regulated transactivation. We also characterized a bidirectional transactivation system based on the ER and demonstrated its utility in titrating both reporter and therapeutic gene expression. The ligand-regulated transactivation system developed by using a mutant form of the ER (G521T, lacking affinity for the endogenous ligand 17β-estradiol, whereas maintaining affinity for other ligands) showed efficient activation by the ligand raloxifene in living mice without significant interference from the circulating endogenous ligand. The ligand-regulated transactivation system was used to test the therapeutic efficiency of the tumor suppressor protein p53 in HepG2 (p53+/+) and SKBr3 (p53−/−/mutant-p53+/+) cells in culture and tumor xenografts in living mice. The multifunctional capabilities of this system should be useful for gene therapy applications, to study ER biology, to evaluate gene regulation, ER ligand screening, and ER ligand biocharacterization in cells and living animals.

Introduction

Gene- and cell-based therapies hold significant potential in treating several genetic as well as noninherited disorders. Although a significant level of progress has been made to achieve efficient, less toxic, and less immunogenic gene delivery vehicles to facilitate long-term expression of delivered transgenes, titration of expression still remains a key issue.1,2 As gene therapy research continuously progresses, the need for regulatable gene expression systems becomes evermore apparent. An efficient regulatable gene expression system should have the ability to control the level of expressed transgenes in a dose-dependent manner in response to externally administered pharmacological agents. In addition, the regulatable gene expression system should also have no or very low levels of transgene expression before administering any activators/regulators. The use of regulatable gene expression systems are not only restricted to gene therapy applications, but they should also be useful for different functional genomic studies.3

Several early regulatable gene expression systems were developed by using promoters responsive to naturally occurring physical and chemical stimuli such as heat, electric, light, and heavy metal–inducible promoters.4,5,6,7 Although all these natural promoters perform reasonably well in controlling the levels of transgene expression, adopting them for mammalian gene therapy applications is difficult because of the potential hazardous effects associated with their activators/regulators. To overcome this, combinations of elements derived from prokaryotic and eukaryotic systems were used for developing regulatable gene expression systems. Systems in use include the tetracycline-,8,9 mifepristone-,10 ecdysone-,11 rapamycin-, and tamoxifen-regulated system (Gal4-VP16-ER and VP16-Gal4-ER),12 and the ligand-activated site-specific recombination system (Cre-ER).13 Even though all these systems show significant control over transgene expression in response to externally administered pharmacological agents, most of them suffer from high levels of background signal prior to exposure to activators/regulators.14

The human estrogen receptor (ER) is a key modulator of reproductive functions in females in response to its endogenous ligand 17β-estradiol. The ER is activated upon binding of estrogen and a wide variety of other chemicals.15 The binding of hormones or other chemical ligands to the ligand-binding domain (LBD) of ER results in a series of molecular events that include ligand-induced structural change.16,17 The LBD of ER is folded into a three-layered antiparallel α-helical sandwich comprising a central core layer of three helices (H5/6, H9, and H10), a small two-stranded antiparallel β-sheet (S1 and S2) and helix 12 (ref. 16,18). The helix 12 is mainly located in the ligand-binding pocket and determines the structural conformation of ER in response to ligands. The structures of LBD complexes with either the ligand 17β-estradiol or raloxifene show different conformations in helix 12 (ref. 19), despite both ligands binding at the same site within the core of the LBD.15 Previously, we took advantage of the occurrence of these varying conformations in the ER by using split reporter protein complementation and studied ER intramolecular folding to differentiate between various agonists/antagonists in cell culture and in living mice.20

In the current study, we made use of the property of ligand-induced conformational changes in ER that specifically bring the N- and C-termini closer to each other, in combination with the HSV1-VP16 transactivator and the yeast DNA-binding domain (GAL4DBD), to develop a novel ligand-regulated transactivation system (Figure 1b). A mutant form of ER (G521T) that specifically shows no affinity for the endogenous ligand 17β-estradiol was also used in order to extend the application of this current system in living animals without much interference from circulating endogenous ligand. This strategy was validated using a bidirectional system in which two genes can be titrated jointly. The bidirectional system was further applied to study the therapeutic efficiency of the biologically important tumor suppressor protein p53 along with the reporter gene firefly luciferase in cells and tumor xenografts in living animals.

Figure 1.

Figure 1

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Schematics of the constitutive and the ER ligand–regulated transactivation systems. (a) In the constitutive transactivation system, GAL4DBD is directly fused to the VP16 to express a fusion protein GAL4DBD-VP16. The GAL4DBD-VP16 binds to specific DNA sequence in the co-delivered reporter plasmid vector p(GAL4DNA)5-E4TATA-FLUC and the VP16 activates the minimal promoter (E4TATA) adjacent to it. (b) In the ligand-regulated transactivation system, the gene coding for the yeast GAL4-DNA-binding domain (DBD) is fused to the VP16-transactivation domain through a ligand-binding domain of ER [ER(LBD)] amino acids 281–595 to express a fusion protein GAL4DBD-ER(LBD)-VP16. The GAL4DBD from the expressed fusion protein binds to a specific DNA sequence present in the co-delivered reporter plasmid vector. The VP16 portion of the fusion protein is kept away from the minimal promoter (E4TATA) by the ER before binding to ligand 17β-estradiol. When the cells are exposed to ligand 17β-estradiol, it binds with the ER portion of the fusion protein and leads to conformational change in the ER(LBD) that brings the VP16 closer to the minimal promoter that in turn activates gene expression. (c) Comparison of the ER ligand–regulated and the constitutive transactivation systems in a transiently co-transfected 293T cells. The 293T cells co-transfected with the reporter plasmid and the activator plasmid expressing either GAL4DBD-ER(LBD)-VP16 or GAL4DBD-VP16 were assayed for activated Fluc activity before and after exposure to 1 µmol/l of ligand 17β-estradiol. The ligand-regulated transactivation system shows significant (P < 0.001) levels of luciferase activity only when cells are exposed to 17β-estradiol. The error bars are the SEM of triplicate determinations. ER, estrogen receptor; RLU, relative light units; VP16, transactivator domain of herpes simplex viral protein 16.

