Mol Cancer Ther. 2006;5:1927-1934
© 2006 American Association for Cancer Research
Research Articles: Targets
A retroviral expression system based on tetracycline-regulated tricistronic transactivator/repressor vectors for functional analyses of antiproliferative and toxic genes
Michael J. Ausserlechner1,
Petra Obexer3,
Andrea Deutschmann1,3,
Kathrin Geiger3 and
Reinhard Kofler2,3
1 Molecular Biology Research Laboratory, Department of Pediatrics, Medical University Innsbruck; 2 Division of Molecular Pathophysiology, Biocenter, Medical University Innsbruck; and 3 Tyrolean Cancer Research Institute, Innsbruck, Austria
Requests for reprints: Michael J. Ausserlechner, Molecular Biology Research Laboratory, Pediatric Department, Medical University Innsbruck, Innrain 66, A-6020 Innsbruck, Austria. Phone: 43-512-504-27748; Fax: 43-512-504-27750. E-mail: michael.j.ausserlechner{at}uibk.ac.at
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Abstract
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Establishment of stably transfected mammalian cells with conditional expression of antiproliferative or proapoptotic proteins is often hampered by varying expression within bulk-selected cells and high background in the absence of the inducing drug. To overcome such limitations, we designed a gene expression system that transcribes the tetracycline-dependent rtTA2-M2-activator, TRSID-silencer, and selection marker as a tricistronic mRNA from a single retroviral vector. More than 92% of bulk-selected cells expressed enhanced green fluorescent protein or luciferase over more than three orders of magnitude in an almost linear, dose-dependent manner. To functionally test this system, we studied how dose-dependent expression of p27Kip1 affects proliferation and viability of SH-EP neuroblastoma cells. Low to moderate p27Kip1 expression caused transient G0-G1 accumulation without reduced viability, whereas high p27Kip1 levels induced significant apoptosis after 72 hours. This proves that this expression system allows concentration-dependent analysis of gene function and implicates p27Kip1 as a critical regulator of both proliferation and apoptosis in SH-EP neuroblastoma cells. [Mol Cancer Ther 2006;5(8):1927–34]
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Introduction
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Regulated gene expression in mammalian cells has a tremendous potential in both basic science and clinical medicine. Earlier expression systems were based on endogenous elements that responded to physiologic compounds or cellular stress, such as hormones (1, 2) and heat shock (3), but the use of these expression systems was associated with pleiotropic effects due to the physiologic function of the inducers. More advanced systems have been developed that use artificial transcription factor systems based on nonmammalian control elements, such as the ectysone-inducible system (4, 5), lac repressor/operator (6), tetracycline (Tet)–inducible system (7), tamoxifen-regulated cre-recombinases (8), and a large number of other systems.
The Tet-inducible system is based on the regulatory Tet-repressor protein, which is switched off in the absence of Tet by the Tet-repressor protein. Upon binding of Tet or of the more stable analogue doxycycline, the repressor protein undergoes a conformational change and loses its ability to bind to the respective DNA sequences (tetO) of the Tet-resistance operon. Doxycycline is a nontoxic, widely used drug that efficiently traverses the cytoplasmic membrane. Due to the evolutionary distance, it is unlikely that the prokaryotic transcriptional elements will interfere with gene regulation in mammalian cells. A mutant of the bacterial Tet repressor containing a four-amino-acid exchange exhibits a reverse phenotype (reverse Tet-transactivator, rtTA) when fused to a transcriptional activation domain; that is, it induces transcription in the presence of doxycycline (9).
However, some shortcomings of the Tet-regulated system have become apparent over the last years. Because the rtTA transcription factor has residual affinity to the Tet-operator sequences in the absence of the drug, the leakiness of currently available Tet-regulated systems makes it difficult to express toxic, antiproliferative, or apoptosis-inducing proteins in some cell types. To circumvent this drawback, the Tet-regulated repressor (tTR) protein tetRKrab (10) and, more recently, the repressor tTS-Kid have been coexpressed with rtTA variants (11–14). However, usually only a limited number of cells show Tet-responsive gene expression with conventional Tet vectors (15), requiring selection and testing of individual clones for conditional transgene expression.
