Endonucleases induced TRAIL-insensitive apoptosis in ovarian carcinoma cells

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Chapter 6 Endonucleases induced TRAIL-insensitive apoptosis in ovarian carcinoma cells T. M. Geel1, G. Meiss2, B.T. van der Gun1, B. J. Kroesen1, L. F. de Leij1, M. Zaremba3, A. Šilanskas3, M. Kokkinidis4, A. Pingoud2, M. H. Ruiters1,5, P. M. McLaughlin1, #, M. G. Rots1,#, *

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2. 3. 4. 5.

Department of Pathology and Medical Biology, Groningen University Institute for Drug Exploration (GUIDE), University Medical Center Groningen (UMCG), 9713 GZ, Groningen, the Netherlands Institute of Biochemistry, Justus-Liebig-University Gießen, D-35392 Giessen, Germany Institute of Biotechnology, Vilnius LT-02241, Lithuania IMBB/FORTH & University of Crete/Dept. of Biology, GR-71409 Heraklion/Crete, Greece Synvolux therapeutics, Groningen, The Netherlands

Exp Cell Res. 2009; 315(15): 2487-2495

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ABSTRACT TRAIL induced apoptosis of tumor cells is currently entering phase II clinical settings, despite the fact that not all tumor types are sensitive to TRAIL. TRAIL resistance in ovarian carcinomas can be caused by a blockade upstream of the caspase 3 signaling cascade. We explored the ability of restriction endonucleases to directly digest DNA in vivo, thereby circumventing the caspase cascade. For this purpose, we delivered enzymatically active endonucleases via the cationic amphiphilic lipid SAINT18“:DOPE to both TRAIL sensitive and insensitive ovarian carcinoma cells (OVCAR and SKOV-3, respectively). Functional nuclear localization after delivery of various endonucleases (BfiI, PvuII and NucA) was indicated by confocal microscopy and genomic cleavage analysis. For PvuII, analysis of mitochondrial damage demonstrated extensive apoptosis both in SKOV-3 and OVCAR. This study clearly demonstrates that cellular delivery of restriction endonucleases holds promise to serve as a novel therapeutic tool for the treatment of resistant ovarian carcinomas.

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INTRODUCTION Dysfunction of apoptosis contributes to many diseases including cancer. Two major apoptotic pathways have been identified. The receptor mediated pathway, or death-receptor pathway, is triggered by the death receptor super family and responds mainly to extracellular stimuli. The receptor independent, or mitochondrial pathway, is activated by intracellular signals such as DNA damage and growth factor depletion. These two pathways converge at the level of caspase 3 activation, resulting in the execution of apoptosis 1,2. Anticancer therapies currently in clinical use induce apoptosis of the target cell either way. A potent death-receptor mediating anticancer agent is TRAIL (tumor necrosis factor-related apoptosis inducing ligand), which has been shown to induce apoptosis in many tumor cells via binding to death-receptors 4 and 5. Because TRAIL exhibits no apoptotic activity towards normal human cells3-7, it is an attractive candidate for cancer treatment. However, some tumor types, including some ovarian carcinomas, are resistant to TRAIL 8-18. Ovarian cancer represents the most fatal gynecologic type of cancer in the world. Current treatments involve surgery followed by chemotherapy. However, development of chemo-resistance is frequently observed posing a serious limitation to the success of current therapeutic approaches. Therefore, development of novel therapeutic entities, such as TRAIL, is highly warranted. However, resistance of ovarian carcinomas to TRAIL has been reported, and is partly associated with defects located upstream of the caspase 3 signaling cascade10. Treatment strategies for TRAIL-resistant ovarian carcinomas should therefore include a strategy to bypass this blockade. Caspase 3 activation is followed by DNA cleavage effected by endogenous nucleases leading to DNA laddering, a feature of apoptotic cells. Direct induction of DNA cleavage therefore provides a good alternative to circumvent the caspase 3 activation step. In this context, endogenous endonucleases, although extensively used as laboratory tools for molecular biological purposes, have not been used as pharmaceutical agents. So far, no direct delivery of restriction endonucleases has been investigated as anti-cancer agent. In this study, we demonstrate functional delivery of restriction endonucleases to ovarian carcinoma cell lines using SAINT-18“:DOPE. We tested the randomly cutting endonuclease NucA, and its inactive forms19, and two six-base-pair cutting restriction endonucleases PvuII and BfiI including the mutant variant BfiI-K107A20,21,21 and identified especially PvuII as a strong inducer of apoptosis in both TRAIL sensitive and insensitive cell lines. MATERIAL AND METHODS Reagents PvuII, which cleaves the palindromic sequence 5’-CAGCTG-3’ in a Mg2+-dependent manner resulting in double strand breaks with blunt-ends21 and NucA, a non-specific endonuclease from the EEDMe-finger nuclease super family, which nicks the DNA19, its variant NucA-H124A, and the inactive nuclease-inhibitor complex NucA-NuiA, were kindly provided by Dr. W. Wende (Gießen, Germany). The mutant NucA-H124A possesses a His to Ala mutation at residue 124, positioned in the catalytic