Results

Design of an ER intramolecular folding–based ligand regulatable gene activation system

An ER ligand–regulated transactivation system was developed by constructing a series of plasmid vectors that constitutively expresses fusion protein chimeras containing the yeast GAL4-DNA-binding domain (GAL4DBD), human herpes simplex virus type 1 transactivator peptide (HSV1-VP16), and an ER-LBD of different lengths (GAL4DBD-ER(LBD)-VP16) (Figure 1b). These vectors were used in combination with a reporter plasmid vector flanking five time repeats of a yeast GAL4-binding nucleotide sequence [(GAL4DNA)5], E4 minimal promoter (E4TATA) derived from adenovirus, and the reporter gene firefly luciferase (FLUC) [(GAL4DNA)5-E4TATA-FLUC], in different co-transfection experiments. A vector constitutively expressing the fusion protein containing the GAL4DBD directly fused to the HSV1-VP16 (GAL4DBD-VP16) (constitutive transactivation system) was used as a control system (Figure 1a). Both these systems were subsequently studied in cultured cells and cell implanted xenografts in mouse models. Comparison of the ER ligand–regulated transactivation system with the constitutive transactivation system confirms the inducible nature of the ligand-regulated transactivation system (Figure 1c). The constitutive transactivation system showed luciferase signal both in the presence and the absence of 17β-estradiol as expected (statistically not significant), but the cells co-transfected with the ER ligand–regulated transactivation system showed activated firefly luciferase (Fluc) signal only after the cells were exposed to 17β-estradiol (P < 0.001).

The evaluation of ER ligand–regulated transactivation system

The efficiency of the ER ligand–regulated transactivation system was additionally evaluated with minimal promoters of different nucleotide sequences originated from adenovirus [adenoviral early (AdE) and adenoviral late (AdL)] using different concentrations of the ligand 17β-estradiol and also in different cell lines. The comparison of the ER ligand–regulated transactivation system with the adenoviral early [(GAL4DNA)5-AdE-E4TATA-FLUC] and late minimal [(GAL4DNA)5-AdL-E4TATA-FLUC] promoters showed that the late promoter had significantly increased efficiency (Supplementary Figure S1a). The transiently co-transfected 293T and CHO cells with the system showed a specific ligand (17β-estradiol) concentration–dependent increase in the level of assayed Fluc signal by both the cells (Supplementary Figure S1b).

The ER ligand–regulated transactivation system responds to different ER ligands tested in this study

In addition to the ligand concentration–dependent activation of gene expression by 17β-estradiol, the system was also studied for its response to several other ER ligands (agonists and antagonists). A known anticancer drug cisplatinum reported with no binding affinity for ER was used as a control. The results showed a significant (P < 0.001) level of activated Fluc expression by all ER ligands used for the study. Even though all ER ligands induced the expression of luciferase gene, the absolute level of Fluc activity calculated for each ligand was different (see Supplementary Figure S2a for details). To confirm the specificity of the system in response to different ER ligands, western blot analysis of different samples were performed to detect activated Fluc, GAL4DBD-ER(LBD)-VP16, and the control α-tubulin proteins. The result confirmed that the increase in the level of reporter protein expression was not due to the increase in the expression level of GAL4DBD-ER(LBD)-VP16 protein. The increase was primarily due to the change in the folding pattern of ER(LBD) in response to ligand binding and the subsequent activation of luciferase gene expression through the activator peptide VP16 (Supplementary Figure S2b).

The ER ligand–regulated transactivation system in a bidirectional vector shows efficient control in regulating the level of transgene expression in both directions. Next, we extended the use of the ligand-regulated transactivation system in regulating two different reporter genes expressed in a single bidirectional vector that can eventually be used for indirectly monitoring the efficiency of nonreporter therapeutic genes in gene therapy studies. The plasmid vector expressing the activator fusion protein chimera GAL4DBD-ER(LBD)-VP16 was used along with a bidirectional vector previously developed21 that expresses firefly and renilla luciferase genes [FLCU-E4TATA-(GAL4DNA)5-E4TATA-RLUC]. The co-transfected 293T cells were assayed for both firefly and renilla luciferase activities after exposure to increasing concentrations of 17β-estradiol (0–1 µmol/l). The result showed a linear ligand concentration–dependent increase in the levels of both the reporter proteins (R2 = 0.994) (Figure 2a,b).

Figure 2.