In this article, we describe a retroviral expression system combining recently published rtTA proteins with tTRs in single retroviral vectors that showed greatly diminished leakiness, an improved regulatory range and, interestingly, a strictly dose-dependent gene expression within the entire bulk population of infected cells. We used this system to analyze the effect of p27Kip1 on cell cycle progression in SH-EP neuroblastoma cells. p27Kip1 has been described to have a dual function during G1—it is essential for cyclin D/CDK4/6 import into the nucleus; however, increased p27Kip1 expression also blocks the activity of cyclin E/CDK2 kinase complexes causing cell cycle arrest at the G1-S boundary (reviewed in ref. 16). We found that the expression of p27Kip1 not only induces cell cycle arrest but that the level of expression also determines whether the cells undergo apoptotic cell death or not. In neuroblastoma, several substances, such as retinoic acid (17, 18), polyamine (19), and thyroid hormones (20) induce cell cycle arrest and apoptosis associated with elevated expression of p27Kip1. Understanding how distinct levels of p27Kip1 modulate the cellular response might, therefore, be essential for the therapeutic use of such substances. The herein described experiments on p27Kip1 and our finding that the system also efficiently works in a variety of other cell types, such as cervix carcinoma cells, human breast cancer cells, and in vivo for suicide gene therapy studies (21), further underline that this tightly regulated retroviral expression system represents a significant step forward for a wide range of gene function studies in vitro and in vivo. The observation that increasing p27Kip1 levels over a critical cellular threshold represses proliferation, and, if more highly expressed, induces apoptotic cell death in neuroblastoma, underlines that p27Kip1 is a critical molecular target for the pharmacologic treatment of this malignancy.
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Materials and Methods
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Cell Lines, Culture Conditions, and Reagents
SH-EP is an established neuroblastoma cell line (22, 23). Phoenix retroviral packaging cells were kindly provided by G. Nolan (24). All cells were maintained in RPMI 1640 containing 10% FCS (Life Technologies, Paisley, United Kingdom), 100 units/mL penicillin, 100 µg/mL streptomycin, and 2 mmol/L L-glutamine (Life Technologies) at 5% CO2 and 37°C in saturated humidity. Doxycycline was maintained as a 10 mmol/L solution dissolved in PBS. The cell lines were tested for Mycoplasma infection and their authenticity was verified by DNA fingerprinting using the PowerPlex 16 System (Promega Corporation, Madison, WI; Courtesy of W. Parson, Institute of Legal Medicine, Innsbruck, Austria).
Construction of Retroviral Vectors
Plasmids coding for the reverse Tet-transactivator proteins rtTA (9), rtTA2-M2, and rtTA2-S2 (25); and the Tet-regulated silencers TRSID (26), tetRKrab (10), and the pUHD13.2luc reporter vector (7) have been described previously. pLIB-MCS2iresPuro was constructed by inserting a multicloning site, an encephalomyelitis virus internal ribosome entry site (IRES) element and a puromycin resistance gene into the retroviral vector pLIB (BD Clontech, Mountain View, CA).
To obtain pLIB-rtTAiresPuro, rtTA was PCR amplified from pUHD17-1 (9) and cloned into the EcoRI site of the pLIB-MCS2iresPuro plasmid. For construction of pLIB-rtTA2-M2iresPuro and pLIB-rtTA2-S2iresPuro, the cDNAs coding for rtTA2-M2 and rtTA2-S2 were excised with EcoRI and BamHI from pUHrT62-1 and pUHrT61-1 (25), respectively, and cloned into the EcoRI-BamHI sites of pLIB-MCS2iresPuro. The coding region of tetRKrab was amplified from pCMV-tetR-KRAB (10) fused to an IRES element, digested with BglII and BamHI and inserted into the BamHI site of pUHrT62-1 downstream of the rtTA2-M2 transactivator coding region. A fragment containing rtTA2-M2, IRES-element and tetRKrab was then excised with SacII (blunted) and BamHI and inserted into the EcoRI (filled in) and BamHI sites of pLIB-MCS2iresPuro, generating the retroviral vector pLIB-rtTA2-M2iresKRABiresPuro. To construct pLIB-rtTA2-M2iresTRSIDiresPuro, the coding region of TRSID was amplified from pTHE (26) fused to an IRES element and cloned into pUHrT62-1, as described above. The rtTA2-M2-IRES-TRSID cassette was excised and cloned into the EcoRI-BamHI sites of pLIB-MCS2iresPuro.
Retroviral, self-inactivating reporter vectors were constructed by inserting the PTet-cytomegalovirus promoter containing the XhoI-EcoRI fragment from pUHD10-3 (9) into the pSIR vector (BD Clontech) generating pSITV-Neo. Enhanced green fluorescent protein (EGFP) was inserted as a PCR fragment amplified from pEGFP-N1 (BD Clontech) into the BamHI-EcoRI sites of pSITV (pSITV-EGFP-Neo) and firefly luciferase was excised from pGL3 (Promega, Mannheim, Germany) and cloned into the BamHI-EcoRI sites generating pSITV-luc-Neo. pSITV-p27Kip1-Neo was generated by inserting the coding region of human p27Kip1 from pUHD-p274 into the BamHI-EcoRI sites of pSITV-Neo.
Transient Transfection and Luciferase Activity Assays
For transient transfection experiments, 1.5 µg pLIB vectors containing the Tet-regulated transcription factors, 1 µg of the luciferase reporter plasmid pUHD13.3-luc, and 0.5 µg of the reporter vector pRL-SV40 renilla (Promega) were transfected into human embryonic kidney cells (HEK293T) using 12 µL metafectene (Biontex Laboratories GmbH, Munich, Germany) according to the instructions from the manufacturer. After 24 hours, the cells from each transfection were split into two six-well plates and cultured with or without 500 ng/mL doxycycline for another day. Then, cells were harvested and the activity of firefly and renilla luciferase was quantified using a dual luciferase activity assay (Promega). Light emission was measured in a standard tube luminometer (Berthold Technologies, Bad Wildbad, Germany).