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centre of the enzyme19. In the inactive nuclease/inhibitor complex NucA-NuiA, the inhibitor NuiA binds NucA with picomolar affinity, thereby preventing digestion of nucleic acids by NucA. BfiI, which cleaves the DNA at fixed positions downstream of an asymmetric sequence (5’-ACTGGG-3’) in a double strand break but acts without requiring metal ions 20, and its mutant variant BfiI-K107A, which possesses a Lys to Ala mutation at residue 107, were kindly provided by V. Siksnys (Vilnius, Lithuania). SAINT-18“:DOPE (commercially available as SAINT:MIX) was purchased from Synvolux Therapeutics Inc. (Groningen, The Netherlands). The caspase inhibitors Z-VAD-fmk was obtained from Calbiochem (San Diego, CA, USA). Actinomycin D was obtained from USB Corporation (Cleveland, USA). DioC6 (3,3’dihexylocarbocyanine) was purchased from Molecular Probes (Eugene, OR, USA). scFv425:sTRAIL was kindly provided by Dr. W. Helfrich (University Medical Center Groningen, the Netherlands). Cell Culture The human ovarian cancer cell lines SKOV-3 and OVCAR were obtained from the ATCC (Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 50μg/ml gentamycine sulfate, 2mM L-glutamine, 10% FBS (BioWhittaker Inc, Walkersville, MD) at 37qC under humidified conditions. Primary isolated Fetal Lung Fibroblasts (FLF) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 50μg/ml gentamycine sulfate, 2mM L-glutamine, 10% FBS (BioWhittaker Inc, Walkersville, MD) at 37qC under humidified conditions. HUVEC (human umbilical vein endothelial cells) were obtained from the UMCG Endothelial Cell Facility and cultured in RPMI 1640 supplemented with 20% heat inactivated FBS, 2 mM L-glutamine, 5 U/ml heparin (Leo, Weesp, The Netherlands), 100 U/ml penicillin (Yamanouchi Pharma, Leiderdorp, The Netherlands), 100 μg/ml streptomycin, and 50 μg/ml crude endothelial cell growth factor (home isolated) in 1% gelatin-coated tissue culture flasks at 37˚C under humidified conditions. Anonymized primary human skin fibroblasts were kindly provided by Dr. K.E. Niezen-Koning (University Medical Center Groningen, the Netherlands) and were cultured in F10HAM medium supplemented with 10% FCS and 2% Penicillin-Streptomycin (Invitrogen, Breda, The Netherlands). Cleavage of endogenous genomic DNA SAINT-18“:DOPE (SD) was used to deliver restriction endonucleases as protein (profection, in detailed described in22). Cells were seeded at 0.3 × 106/6-well and protein delivery was performed at 50-80% confluency. Cells were treated with 1, 2 or 5µg endonuclease alone or in complex with SD. As positive controls for inducing cell death Actinomycin D at a final concentration of 0.2µg/ml in serum-containing medium and scFv425:sTRAIL23 at a final concentration 2.7µg/ml were used. The cells were incubated for 24 or 48 h at 37ºC in humidified 5% CO2. To inhibit caspase activity, 20µM (final concentration) Z-VAD-fmk was added.