Figure 2

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The efficiency of ER ligand–regulated transactivation system in controlling the levels of two transgenes expressed in a bidirectional vector in two different orientations. (a) The 293T cells co-transfected with the bidirectional reporter plasmid [FLUC-E4TATA-(GAL4DNA)5-E4TATA-RLUC] and the activator plasmid expressing GAL4DBD-ER(LBD)-VP16 fusion protein were assayed for renilla and firefly luciferase activity after exposure to 12 different concentrations of 17β-estradiol (0–1.5 µmol/l). The result shows ligand concentration–dependent increase in the expression level of both firefly and renilla luciferases. (b) The result shows significant correlation (R2 = 0.994) between the ligand concentrations, and the level of expressed renilla and firefly luciferases. The error bars are the SEM of triplicate determinations. DBD, DNA-binding domain; ER, estrogen receptor; LBD, ligand-binding domain; VP16, transactivator domain of herpes simplex viral protein 16.

The ER ligand–regulated transactivation system studied with the activator fusion protein without the F-domain (GAL4DBD-ER281–549-VP16) and with the mutant-ER (ERG521T). From the previous study, we found that an ER-LBD of different lengths (with or without the F-domain) can yield different degrees of intramolecular folding–based complemented luciferase signal for various ligand agonists and antagonists.20 A similar strategy was adopted in this ER ligand–regulated transactivation system, which produced a similar pattern of results to those expected. We constructed an activator plasmid expressing the fusion protein chimera GAL4DBD-ER(LBD)-VP16 containing the ER-LBD without the F-domain (amino acids 281–549 instead of 281–595). The system, transiently co-transfected 293T cells assayed for luciferase activity after exposure to 1 µmol/l concentration of different ER ligands (agonists: 17-β estradiol and diethylstilbestrol; antagonist: ICI 182,780; and selective estrogen receptor modulators: tamoxifen, raloxifene, and 4-hydroxytamoxifen), showed a significantly greater level of induction by selective estrogen receptor modulators as compared to agonists (P < 0.01). The system also distinguished ER ligands based on their properties (Figure 3a,b). To extend the use of the ER ligand–regulated transactivation system in living animals, the expected problem of competitive binding from the endogenous ligand 17β-estradiol was considered, therefore used a mutant form of ER (ERG521T) identified from our previous study20 that specifically shows no affinity for the endogenous ligand 17β-estradiol. The system constructed with the activator plasmid containing the mutant form of the ER in transiently transfected 293T cells showed no ligand-induced Fluc activity in the presence of 17β-estradiol as compared to various other ER ligands (Figure 3c). As ER-LBD of amino acids 281–549 can produce variable activation with different ligands, therefore the remaining studies were performed with the activator plasmid expressing the fusion protein containing the LBD of amino acids of 281–549.

Figure 3.

Figure 3

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Evaluation of the ER ligand–regulated transactivation system with the ER(LBD) of amino acids 281–549. (a) Schematic diagram of reporter and the activator plasmids used in this study. (b) The 293T cells co-transfected with the reporter plasmid (GAL4DNA)5-E4TATA-FLUC and the activator plasmid expressing GAL4DBD-ER(LBD281–549)-VP16 were assayed for Fluc activity after exposure to 1 µmol/l of different ligands. The system shows ligand-dependent activation of reporter gene expression that can distinguish ER ligand agonists from SERMs. The error bars are the SEM of triplicate determinations. (c) The ER ligand–regulated transactivation system with the mutant-ER(LBD). To extend the application of the ER ligand–regulated transactivation system in living animals, the activator plasmid containing the mutant form of ER(LBDG521T) that has very low affinity for the endogenous ligand 17β-estradiol was studied in transiently co-transfected 293T cells. The cells were assayed for Fluc activity after exposure to different ER ligands (DES, OHT, Tam, and Ral) along with the endogenous ligand 17β-estradiol. The error bars are the SEM of triplicate determinations. ER, estrogen receptor; LBD, ligand-binding domain; SERMs, selective estrogen receptor modulators; VP16, transactivator domain of herpes simplex viral protein 16.

The ER ligand–regulated transactivation system shows ligand-specific induction of luciferase signal in living mice by optical CCD camera imaging. To study the ligand-regulated transactivation system in living mice, 293T cells transiently co-transfected with the reporter plasmid, and the activator plasmid expressing the fusion protein chimera containing the mutant form of ER (GAL4DBD-ERG521T/281–595-VP16) were subcutaneously implanted in living female nude mice. Similarly, 293T cells co-transfected with the constitutive active system (GAL4DBD-VP16) were used as a control. The animals (n = 4 each for control and experiment groups) were imaged at 24-hour intervals by the administration of the ligand raloxifene every alternate day only to the experimental animals. The results showed no Fluc signal immediately after implantation from either of the cell populations. After 24 hours, the group of animals that received the ligand raloxifene showed significant levels of Fluc signals compared to the control group (P < 0.001). The site implanted with the cells transfected with the constitutively active system showed Fluc signal both before and after administration of the ligand raloxifene, in both the experimental and control groups. The system showed efficient ligand-regulated transactivation of reporter gene expression in mice implanted with 293T cells as imaged by the optical CCD camera. The level of signal achieved before induction was not significantly different from background. When induced with 0.5 mg (20 mg/kg body weight) of raloxifene, the luciferase signal increased 15 ± 5-fold (P < 0.05) as compared to the noninduced group. The intermittent injection of raloxifene showed the ability of the system to respond to the ligands in living mice (Figure 4).

Figure 4.

