Efficient genetic manipulation of 1321N1 astrocytoma cells using lentiviral gene transfer

Journal of Neuroscience Methods 206 (2012) 138–142

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Efficient genetic manipulation of 1321N1 astrocytoma cells using lentiviral gene transfer Anja Keim, Isabelle Müller, Gerald Thiel ∗ Department of Medical Biochemistry and Molecular Biology, University of Saarland Medical Center, D-66421 Homburg, Germany

a r t i c l e

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Article history: Received 4 January 2012 Received in revised form 14 February 2012 Accepted 15 February 2012 Keywords: Lentivirus Small hairpin RNA Reporter gene Egr-1 Elk-1

a b s t r a c t 1321N1 astrocytoma cells are frequently used to analyze stimulus-induced intracellular signaling. These experiments require genetic manipulation of the cells and several chemical and physical methods have been employed in the past. Recently, microporation has been suggested as the best method to transfect 1321N1 astrocytoma cells. Here, we demonstrate that lentiviral gene transfer into 1321N1 cells is highly efficient, cheap and non-toxic. In addition, lentiviral gene transfer efficiently facilitates stable expression of small hairpin RNAs. Finally, lentiviral gene transfer can be used to implant promoter/luciferase reporter genes into the chromatin of the cells, allowing promoter studies using templates that are embedded into the nucleosomal structure of the chromatin. © 2012 Elsevier B.V. All rights reserved.

1. Introduction

2. Materials and methods

1321N1 astrocytoma cells express a variety of receptors, including M3 muscarinic acetylcholine receptors, protease-activated receptors, thromboxane A2 receptors and EGF receptors (Trejo et al., 1992; Rössler and Thiel, 2009; Saito et al., 2010). Analyzing the stimulus-induced intracellular signaling requires genetic manipulation of the cells. Unfortunately, these cells are difficult to transfect. One way to overcome this is generation of stable cell lines as has been done to investigate the signaling of ionotropic P2X7 receptors in 1321N1 astrocytoma cells (Gendron et al., 2003). A recent study compared different transfection methods for 1321N1 astrocytoma cells. The authors concluded that microporation of the cells reached an efficiency of approximately 95%. However, the viability of the cells was less than 80%. In addition, microporation is an expensive technique that requires a special buffer and a specific electroporator (Marucci et al., 2011). Here, we show that gene transfer using recombinant lentiviruses overcomes the limitations of the classic delivery systems of genetic information, such as calcium phosphate transfection, lipofection or electroporation.

2.1. Cell culture

∗ Corresponding author. Tel.: +49 6841 1626506; fax: +49 6841 1626500. E-mail address: [email protected] (G. Thiel). 0165-0270/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jneumeth.2012.02.016

The human brain astrocytoma cell line 1321N1 was obtained from the European Collection of Cell Cultures (ECACC # 86030402). The cells were maintained in Dulbecco’s modified Eagles medium supplemented with 10% heat inactivated fetal calf serum, 100 U/ml penicillin, 100 ␮g/ml streptomycin and 2 mM glutamine at 37 ◦ C in 5% CO2 . Cells were incubated for twenty-four hours in medium containing 0.05% serum before stimulation. Stimulation with EGF (10 ng/ml, Promega, Mannheim, Germany, # G5021, dissolved in H2 O as a 100 ␮g/ml stock solution) was performed as indicated. 2.2. Lentiviral gene transfer and reporter gene analysis The viral particles were produced by transient transfection of 2.0 × 106 293T/17 cells using the calcium phosphate coprecipitation technique. Three plasmids were transfected into the cells plated on 60-mm plates: 6.6 ␮g of the transfer vector, 5 ␮g of the pCMVR8.91 packaging vector, and 2.3 ␮g of plasmid pCMVG, encoding the vesicular stomatitis virus glycoprotein. Transfections were performed in the presence of 25 ␮M chloroquine. Viral supernatants were harvested 60 h after transfection, filtered through a 0.45 ␮m filter, and used to infect 1321N12 astrocytoma cells in