Immunoblotting
Cells were lysed in CelLytic-Mammalian cell lysis/extraction buffer (Sigma, Vienna, Austria). Samples were separated by SDS-PAGE on 15% polyacrylamide gels and transferred to nitrocellulose membranes by a semidry transfer apparatus. The membranes were blocked with TBS containing 1% Tween20 and 5% nonfat dry milk, incubated with an antibody directed against the Tet-repressor DNA-interacting domain (kindly provided by S. Geley), p27Kip1 or 
-tubulin (BD PharMingen, Heidelberg, Germany) and detected with a horseradish peroxidase–conjugated secondary antibody (Amersham, Braunschweig, Germany). The blots were developed with enhanced chemiluminescence (Amersham) and quantified in a chemiluminescence detection system (UVP, Cambridge, United Kingdom).
Retroviral Infection and Characterization of Double-Transfected Bulk Cells
Retroviral supernatants were produced as previously described (27, 28). Briefly, 1 x 106 Phoenix packaging cells were transfected with 2 µg of the retrovirus vector and 1 µg pMD2.VSV-G (29) using 12 µL metafectene (Biontex Laboratories). Retrovirus-containing supernatants were filtered through 0.45-µm syringe filters (Sartorius, Göttingen, Germany) and used to infect SH-EP neuroblastoma cells. Doxycycline-dependent expression of EGFP and luciferase was measured in a Becton Dickinson FACScan or with a luciferase quantification kit (Promega) in a tube-luminometer (Berthold Technologies), respectively.
Immunofluorescence
Approximately 1 x 104 SH-EP cells were seeded onto LabTec slides (NUNC, Rochester, NY), fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100. The cells were incubated with monoclonal p27Kip1 antibody (BD PharMingen) diluted in PBS/1% bovine serum albumin for 30 minutes, washed, and detected with an Alexa 488–conjugated secondary antibody (Invitrogen, Carlsbad, CA). Analysis was done in an Axiovert200 M microscope (Zeiss, Oberkochen, Germany) using AxioVision Software (Zeiss).
Fluorescence-Activated Cell Sorting Analysis and [3H]Thymidine Incorporation Assays
Apoptosis and cell cycle distribution was quantified by nuclear staining with propidium iodide in concert with forward/sideward scatter analysis (30). Nuclei in the sub-G1 marker window were considered to represent apoptotic cells. For quantification of S-phase progression, cells were pulse labeled for 4 hours using a FITC-bromodeoxyuridine (BrdUrd) kit (BD PharMingen) according to the instruction of the manufacturer. Cell cycle distribution and BrdUrd incorporation were measured in concert with forward/sideward scatter analysis in a Cytomix FC500 (Beckman Coulter, Fullerton, CA). DNA synthesis was also determined by incubating 1.4 x 104 cells with 4 µCi/mL [3H]thymidine (Hartmann Analytics, Braunschweig, Germany) for 6 hours after which incorporated radioactivity was counted by liquid scintillation.
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Results and Discussion
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Transcriptional Activity of Retroviruses Expressing Tetracycline-Regulated Transactivator and Repressor Proteins
The rtTA (9) and its variants, rtTA2-M2 and rtTA2-S2 (25), were cloned into a modified pLIB retroviral vector containing an IRES element and a puromycin resistance marker. The rtTAs and the puromycin resistance marker genes were transcribed as bicistronic mRNAs driven from the viral 5' long terminal repeat promoter (Fig. 1A
). The ability of these vectors to induce Tet-dependent gene expression was analyzed by transient transfection, as shown in Fig. 1B. HEK293T cells transfected with pLIB vectors expressing rtTA, rtTA2-M2, rtTA2-S2, or control vectors; the reporter plasmid pUHD13.3; and the luciferase control plasmid pRL-SV40renilla were split after 24 hours and treated with 500 ng/mL doxycycline for another 24 hours. The cells were then subjected to a dual luciferase activity assay. Compared with the original rtTA, the rtTA2-S2 showed about equal levels of basal expression in the absence of doxycycline (leakiness), and lower absolute values of induced activity. In contrast, rtTA2-M2 had about twice as high absolute levels of induction, but 4-fold higher leakiness (Fig. 1B and D; Table 1
). This was in contrast to earlier studies showing that rtTA2-S2 and rtTA2-M2 exhibited significantly reduced leakiness (25, 31). Differences may be ascribed to the use of different cell lines (HEK293T), as Urlinger et al. (25) did their experiments with HeLa cells containing a stably integrated pUHD-luc reporter vector. However, similar to our data, Lamartina et al. (31) reported increased basal activity of the rtTA2-M2 when transiently transfected with a tetO/Epo reporter vector. To solve the problem of basal transcription, we designed a second set of retroviral vectors combining the transactivator rtTA2-M2 with the transcriptional repressors tetRKrab or TRSID, which allows simultaneous translation of all three coding regions from one mRNA and facilitates efficient selection of cells that express rtTA and tTR proteins at high level (Fig. 1C). TetRKrab is a transcriptional repressor containing the KRAB domain of the Kox1 gene (10), thereby recruiting the corepressor Kap-1, which modulates epigenetic gene silencing (32). TRSID was generated by fusing the bacterial Tet-repressor to the mSIN-interacting domain of Mad1 (26). This allows TRSID to interact and recruit histone deacetylases, thereby exploiting the potent gene silencing function of chromatin remodeling. The Tet-regulated transcriptional activity was analyzed by transient transfection, as described above. Compared with rtTA2-M2 without tTR genes, both rtTA2-M2 constructs containing tTR genes revealed marked reduction in leakiness with similar absolute levels of gene induction (Fig. 1D). This suggests that a formation of nonfunctional heterodimers between rtTA2-M2 and tTR proteins (33, 34) either did not occur in this experimental setting or had no effect on leakiness and inducibility of the system (Table 1). The expression of tetRKrab or TRSID even repressed the basal luciferase activity to 30% of the transfection control (containing only the empty pLIB-iresPuro vector and the reporter plasmid pUHD 13.3).