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Apoptosis Analyses DNA fragmentation analysis. Cells were collected 48 h after treatment and resuspended in lysis buffer (0.5% N-Lauroyl sarcosine (Sigma), 0.5mg/ml RNase A, 1mg/ml Proteinase K (Invitrogen) in 50mM Tris-HCl, pH, 8.0) and incubated at 50°C for 2 h. The samples were sheared, loaded on a 1.3% agarose gel and run for 15min at 100V (dry, after 15min 1×TAE was added). DNA was visualized by ethidium bromide fluorescence on exposure to ultra-violet light. For analysis of DNA fragmentation by flow cytometry, cells were collected and pellets resuspended in 100µl PBS. After addition of 3ml ice-cold (-20°C ) 70% EtOH, cells were incubated for 1 h at 4°C, washed twice with PBS and centrifuged for 5 min at 1800 rpm. After which 100µl 1:10 RNase buffer (10mM Tris, 15mM NaCl, RNase (10mg/ml) was added followed by 10 min of boiling (to deactivate DNases). After 30 min of incubation at 37°C 200µl PI solution (0.1µg/µl in PBS) was added. After another 30 min incubation at 4°C, cells were analyzed using a Calibur flow cytometer (BD Biosciences, Breda, the Netherlands). Mitochondrial-membrane potential analysis. Apoptosis was assessed by loss of mitochondrialmembrane potential using the cell permanent green-fluorescent lipophilic dye DioC6. Cells were harvested by centrifugation (1500 rpm for 5 min), incubated for 20min at 37°C with 0.1mM DiOC6 in fresh medium, harvested (1500 rpm for 5 min), resuspended in 250µl fresh medium and analyzed for DioC6 staining using a Calibur flow cytometer.

6 Confocal microscopy To visualize intracellular delivery of the restriction endonuclease PvuII confocal microscopy was performed. For profection, SKOV-3 cells were seeded 0.3 × 10 5/8-well (8 well 1µ-Slide IBIDI treat from Ibidi) and protein delivery was performed at 40% confluency. Cells were treated with 0.1 µg PvuII labelled with the fluorescent dye alexa-488 with or without SD. 48 h after profection cells were fixed in 4% formaldehyde/DMEM+ for 15 minutes at 37˚C, after washing cells were permeabilized with 0.2% Triton X100/PBS for 10 minutes at RT. Finally, nuclei were counterstained with To-Pro3 (Invitrogen) / PBS solution (1:5000) for 15 minutes at RT. Cells were imaged using a Leica TCS SP2 (AOBS) inverted confocal microscope with a 40x optical lens. Analysis was performed using Image J software. Phosphorylated Histone 2AX (γH2AX) detection γH2AX Western Blot. Cells were lysed in 200 μl buffer (20mM Tris-HCl, 5mM EDTA, 2mM EGTA, 100mM NaCl, 0.05% SDS, 0.5% NP-40, 1mM Phenylmethylsulfonyl Fluoride (PMSF), 10μg/ml Aprotein, 10μg/ ml Leupeptin and loading buffer with 2-mercapto-ethanol). Proteins were separated by SDS-PAGE and electroblotted onto 0.45μm nitrocellulose transfer membranes (Schleicher & Schuell, Dassel, Germany). Immunodetection was done with phosphorylated Histone 2AX (Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb (alexa fluor 488 conjugate) #9719, Cell Signaling, Leiden, The

