Modifications Enhance the Apoptosis-Inducing Activity of FADD

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Modifications enhance the apoptosis-inducing activity of FADD

Tomoki Takashina1 and Manabu Nakayama1,2 origins have been used to analyze apoptosis in mammals; thus, it is known that the susceptibility of different cell 1 Laboratory of Pharmacogenomics, Graduate School of types to apoptosis varies. The method used to induce Pharmaceutical Sciences, Chiba University and 2Department of Human Genome Research, Kazusa DNA Research Institute, apoptosis must be appropriate for a given cell line. For Kisarazu, Chiba, Japan effective induction of apoptosis in cultured cells by an apoptosis-related protein, excessive overproduction of the protein, serum starvation, or treatment with cyclo- Abstract heximide may be necessary (2, 3). Moreover, long times The ability to enhance apoptosis-inducing activity in (24–48 h) are often required for apoptosis to be com- specific cells, despite the presence of cellular antiapoptotic pleted. The susceptibility of cultured cells to apoptosis proteins, would allow the removal of target cells from a cell is similar to that exhibited in vivo by cells of the tissue population. Here, we show that modification of Fas- from which the cell line was derived. Cell lines derived associated protein with death domain (FADD) by fusing from Tor B cells have a strong propensity to undergo the tandem death effector domains (DED) of FADD to the E apoptosis, which is consistent with their role in vivo,as protein of L phage, a head coat protein with self-assembly most unnecessary Tand B cells are selected by apoptosis. activity, greatly increases the apoptosis-inducing activity Cells from most other tissues only die when damage of FADD in both adherent NIH3T3 and HEK293 cells. occurs (e.g., apoptosis in virus infection and necrosis in Induction of apoptosis in cell lines that stably express injury), or if they have reached a particular developmen- modified FADD (2DEDplusE) resulted in rapid blebbing, and tal stage. This suggests that commonly used cell lines may most cells detached from the flask within 5 h. In contrast, be resistant to cell death due to the innate features of following induction of apoptosis, it took over 24 h for the the cells from which they were derived, in addition to the cells expressing unmodified FADD to exhibit these signs. absence of receptors for apoptosis. Moreover, inhibitor of The cells expressing the modified FADD underwent apoptosis protein gene amplification and protein over- apoptosis through the typical apoptosis cascade via expression have been identified in several human cancers, activation of caspase-3, and apoptosis was inhibited by a suggesting a means by which cancer cells escape apop- caspase inhibitor (i.e., z-VAD-fmk). Theoretically, as our tosis during tumorigenesis and become resistant to adhesive stable cell lines undergo apoptosis rapidly and chemotherapy and radiation treatments (4). in synchrony following mifepristone- or tetracycline- We aim to develop a technique whereby we can remove controlled production of a single apoptosis protein without desirable or unnecessary cells in vivo, such as cancer cells or affecting any other cellular pathways, they provide excel- infected cells, by inducing apoptosis specifically in the lent model systems in which to analyze the phenomenon of target cell population. Such technology could have many apoptosis in adhesive cell lines, in particular, blebbing and applications not only in medical therapy but also in basic detachment. [Mol Cancer Ther 2007;6(6):1793–803] biological research, including the production of mice that lack a particular cell type. Many cellular antiapoptotic Introduction proteins have been identified (5, 6), and it is likely that As abnormal regulation of apoptosis is believed to be a more will be found. We therefore believe that enhancement direct cause of many diseases, there has been much of apoptosis-inducing activity will be required to achieve research aimed at understanding the mechanisms by complete apoptosis of a particular cell population, partic- which apoptosis occurs (1). Cultured cells of various ularly as production of an apoptosis-related protein in vivo would be relatively low even when expressed under the control of a strong promoter (7–10). Fas/APO-1/CD95 is a member of the tumor necrosis Received 8/25/06; revised 3/10/07; accepted 4/26/07. factor receptor superfamily. Binding of the Fas ligand Grant support: Kazusa DNA Research Institute and grants-in-aid for induces trimerization of Fas in the target cell membrane. Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government. Activation of Fas causes the recruitment of Fas-associated The costs of publicationof this article were defrayed inpart by the protein with death domain (FADD) via interactions payment of page charges. This article must therefore be hereby marked between the death domain of Fas and FADD (11). advertisement inaccordancewith 18 U.S.C. Section1734 solely to Procaspase-8 binds to Fas-bound FADD via interactions indicate this fact. between the death effector domains (DED) of FADD and Requests for reprints: Manabu Nakayama, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba 292-0818 Japan. Phone: 81-438-52-3909; procaspase-8, leading to the activation and processing of Fax: 81-438-52-3946. E-mail: [email protected] caspase-8 (12). This complex is called the death-inducing Copyright C 2007 AmericanAssociationfor CancerResearch. signaling complex (13). The concentration of caspase- doi:10.1158/1535-7163.MCT-06-0522 8 determines which of two paths are taken towards