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Figure 1. Schematic presentation and regulatory properties of retroviral vectors for tandem expression of rtTA and tTR proteins. Structures of the retroviral vectors coding for the Tet-regulated transactivator (rtTA) proteins rtTA, rtTA2-M2, and rtTA2-S2; and Tet-repressor (tTR) proteins TRSID and tetRKrab. rtTAs and tTRs are arranged together with the puromycin selection marker gene as bicistronic and tricistronic transcription units (A and C). LTR, long terminal repeat. Doxycycline (Dox)–induced expression of luciferase in transiently transfected HEK293T cells. HEK293T cells transfected with rtTA/tTR vectors and the reporter plasmid pUHD13.3 were split into two wells 24 h after transfection and exposed to 500 ng/mL doxycycline for another 24 h. Reporter gene activation was measured by a dual-luciferase activity assay. Columns, means of two independent experiments (B and D). Ctr., control. E, cells transfected with rtTA/tTR retrovirus vectors, an empty retroviral control vector, or expression plasmids coding for TRSID (pTHE) and tetRKrab were subjected to immunoblot analysis with an antibody directed against the TetR-binding domain.
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When analyzing the cellular expression of transactivator and repressor proteins by immunoblot with an antibody directed against the DNA-binding domain of these proteins, we found that in rtTA2-M2/TRSID–transfected cells, transactivator and repressor were expressed at equal levels (Fig. 1E), whereas in rtTA2-M2/KRAB–transfected cells, the tetRKrab repressor was significantly more highly expressed than the rtTA2-M2 transactivator.
Comparison of rtTA/tTR Retrovirus–Regulated Gene Expression in Stably Infected Bulk Cells
To assess whether the results of the transient transfection experiments were also reflected in cells with the Tet-responsive promoter and the transactivator/repressor–expressing retrovirus stably integrated in the host genome, we constructed self-inactivating retrovirus vectors for Tet-regulated expression of EGFP and firefly luciferase, respectively (Fig. 2A
). In these retroviral vectors, inactivation of the 5' long terminal repeat during reverse transcription eliminates the interference between the viral promoter and the PTet-CMV minimal promoter.
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Figure 2. Regulatory range of rtTA/tTR transcription systems measured by stably integrated viral EGFP and luciferase reporter vectors. SH-EP neuroblastoma cells were infected with retroviruses coding for the transcription units rtTA2-S2, rtTA2-M2, rtTA2-M2/tetRKrab, and rtTA2-M2/TRSID, and the self-inactivating retroviral reporter vectors pSITV-EGFP-Neo and pSITV-Luc-Neo. pSITV-EGFP/Luc-Neo vectors contain a constitutive histone H4 promoter, a G418 resistance marker cassette, and the reporter gene (EGFP or firefly luciferase) under the control of a PTet-CMV minimal promoter (A). After selection, cells were exposed to doxycycline concentrations as indicated (B–D). EGFP expression was quantified by FACS analysis (left panels). For quantification of luciferase expression, equal amounts of protein were subjected to a standard luciferase activity assay (right panels). One representative experiment out of three. A linear regression curve was calculated using MS-Excel (dashed line, D).