Figure 4

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Imaging of ER ligand–regulated transactivation in living animals. (a) Mice (n = 4) implanted with 5 million 293T cells transiently co-transfected with reporter plasmid (GAL4DNA)5-E4TATA-FLUC and the activator plasmid expressing fusion protein GAL4-ERLBD/G521T-VP16 (site A), and 5 million 293T cells transiently co-transfected with reporter plasmid (GAL4DNA)5-E4TATA-FLUC and the activator plasmid expressing fusion protein GAL4-VP16 (site B) were imaged immediately after implantation, and every 24 hours with or without injecting the ligand raloxifene (20 mg/kg body weight). A significant (P < 0.05) level of reporter gene expression was observed from site A implanted with the ER ligand–regulated transactivation system only when the animals receive raloxifene. Site B implanted with the cells expressing the constitutively active system show luciferase signal both in the presence and the absence of raloxifene. (b) Quantitative analysis of result from the animals studied at different conditions. The error bars are the SEM of four determinations. ER, estrogen receptor; LBD, ligand-binding domain; LBDF, ligand-binding domain with F-domain; VP16, transactivator domain of herpes simplex viral protein 16.

Comparison of the ER ligand–regulated transactivation system with the TET-ON system

To further scrutinize the efficiency of the ER ligand–regulated transactivation system, the well-studied TET-ON system was selected for comparison. Both these systems were studied in transiently co-transfected 293T cells. The transfected cells were assayed for luciferase activity after exposure to increasing concentrations of the respective ligands [ER ligand, OHT (10−7 to 100 µmol/l) for the ER ligand–regulated transactivation system, and doxycycline (4 × 10−6 to 4 × 101 µg/ml) for the TET-ON system]. The results showed ligand concentration–dependent increase in the luciferase signal by both the systems (Supplementary Figure S3). The ER ligand–regulated transactivation system showed a significantly (P < 0.05) lower level of background luciferase signal when compared to the TET-ON system before exposure to their respective ligands (Supplementary Figure S3 inset). We observed similar findings from HepG2, SKBr3, NIH3T3, and CHO cells in transient transfection experiments (data not shown).

Application of ER ligand–regulated transactivation system to regulate the expression level of a reporter (Fluc) and a therapeutic gene (p53) in a bidirectional vector to indirectly monitor the therapeutic efficiency of p53 in living animals. To evaluate the efficiency of the ER ligand–regulated transactivation system in a biological application particularly relevant to cancer research, a bidirectional vector was constructed to express a reporter (Fluc) and a therapeutic gene (p53), so as to indirectly monitor p53-mediated cell cycle arrest, apoptosis, and cell death, by imaging luciferase expression (Figure 5a). The system was studied in stably co-transfected SKBr3 (expressing endogenous mutant-p53) and HepG2 (expressing endogenous wt-p53) cells. The cells were confirmed for the stably integrated bidirectional vector by PCR amplification of the Fluc DNA from the extracted genomic DNA (Figure 5b). The cells were studied by induction with different ER ligands (E2, OHT, RAL, DES, DPN, MPP, PPT, and GEN) and also with different concentrations of the ligand OHT. The ligand-mediated activation of Fluc expression was assessed by luminometry and optical CCD camera imaging, and p53 expression by western blot analysis (Figure 5c, Supplementary Figures S4 and S5). The therapeutic effect of p53 was assessed by fluorescence-activated cell sorting analysis of cells after propidium iodide staining, Trypan blue exclusion assay, and the activated signal proteins p21 and MDM2 by western blot analysis (Supplementary Figures S5–S7). The results showed a significant (P < 0.001) level of induction of both Fluc and p53/mutant-p53 expression, as measured by optical imaging and western blots, respectively. There was a significant level of p53-induced cell death in both HepG2 and SKBr3 cells (P < 0.01). The endogenous p53+ HepG2 cells transfected to overexpress mutant-p53 showed no cell death, but the SKBr3 cells expressing endogenous mutant-p53 showed a significantly higher level of cell death (P < 0.01). To further demonstrate the ligand-induced expression of p53 and the associated cell death, Molecular Probes Live (green) and Dead (red) cell staining kit was used. The results showed good correlation with the induced p53 level and the associated dead cells stained by the kit (Figure 5d). A significant percentage (40–50%) of cells died in the first 24 hours, and up to 90% did so within 72 hours (Supplementary Figures S6–S8).

Figure 5.

Figure 5

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Bidirectional ER ligand–regulated transactivation system express reporter gene Fluc and therapeutic tumor suppressor gene p53. (a) Schematics of reporter and the activator plasmids used for the study. (b) PCR confirmation of HepG2 cells stably co-transfected with the reporter and the activator plasmids for the integrated DNA. (c) ER ligand–induced activation of p53 and Fluc in stable HepG2 cells assayed by western blot and optical CCD camera imaging. (d) HepG2 cells stably expressing either wt-p53 or mutant-p53 assayed for p53-mediated apoptotic cells by Molecular Probes Live (green) and Dead (red) cell staining kit 24 hours after induction with ligand OHT. (e) Imaging of ER ligand–induced activation of p53 protein expression in HepG2 tumor xenograft model. A representative mouse from group of animals imaged on day 2, 5, 10, and 20 after induction with 0.5 mg of ligand raloxifene. The data across several mice (n = 10 each for wt-p53- and mutant-p53-induced group; n = 6 each for wt-p53- and mutant-p53-uninduced group) were obtained after imaging for a period of 20 days. The results showed wt-p53-mediated tumor regression as measured by tumor size and their associated drop in the Fluc signal (site B). The results across several mice are presented as a graph in Supplementary Figure S11. The tumor xenograft from cells expressing mutant-p53 was used as control (site A). ER, estrogen receptor; VP16, transactivator domain of herpes simplex viral protein 16.