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Fig. 1. Lentiviral gene transfer in 1321N1 cells. (A) Diagram of the lentiviral transfer vector HIV-7/␤-gal. The ␤-galactosidase coding region is inserted downstream of the CMV promoter/enhancer. The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) is indicated. (B) 1321N1 astrocytoma cells were infected with a ␤galactosidase encoding lentivirus. The viral particles were produced by triple transfection of 293T/17 cells with the gag-pol-rev packaging plasmid, the env plasmid encoding VSV glycoprotein and the transfer vector. ␤-galactosidase activity was visualized by X-gal histochemistry. (C) 1321N1 astrocytoma cells were infected with a ␤-galactosidase encoding lentivirus. As a control 1321N1 cells were infected with lentiviral stocks prepared with the lentiviral transfer vector pFUW (mock). Uninfected 1321N1 cells were treated with C2-ceramide (25 ␮M) (black bars). Cytoplasmic extracts were prepared 48 h later and analyzed for caspase-3/7 activity using “CaspaseGlo-Substrate”. (D) Diagram of the lentiviral transfer vector pLentiLox3.7. The coding region for the shRNA is inserted downstream of the U6 promoter. The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) is indicated. A second transcription unit encodes EGFP under the control of the CMV promoter/enhancer. (E) Phase contrast and fluorescence images of 1321N1 cells that had been infected with a lentivirus produced with the lentiviral transfer vector pLentiLox3.7. (F) 1321N1 astrocytoma cells were infected for 48 h with a lentivirus that encoded for a p53-specific shRNA. As a control, cells were infected with lentivirus prepared with the lentivial transfer vector pLL3.7 (mock). Nuclear extracts were prepared and subjected to Western blot analysis using antibodies directed against p53, or histone deacetylase-1 (HDAC1).

the presence of 8 ␮g/ml polybrene at 37 ◦ C. The lentiviral transfer vectors pFUW, pFUW-REST/Elk-1C and pFUW-REST/CREB have been described elsewhere (Lois et al., 2002; Mayer et al., 2008; Rössler and Thiel, 2009). The lentiviral vector pLentiLox3.7 (pLL3.7) was purchased from American Type Culture Collection (Manassas, VA). The sequence used to knock down human p53 has been described (Brummelkamp et al., 2002). The oligonucleotides for creating RNAi stem loops for pLL3.7 were designed as described (http://mcmanuslab.ucsf.edu/protocols/ll37stemloop design.pdf). The lentiviral transfer vector pFWEgr-1.2luc, encoding the luciferase reporter gene under the control of 490 nucleotides of the human Egr-1 5 -flanking region has been described recently (Rössler et al., 2008). Cell extracts of stimulated cells were prepared using reporter lysis buffer (Promega, Mannheim, Germany) and analyzed for luciferase activities as described (Thiel et al., 2000). Luciferase activity was normalized to the protein concentration.

2.3. Caspase-3/7-activity Caspase 3/7 activities were measured using the Caspase-Glo assay kit (Promega, Mannheim, Germany) as described (Spohn et al., 2010).

2.4. Western blots Nuclear cell extracts were prepared. Proteins were separated by SDS-PAGE, blotted and incubated with antibodies directed against either p53 (Santa Cruz, Heidelberg, Germany, # sc-126), Egr-1 (Santa Cruz, Heidelberg, Germany, # sc-110), or HDAC1 (Upstate Biotechnology, Lake Placid, NY # 05-100). The antibody directed against HDAC1 was used as a loading control. To detect FLAGtagged proteins, we used the M2 monoclonal antibody directed against the FLAG epitope (Sigma–Aldrich, Steinheim, Germany, # F3165). Immunoreactive bands were detected via enhanced chemiluminescence as described (Spohn et al., 2010).

3. Results 3.1. Expression of ˇ-galactosidase in 1321N1 astrocytoma cells using lentiviral gene transfer We tested the delivery of genetic sequences into 1321N1 astrocytoma cells using lentiviral gene transfer. The cells were infected with a recombinant lentivirus expressing ␤-galactosidase (Kowolik and Yee, 2002) (Fig. 1A). The efficiency of gene transfer was >99% (Fig. 1B).