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SH-EP neuroblastoma cells infected with rtTA/tTR retroviruses and the reporter virus pSITV-EGFP-Neo were analyzed for dose-dependent EGFP expression by fluorescence-activated cell sorting (FACS). As shown in Fig. 2B, cells expressing rtTA2-S2 achieved moderate EGFP fluorescence at 1,000 ng/mL doxycycline (Fig. 2B, left), in agreement with our transient experiments (Fig. 1B) and those of Urlinger et al. (25), who found rtTA2-S2 to be responsive only to high doses of the drug. In contrast, cells expressing rtTA2-M2 had a very steep induction of EGFP and reached a plateau at 200 to 500 ng/mL doxycycline. EGFP mean fluorescence intensities, as quantified by FACS analysis of bulk-selected SH-EP cells, were 2-fold higher than with rtTA2-S2 (Fig. 2C, left). SH-EP cells infected with the pLIB-rtTA2-M2iresKRABiresPuro retrovirus responded only weakly to doxycycline treatment (data not shown), whereas the Tet-transactivator/repressor vector pLIB-rtTA2-M2iresTRSIDiresPuro had excellent regulatory properties although the reporter vector was stably integrated into the host genome (Fig. 2D, left). Most impressively, expression of EGFP in rtTA2-M2-TRSID cells was in a 99% linear correlation to the doxycycline dose over the whole range of concentrations in three independent experiments. Such a linear dose-response curve was not achieved with any of the rtTAs alone.
Because low-level EGFP expression may be obscured by cellular autofluorescence, it was not possible to study regulation in the very low expression ranges using EGFP as a reporter. Therefore, to assess the full regulatory range of these expression systems, we next used the vector pSITV-luc-Neo; hence, autofluorescence does not interfere with luciferase activity. With this reporter system, it became apparent that basal expression was reduced 10- to 20-fold by TRSID without reducing maximal induction levels (Fig. 2, right; Table 2
). Thus, an induction rate over more than three orders of magnitude was achieved with the rtTA2-M2-TRSID retroviral system.
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Table 2. Absolute basal and doxycycline-induced expression levels of luciferase activity in stably infected SH-EP cells
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To investigate how many cells in a double-selected bulk population responded to addition of doxycycline, and whether these cells showed dose-dependent gene regulation or an on/off switch, EGFP expression and its distribution within the cell population was analyzed by flow cytometry and fluorescence microscopy (Fig. 3A and B
). In the absence of doxycycline, we observed a homogenous cell population without EGFP expression. However, upon addition of increasing doxycycline concentrations, the entire population (>90%) gradually shifted toward higher levels of EGFP fluorescence within 48 hours. There was almost no intermediate EGFP expression between the sharply circumscribed population of responders and the few nonreacting cells. Obviously, all cells that were competent for transgene expression reached a similar, dose-dependent level of EGFP after 48 hours.
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Figure 3. Quantification of bulk-selected cells conditionally expressing EGFP. SH-EP neuroblastoma cells infected with the self-inactivating retroviral reporter vector pSITV-EGFP-Neo and pLIB-rtTA2-M2iresTRSIDiresPuro were cultured in the presence of various doxycycline concentrations as indicated. The percentage of cells with doxycycline-inducible EGFP expression was quantified by FACS analysis (A) and fluorescence microscopy (B) 48 h after addition of the drug.
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Coexpression of transactivators and repressors that recognize the same promoter sequence has been shown to convert rheostat regulation observed with either the transactivator or the repressor alone into a binary on/off switch, thereby losing dose-dependent transcriptional regulation (13). However, in contrast to these data, our rtTA2-M2/TRSID transactivator-repressor system provides both significantly reduced leakiness in the absence of the drug and dose-dependent, almost linear, transgene expression over a wide regulatory range. Lamartina et al. (31) also reported a dose-dependent response when coordinately expressing rtTA-M2 and the silencer TSKid in a bicistronic plasmid, although not on the level of single cells. As also suggested by our observations with the rtTA2-M2/tetRKrab expression vector, the ratio of transactivator and silencer protein within a single cell might influence the regulatory properties of rtTA/tTR expression systems. Our results further support the implication that this system allows gene function studies in double-selected bulk populations without the necessity of raising and characterizing single cell clones. We next tested this optimized Tet system for the expression of the cell cycle inhibitor p27Kip1 in SH-EP neuroblastoma cells.