ER ligand–regulated transactivation system in a bidirectional vector to monitor the therapeutic efficacy of p53 in living animals by imaging luciferase expression. The HepG2 cells stably transfected with the ER ligand–regulated transactivation system and the bidirectional vector expressing p53 and Fluc were used for monitoring p53-mediated therapeutic efficiency in tumor xenograft models in mice. These stable cells were in vitro characterized for their response (Supplementary Figures S4–S9). The cells were then studied by creating tumor xenografts in nude mice. The animals were imaged every 24 hours after induction with the ligand raloxifene (20 mg/kg body weight) once every 48 hours over a period of 20 days. The scheme of experiments used was as indicated in the flow chart (Supplementary Figure S10). The results showed a significant (P < 0.05) level of Fluc induction and p53-induced tumor regression in mice implanted with tumor xenografts expressing wild-type p53 but not with mutant-p53 (Figure 5e and Supplementary Figure S11). The group of mice that periodically received the ligand raloxifene showed an increase in the luciferase signal during the complete period of imaging, with tumor growth–associated net increase in the Fluc signal from the xenografts expressing mutant-p53. However, in the same animals, the tumor xenograft–expressing p53 showed a Fluc signal increase in the first 4 days, followed by a subsequent decrease. An independent measurement of tumor size showed a consistent regression (Supplementary Figure S11).

Discussion

Despite the current use of several regulatable gene expression systems in different biological applications,2 only the tetracycline-regulated system has been successfully extended to preclinical experiments,22 and there are no examples of regulatable systems that have been employed in clinical trials.22 A steroid hormone receptor–based regulatory system would be promising for gene therapy applications because endogenous steroids easily cross epithelial barriers and plasma membranes.1 The steroid hormones or their analogues bind to their receptors in the cytoplasm and these ligand–receptor complexes are then translocated to the nucleus where they regulate gene expression.23 The key advantage of steroid hormone receptors is that the major components of this system are modified human proteins and therefore should not be potent activators of the immune system.1,2

In this study, we successfully adopted steroid/steroid–receptor interactions to develop a regulatable gene expression system by using the ER, one of the most studied receptors among the steroid receptor superfamily of receptors. From a prior study of ours using a split Fluc complementation system, we found that the ER can undergo a large change in its protein structural conformation when it binds to ligands.20 This folding leads to a change in the distance between the NH2- and COO- terminals of this receptor protein. This property was made use of in the conception and development of the current ligand-regulated transactivation system in which a plasmid vector was constructed to express the fusion protein GAL4DBD-ER(LBD)-VP16 that folds in response to the binding of ligands and activates gene expression through the VP16-transactivator (Figure 1a).

The ER-transactivation system was studied by expressing the Fluc reporter gene in different cell lines and in living mice implanted with cells transfected to express the system. The system studied in transiently co-transfected 293T cells in the presence of 17β-estradiol showed approximately twice the expression levels of those in the corresponding constitutive expression system. The expression level of transgene in both the systems described (constitutive and ER ligand regulated) in this study is not regulated at the promoters' level. It is mainly controlled by the amount of activators (Gal4-VP16/Gal4-ER-VP16) and their binding efficiencies to the Gal4-DNA sequence, and also the folding pattern in which the VP16 portion of the fusion activator protein is positioned to the minimal promoter (E4) used in the study. Somehow the ER favors the VP16 portion of the Gal4-ER-VP16 fusion protein, to allow better positioning when binding with the ligand estradiol. There are several commercially available drugs that can serve as ligand agonists or antagonists in modulating the ER and other steroid receptors.16,24,25 Therefore, the system has the advantage of being inducible with several ligands each of which has its own range of concentrations leading to different signal levels. The study of this system in living animals showed efficient ER ligand–regulated transactivation of reporter gene expression, and also followed a rapid “on” and “off” switch mechanism as demonstrated by a decrease of the activated Fluc signal a few hours later when no further dose of ligand was injected, owing to a decrease in the concentration of the ligand inducer (Figure 4). To overcome the issue of activation by endogenous ligands, the mutant-ER (G521T), developed from our previous study that has selectively no binding affinity for the endogenous ligand 17β-estradiol (Figure 3c), was used in this system.

A few limitations associated with steroid hormone receptor–based regulatory systems are the physiological changes in the level of endogenous ligand that may affect the expression level of the transgene, and the activation of endogenous ER in nontarget cells by ligand inducers used for controlling the transgene expression. Several groups have successfully designed strategies to overcome some of these problems.10,26,27 Moreover, it is also possible to use nonmammalian steroid receptors using the intramolecular folding strategy from the current study, in combination with the development of a synthetic compound that could potentially solve many of these problems.