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apoptosis. If caspase-8 levels are high, it directly cleaves The resulting two PCR products were digested with BglII, caspase-3. Caspase-3 inactivates the inhibitor of caspase- ligated to construct a full-length FADD open reading activatable DNase, and caspase-activatable DNase is then frame, and cloned into the pGEM-TEasy Vector (Promega). free to enter the nucleus and cleave DNA into lengths of For construction of the expression vector pGene5CM- f180 bp (14). If caspase-8 concentration is low, it truncates VEGFP, we combined the 3.4-kb NaeI DNA fragment of BID into tBID, a form of BID that is capable of releasing pGene/V5-HisB and the 1.6-kbp AseI-MluI DNA fragment cytochrome c from mitochondria. Cytochrome c interacts of pEGFP-C3 (Clontech). For construction of pGeneEGFP- with proteins such as Apaf-1, dATP, and procaspase-9 to FADD, the full-length FADD in pGEM-TEasy was digested produce active caspase-9 (15). Caspase-9 then cleaves with NcoI and SalI and ligated into the SpeI/NotI site of caspase-3, which activates caspase-activatable DNase in pGene5CMVEGFP. We used an upstream primer the same way as it does in the first pathway. Fas-induced (5¶-GGAGATCTGATGTCGATGTACACAACCGCC-3¶) apoptosis can be effectively blocked at several stages. and a downstream primer (5¶-CCGTCGACTTACGC- ¶ Apoptosis inhibitors Bcl-2 and Bcl-XL block apoptosis CAGTTGTACGGACACG-3 ) to amplify the head coat through the BID-mediated pathway but not the direct protein (i.e., E protein) from E phage cIind 1 ts857 Sam7 (caspase cascade) pathway (6, 16, 17). Another group of DNA (Invitrogen Corp.). Although full-length FADD is 205 inhibitors, known as FLIPs, contains two DEDs capable of amino acids, 2DED2DD was a fusion protein composed of binding to the Fas-FADD complex (5, 18). In this way, they amino acids 1 to 96, 1 to 205, and 95 to 205 of FADD. prevent the recruitment of caspase-8 and inhibit both Another fusion protein, 2DEDplusE was composed of two apoptosis pathways. repeats of amino acids 1 to 96 from FADD and the full- Although addition of staurosporine or etoposide is length E protein from E bacteriophage. These two modified often used as a general method for inducing apoptosis, FADDs were ligated into the SpeI/NotI site of pGene5CM- they are inhibitors of protein kinase and topoisomerase II, VEGFP to construct the plasmids pGeneEGFP-2DED2DD respectively (19, 20). These are very strong inducers of and pGeneEGFP-2DEDplusE, respectively. apoptosis but never act only at apoptosis cascades (e.g., The plasmid pT-REx-EGFP-FRT-DEST was constructed caspase cascade) and are sure to affect many pathways in from pT-Rex-DEST30 (Invitrogen), pEF5/FRT/V5-DEST cellular function in addition to apoptosis. We have made (Invitrogen), and pEGFP-C3 (Clontech) and was kindly some stable cell lines to analyze the enhanced apoptotic- provided by Dr. Nakajima (DNA sequences are available inducing activity of modified FADD proteins. Consider- upon request). The expression plasmid allows produc- ing that in these adhesive stable cells lines apoptosis may tion of a foreign protein under the control of a be induced rapidly and synchronously by expression of tetracycline repressor system and constitutively produ- just one protein in the apoptosis cascade, they are an ces enhanced green fluorescent protein (EGFP) from a excellent model system in which to analyze the phenom- cytomegalovirus (CMV) promoter. For construction of enon of apoptosis, in particular, blebbing and detachment pTREx-FADD, pTREx-2DED2DD, and pTREx-2DED- of cells. We have analyzed in detail a system in which the plusE, KpnI/NheIDNAfragmentsofpGeneEGFP- induction of apoptosis is enhanced by modification of FADD, pGeneEGFP-2DED2DD, and pGeneEGFP-2DED- FADD. This has been done in detail for a number of plusE, respectively, were ligated into the EcoRV sites of reasons: (a) to confirm that our modifications to FADD pT-REx-EGFP-FRT-DEST. All constructs were confirmed enhance apoptosis through the apoptotic cascade; (b)to by DNA sequencing. examine whether stable cell lines expressing modified Measuring Cell Death ActivitybyTransient Transfec- FADD are suitable to use for analysis of the phenomenon tion of Original and Modified FADD of apoptosis in adhesive cells, in particular, to study Geneswitch-3T3 cells, originally from the mouse NIH3T3 blebbing and detachment of cells; (c) to evaluate whether cell line, were obtained from Invitrogen. The Geneswitch- our system is useful as an assay system for screening a 3T3 cells were grown in DMEM supplemented with 10% variety of antiapoptotic proteins, in particular, large fetal bovine serum (Invitrogen). Expression plasmids proteins that have not been screened extensively by other encoding unmodified or modified FADD were transfected groups. into Geneswitch-3T3 cells using FuGene 6 Transfection Reagent (Roche) in poly-D-lysine–coated glass-bottomed dishes (MatTek Corp.). Twenty-four hours after transfec- Materials and Methods tion, EGFP signal was observed by fluorescence microsco- Construction of Plasmids Expressing Original FADD py (Olympus IX71) with a UPlanFI Â10/0.30 Ph lens, and and Two Modified FADD nine fields of cells per dish were recorded using a CCD Two pairs of oligos (5¶-CCCATGGACCCATTCCTGGTG camera (Hamamatsu ORCA-ER) under fixed conditions -3¶ and 5¶-GCAGATCTGCCTCCCCCGGGGGCGCAGC-3¶, (typically 1 s, no gain). The number of cells with fluorescent 5 ¶-AGATCTGCAGGTGGCATTTGA-3¶ and 5¶- EGFP signals was counted. Mifepristone was then added TCAGGGTGTTTCTGAGGAAGA-3¶) were used for PCR into the medium to a final concentration of 10 nmol/L. amplification of the FADD coding sequence from the Twenty-four hours after mifepristone addition, the fluores- genomic DNA of R1 embryonic stem cells (which origina- cent signal of the cells was again observed and recorded ted from the F1 offspring of 129X1/SvJ and 129S1/Sv mice). with the CCD camera under the same conditions. The