Conditional Expression of p27Kip1 Reveals Gene Dose–Dependent Effects on Cell Cycle and Cell Death Regulation
SH-EP neuroblastoma cells are highly proliferative despite moderate expression of the cell cycle inhibitors p27Kip1 and p21Cip1 (data not shown). This raises the question whether loss of G0-G1 checkpoint control and unrestricted proliferation results from the inability of these cells to elevate the cellular level of p27Kip1 above a certain threshold. We inserted the coding region of human p27Kip1 into the pSITV-Neo vector and analyzed bulk-selected SH-EP neuroblastoma cells for p27Kip1 expression by immunofluorescence. Upon addition of doxycycline, massive induction and accumulation of p27Kip1 in the nucleus was observed within 24 hours in almost all bulk-selected SH-EP neuroblastoma cells (Fig. 4A
). This again confirmed our results on EGFP-expressing, bulk-selected cells, where >90% of the cells expressed EGFP in a Tet-dependent manner (Fig. 3). We next studied whether cell cycle arrest and/or cell death is critically regulated by the cellular amount of p27Kip1. For this purpose, the cells were cultured in various concentrations of doxycycline ranging from 10 to 500 ng/mL. As expected from our earlier experiments, the [3H]thymidine incorporation of p27Kip1-transgenic cells was repressed in a dose-dependent manner, suggesting that checkpoint control in these cells can be activated by sufficient levels of p27Kip1 (Fig. 4B). This further underlines the ability of the rtTA2-M2/TRSID expression system to study dose-dependent target gene function in bulk-selected cells. In pilot experiments, we observed that expression of p27Kip1 not only induced cell cycle arrest but also cell death. Therefore, we next studied whether the kinetics of cell cycle arrest and cell death induction also vary with the cellular p27Kip1 expression level. SH-EP cells were cultured at concentrations in the range from 50 to 500 ng/mL and dose-dependent expression of p27Kip1 was verified by immunoblot analysis (Fig. 4C). In concordance with the [3H]thymidine incorporation assay, G1 accumulation was more prominent at higher doses of doxycycline after 24 hours. However, after 48 hours, the entire cell population slowly shifted into S-phase and finally underwent apoptotic cell death after 72 hours at higher doxycycline concentrations (Fig. 4C). Quite unexpectedly, cells with strong p27Kip1 expression seemed to appear earlier in S phase (Fig. 4D). To specifically address this observation, BrdUrd pulse labeling was done at 50 and 500 ng/mL doxycycline (Fig. 4E). Both concentrations of doxycycline reduced BrdUrd incorporation from 20% to 10% within 4 hours (Fig. 4E). Sixteen hours after addition of doxycycline, 
1% of the cells still proliferated at 500 ng/mL doxycycline in contrast to 7% at low doxycycline dose. After 40 hours in the presence of 50 ng/mL doxycycline, however, a large number of cells reentered cell cycle and incorporated BrdUrd, whereas those cells expressing higher levels of p27Kip1 mainly remained arrested (25% versus 12%; Fig. 4E). These FACS data suggest that p27Kip1 causes a transient G1 arrest as soon as a critical threshold is reached and that the cellular level of p27Kip1 determines the duration of G1 cell cycle arrest in SH-EP neuroblastoma cells. Bulk-selected cells with lower p27Kip1 expression enter S phase earlier, progress through the cell cycle in a semisynchronized manner, and return to G1 at 48 hours after addition of doxycycline. In addition, we find that moderate levels of p27Kip1 induce cell cycle arrest without apoptosis induction, whereas strong expression of p27Kip1 was associated with activation of the cell death machinery. These data may also explain discrepancies concerning the proapoptotic and antiapoptotic effects of p27Kip1 in various overexpression studies (reviewed in refs. 35, 36) and imply that pharmacologic substances known to elevate p27Kip1 not only counteract tumor growth but also efficiently activate apoptotic cell death in neuroblastoma tumors.
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Figure 4. Conditional expression of p27Kip1 reveals gene-dose–dependent effects on cell cycle and cell death regulation. Bulk-selected SH-EP cells stably infected with pLIB-rtTA-M2iresTRSIDiresPuro and the pSITV-p27Kip1-Neo retrovirus for conditional expression of the cell cycle inhibitor p27Kip1 were cultured in the presence of 500 ng/mL doxycycline for the times indicated and subjected to immunofluorescence analysis (A). DAPI, 4',6-diamidino-2-phenylindole. To analyze dose-dependent effects of p27Kip1 on cell cycle progression and cell death induction, cells were cultured in the presence of various doxycycline concentrations (as indicated) for 24 h (B and C) or for the times indicated (D), and subjected to [3H]thymidine incorporation analysis (B), immunoblot (C), and FACS analysis of propidium iodide (PI)–stained nuclei (D). BrdUrd pulse labeling was done for 4 h at the times indicated and analyzed by FACS (E). 7-AAD, 7-amino-actinomycin D.
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In conclusion, this study describes a significant improvement of current Tet-regulated expression systems with respect to regulatory range, leakiness, and efficient delivery into target cells. The striking advantages of the retroviral vectors expressing rtTA2-M2, TRSID, and selection marker genes as tricistronic transcription units are reflected by an excellent inducibility of the transgene in over 90% of infected cells, which allows the analysis of physiologic effects on bulk-selected cells. This proves that the isolation of individual cell clones is dispensable with this system. The combination of rtTA2-M2 and the histone deacetylase–recruiting repressor TRSID in a single retroviral expression vector yielded an efficiently transducible, Tet-regulated system with superior properties allowing an almost linear dose-response titration of transgene expression. This surprising effect was also reflected when studying gene-dose effects of the cell cycle inhibitor p27Kip1. With this system, we could show that the antiproliferative effect of p27Kip1 and its apoptosis-inducing ability depend on the cellular expression level in SH-EP neuroblastoma cells. This implicates p27Kip1 as a critical molecular target for the pharmacologic treatment of this malignancy.