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Netherlands), or 1:1000 of caspase-8 antibody (1C12) (#9746, Cell Signaling) followed by RDM-PO and visualized using the ECL chemoluminescence detection kit (Pierce, Rockford, IL). γH2AX FACS analysis. At indicated time points, cells were harvested and washed with PBS. Cells were fixed in 4% Formaldehyde (Merck) / PBS for 10 minutes at 37°C. After addition of 1 ml PBS and chilling on ice for 1 minute, cells were washed and permeabilized in 90% methanol / PBS for 30 minutes on ice. Staining of phosphorylated H2AX was performed as follows: permeabilized cells were washed by addition of 2 ml of incubation buffer (0.5% BSA in PBS). Cells were resuspended in 85 µl incubation buffer followed by addition of 10µl of conjugated antibody, 4µl of PI (final conc. 10µg/ml; Nexins, Kattendijke, The Netherlands) and 1µl of RNase (final conc. 100µg/ml) and were incubated for 30 minutes in the dark at RT. Cells were washed again with incubation buffer, resuspended in 200µl PBS and analyzed for phosphorylated H2AX staining using a Calibur flow cytometer. Graphs were made using WinMDI software. Statistical analysis Statistical comparisons between untreated cells (BL) and cells treated with SD + restriction endonuclease, Actinomycin D or scFv425-TRAIL were performed using the paired t-test. Statistical significance was indicated by P < 0.05. RESULTS Analysis of genomic DNA cleavage by profected restriction endonucleases Functional delivery of different types of restriction endonucleases was studied using SAINT18“:DOPE as delivery device. We tested the randomly cutting non-specific endonuclease NucA, and its less active forms, as well as three less frequently cutting type II REases, PvuII and BfiI and the BfiI variant K107A. The restriction endonucleases were delivered into SKOV-3 cells with or without SD and DNA fragmentation gel analysis was performed on DNA isolated from treated cells (Figure 1). 48 h after profection, NucA was able to completely fragment the DNA (Figure 1a). BfiI caused fragmentation of the DNA already 4 h after profection, but the nicks seemed to be repaired after 24 h (Figure 1b and Figure 1c respectively). PvuII was most effective in inducing DNA fragmentation 48 h after profection (Figure 1d). These data indicate that the endonucleases are able to exert their catalytic function after intracellular delivery. Apoptotic effects of endonucleases after delivery with SD After establishing intracellular and functional delivery of the restriction endonucleases, we compared the observed restriction endonuclease mediated apoptosis with apoptosis induced by Actinomycin D and scFv425:sTRAIL 23 (Figure 2). In concordance with literature, the ovarian carcinoma cell lines used in this study respond differently to the potent apoptosis-inducer TRAIL: SKOV-3 is TRAIL resistant, whereas OVCAR is sensitive for TRAIL induced apoptosis. scFv425:sTRAIL

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Figure 1. Cleavage pattern of Restriction endonucleases on genomic DNA after delivery of restriction enzymes using SD. SKOV-3 cells were profected for indicated time points with: a. 10µg of the non-specific endonuclease NucA, and inactive forms of the enzyme for 48 h. lane 1, Blanco; 2, SD; 3, SD+NucA; 4, SD+H124A; 5, SD+NucD; 6, Actinomycin D. b. 2µg BfiI (type IIS REase) and the BfiI variant K107A for 4 h. 1, Marker; 2, Blanco; 3, SD; 4, BfiI; 5, SD+BfiI; 6, K107A; 7, SD+K107A. c. 2µg BfiI (type IIS REase) and the BfiI variant K107A for 24 h. 1, Blanco; 2, SD; 3, BfiI; 4, SD+BfiI; 5, K107A; 6, SD+K107A. d. 1µg of PvuII (type IIP REase), BfiI (type IIS REase) and NucA for 48 h. 1, Blanco; 2, SD; 3, PvuII; 4, SD+PvuII; 5, BfiI; 6,SD+BfiI; 7, NucA; 8, SD+NucA; 9, Act.D; 10, scFv425:TRAIL. After profection, the fragmented DNA was sheared and analyzed by 1.3% agarose gel electrophoresis as described in “Material and Methods”.