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number of fluorescent cells following treatment was CCD camera (Olympus DP70). Alternatively, the cells were calculated as a ratio to the number of fluorescent cells observed using a fluorescence microscope (Olympus IX71; counted before treatment. Three independent experiments lens UPlanApo Â40/0.85) and recorded using a CCD were done. Representative results are given. camera (Hamamatsu ORCA-ER). Generation of Stable Expression Cell Lines Northern Blot Analysis Geneswitch-3T3 cells were transfected with 11 Agof Cells were plated at a density of 7 Â 106 per 100-mm dish expression plasmid pGeneEGFP-2DEDplusE or empty with appropriate DMEM. Twenty-four hours after subcul- vector pGene5CMVEGFP with 1.1 AgpMC1neopolyA ture, mifepristone was added to the medium. At the (Stratagene) using FuGene 6 transfection reagent. The indicated time, total RNA was prepared from the cells transformants were grown in DMEM containing 10% using TRIZOL reagent (Invitrogen). Northern blot analysis fetal bovine serum with 50 Ag/mL Hygromycin B and was done using a 32P-CTP-labeled probe according to 100 Ag/mL neomycin. Forty neomycin-resistant colonies standard methods that have been described previously that expressed EGFP were isolated and expanded. After (21). Signals were detected using BAS2000 (Fujifilm). On subculture of the stable cell line, we added mifepristone the basis of the nucleotide sequence of the bacteriophage E to a final concentration of 10 nmol/L and incubated the genome (DNA Database of Japan/Genbank/European j cells at 37 CinaCO2 incubator to examine expression of Molecular Biology Laboratory DNA databases under the gene of interest. Just a few of the hygromycin- accession no. NC_001416), DNA fragments from 6499 to resistant clones were used for the various experiments, 6879 were used as probes for Northern blot analysis. but the other clones also gave similar results when Immunoblot Analysis tested. Cells were plated at a density of 2 Â 105 per 35-mm dish Flp-In T-REx-293 cells (Invitrogen) were cotransfected with appropriate DMEM. Twenty-four hours after subcul- with expression vector (pTREx-FADD, pTREx-2DED2DD, ture, mifepristone was added. At the indicated time, cell and pTREx-2DEDplusE) and pOG44, which encoded Flp extracts were prepared by washing the cells with PBS and recombinase, using FuGene 6 (Roche). Because the Flp-In T- adding 100 AL of Chaps Cell Extract Buffer (Cell Signaling REx-293 cells contain a single integrated Flp recombination Technology). The protein concentrations of the extracts target (FRT) site, all of the hygromycin-resistant clones were determined using a BCA kit (Pierce). Forty micro- should be isogenic. However, some clones with h-galacto- grams of each extract were separated on a 15% polyacryl- sidase activity were detected in the pooled polyclonal cells, amide gel and blotted onto a nitrocellulose transfer although the manufacturer’s instructions were followed. To membrane (PROTRAN, Schleicher & Schuell) using obtain truly isogenic stable cell lines, the hygromycin- a semi-dry system (ATTO Co.). Membranes were blocked resistant clones were picked and expanded to generate with 1% blocking reagent (Beringer) in PBS and then individual monoclonal cell lines. We verified that the incubated with 1:1,000 anti–cleaved caspase-3 (Asp175; expression construct had integrated into the FRTsite by 5A1) rabbit monoclonal antibody (Cell Signaling Technol- testing each clone for lack of h-galactosidase activity. After ogy) or 1:5,000 anti-actin (Ab-5) mouse monoclonal subculture of the stable cell line, we added tetracycline to a antibody (BD Biosciences) in 1% bovine serum albumin/ final concentration of 1 Ag/mL and incubated the cells at PBS overnight at 4jC. After incubation, the membrane j 37 CinaCO2 incubator to induce expression of the gene of was washed thrice with PBS containing 0.1% Tween 20 interest. One of the hygromycin-resistant clones was used and then incubated with 1:2,000 horseradish peroxidase– for the experiments, but the other clones also gave similar conjugated anti-rabbit or anti-mouse IgG antibody (Prom- results. ega) in 1% bovine serum albumin/PBS, respectively. After Analysis by Fluorescence Microscopy washing, the bands were detected using the ECL plus The cells were plated in appropriate medium on poly-D- Western blotting detection system (Amersham Bioscien- lysine–coated glass-bottomed dishes. Twenty-four hours ces) according to the manufacturer’s instructions and after subculture, mifepristone was added. At the indicated visualized with a Luminescent Image Analyzer LAS1000 time, the cells were washed with PBS and fixed with 4% (Fujifilm). paraformaldehyde in 0.1 mol/L phosphate buffer and 3.4% Live Cell Imaging byTime-Lapse Microscopy sucrose for 30 min at room temperature. The cells were Cells were observed using phase contrast microscopy then incubated with blocking solution (10% goat serum, 1% (Olympus IX71) with a LCPlanFI Â40/0.60 Ph objective bovine serum albumin in PBS) for 1 h at room temperature lens and a CO2 incubator for microcopy (Olympus MI-IBC-I) and incubated with primary antibodies [anti–cleaved and recorded using a CCD camera (Hamamatsu ORCA-ER) caspase-3 (Asp175; 5A1) rabbit monoclonal antibody; Cell and AQUA-C imaging software (Compix, Inc.). Typically, 4 Signaling Technology, Inc.] in 1% bovine serum albumin/ 5 Â 10 cells were plated per poly-D-lysine–coated glass- PBS overnight at 4jC. After washing, the cells were bottomed 35-mm dish. Twenty-four hours after subculture, incubated with Cy3-conjugated anti-rabbit IgG antibodies mifepristone was added, and the cells were then recorded (Amersham). Some samples were incubated with 4¶,6- continuously for 10-min intervals. diamidino-2-phenylindole (Roche). The cells were observed Caspase-3 Assay using an inverted microscope (Zeiss Axiovert S100; lens LD Cells (1 Â 104) were plated in an appropriate medium. ACHROPLAN Â20/0.40 Korr Ph) and recorded using a The next day, before the indicated time, tetracycline was