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3.2. Lentiviral infection of 1321N1 cells does not activate caspase-3/7 activity A recent report suggested the use of microporation to genetically manipulate 1321N1 cells. However, the viability of the cells was less than 80% following microporation (Marucci et al., 2011). We measured the activity of caspases 3 and 7, enzymes that play a critical role in the execution phase of apoptosis, to test whether lentiviral infection induces cell death in 1321N1 astrocytoma cells. Fig. 1C shows that the caspase-3/7 activity is similar in mockinfected cells, and in cells infected with a ␤-galactosidase-encoding lentivirus, in comparison to non-infected cells. As a positive control, we show that treatment of the cells with C2-ceramide leads to an upregulation of caspase-3/7 activity. 3.3. Expression of small-hairpin RNAs in 1321N1 astrocytoma cells using lentiviral gene transfer We tested whether short hairpin RNAs (shRNAs) can be expressed in 1321N1 cells using lentiviral gene transfer. Infection with lentiviruses leads to the integration of the provirus into the genome, thus triggering a constitutive synthesis of shRNAs in the cells. We expressed a p53-specific shRNA in 1321N1 cells using the lentiviral transfer vector pLentiLox3.7 (Fig. 1D). The vector contains two transcription units: the shRNA is expressed under the control of the U6 promoter, while EGFP is expressed under the control of the CMV promoter/enhancer. We made recombinant lentiviruses using the pLentiLox3.7 transfer vector and infected 1321N1 astrocytoma cells. Fig. 1E shows that the infected cells expressed EGFP. Infection with a lentivirus that expressed a p53-specific shRNA induced a significant knockdown of p53 in the cells (Fig. 1F). 3.4. Upregulation of Egr-1 expression in epidermal growth factor (EGF)-stimulated 1321N1 astrocytoma cells require ternary complex factors As a “proof of principle”, we demonstrated the utility of lentiviral gene transfer of 1321N1 astrocytoma cells by analyzing the regulation of Egr-1 expression. The biosynthesis of Egr-1, a zinc finger transcription factor, is induced by many extracellular signaling molecules, including EGF (Kaufmann and Thiel, 2001; Rössler and Thiel, 2009). Fig. 2A shows that stimulation of 1321N1 cells with EGF leads to the biosynthesis of Egr-1. The regulatory region of the Egr-1 gene contains a cyclic AMP response element (CRE) and several serum response element (SRE). We used lentiviral gene transfer to express dominant-negative mutants of Elk-1 and CREB in 1321N1 astrocytoma cells to clarify whether the EGFresponsiveness of Egr-1 gene transcription relies on Elk-1 and/or CREB. The modular structure and expression of the mutants is depicted in Fig. 2B and C. Expression of REST/Elk-1C completely abolished the EGF-induced upregulation of Egr-1 expression, while expression of REST/CREB had no effect on the biosynthesis of Egr-1 (Fig. 2D). These data show that EGF stimulation induced the biosynthesis of Egr-1 via activation of ternary complex factors. 3.5. Upregulation of Egr-1 promoter activity in EGF-stimulated 1321N1 astrocytoma cells requires ternary complex factors The previous data were corroborated by analyzing the activity of the Egr-1 promoter in 1321N1 astrocytoma cells. Reporter genes are frequently introduced into cultured cells via transient transfection of plasmids. This approach has the disadvantage that the structure of these plasmids may be incompletely organized in comparison to cellular chromatin, and may thus resemble a prokaryotic gene organisation including a nonrestrictive transcriptional ground state. In contrast, the chromatin structure in eukaryotes causes

a restrictive ground state, occluding proteins such as RNA polymerases and transcriptional regulators from binding to DNA. Hence, an Egr-1 promoter/luciferase reporter gene was introduced into the chromatin of 1321N1 astrocytoma cells using lentiviral gene transfer (Fig. 2E). Expression of REST/Elk-1C completely abolished upregulation of reporter gene transcription in EGF-stimulated astrocytoma cells. In contrast, expression of REST/CREB did not attenuate stimulation of the Egr-1 promoter in EGF-treated astrocytoma cells (Fig. 2F).

4. Discussion We have shown that lentiviral gene transfer is very efficient to genetically manipulate 1321N1 astrocytoma cells. The efficiency is almost 100% allowing the expression of dominant-negative mutants or shRNAs in the cells. The high infection rate allowed us to investigate the regulation of Egr-1 biosynthesis in EGF-stimulated 1321N1 astrocytoma cells. The human Egr-1 promoter contains five SREs encompassing the consensus sequence CC[A/T]6 GG, known as the CArG box. In addition, multiple binding sites for Elk-1 and other ternary complex factors are adjacent to the CArG boxes having the Ets consensus core sequence GGAA/T (Bauer et al., 2005). Transcriptional activation of Egr-1 is often preceded by an activation of Elk-1, indicating that the SREs within the Egr-1 promoter mediate signalinduced activation of Egr-1 gene transcription. In addition, a cAMP response element (CRE) is present in the Egr-1 5 -upstream region and binding of CREB to the Egr-1 promoter has been shown (Mayer and Thiel, 2009). We performed loss-of-function experiments to unequivocally prove the role of ternary complex factor or CREB activation for EGF-induced upregulation of Egr-1 expression in 1321N1 astrocytoma cells. Genetic inactivation of Elk-1 in transgenic mice revealed minimal changes of the phenotype (Cesari et al., 2004), suggesting that functional redundancy between the proteins of the ternary complex factor family exists. Therefore, we have assessed the necessity of ternary complex factor activation for EGF-induced Egr-1 biosynthesis by using a dominant negative version of Elk-1 in loss-of-function experiments. Due to its binding to DNA and SRF, the Elk-1 mutant REST/Elk-1C most likely also inhibits the activity of two other ternary complex factors, SAP-1 and SAP-2. The Elk-1 mutant additionally contains the N-terminal repression domain of REST to recruit histone deacetylases to Elk-1 regulated genes. However, experiments involving a N-terminal truncated Elk-1 mutant lacking the REST repression domain (Bauer et al., 2005) revealed that blocking the DNA-binding site of Elk-1 to target genes as well as inhibiting the TCF/SRF interaction are of main importance for the biological activity of the mutant. The experiments described here revealed that expression of REST/Elk-1C completely blocked the stimulus-induced biosynthesis of Egr-1, while expression of a dominant-negative mutant of CREB did not change the Egr1 expression level in EGF-stimulated astrocytoma cells. Thus, ternary complex factor activation is a key step in connecting EGF-stimulation with enhanced Egr-1 biosynthesis. Using a chromatin-integrated reporter gene, controlled by the Egr-1 promoter, we directly showed that expression of the dominantnegative mutant of Elk-1 impaired reporter gene transcription after stimulation of the cells with EGF. The high infection efficiency of 13231N1 cells also allows the use of RNA interference to analyze signaling pathways. Here, we have shown that p53 expression is strikingly reduced following expression of a p53-specific shRNA. Thus, the expression of shRNAs would be valuable tool to analyze apoptotic signaling pathways in these cells as well. Lentiviral transfer vectors expressing gene-specific