was able to induce apoptosis in the TRAIL-sensitive ovarian carcinoma cell line OVCAR (73% ± 0.7 %), but not in SKOV-3 cells (12% ± 4.2%). The positive control Act.D led to induction of apoptosis in both cell lines (48% ± 3.8% in OVCAR and 35% ± 18% in SKOV-3). Treatment of OVCAR (Fig 2a) and SKOV-3 (Fig 2b) cells with NucA:SD hardly induced any apoptosis, whereas treatment with BfiI:SD induced apoptosis in 54 (± 5.3%) and 21% (± 4%) of OVCAR and SKOV-3 cells, respectively. Treatment of PvuII:SD induced the highest rates of apoptosis in both OVCAR (50% ± 0.5%) and SKOV-3 cells (77% ± 3.4%). These findings expose PvuII, as low as 1µg/ml (data not shown), as an efficient endonuclease for the induction of apoptosis also in the TRAIL-resistant cell line. Analysis of nuclear localization of the restriction endonuclease PvuII To demonstrate that the observed cell death is due to nuclear activity of the restriction endonucleases, we visualized the subcellular localization of PvuII labelled with the fluorescent dye alexa 488 using confocal microscopy. The pictures indicate nuclear localization of the restriction endonuclease PvuII-alexa 488 using ToPro-3 to counterstain the nucleus (Figure 3). PvuII-alexa 488 alone was also efficiently taken up by cells, but remained localized in the cytoplasm. The presence of vesicles were observed by both PvuII-alexa 488 + SD and PvuII-alexa 488 alone, suggesting the involvement of an endocytic pathway in the internalization. To further confirm functional intranuclear delivery, PvuII was delivered into SKOV-3 cells using SD and cells were analyzed by intranuclear DNA fragmentation. FACS analysis of cellular DNA 83

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Figure 2. Apoptosis induction after delivery of endonucleases with SD as determined by loss of mitochondrial potential. OVCAR (Fig 2a) and SKOV-3 (Fig 2b) were profected with the endonucleases NucA, PvuII and BfiI (5µg) in complex with SD. After 48 h, the cells were stained with DioC6 and analyzed by flow cytometry.

Figure 3. Intranuclear delivery as assessed by confocal imaging. SKOV-3 cells were profected with 0.1 µg PvuII-alexa 488 with or without SD. 48 h after treatment, cells were fixed and nuclei were counterstained using the dye ToPro-3. Cells were imaged using a Leica TCS SP2 (AOBS) inverted confocal microscope. (see for color image page 157)

content, demonstrated fragmentation 48 h after delivery of PvuII in SKOV-3. Treatment of the cells with SD or PvuII alone did not cause fragmentation of the DNA (Figure 4). Next, we investigated the kinetics of PvuII delivery in SKOV-3. Upon induction of double strand breaks, the histone H2AX becomes rapidly phosphorylated and is recruited to the site of DNA damage. SKOV-3 cells were analyzed for phosphorylated H2AX using FACS analysis, 4, 8, 24 and 84

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Figure 4. In vitro DNA fragmentation after profection of PvuII using SD. FACS analysis was performed on SKOV-3 cells after treatment with SD+PvuII (10 µg) to confirm intranuclear DNA fragmentation after 48 h. The peak arising in front of the G0 peak (SD + 10µg) represents DNA fragments.

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Figure 5. Flow cytometric analysis of phosphorylated H2AX in SKOV-3 treated with PvuII with or without SD. SKOV-3 cells were treated with PvuII with or without SD. At indicated time points, cells were collected and stained for phosphorylated H2AX and PI to counterstain cellular DNA. FACS analysis was performed to analyse phosphorylation of H2AX. Graphs were made using WinMDI software. Figure 5a shows the results 4, 8, 24 and 30 after treatment with PvuII:SD. Figure 5b shows the results 30 h after treatment with PvuII, PvuII:SD and Act.D. Cut off for true γH2AX positive cells is shown by line at 10 2 MFI.

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Figure 6. Determination of differential expression levels of γH2AX and caspase 8 after treatment of TRAIL resistant SKOV-3 and TRAIL sensitive OVCAR cells. Expression levels of phosphorylated H2AX (Fig 6a for nuclear extracts and Fig 6b for whole cell lysate), and expression and activation of caspase 8 (Fig 6c, whole cell lysate) in SKOV-3 and OVCAR profected cells with 1µg of PvuII were analyzed by Western Blot. Nuclear extracts and whole cell lysates were collected and were separated by SDS-PAGE 48 h after profection.