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then be converted to the active form and induce a strong apoptosis signal. The activity of this modified factor was tested by transiently expressing them in cultured cells and counting the number of cells that died. To prevent constitutive production of the apoptosis-inducing factors in transfected cells, we used an inducible mammalian expression system (i.e., the GeneSwitch system), which has extremely low levels of leaky expression. Unmodified FADD and 2DEDplusE expression was controlled by an inducible promoter under the control of mifepristone, and EGFP expression was driven by a constitutive CMV promoter. As EGFP was constitutively expressed, it was able to be used to estimate cell number and therefore cell survival. As transient expression of the modified factors was done to compare apoptosis-inducing activity, it was necessary to establish whether a low number of GFP- positive cells resulted from cell death following apoptosis Figure 1. Schematic diagram of modified apoptosis inducers and induction or from low transfection efficiency. GeneSwitch- enhancement of cell death-inducing activity. A, two DED from FADD were fused to the coat E proteinof E phage (2DEDplusE). Two death 3T3 cells that express the GeneSwitch regulatory protein domains (DD) from FADD were fused to the COOH terminus of two DEDs were transfected with the plasmids. Twenty-four hours from FADD (2DED2DD). The DNA fragments encoding the two types of after transfection, the number of GFP-positive cells was modified FADD and original FADD were inserted downstream of a mifepristone-inducible promoter. B, enhancement of the cell death- counted with the aid of a fluorescence microscope. Mifep- inducing activity of FADD by modification. The GeneSwitch-3T3 cells, ristone was then added to the medium to induce expression plated onpoly- D-lysine – coated glass dishes, were transiently transfected of the modified proteins. At the indicated time after adding with the FADD constructs or empty vector using FuGene 6. Twenty-four mifepristone, the number of GFP-positive cells was counted hours after transfection, the number of cells expressing EGFP were counted as described in Materials and Methods. Mifepristone was added again. Figure 1B shows the number of cells that survive per to induce expression of the factors. At the indicated time after induction, GFP-positive transfectant. The number of GFP-positive the number of cells expressing EGFP was counted using the same control cells, which had been transfected with empty conditions as before. A reduction in the number of cells following induction indicated that the induced factor resulted in cell death. In the expression vector, was approximately the same before control experiment, an increased number of cells was detected due to and after mifepristone treatment. The increase in the accumulation of EGFP during the 24 h following mifepristone treatment. number of GFP-positive cells was due to accumulation of EGFP during the additional 24 h. When FADD protein was expressed, f75% of the transfectants had died 24 h after added to the medium to a final concentration of 1 Ag/mL, induction. In contrast, all transformants expressing 2DED- and the cells were incubated at 37jC for the indicated time plusE protein were dead 24 h after induction. It is clear in a CO2 incubator. One hundred microliters of Caspase- from this experiment that expression of the 2DEDplusE Glo 3/7 reagent (Promega) were added to each well of 96- protein induced stronger apoptosis activity than unmodi- well assay plates containing the sample in 100 AL of culture fied FADD. We also constructed another modified FADD medium per well. After 1 h of incubation at 25jC, the using two DEDs and two death domains as shown in luminescence of each sample was measured by a plate Fig. 1A. The 2DED2DD induced medium apoptosis activity reader luminometer (Perkin-Elmer). Three independent (data not shown). experiments were done. To analyze this cell death in detail, we isolated stable transfectants expressing 2DEDplusE protein under the control of an inducible promoter or expressing GFP alone Results from stable integration of empty vector DNA. GeneSwitch- Expression of Modified FADD Triggers Cell Death 3T3 cells were cotransfected with the pGene5CMVEGFP- To produce a trigger protein with strong apoptosis- based plasmids described above and the pMCneo1 plasmid inducing activity by modification, we attempted to assem- for the neomycin resistance gene. The neo-resistant and ble procaspase-8 because procaspase-8 contains a weak GFP-positive colonies were isolated. Figure 2 shows the proteinase activity even before it is processed and can self- effect of mifepristone-induced 2DEDplusE expression activate to form caspase-8. The DED of FADD binds to the compared with control cell lines stably transfected with DED of procaspase-8. Two FADD DEDs were fused to the E empty vector (Fig. 2A). Three hours after the addition of protein of E phage, which is a head coat protein with self- mifepristone to cells stably transfected with 2DEDplusE, assembly activity (22, 23), to form 2DEDplusE (Fig. 1A). more than half of the cells had detached from the surface of The idea was that when the 2DEDplusE protein molecules the dish and exhibited cell death. On the other hand, no are produced in a cell, they should assemble automatically effect was observed in control cells that were stably as the E protein should self-associate, and each DED should transfected with empty vector. Results were comparable bind to procaspase-8. The assembled procaspase-8 should for four independently isolated clones (data not shown).

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Cells Expressing the Modified Factor 2DEDplusE induction, strong signals were detected in most cells. The Undergo Apoptosis via the Typical Apoptosis Cascade, signals from shrunken cells tended to be stronger than byActivation of Caspase-3 those of healthy-looking cells. The shrunken cells were To confirm that the observed cell death was typical almost detached from the glass and entering the final stages apoptosis, indirect immunofluorescence analysis using of apoptosis. There were some cells that progressed antibodies against the active form of caspase-3, which do through the stages of cell death faster than the majority of not recognize procaspase-3, was done on stable cell lines at cells; however, the strength of the signal from the active- various times after the addition of mifepristone. As shown form of caspase-3 was observed to be uniform in most cells. in Fig. 2B, signals were hardly detected until 1.5 h after The active form of caspase-3 was not detected in control induction. After 2 h, weak signals were detected in the cells bearing empty vector 3 h after mifepristone addition. cytoplasm. Two and a half hours after induction, in Figure 2C shows a high magnification image of stained addition to weak signals from most cells, strong signals cells 3 h after induction. After induction with mifepristone, were detected in some shrunken cells. Three hours after healthy and well-extended cells shrunk rapidly. Each