As shown by our experiments on p27Kip1 and the in vivo expression of the highly toxic bacteriophage protein 
-holin (21), the rtTA2-M2/TRSID system will open new avenues in studying the function of apoptosis-inducing, antiproliferative, or differentiation-associated genes as well as toxic genes that might serve as candidates for suicide gene therapy. In addition, analysis of gene-dose effects in various cell types may be efficiently addressed due to the wide, but strictly controlled, regulatory range of transgene expression. The retroviral Tet system presented herein will significantly advance studies of gene function in various cell types and may be useful for gene therapy approaches.
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Acknowledgments
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We thank S. Geley (Institute of Pathophysiology, Medical University Innsbruck, Innsbruck, Austria), M. Gossen (Max Delbruck Center, Berlin, Germany), W. Hillen, T.C. He (Molecular Oncology Laboratory, University of Chicago, Chicago, IL), and L. Naldini (San Rafaele-Telethon Institute for Gene Therapy, Milan, Italy) for donating plasmids and antibodies; G.P. Nolan (Department of Microbiology and Immunology, Stanford University, Stanford, CA) for the Phoenix cell line; S. Lobenwein and S. Wuehl for technical assistance; and M.K. Occhipinti-Bender for editorial assistance.
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Footnotes
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Grant support: Austrian Science Fund (SFB-F021) and P18571, the Children Cancer Society of Tyrol and Vorarlberg, the Kinderkrebshilfe Südtirol Regenbogen, the Children's Cancer Research Institute, SVP-Women Organisation, the OeNB Anniversary Fund Project 11436, Provita Kinderleukämiestiftung, MFF-Tirol, Tiroler Landeskrankenanstalten Ges.m.b.H., the Tyrolean Cancer Society, and the Department of Health-Care, Autonomy of South Tyrol.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: M.J. Ausserlechner and P. Obexer contributed equally to this work.
4 Unpublished data. 
Received 11/30/05;
revised 6/11/06;
accepted 6/22/06.
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References
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Israel DI, Kaufman RJ. Highly inducible expression from vectors containing multiple GRE's in CHO cells overexpressing the glucocorticoid receptor. Nucleic Acids Res 1989;17:4589–604.[Abstract/Free Full Text] Ko MS, Takahashi N, Sugiyama N, Takano T. An auto-inducible vector conferring high glucocorticoid inducibility upon stable transformant cells. Gene 1989;84:383–9.[CrossRef][Medline] Schweinfest CW, Jorcyk CL, Fujiwara S, Papas TS. A heat-shock-inducible eukaryotic expression vector. Gene 1988;71:207–10.[CrossRef][Medline] No D, Yao TP, Evans RM. Ecdysone-inducible gene expression in mammalian cells and transgenic mice. Proc Natl Acad Sci U S A 1996;93:3346–51.[Abstract/Free Full Text] Hoppe UC, Marban E, Johns DC. Adenovirus-mediated inducible gene expression in vivo by a hybrid ecdysone receptor. Mol Ther 2000;1:159–64.[CrossRef][Medline] Takekoshi M, Maeda-Takekoshi F, Ihara S, Sakuma S, Watanabe Y. Inducible expression of a foreign gene inserted into the human cytomegalovirus genome. J Gen Virol 1993;74:1649–52.[Abstract/Free Full Text] Gossen M, Bujard H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci U S A 1992;89:5547–51.[Abstract/Free Full Text] Feil R, Wagner J, Metzger D, Chambon P. Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains. Biochem Biophys Res Commun 1997;237:752–7.[CrossRef][Medline] Gossen M, Freundlieb S, Bender G, et al. Transcriptional activation by tetracyclines in mammalian cells. Science 1995;268:1766–9.[Abstract/Free Full Text] Deuschle U, Meyer WK, Thiesen HJ. Tetracycline-reversible silencing of eukaryotic promoters. Mol Cell Biol 1995;15:1907–14.[Abstract] Forster K, Helbl V, Lederer T, et al. Tetracycline-inducible expression systems with reduced basal activity in mammalian cells. Nucleic Acids Res 1999;27:708–10.[Abstract/Free Full Text] Freundlieb S, Schirra-Muller C, Bujard H. A tetracycline controlled activation/repression system with increased potential for gene transfer into mammalian cells. J Gene Med 1999;1:4–12.[CrossRef][Medline] Rossi FM, Kringstein AM, Spicher A, Guicherit OM, Blau HM. Transcriptional control: rheostat converted to on/off switch. Mol Cell 2000;6:723–8.[CrossRef][Medline] Salucci V, Scarito A, Aurisicchio L, et al. Tight control of gene expression by a helper-dependent adenovirus vector carrying the rtTA2(s)-M2 tetracycline transactivator and repressor system. Gene Ther 2002;9:1415–21.