30 h after profection with PvuII (Figure 5). As untreated cells are known to contain background levels of γH2AX, we set the cut off for γH2AX at 10 2 (0.1% of normal cells). Delivery of PvuII resulted in phosphorylation of H2AX already 4 h after profection: 32% of the cells were positive for phosphorylated H2AX and this percentage increased to 50% for 30 h after treatment (Fig 5a). Actinomycin D treatment also resulted in phosphorylated H2AX and increased over time although in a lesser extent (to 9% after 4 h (data not shown) and 34% after 30 h) (Fig 5b). Using Western Blot analysis (Figure 6) we showed that scFv425:sTRAIL treatment after 48 h resulted in phosphorylation of H2AX and in caspase 8 activation in the TRAIL sensitive cell line OVCAR, but not in the TRAIL resistant cell line SKOV-3. Treatment with PvuII and Actinomycin D led to phosphorylation of H2AX and to caspase 8 activation in both cell lines. These data demonstrate that we efficiently induced double strand breaks and caspase activation for PvuII as well as for Actinomycin D in both SKOV-3 and OVCAR, showing that PvuII induces apoptosis in a TRAIL independent manner.

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Figure 7. Effect of caspase inhibition on treatment with PvuII:SD. The inhibition of z-VAD-fmk (20µM) on endonucleasemediated apoptosis was analyzed by flow cytometry. SKOV-3 and OVCAR cells were profected with 1µg of PvuII and SD in the absence or presence of 20µM Z-VADfmk. As a control, Actinomycin D and scFv425-sTRAIL were used. After 48 h, apoptosis was assessed by loss of mitochondrial-membrane potential.

To further demonstrate this, we performed PvuII profection experiments in SKOV-3 and OVCAR cells with or without the total caspase inhibitor Z-VAD-fmk. After 48 h, cells were harvested and were analyzed for induction of apoptosis by FACS analysis (Figure 7). Treatment with PvuII:SD resulted in induction of apoptosis in both OVCAR as well as SKOV-3 cells even in the presence of Z-VAD-fmk. Treatment with scFv425:sTRAIL induced apoptosis and was efficiently blocked in the TRAIL-sensitive cell line OVCAR in the presence of the caspase inhibitor. A Alexa 488 ToPro-3 Overlay

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Figure 8. Effects of PvuII on normal human cells. Primary human skin fibroblasts (Fig 8a), HUVEC (Fig 8b) and Fetal Lung Fibroblasts (Fig 8c) were used as model system to asses the effect of SD + PvuII on normal cells. Cells were profected with PvuII (1µg) in complex with SD. After 48 h cells were stained with DioC6 and analyzed by flow cytometry. The insert in Fig 8a shows primary human skin fibroblasts profected with 0.1 µg PvuII-alexa 488 with or without SD. 48 h after treatment cells were fixed, nuclei were counterstained with ToPro-3 and nuclear delivery was analyzed using confocal microscopy (included in Fig 8a).