Figure 2. Activation of caspase-3 and condensation of nuclei after induction of 2DEDplusE expression. A, the adhesive stable cell line underwent apoptosis depending on modified factor under the control of mifepristone. NIH3T3 cells stably expressing 2DED- plusE under the control of the GeneSwitch expression system were subcultured inpoly- D-lysine – coated glass- bottomed dishes. Twenty-four hours after subculture, mifepristone was added to induce expression of 2DED- plusE protein. Without any fixation, the cells stably transfected with 2DEDplusE DNA before (c) or after (d) treatment with mifepristone were observed using phase- contrast microscopy. Cells stably transfected with empty vector DNA before (a) and after (b) mifepristone treatment. Expression of 2DEDplusE resulted in rapid induction of apoptosis. Bar, 100 Am. B, the stable cell lines expressing 2DEDplusE under control of Gene- Switch were subcultured onthe surface of poly- D- lysine – coated glass-bottomed dishes. At the indicated times after mifepristone addition, the cells were fixed with 4% paraformaldehyde and then immunostained using anti-caspase antibodies that recognize the processed, active form of caspase-3 and Cy3-labeled secondary antibodies. The cells were observed using fluorescence microscopy. a–e, images of cells stained with anti-caspase-3/Cy3 visualized by fluorescent microscopy; f–j, phase-contrast images the of same fields of view as the respective fluorescent images. Bar, 100 Am. C, higher-magnification image of anti-caspase- 3/Cy3-stained cells 3 h after addition of mifepristone (a) and the same field of view shown by phase-contrast microscopy (b). Arrowheads indicate blebbing structures that are oftenseeninapoptosis. Bar, 10 Am. D, cells stably transfected with 2DEDplusE stained with 4¶,6- diamidino-2-phenylindole 6 h after mifepristone addition (a) and the same field viewed using phase-contrast microscopy (b). Closed arrowheads indicate condensa- tionof nuclei that is oftenseeninapoptosis. Open arrowheads indicate nuclei that appear normal. Bar, 10 Am.

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nuclei. The stage of fragmentation of nuclei tended to be significantly more advanced than that of still adherent cells. The cells and nuclei were so fragile that they were easily broken, and the strings of DNA spread following the cell fixing and washing procedure. When condensation of chromatin and fragmentation of nuclei were observed in the shrunken and floating cells, the DNA was extracted and separated by agarose gel electrophoresis. This was done to see if the DNA formed a nucleosome ladder, which is a series of DNA fragments containing multiple numbers of nucleosomes and results from the breaking of chromo- somes in typical apoptosis. A small amount of nucleosome ladder was detected relative to the amount of long chromosomal DNA (data not shown). Even when other types of apoptosis inducers are used on 3T3 cells, nucleosome ladders are rarely detected in DNA from 3T3 cells. The fact that only a small amount of nucleosome ladder was observed in the samples is, therefore, likely to be because 3T3 cells were used. Taken together, the data Figure 3. Inhibition of modified FADD, 2DEDplusE-mediated apoptosis by inhibitors of caspase. Cells stably transfected with 2DEDplusE were subcultured inpoly- D-lysine – coated glass-bottomed dishes. Twenty-four hours after subculture, mifepristone was added to the medium. The cells were treated with z-VAD-fmk or Ac-DEVD-CHO 2 h before the additionof mifepristone. The cells were fixed at the indicated times and immunostained with anti-caspase-3 primary antibody/Cy3-labeled secondary antibody and observed using fluorescence microscopy. a–c, images of the control experiment without any inhibitor; d–f,images of anti-caspase- 3/Cy3-stained cells that had been treated with z-VAD-fmk, which can inhibit all types of caspase; g–i, images of anti-caspase-3/Cy3-stained cells that had been treated with Ac-DEVD-CHO, which mainly inhibits caspase-3 and caspase-7. Bar, 50 Am.

shrinking cell body formed f10 clustered small balls (i.e., exhibited blebbing). A strong caspase-3 signal was detected in these small balls. This suggests that the small balls contained a large amount of the active form of caspase-3. At that time, abnormal nuclei had not yet been observed using phase-contrast microscopy. Then, the small balls disappeared, the cell bodies became completely round, and the cells parted from the surface. To confirm changes in nuclei in addition to the observed changes in the cytoplasm and cell adhesion, 4¶,6-diamidino-2-phenylindole staining was done (Fig. 2D). Several hours after mifepristone addition, condensation of chromatin and fragmentation of nuclei were observed. Although this was rare after 6 h, as for one of the stable cell lines, which showed a relatively slow progression of apoptosis, the nuclei of some relatively healthy and well-spread cells looked large and relatively light by 4¶,6-diamidino-2-phenylindole staining. 4¶,6-Dia- midino-2-phenylindole staining showed that the nuclei of Figure 4. Time course of the induction of 2DEDplusE expression and activationof caspase-3. A, Northern hybridization analysis was done using shrunken cells with blebbing appeared shrunken, and that total RNA from cells stably transfected with 2DEDplusE (lanes 1 – 6)or strongly stained masses were apparent in the nuclei, which empty vector (lanes 7 – 12) at 0, 1, 2, 2.5, 3, 3.5, and 24 h (lanes 1 – 6 and correspond to condensed chromatin. It was also observed 7–12, respectively) after mifepristone addition. Numbers on the right side of the gel refer to the positions of the RNA molecular weight markers that the nucleus was fragmented in the round cells. Six (Novagen). B, immunoblot analysis was done using extracts of 2DEDplusE hours after induction, the majority of cells had peeled off (lanes 8 – 14) and empty vector (lane 1 – 7) stably transfected cells at 0, 1, from the surface of the dish and detached. The detached 2, 2.5, 3, 3.5, and 4 h (lanes 1 – 7 and 8–14, respectively) after cells were collected by centrifugation and stained with 4¶,6- mifepristone addition. Caspase-3 was detected using anti-caspase-3 antibody and the ECL plus Western blotting detection system. Numbers diamidino-2-phenylindole. The nuclei of detached cells also on the right side of the gel indicate the positions of the protein molecular showed condensation of chromatin and fragmentation of weight markers (Amersham).