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Fig. 2. EGF-induced upregulation of Egr-1 promoter activity and Egr-1 expression in 1321N1 astrocytoma cells requires ternary complex factors. (A) 1321N1 astrocytoma cells were serum-starved for 24 h and then stimulated with EGF (10 ng/ml) for the indicated periods. The cells were harvested, nuclear extracts were prepared and subjected to Western blot analysis using antibodies against Egr-1, and HDAC-1. (B) Schematic representation of the modular structure of Elk-1, the dominant-negative mutant REST/Elk1C, CREB, and the dominant-negative mutant REST/CREB. The Elk-1 mutant lacks the phosphorylation-regulated activation domain, but retains the DNA and serum response factor (SRF) binding domains. The dominant-negative mutant REST/CREB lacks this phosphorylation-regulated activation domain, but retains the DNA and dimerization domains. The bZIP domain is shown as well as the phosphorylation-dependent transcriptional activation domain CREB (kinase-inducible domain, KID). The truncated Elk-1 and CREB mutants are expressed as fusion proteins together with a transcriptional repression domain derived from the transcriptional repressor REST. (C) Western blot analysis of mock-infected 1321N1 cells or cells infected with a recombinant lentivirus encoding either REST/Elk-1C or REST/CREB. The Western blot was probed with an antibody against the FLAG-tag. Molecular-mass markers in kDa are shown on the left. (D) Expression of REST/Elk-1C abolishes EGF-induced upregulation of Egr-1 in 1321N1 cells. Cells were either mock infected or infected with a recombinant lentivirus encoding either REST/Elk-1C or REST/CREB. The cells were stimulated with EGF (10 ng/ml) as indicated. Nuclear extracts were prepared and subjected to Western blot analysis. The blot was incubated with antibodies directed against either Egr-1 or HDAC1. (E) Schematic representation of integrated proviruses encoding an Egr-1 promoter/luciferase reporter genes. The cyclic AMP response element (CRE), and the serum response elements (SREs) are depicted. (F) Expression of a dominant-negative mutant of Elk-1 reduces Egr-1 promoter activity in EGF-stimulated 1321N1 astrocytoma cells. The cells were double-infected with a lentivirus encoding an Egr-1 promoter/luciferase reporter gene and with a lentivirus that encoded either REST/Elk-1C or REST/CREB. As a control 1321N1 cells were infected with lentiviral stocks prepared with the lentiviral transfer vector pFUW (mock). The cells were serum-starved for twenty-four hours. Stimulation with EGF (10 ng/ml) was performed for 24 h. Cell extracts were prepared and analyzed for luciferase activities. Luciferase activity was normalized to the protein concentration.

shRNAs are commercially available, thus allowing a quick analysis of putative pro-apoptotic proteins.

Conflict of interest statement None of the authors have any conflict of interest to disclose.

5. Conclusion Acknowledgements We conclude that lentiviral gene transfer is highly efficient, cheap and non-toxic, and represents the best system to introduce genetic information into 1321N1 astrocytoma cells. In addition, shRNAs and promoter/reporter genes can be expressed in the cells following lentiviral infection, offering further tools to analyze stimulus-induced intracellular signaling.

We thank Jiing-Kuan Yee, Department of Virology, Beckman Research Institute, City of Hope, California, USA, for the HIV-7/␤gal transfer vector, Karl Bach for excellent technical help, and Libby Guethlein for critical reading of the manuscript. This work was supported by the University of Saarland.

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