We also explored the properties of the ER and modified the system in such a way that it can differentiate ER ligands as agonists, antagonists, and selective estrogen receptor modulators. The length-adjusted ER ligand–regulated transactivation system used with amino acids 281–549 instead of 281–595 leads to changes in the level of transactivated Fluc signal, which selectively differentiates ER ligands as agonists and selective estrogen receptor modulators (Figure 3b). This may be useful as a strategy to study different ER ligands and to distinguish agonists from antagonists as we had previously accomplished using other strategies.20

To demonstrate the application of this gene regulation system, we selected wt-p53 as an example of a therapeutically important endogenous protein that is impaired in more than 50% of cancers. The system was studied in cell culture by regulating the level of expressed wt-p53 protein (along with endogenous protein) in cells expressing either wt-p53 (HepG2) or mutant p53 (SKBr3). The results showed ligand dose–dependent expression of wt-p53 protein and associated induction of cell death. HepG2 cells expressing endogenous wt-p53 showed rapid induction of cell death even at very low dose of ligand used to induce the wt-p53. Moreover, when HepG2 cells were induced to express mutant-p53, there was sparse cell death at ligand doses of up to 1 µmol/l; however, this was significant at a dose of 5 µmol/l (Supplementary Figures S5–S8). The tumor xenograft of HepG2 cells with the ER ligand–regulatable system showed p53-mediated tumor regression only with the tumor-expressing wt-p53 and not with mutant-p53 (Figure 5e and Supplementary Figure S11). The group of animals received solvent as control showed no activation of either Fluc expression or tumor regression. This clearly indicates the efficiency of the system's response in living animals.

In summary, in this study, an efficient ligand-regulated transactivation system was developed and validated that can be used to control the expression of transgenes in cells and in living animals. The use of a shortened ER-LBD (truncated by 46 amino acids) has helped to differentiate between ER ligands, which, in turn, can now permit the use of this system to screen for ER ligands. The developed system is sensitive and efficient when studied in therapeutic xenograft models implanted in living animals. Forthcoming studies will focus on further evaluation of this system by screening small molecule libraries to potentially identify new compounds that may address many of the above-mentioned limitations. Future studies with transgenic models and gene therapy models incorporating the vectors developed in this work should allow for the study of estrogen biology, including issues related to the tissue-specific action of ERs in response to different ligands.

Materials and Methods

Construction of plasmids. The unidirectional and bidirectional reporter vector containing five GAL4-DNA binding sites and the adenovirus early minimal promoter E4 were from our previous studies.21,28 The reporter plasmid containing five GAL4-DNA binding sites and the major late promoter of adenovirus from Promega (Madison, WI) was used for comparison. The vector expressing the fusion protein containing GAL4DBD-ER-VP16 was constructed by inserting PCR-amplified fragments of ER to the EcoRI and BamHI digested activator plasmid developed previously.28 The mutant form of ER (ERG521T) from our previous study was used for constructing the activator plasmid expressing the fusion protein GAL4DBD-ERG521T-VP16 (Figure 1). Similar strategy was used for constructing bidirectional vectors with wt-p53 and mutant-p53.

Studying the efficiency of the ER ligand–regulated transactivation system with the constitutive active system in transiently transfected 293T cells. To compare the efficiency of the ER ligand–regulated transactivation (GAL4DBD-ER-VP16) system with the previously used constitutive (GAL4DBD-VP16) system, the 293T cells co-transfected with reporter plasmid p(GAL4DNA)5-E4-FLUC either with the activator plasmids expressing fusion protein GAL4DBD-VP16 or with the activator plasmids expressing the fusion protein GAL4DBD-ER-VP16 were assayed for firefly luciferase activity after inducing with the ligand 17β-estradiol for 24 hours.

Studying the ER ligand–regulated transactivation system in response to different ER ligands in transiently transfected 293T cells. To study the ER–ligand-regulated transactivation of reporter gene expression, 293T cells co-transfected with 200 ng/well of the reporter plasmid [p(GAL4DNA)5-E4-FLUC] and 100 ng/well of the activator plasmid expressing the fusion protein GAL4DBD-ER-VP16 were used. The cells were assayed for luciferase activity 24 hours after exposure to 1 µmol/l concentration of different ligands (17β-estradiol, raloxifene, tamoxifen, diethylstilbestrol, genistein, and ICI). The transfection efficiency was normalized by co-transfecting with 10 ng of renilla luciferase driven by a CMV promoter in all the required experiments.

Studying the ER ligand–regulated transactivation system in activating reporter genes expressing in a bidirectional vector in two different orientations. To study the efficiency of the ER ligand–regulated transactivation system in controlling two reporter genes expressing in two different orientations in a single bidirectional plasmid vector, the 293T cells transiently co-transfected with the reporter plasmid pRLUC-E4-(GAL4DNA)5-E4-FLUC and the activator plasmid expressing fusion protein GAL4DBD-ER-VP16 (200 ng and 100 ng, respectively/well in 12-well culture plate) were treated with different concentrations of ligand 17β-estradiol (0–1.5 µmol/l) and assayed for renilla and firefly luciferase activities as mentioned in the previous section. We calculated the R2 coefficient of correlation between the two enzyme levels.

Bidirectional ER ligand–regulated transactivation system expresses optical reporter FLUC and therapeutic tumor suppressor protein p53. The bidirectional vector for the therapeutic efficiency evaluation study was constructed by replacing the renilla luciferase reporter with either wt-p53 or mutant-p53. HepG2 cells stably co-transfected with the reporter and the activator plasmids confirmed for the integrated plasmid by amplifying Fluc gene from genomic DNA and imaging by optical CCD camera was used for xenograft therapeutic tumor model in living nude mice.