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Figure 5. Serial images of cells expressing 2DEDplusE undergoing apoptosis takenby time-lapse microscopy. The stable cell line expressing 2DEDplusE was plated inpoly- D-lysine– coated glass-bottomed dishes. One day after plating, mifepristone was added. Serial images of the 2DEDplusE expressing cells were acquired by time-lapse microscopy at intervals of 30 min. The movie is available as Supplementary Material at Molecular Cancer Therapeutics online (http://mct.aacrjournals.org/). Bar, 20 Am.

indicate that 2DEDplusE protein induces apoptosis via gests that another cascade that does not involve caspase-3 is activation of caspase-3. involved in apoptosis execution. The 2DEDplusE protein Blocking the Effects of Modified FADD byCaspase binds procaspase-8 and activates caspase-8, and the Inhibitors activated caspase-8 mainly processes procaspase-3 imme- Next, to determine whether apoptosis induced by the diately and induces the caspase-8/caspase-3 cascade 2DEDplusE protein can be inhibited by caspase inhibitors, directly. Besides the main cascade, however, activated experiments using two caspase inhibitors were done. z- caspase-8 processes and activates BID protein. Through VAD-fmk, which can inhibit all known caspases, was activation of BID, the apoptosis signal was amplified in added to the cell medium 2 h before induction (24). After mitochondria and then transmitted to the caspase-9– addition of mifepristone to induce expression of 2DED- mediated cascade, and then apoptosis was executed. We plusE protein, at the times indicated in Fig. 3, the state of also confirmed that the unprocessed BID was decreased the cells was observed, and caspase-3 staining was done. based on the induction of 2DEDplusE protein as shown by No change in the state of the inhibitor-treated cells was immunoblot analysis and anti-BID antibodies (data not observed during the first 3.5 h, although dramatic changes shown). This indicates that the 2DEDplusE protein will were observed without inhibitor. Moreover, no cleaved activate not only the caspase-8/caspase-3 cascade but also caspase-3 was detected, and no activation of caspase-3 was the caspase-9 cascade. When caspase-8 activity was blocked observed (Fig. 3). z-VAD-fmk was able to completely with z-VAD-fmk during the initial stages of apoptosis inhibit the apoptosis-inducing properties of 2DEDplusE. induction by the 2DEDplusE protein, all reactions of This result shows that 2DEDplusE activates caspase-8 via apoptosis after caspase-8 activation were blocked. the mechanism that we expected, and that there are no Time Course of Transcriptional Induction of 2DED- unexpected artificial effects caused by 2DEDplusE. Ac- plusE and Activation of Caspase-3 DEVD-CHO, which can specifically inhibit caspase-3 and Next, we did a time course experiment in which apoptosis caspase-7, was added to the cell medium 2 h before was observed in cells following mifepristone addition. The induction (25). Inhibition was exhibited, although it was Northern blotting results in Fig. 4A show that 2DEDplusE not complete. The progression of apoptosis in these cells mRNA levels had increased after 1 h and reached maximum was weak and slow compared with the cells without levels after 2 h. It is worthy to note that 2DEDplusE mRNA is inhibitor. In addition, 3 h after induction, a few strong present at moderate levels even after mifepristone induction, signals were observed in shrunken cells. Four hours after and that this result strongly suggests that even a moderate induction, f30% of cells had executed apoptosis and had amount of 2DEDplusE mRNA can induce strong apoptosis detached from the surface and were floating in the activity. The amount of the active form of caspase-3 was medium. This partial inhibition by Ac-DEVD-CHO sug- assessed by Western blot analysis. Although no signal was

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detected 1 h after induction or without mifepristone, a weak The first shrunken cell was observed after 2.5 h. In the image signal was detected after 2 h, and the strength of the signal taken after 2.5 h, rounded cells that were undergoing cell rapidly reached a maximum level after 2.5 h and remained at division were observed, but these were easily distinguished a high level until the 4-h time point. The active form of from rounded cells undergoing apoptosis. Five hours after caspase-3 was detected in control cells transfected with empty induction, most cells had entered apoptosis and shrunk. vector, even after mifepristone addition. Two minor bands Less time was taken for cells to appear round after induction corresponding to f50 and 30 kDa were observed in the of apoptosis by 2DEDplusE than for cells treated with a Western blots of 2DEDplusE-expressing cells. The intensities typical strong apoptosis inducer (i.e., etoposide). When the of these two bands were greatest in samples taken 2.5 and parent GeneSwitch-3T3 cells were treated with etoposide 4 h after mifepristone treatment, respectively. Because these and recorded with a time-lapse system, the same amount of bands are dependent on mifepristone treatment and are not apoptosis induction was observed over 12 h (data not detected in control cells, they may be procaspase-3. It is shown). This rapid execution of apoptosis is also evidence possible that they are other members of the caspase family. that the 2DEDplusE protein has enhanced apoptosis- However, although a detailed analysis has not yet been done, inducing activity relative to FADD. In contrast, in control the anti-caspase-3 antibodies should not cross-react with cells treated with mifepristone, the only rounded cells other proteins. Similar results were obtained using indepen- observed were undergoing cell division, not apoptosis. On dent isolated clones expressing 2DEDplusE and independent average, the number of small balls attached to shrunken control cells (data not shown). cells as a result of apoptosis (blebbing) was 14. The Modified Factor 2DEDplusE Induced Rapid and Modified FADD EffectivelyInduces Apoptosis not Synchronous Blebbing and Detachment onlyin NIH3T3 but also in HEK293 Cells Figure 5 shows a series of phase-contrast images of Finally, we determined whether the enhanced apoptotic mifepristone-treated adhesive 2DEDplusE-expressing cells activity of modified FADD that was observed in NIH3T3 undergoing apoptosis. In summary, at the indicated time cells was also effective in inducing apoptosis in another after induction, the extended and attached cells exhibited cell line, the human embryonic kidney 293 (HEK293) cells. blebbing (i.e., they shrunk into f10 small balls). The The Flp-In T-Rex 293 cell line stably expresses the Tet shrunken cells then became compact and round. The repressor, and the cells contain a single integrated FRTsite rounded cells remained on the surface of the dish if it was in the genome. Flp recombinase mediates insertion of not shaken. Following this, the rounded cells detached from the expression construct carrying a FRTsite into the genome the bottom of dish and disappeared from the field of view. at the integrated FRTsite through site-specific DNA

Figure 6. Modified FADD also induced rapid apoptosis inHEK293 cells. A, HEK293 cells stably transfected with FADD, modified 2DED2DD, and 2DEDplusE under the control of tetracycline-inducible promoters were established using the Flp-in T-Rex 293 system. They were subcultured incoated glass-bottomed dishes. Twenty-four hours after subculture, tetracycline was added. At the indicated times after additionof tetracycline,the cells were observed by phase-contrast microscopy. Bar, 100 Am. B, caspase-3 activity was measured by a Caspase-Glo 3/7 assay. The Flp-inT-Rex 293 stable cell line carrying DNA encoding FADD, modified 2DED2DD, and 2DEDplusE, expressed under the control of the tetracycline promoter, were subcultured in96-well microtiter plates. Twenty-four hours after subculture, tetracycline was added. At the indicated times after addition of tetracycline, caspase-3/7 activity was measured using a Caspase-Glo 3/7 assay.

