A Novel Estrogen Receptor Intramolecular Folding–based Titratable Transgene Expression System
Authors: Ramasamy Paulmurugan, Parasuraman Padmanabhan, Byeong-Cheol Ahn, Sunetra Ray, Juergen K Willmann, Tarik F Massoud, Sandip Biswal, Sanjiv S Gambhir
Journal: Molecular Therapy (2009)

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. 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. IntroductionGene- 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.1Vilaboa N Voellmy R Regulatable gene expression systems for gene therapy.Curr Gene Ther. 2006; 6: 421-438Crossref PubMed Scopus (50) Google Scholar,2Goverdhana S Puntel M Xiong W Zirger JM Barcia C Curtin JF et al.Regulatable gene expression systems for gene therapy applications: progress and future challenges.Mol Ther. 2005; 12: 189-211Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar 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.3Zhao HF Boyd J Jolicoeur N Shen SH A coumermycin/novobiocin-regulated gene expression system.Hum Gene Ther. 2003; 14: 1619-1629Crossref PubMed Scopus (43) Google ScholarSeveral 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.4Rubenstrunk A Orsini C Mahfoudi A Scherman D Transcriptional activation of the metallothionein I gene by electric pulses in vivo: basis for the development of a new gene switch system.J Gene Med. 2003; 5: 773-783Crossref PubMed Scopus (8) Google Scholar,5Mayo KE Warren R Palmiter RD The mouse metallothionein-I gene is transcriptionally regulated by cadmium following transfection into human or mouse cells.Cell. 1982; 29: 99-108Abstract Full Text PDF PubMed Scopus (157) Google Scholar,6Bienz M Pelham HR Heat shock regulatory elements function as an inducible enhancer in the Xenopus hsp70 gene and when linked to a heterologous promoter.Cell. 1986; 45: 753-760Abstract Full Text PDF PubMed Scopus (130) Google Scholar,7Shimizu-Sato S Huq E Tepperman JM Quail PH A light-switchable gene promoter system.Nat Biotechnol. 2002; 20: 1041-1044Crossref PubMed Scopus (482) Google Scholar 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-,8Baron U Freundlieb S Gossen M Bujard H Co-regulation of two gene activities by tetracycline via a bidirectional promoter.Nucleic Acids Res. 1995; 23: 3605-3606Crossref PubMed Scopus (266) Google Scholar,9Miller N Whelan J Progress in transcriptionally targeted and regulatable vectors for genetic therapy.Hum Gene Ther. 1997; 8: 803-815Crossref PubMed Scopus (180) Google Scholar mifepristone-,10Sirin O Park F Regulating gene expression using self-inactivating lentiviral vectors containing the mifepristone-inducible system.Gene. 2003; 323: 67-77Crossref PubMed Scopus (29) Google Scholar ecdysone-,11Galimi F Saez E Gall J Hoong N Cho G Evans RM et al.Development of ecdysone-regulated lentiviral vectors.Mol Ther. 2005; 11: 142-148Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar rapamycin-, and tamoxifen-regulated system (Gal4-VP16-ER and VP16-Gal4-ER),12Lee HS Aumais J White JH Hormone-dependent transactivation by estrogen receptor chimeras that do not interact with hsp90. Evidence for transcriptional repressors.J Biol Chem. 1996; 271: 25727-25730Crossref PubMed Scopus (21) Google Scholar and the ligand-activated site-specific recombination system (Cre-ER).13Kemp R Ireland H Clayton E Houghton C Howard L Winton DJ Elimination of background recombination: somatic induction of Cre by combined transcriptional regulation and hormone binding affinity.Nucleic Acids Res. 2004; 32: e92Crossref PubMed Scopus (67) Google Scholar 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.14Stebbins MJ Urlinger S Byrne G Bello B Hillen W Yin JC Tetracycline-inducible systems for Drosophila.Proc Natl Acad Sci U S A. 2001; 98: 10775-10780Crossref PubMed Scopus (82) Google ScholarThe 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.15Legler J van den Brink CE Brouwer A Murk AJ van der Saag PT Vethaak AD et al.Development of a stably transfected estrogen receptor-mediated luciferase reporter gene assay in the human T47D breast cancer cell line.Toxicol Sci. 1999; 48: 55-66Crossref PubMed Scopus (367) Google Scholar 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.16Brzozowski AM Pike AC Dauter Z Hubbard RE Bonn T Engström O et al.Molecular basis of agonism and antagonism in the oestrogen receptor.Nature. 1997; 389: 753-758Crossref PubMed Scopus (2918) Google Scholar,17Logie C Nichols M Myles K Funder JW Stewart AF Positive and negative discrimination of estrogen receptor agonists and antagonists using site-specific DNA recombinase fusion proteins.Mol Endocrinol. 1998; 12: 1120-1132Crossref PubMed Google Scholar 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. 16Brzozowski AM Pike AC Dauter Z Hubbard RE Bonn T Engström O et al.Molecular basis of agonism and antagonism in the oestrogen receptor.Nature. 1997; 389: 753-758Crossref PubMed Scopus (2918) Google Scholar,18Pike AC Brzozowski AM Walton J Hubbard RE Bonn T Gustafsson JA et al.Structural aspects of agonism and antagonism in the oestrogen receptor.Biochem Soc Trans. 2000; 28: 396-400Crossref PubMed Google Scholar). 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. 19Shiau AK Barstad D Loria PM Cheng L Kushner PJ Agard DA et al.The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen.Cell. 1998; 95: 927-937Abstract Full Text Full Text PDF PubMed Scopus (2225) Google Scholar), despite both ligands binding at the same site within the core of the LBD.15Legler J van den Brink CE Brouwer A Murk AJ van der Saag PT Vethaak AD et al.Development of a stably transfected estrogen receptor-mediated luciferase reporter gene assay in the human T47D breast cancer cell line.Toxicol Sci. 1999; 48: 55-66Crossref PubMed Scopus (367) Google Scholar 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.20Paulmurugan R Gambhir SS An intramolecular folding sensor for imaging estrogen receptor-ligand interactions.Proc Natl Acad Sci U S A. 2006; 103: 15883-15888Crossref PubMed Scopus (84) Google ScholarIn 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.ResultsDesign of an ER intramolecular folding–based ligand regulatable gene activation systemAn 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 systemThe 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 studyIn 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 developed21Ray S Paulmurugan R Hildebrandt I Iyer M Wu L Carey M et al.Novel bidirectional vector strategy for amplification of therapeutic and reporter gene expression.Hum Gene Ther. 2004; 15: 681-690Crossref PubMed Scopus (41) Google Scholar 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 2The 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)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.20Paulmurugan R Gambhir SS An intramolecular folding sensor for imaging estrogen receptor-ligand interactions.Proc Natl Acad Sci U S A. 2006; 103: 15883-15888Crossref PubMed Scopus (84) Google Scholar 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 study20Paulmurugan R Gambhir SS An intramolecular folding sensor for imaging estrogen receptor-ligand interactions.Proc Natl Acad Sci U S A. 2006; 103: 15883-15888Crossref PubMed Scopus (84) Google Scholar 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 3Evaluation 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)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 4Imaging 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Comparison of the ER ligand–regulated transactivation system with the TET-ON systemTo 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 5Bidirectional 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