In vitro evaluation of stable HepG2 and SKBr3 cells expressing the bidirectional vector with the activator plasmid and the reporter plasmid with FLUC and p53/mutant-p53. The HepG2 and SKBr3 cells stably co-transfected with the bidirectional reporter vector expressing either wt-p53 or mutant-p53 with the activator plasmid were induced with different concentrations of ligand OHT (0–1 µmol/l) and imaged by adding substrate D-luciferin to quantitate Fluc level at 24, 48, 72, and 96 hours postinduction. Similarly, the cells were analyzed by flow cytometry following propidium iodide staining for the induced wt-p53-mediated apoptosis at different time points after inducing with ligand OHT. The cells were also further confirmed by staining with the Live–Dead cell staining kit from Molecular Probes (Invitrogen, Carlsbad, CA) as per the manufacturer's protocol.

Imaging the ER ligand–regulated transactivation system in living animals. All animals' handling was performed in accordance with Stanford University Animal Research Committee guidelines. For imaging in living female nude mice (nu/nu), 293T cells transiently co-transfected with reporter plasmid either with the activator plasmid expressing fusion protein GAL4DBD-VP16 (constitutive) or with the activator plasmid expressing fusion protein GAL4DBD-ER-VP16 (ligand-regulated system) were used. HepG2 cells stably expressing the regulated system were used for tumor xenograft model in mice. All mice were imaged by injecting 3 mg of substrate D-luciferin by following the protocol published previously.19

SUPPLEMENTARY MATERIALFigure S1. Evaluation of ER ligand–regulated transactivation system with adenoviral early and late minimal promoters.Figure S2. The ER ligand regulated transactivation system evaluated with different ER ligands.Figure S3. Comparison of the ER ligand–regulated transactivation system with the TET-ON system.Figure S4. The HepG2 cells stably co-transfected with the bidirectional reporter vector expressing either p53 or mt-p53 with the activator plasmid, were induced with different concentrations of ligand OHT (0 to 1 µmol/l) and imaged to quantitate Fluc level at 24, 48, 72 and 96 hours postinduction.Figure S5. Monitoring of p53-mediated cell death and Fluc expression in HepG2 cells stably expressing ER ligand–regulated transactivation system.Figure S6. Flow cytometry analysis of HepG2 cells stably co-transfected with ER-ligand regulated transactivation system expresses p53 (HepG2-p53) and mutant p53 (HepG2-mt-p53) along with control HepG2 cells analyzed for p53 mediated apoptosis/cell death by PI staining after induction with 1.0 µmol/l of ER-ligand 4-hydroxy tamoxifen (OHT) along with solvent DMSO as control for a period of 24 hours.Figure S7. Flow cytometry analysis of HepG2 cells stably co-transfected with the ER-ligand regulated transactivation system expresses p53 (HepG2-p53) and mutant p53 (HepG2-mt-p53) along with control HepG2 cells analyzed for p53 mediated apoptosis/cell death by PI staining after induction with different concentration (0.1, 1.0 and 5.0 µmol/l) of ER-ligand 4-hydroxy tamoxifen (OHT) along with solvent DMSO as control for a period of 48 hours.Figure S8. Flow cytometry analysis of HepG2 cells stably co-transfected with the ER-ligand regulated transactivation system expresses p53 (HepG2-p53) and mutant p53 (HepG2-mt-p53) along with control HepG2 cells analyzed for p53 mediated apoptosis/cell death by PI staining after induction with different concentrations (0.1, 1.0 and 5.0 µmol/l) of ER-ligand 4-hydroxy tamoxifen (OHT) and DMSO as solvent control analyzed at 24, 48 and 72 hours.Figure S9. Western blot analysis of p53, p21and β-actin from HepG2, HepG2-p53 and HepG2-mt-p53 cells 24 and 72 hours after induction with ligand 4-hydroxy tamoxifen and DMSO as solvent control.Figure S10. Schematic flow chart showing of the various animal studies performed.Figure S11. Twenty-day imaging signal from mice with tumor xenografts of HepG2 cells expressing p53 and mt-p53 under ER-ligand regulated transactivation system.

Supplementary Material

Figure S1.

Evaluation of ER ligand–regulated transactivation system with adenoviral early and late minimal promoters.

Click here for supplemental data (105KB, doc)
Figure S2.

The ER ligand regulated transactivation system evaluated with different ER ligands.

Click here for supplemental data (199KB, doc)
Figure S3.

Comparison of the ER ligand–regulated transactivation system with the TET-ON system.

Click here for supplemental data (63.5KB, doc)
Figure S4.

The HepG2 cells stably co-transfected with the bidirectional reporter vector expressing either p53 or mt-p53 with the activator plasmid, were induced with different concentrations of ligand OHT (0 to 1 µmol/l) and imaged to quantitate Fluc level at 24, 48, 72 and 96 hours postinduction.

Click here for supplemental data (1.4MB, doc)
Figure S5.

Monitoring of p53-mediated cell death and Fluc expression in HepG2 cells stably expressing ER ligand–regulated transactivation system.

Click here for supplemental data (709KB, doc)
Figure S6.