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recombination. The advantage of this system is that it cycloheximide and serum starvation. Generally, it is difficult allowed us to make direct comparison of the apoptotic to induce apoptosis completely in most cell types, with the activity of modified proteins, as there is no variation in exception of cells with a T- or B-cell origin. This situation plasmid copy number in the cells, even if they are stably motivated us to make an enhanced apoptosis-inducing transfected. Figure 6A shows time courses of the stable cell factor capable of inducing apoptosis when expressed in cells lines expressing the three different proteins after tetracycline relatively resistant to apoptosis. We aim to develop addition. When expression of unmodified FADD was technology that would facilitate the removal of selected induced in a stable cell line by the addition of tetracycline, target cells by apoptosis. In the present study, we have few, if any, apoptotic cells were observed. Twenty-five hours succeeded in enhancing the apoptosis-inducing activity of after induction, f10% of cells were affected and detached FADD such that apoptosis is rapidly and almost completely from the surface of the dish. In contrast, when 2DEDplusE executed by modified FADD. FADD contains two f90- expression was induced, after 3 h, f80% of the cells were amino-acid protein interaction modules (an NH2-terminal affected and had detached from the bottom of the dish, DED and a COOH-terminal death domain), each of which whereas no significant effect was observed 2 h after adopts a structurally similar six a-helical bundle. Interest- tetracycline addition. After 4 h, most of the cells had detached ingly, FADD does not contain an enzymatic domain. Fas completely. No cells adhered to the bottom of the dish. The ligands trimerize through the binding of Fas receptors. The apoptosis-inducing activity of the 2DED2DD protein is death domain of FADD can bind to the death domain in the stronger than that of unmodified FADD but weaker than cytoplasmic domain of Fas receptors. The DED of FADD can that of the 2DEDplusE protein. No apoptotic cell death was bind the DED of procaspase-8. This complex is called death- observed in the stable cell line expressing 2DED2DD protein inducing signaling complex and can activate initiator 3 h after the addition of tetracycline. Five hours after caspase-8 by autoprocessing of procaspase-8. We fused induction, f40% of cells were affected and had detached, DED of FADD to E protein, which is able to self-assemble and 6 h after induction, only f10% of cells still adhered to the and is the coat protein of E phage, so that the modified factor bottom of the dish. These results also were consistent with the can construct artificial death-inducing signaling complex results of caspase-3/7 assay. At the indicated times after without Fas ligand and receptor. We expected that the self- addition of tetracycline, caspase-3/7 activity was measured assembled modified factor 2DEDplusE would bind procas- by a caspase-Glo 3/7 assay as shown in Fig. 6B. Interestingly, pase-8. This was expected to increase the procaspase-8– 25 h after induction of 2DED2DD expression, the number of activated autoprocessing activity of procaspase-8 more adherent cells increased. It is possible that this increase was effectively. In fact, compared with unmodified FADD, the due to reattachment of cells or proliferation of surviving cells. modified factor can induce strong apoptosis in adhesive However, in stable cell lines expressing 2DEDplusE, the NIH3T3 and HEK293 cells, which are known to be relatively number of adherent cells detected did not increase. This resistant to apoptosis but are typically used in cellular suggests that the 2DEDplusE-induced apoptosis was com- studies. plete. Taken together, the data indicate that the modified There are two pieces of evidence that suggest apoptosis is FADD (2DEDplusE) has enhanced apoptotic activity com- enhanced. The first is based on the quantitative examina- pared with unmodified FADD in at least two types of cell tion of transient expression. The ratio of cell death in cells lines (NIH3T3 and HEK293), which are used as standard transfected with the modified FADD 2DEDplusE 24 h after cell lines in the field of cellular biology but are known to be induction of expression is 100%, which is higher than in quite resistant to the induction of apoptosis. Additionally, cells transfected with unmodified FADD. The other when we tested the activities of these modified factors by evidence is that the cells expressing 2DEDplusE rapidly transient transfection into other human cell lines (HeLa underwent the morphologic changes that accompany cells, which originate from cervical cancer; Saos2 cells, which apoptosis (i.e., blebbing and detachment) and had a higher originate from osteosarcoma; and HepG2 cells, which ratio of apoptotic cells at the various time points relative to originate from hepatocellular carcinoma), we obtained cells expressing unmodified FADD. A strong inducer of almost the same results as with NIH3T3 and HEK293 cells apoptosis (etoposide) takes about 12 h to result in (data not shown). Moreover, in a caspase-Glo 3/7 assay detachment of adhesive cells. It should be emphasized that based on the induction of 2DEDplusE, we observed a almost 100% of cells underwent apoptosis. The noted significant increase in caspase-3 activity in the three cell effects of 2DEDplusE on apoptosis make it suitable for use lines (HEK293, NIH3T3, and HeLa cells) we tested. in future experiments in which we will express this protein in vivo because we predict various restrictions in the in vivo situation: low promoter strength or lower amount of Discussion protein production. Because FADD is upstream of the Transfection of certain types of cells with a construct apoptosis cascade, we expected modification of FADD to encoding an apoptosis-related protein can induce weak or amplify the signal more effectively than modification of the moderate amounts of apoptosis when cultured under downstream caspase-3 or caspase-9. Previously, several suitable conditions. However, often more than overproduc- studies have been reported in which an apoptosis-related tion of a protein is required, and another stimulus is needed, protein has been deleted or modified into a fusion protein, such as addition of an aggregating anti-Fas antibody and such as serial deletion mutants and fusion proteins in