[CrossRef][Medline] Ausserlechner MJ, Obexer P, Wiegers GJ, et al. The cell cycle inhibitor p16/INK4A sensitizes lymphoblastic leukemia cells to apoptosis by physiologic glucocorticoid levels. J Biol Chem 2001;276:10984–9.[Abstract/Free Full Text] Sherr CJ, Roberts JM. Living with or without cyclins and cyclin-dependent kinases. Genes Dev 2004;18:2699–711.[Abstract/Free Full Text] Borriello A, Pietra VD, Criscuolo M, et al. p27Kip1 accumulation is associated with retinoic-induced neuroblastoma differentiation: evidence of a decreased proteasome-dependent degradation. Oncogene 2000;19:51–60.[CrossRef][Medline] Matsuo T, Seth P, Thiele CJ. Increased expression of p27Kip1 arrests neuroblastoma cell growth. Med Pediatr Oncol 2001;36:97–9.[CrossRef][Medline] Wallick CJ, Gamper I, Thorne M, et al. Key role for p27Kip1, retinoblastoma protein Rb, and MYCN in polyamine inhibitor-induced G1 cell cycle arrest in MYCN-amplified human neuroblastoma cells. Oncogene 2005;24:5606–18.[CrossRef][Medline] Perez-Juste G, Aranda A. The cyclin-dependent kinase inhibitor p27(Kip1) is involved in thyroid hormone-mediated neuronal differentiation. J Biol Chem 1999;274:5026–31.[Abstract/Free Full Text] Agu CA, Klein R, Schwab S, et al. The cytotoxic activity of the bacteriophage -holin protein reduces tumour growth rates in mammary cancer cell xenograft models. J Gene Med 2006;8:229–41.[CrossRef][Medline] Ross RA, Spengler BA, Biedler JL. Coordinate morphological and biochemical interconversion of human neuroblastoma cells. J Natl Cancer Inst 1983;71:741–7.[Medline] Kemshead JT, Ritter MA, Cotmore SF, Greaves MF. Human Thy-1: expression on the cell surface of neuronal and glial cells. Brain Res 1982;236:451–61.[CrossRef][Medline] Pear WS, Nolan GP, Scott ML, Baltimore D. Production of high-titer helper-free retroviruses by transient transfection. Proc Natl Acad Sci U S A 1993;90:8392–6.[Abstract/Free Full Text] Urlinger S, Baron U, Thellmann M, et al. Exploring the sequence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and sensitivity. Proc Natl Acad Sci U S A 2000;97:7963–8.[Abstract/Free Full Text] Jiang W, Zhou L, Breyer B, et al. Tetracycline-regulated gene expression mediated by a novel chimeric repressor that recruits histone deacetylases in mammalian cells. J Biol Chem 2001;276:45168–74.[Abstract/Free Full Text] Ausserlechner MJ, Obexer P, Bock G, Geley S, Kofler R. Cyclin D3 and c-MYC control glucocorticoid-induced cell cycle arrest but not apoptosis in lymphoblastic leukemia cells. Cell Death Differ 2004;11:165–74.[CrossRef][Medline] Riml S, Schmidt S, Ausserlechner MJ, Geley S, Kofler R. Glucocorticoid receptor heterozygosity combined with lack of receptor autoinduction causes glucocorticoid resistance in Jurkat acute lymphoblastic leukemia cells. Cell Death Differ 2004;11:65–72.[CrossRef] Follenzi A, Ailles LE, Bakovic S, Geuna M, Naldini L. Gene transfer by lentiviral vectors is limited by nuclear translocation and rescued by HIV-1 pol sequences. Nat Genet 2000;25:217–22.[CrossRef][Medline] Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 1991;139:271–9.[CrossRef][Medline] Lamartina S, Silvi L, Roscilli G, et al. Construction of an rtTA2(s)-m2/tts(kid)-based transcription regulatory switch that displays no basal activity, good inducibility, and high responsiveness to doxycycline in mice and non-human primates. Mol Ther 2003;7:271–80.[CrossRef][Medline] Ryan RF, Schultz DC, Ayyanathan K, et al. KAP-1 corepressor protein interacts and colocalizes with heterochromatic and euchromatic HP1 proteins: a potential role for Kruppel-associated box-zinc finger proteins in heterochromatin-mediated gene silencing. Mol Cell Biol 1999;19:4366–78.[Abstract/Free Full Text] Baron U, Schnappinger D, Helbl V, et al. Generation of conditional mutants in higher eukaryotes by switching between the expression of two genes. Proc Natl Acad Sci U S A 1999;96:1013–8.[Abstract/Free Full Text] Rossi FM, Guicherit OM, Spicher A, et al. Tetracycline-regulatable factors with distinct dimerization domains allow reversible growth inhibition by p16. Nat Genet 1998;20:389–93.[CrossRef][Medline] Philipp-Staheli J, Payne SR, Kemp CJ. p27(Kip1): regulation and function of a haploinsufficient tumor suppressor and its misregulation in cancer. Exp Cell Res 2001;264:148–68.[CrossRef][Medline] Coqueret O. New roles for p21 and p27 cell-cycle inhibitors: a function for each cell compartment? Trends Cell Biol 2003;13:65–70.[CrossRef][Medline]
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Abstract
Introduction