Flow cytometry analysis of HepG2 cells stably co-transfected with ER-ligand regulated transactivation system expresses p53 (HepG2-p53) and mutant p53 (HepG2-mt-p53) along with control HepG2 cells analyzed for p53 mediated apoptosis/cell death by PI staining after induction with 1.0 µmol/l of ER-ligand 4-hydroxy tamoxifen (OHT) along with solvent DMSO as control for a period of 24 hours.

Click here for supplemental data (164KB, doc)
Figure S7.

Flow cytometry analysis of HepG2 cells stably co-transfected with the ER-ligand regulated transactivation system expresses p53 (HepG2-p53) and mutant p53 (HepG2-mt-p53) along with control HepG2 cells analyzed for p53 mediated apoptosis/cell death by PI staining after induction with different concentration (0.1, 1.0 and 5.0 µmol/l) of ER-ligand 4-hydroxy tamoxifen (OHT) along with solvent DMSO as control for a period of 48 hours.

Click here for supplemental data (205.5KB, doc)
Figure S8.

Flow cytometry analysis of HepG2 cells stably co-transfected with the ER-ligand regulated transactivation system expresses p53 (HepG2-p53) and mutant p53 (HepG2-mt-p53) along with control HepG2 cells analyzed for p53 mediated apoptosis/cell death by PI staining after induction with different concentrations (0.1, 1.0 and 5.0 µmol/l) of ER-ligand 4-hydroxy tamoxifen (OHT) and DMSO as solvent control analyzed at 24, 48 and 72 hours.

Click here for supplemental data (135.5KB, doc)
Figure S9.

Western blot analysis of p53, p21and β-actin from HepG2, HepG2-p53 and HepG2-mt-p53 cells 24 and 72 hours after induction with ligand 4-hydroxy tamoxifen and DMSO as solvent control.

Click here for supplemental data (206.5KB, doc)
Figure S10.

Schematic flow chart showing of the various animal studies performed.

Click here for supplemental data (115.5KB, doc)
Figure S11.

Twenty-day imaging signal from mice with tumor xenografts of HepG2 cells expressing p53 and mt-p53 under ER-ligand regulated transactivation system.

Click here for supplemental data (138KB, doc)

Acknowledgments

This work is supported in part by NIH grants, R01 CA82214 (SSG), ICMIC P50CA114747 (Sanjiv Sam Gambhir), and the Small Animal Imaging Resource Program.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1.

Evaluation of ER ligand–regulated transactivation system with adenoviral early and late minimal promoters.

Click here for supplemental data (105KB, doc)
Figure S2.

The ER ligand regulated transactivation system evaluated with different ER ligands.

Click here for supplemental data (199KB, doc)
Figure S3.

Comparison of the ER ligand–regulated transactivation system with the TET-ON system.

Click here for supplemental data (63.5KB, doc)
Figure S4.

The HepG2 cells stably co-transfected with the bidirectional reporter vector expressing either p53 or mt-p53 with the activator plasmid, were induced with different concentrations of ligand OHT (0 to 1 µmol/l) and imaged to quantitate Fluc level at 24, 48, 72 and 96 hours postinduction.

Click here for supplemental data (1.4MB, doc)
Figure S5.

Monitoring of p53-mediated cell death and Fluc expression in HepG2 cells stably expressing ER ligand–regulated transactivation system.

Click here for supplemental data (709KB, doc)
Figure S6.

Flow cytometry analysis of HepG2 cells stably co-transfected with ER-ligand regulated transactivation system expresses p53 (HepG2-p53) and mutant p53 (HepG2-mt-p53) along with control HepG2 cells analyzed for p53 mediated apoptosis/cell death by PI staining after induction with 1.0 µmol/l of ER-ligand 4-hydroxy tamoxifen (OHT) along with solvent DMSO as control for a period of 24 hours.

Click here for supplemental data (164KB, doc)
Figure S7.

Flow cytometry analysis of HepG2 cells stably co-transfected with the ER-ligand regulated transactivation system expresses p53 (HepG2-p53) and mutant p53 (HepG2-mt-p53) along with control HepG2 cells analyzed for p53 mediated apoptosis/cell death by PI staining after induction with different concentration (0.1, 1.0 and 5.0 µmol/l) of ER-ligand 4-hydroxy tamoxifen (OHT) along with solvent DMSO as control for a period of 48 hours.

Click here for supplemental data (205.5KB, doc)
Figure S8.

Flow cytometry analysis of HepG2 cells stably co-transfected with the ER-ligand regulated transactivation system expresses p53 (HepG2-p53) and mutant p53 (HepG2-mt-p53) along with control HepG2 cells analyzed for p53 mediated apoptosis/cell death by PI staining after induction with different concentrations (0.1, 1.0 and 5.0 µmol/l) of ER-ligand 4-hydroxy tamoxifen (OHT) and DMSO as solvent control analyzed at 24, 48 and 72 hours.

Click here for supplemental data (135.5KB, doc)
Figure S9.

Western blot analysis of p53, p21and β-actin from HepG2, HepG2-p53 and HepG2-mt-p53 cells 24 and 72 hours after induction with ligand 4-hydroxy tamoxifen and DMSO as solvent control.

Click here for supplemental data (206.5KB, doc)
Figure S10.

Schematic flow chart showing of the various animal studies performed.

Click here for supplemental data (115.5KB, doc)
Figure S11.

Twenty-day imaging signal from mice with tumor xenografts of HepG2 cells expressing p53 and mt-p53 under ER-ligand regulated transactivation system.

Click here for supplemental data (138KB, doc)

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