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which GFP is fused to a domain that regulates dimerization some effects that result from the dimerization of two DEDs. under the control of a chemical compound (CIDs or FK506- If one takes this view, it follows that three DEDs might be binding protein; refs. 8, 26). However, these studies do not worth trying. However, large recombinant proteins tend to mention enhancement of apoptosis-inducing activity, be susceptible to attack by proteases. which probably means that activity is decreased or To construct another type of modified FADD, in unchanged. Some reports of in vivo studies in recombinant particular, a much stronger factor or specific regulator, mice have used toxins; however, the side effects of toxins another self-assembling protein can be used in the place of (i.e., necrosis and inflammation) are concerning. An E protein. The pVIII protein, the major coat protein of attempt to induce apoptosis in selected liver cells was bacteriophage M13, is synthesized as a preprotein (73 reported by Mallet et al. (8); however, only some of the residues), which binds to the inner surface of the plasma target cells in the mouse liver died following induction. We membrane and, in the presence of a membrane potential, strongly believe that in vivo expression of 2DEDplusE will subsequently translocates as a loop structure across the induce apoptosis in most cells. membrane (30). Particles of M13 contain f2,700 copies of DED is a protein-interaction module similar to caspase the processed or mature 50-residue a-helical protein, recruitment domain and death domain. DED-containing arranged in a cylindrical sheath around the bacteriophage proteins have been implicated in apoptosis regulation via DNA, whereas a E phage contains 415 copies of E protein. interactions with DED-containing caspases (caspase-8 and The pVIII protein of M13 has the advantage of a small size caspase-10 in humans and caspase-8 in mice). In addition to and may have a greater propensity to self-assemble. The DED-containing caspases, five proteins (FADD, FLIP, mature form of pVIII may, therefore, be a good alternative DEDD, DEDD2, and PEA-15) with classic DEDs and six to E protein for fusion to DED. Both actin and tubulin are proteins (BAP31, BAR, DAP-3, FLASH, HIP-1, and HIPPI) also good candidates because they are in organized with DED-like domains (pseudo-DEDs) were identified in filaments in the cytoplasm and their properties, inhibitors, human and mouse. A unique family of single DED- and stabilizers are well studied. containing proteins, including DEDD and DEDD2, are In the stable cell lines described in this study, expression targeted to the nucleolus (27). Overexpression of DEDD of the transfected genes is controlled by mifepristone containing an NH2-terminal DED in 293Tcells induced (RU486) or tetracycline. They are useful cell lines in which weak apoptosis (28). Overexpression of DEDD2 induced to analyze the phenomenon of apoptosis for two reasons, moderate apoptosis; however, a direct comparison was not which are described below. made (29). The c-FLIP regulator is an enzymatically The first reason is that these stable cell lines allow inactive relative of caspase-8 that binds to death-inducing apoptosis to be studied in isolation (i.e., without the signaling complex. Two major c-FLIP variants result from undesired effects that other typical apoptosis-inducers alternative mRNA splicing: a short protein with two DEDs display). For example, staurosporine, etoposide, campto- (c-FLIPS) and a long caspase-like protein with two DEDs thecin, and cycloheximide are also inhibitors of protein (c-FLIPL). The role of c-FLIPS as an inhibitor of death kinase, topoisomerase II, topoisomerase I, and protein receptor–mediated apoptosis is well established; however, synthesis, respectively. Therefore, they often affect many the function of c-FLIPL remains controversial. Although cellular regulation pathways in addition to the apoptosis overexpression of c-FLIPL inhibits apoptosis, expression at cascade. The use of these agents, therefore, makes it lower levels supports caspase-8 activation and cell death. difficult to distinguish whether differences in mRNA and Although the c-FLIPS containing only two DEDs works as protein levels measured after inducing apoptosis result an inhibitor of apoptosis, it is interesting that our from apoptosis or from inhibitor side effects. In particular, 2DEDplusE containing two DEDs from FADD showed the stable cell lines we have used will be useful in genome- strong apoptosis-inducing activity. This difference can be wide analysis, using DNA microarray and proteomic explained by the specificity of DED for the binding partner. approaches. A cell system in which apoptosis can be Because the balance of original FADD, procaspase-8, and c- studied synchronously and is dependent on the expression FLIP in death-inducing signaling complex or cytoplasm of only one protein is rare in adhesive cell lines if the use of determines the fate of cells (i.e., apoptosis or survival), we other stimuli, such as aggregation anti-Fas receptor anti- propose that the ability of modified 2DEDplusE to induce bodies, tumor necrosis factor-a,serumstarvation, apoptosis is independent of the FADD/procaspase-8/c- or cycloheximide, are excluded. In the suspension cells FLIP balance. We are also interested in whether enhance- (i.e., Jurkat cells), many reports have been published in ment of apoptosis-inducing activity is observed when other which viral vectors are used to introduce genes into cells. types of DED from other DED-containing proteins are We expect our stable cell lines to have few side effects when fused to E protein and introduced into cells. At present, we apoptosis is induced by tetracycline- or mifepristone- do not know if the double DED in 2DEDplusE is really induced expression of only one protein. effective, as we did not obtain comparative data for single The second reason that our cell lines are useful for DEDs fused to E protein. Our primary goal is to further analyzing apoptosis is that it allows synchronization of the enhance apoptosis-inducing activity by protein modifica- events of apoptosis and occurs over a short period of time. tion. We believe using a single DED should result in the This allows detailed analysis of each step of apoptosis. We same or less enhancement than using two DEDs because of can use these stable cell lines to examine the changes in

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Mol Cancer Ther 2007;6(6). June 2007

Tomoki Takashina and Manabu Nakayama

Mol Cancer Ther 2007;6:1793-1803.

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