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Effect of PvuII on normal cells PvuII successfully induces apoptosis in both TRAIL sensitive and TRAIL resistant ovarian carcinomas, however for a clinical setting the potential toxic effect towards normal cells needs to be assessed. Figure 8 depicts the results 48 h after profection of 1µg PvuII in primary human skin fibroblasts (Fig 8a), HUVECs (Fig 8b) and Fetal Lung Fibroblasts (Fig 8c). After treatment with PvuII:SD or scFv425:sTRAIL, no apoptosis induction was observed after 48 h (7%) in primary human skin fibroblasts, although confocal microscopy confirmed nuclear localization. Actinomycin D led to an induction of apoptosis of 57%. In HUVEC cells, already a high background level of apoptosis was observed (35%). Treatment with PvuII:SD only slightly increased apoptosis (50%), while again no effect was observed for scFv425:sTRAIL. Actinomycin D did induce apoptosis in 90% of the HUVECs. Fetal Lung Fibroblast cells showed 60% apoptosis after treatment with PvuII:SD. Actinomycin D led to a strong induction of apoptosis (75%). Interestingly, also scFv425-scTRAIL showed apoptotic activity towards Fetal Lung Fibroblast cells (30%). These data demonstrate that treatment with PvuII:SD does induce apoptosis in HUVEC and Fetal Lung Fibroblast cells, but not in primary human skin fibroblasts. While for the carcinoma cells up to 2 fold higher apoptosis was induced by PvuII compared to Actinomycin D, the extent of apoptosis in “normal” cells, however, is lower then apoptosis induced by Actinomycin D. DISCUSSION Cells and tissues in the human body are maintained in homeostasis by the physiological process of apoptosis. Malfunction of apoptosis is considered to be an important step in the development of cancer and metastasis. Various anticancer therapies thus aim to induce apoptosis of the tumor cells. Although TRAIL is a potent and specific apoptosis inducer and as such a promising anticancer agent3-6, mechanisms of TRAIL resistance have been reported including IL-8 overexpression and altered turnover of caspase 38,11-18,24. To bypass TRAIL resistance, we propose to directly deliver restriction endonucleases as potent apoptosis inducers. In this study, we demonstrate that direct nuclease-mediated cleavage of the DNA indeed results in apoptosis in both TRAIL sensitive as well as in TRAIL resistant ovarian carcinoma cells. Of the three different types of restriction endonucleases tested, PvuII was most efficient in inducing apoptosis not only in TRAIL sensitive but also in TRAIL resistant ovarian cancer cell lines. We confirmed nuclear localization of PvuII after delivery with SAINT-18 “:DOPE (SD) by confocal microscopy. To provide further evidence that the induction of apoptosis is caused by direct DNA cleavage by PvuII, we investigated the H2AX phosphorylation kinetics and the caspase (in) dependency. Upon induction of DNA double strand breaks the histone variant H2AX becomes rapidly phosphorylated and is recruited near the DSB site25-27. FACS analysis showed that treatment with the apoptosis-inducing Actinomycin D in the TRAIL resistant cell line SKOV-3 resulted in phosphorylation of H2AX, while treatment with scFv425:sTRAIL did not lead to phosphorylation of H2AX. As expected, treatment with PvuII:SD resulted in phosphorylation of H2AX, which increased

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over time. The TRAIL sensitive cell line OVCAR demonstrated phosphorylation of H2AX both by scFv425:sTRAIL and SD:PvuII treatment (data not shown). The relative low levels of phosphorylation of H2AX caused by Actinomycin D in both lines might be explained by the fact that Actinomycin D does not directly cleaves the DNA but interacts with the DNA 28-30. To demonstrate that the DSBs are the direct effect of PvuII and not through caspase mediated cleavage, Z-VAD-fmk was used in our experiments to block total caspase activity. PvuII induced apoptosis was slightly blocked by ZVAD in both cell lines, whereas scFv425:sTRAIL induced apoptosis was completely blocked by the caspase inhibitor. The caspase independent mechanism of action of PvuII was further confirmed by the absence of Z-VAD effect on phosphorylation kinetics of H2AX (data not shown). The data presented here clearly demonstrate efficient delivery of the restriction endonucleases using SD, resulting in apoptosis. The apoptotic activity, however, was also observed to some extent for normal cells, requiring targeted delivery for in vivo application. Coupling of antibodies or peptides to SD (manuscript in preparation) and other liposomal formulations has shown promise for targeted delivery strategies towards cancer cells or to sites of chronic inflammation31-33. As such targeting strategies are being optimized, direct delivery of restriction endonucleases offer a promising approach of inducing targeted cell death for otherwise resistant tumors. Acknowledgements The project was funded by the European Union FP6 program, activity NEST, contract number 015509, MenuG. We thank A.J. Noel (Gießen, Germany) for production of NucA, V. Siksnys (Vilnius, Lithuania) for providing BfiI and the mutant BfiI-K107A, W. Helfrich (UMCG) for providing scFv425:sTRAIL and A. Arendzen, H.G. Kazemier, G. Mesander and K. Sjollema (UMCG) for excellent technical assistance. We also thank H. Moorlag and G. Molema (UMCG) for providing HUVEC cells. M.H.J. Ruiters is Associate Professor at the department of Medical Biology, next to his involvement in the company Synvolux which owns the patent of the liposomal compound SAINT-18“:DOPE. Gregor Meiss was supported by the Dr.-Herbert-Stolzenberg-Stiftung of the Justus-Liebig-University Giessen.

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