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Originally published In Press as doi:10.1074/jbc.M401327200 on August 10, 2004

J. Biol. Chem., Vol. 279, Issue 42, 44005-44011, October 15, 2004
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Transduction of the TAT-FLIP Fusion Protein Results in Transient Resistance to Fas-induced Apoptosis in Vivo*

Stefan Krautwald{ddagger}, Ekkehard Ziegler{ddagger}, Karen Tiede, Rainer Pust, and Ulrich Kunzendorf§

From the Department of Nephrology and Hypertension, University of Schleswig-Holstein, Campus Kiel, 24105 Kiel, Germany

Received for publication, February 6, 2004 , and in revised form, August 10, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Although tightly regulated programmed cell death (apoptosis) possesses great importance for tissue homeostasis, several pathologic processes are associated with organ failure due to adversely activated cell apoptosis. Transient increase in apoptosis has been shown to cause organ damage during fulminant hepatitis B, autoimmune diseases, ischemia-reperfusion injury, sepsis, or allograft rejection. A defined and temporary inhibition of cell apoptosis may therefore be of high clinical relevance. Activation of death receptors results in caspase-8 recruitment to the death-inducing signaling complex, which initiates the apoptotic process through cleavage of caspase-8 and downstream substrates. This initial step may be inhibited by the caspase-8 inhibitor FLIP (FLICE inhibitory protein). To specifically inhibit the initiation of death receptor-mediated apoptosis we constructed a fusion protein containing FLIP fused N-terminally to the human immunodeficiency virus TAT domain. This TAT domain allows the fusion protein to cross the cell membrane and thus makes the FLIP domain able to interfere with the death-inducing signaling complex inside of the cell. We observed that incubation of lymphocytic Jurkat or BJAB cells with TAT-FLIPS proteins significantly inhibits Fas-induced activation of procaspase-8 and downstream caspases, preventing cells from undergoing apoptosis. Systemic application of TAT-FLIPS prolongs survival and reduces multi-organ failure due to Fas-receptor-mediated lethal apoptosis in mice. Therefore, application of cellular FLIPS in the form of a TAT fusion protein may open a promising, easily applicable new tool for providing protection against transient, pathologically increased apoptosis in various diseases.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Because of severe organ damage due to misregulated apoptosis in various diseases, intense studies have focused on apoptosis signaling pathways triggered by death receptors and their ligands including the Fas ligand, the tumor necrosis factor, and the tumor necrosis factor-related apoptosis-inducing ligand (1-3). Fas (APO-1/CD95) surface expression does not necessarily render cells susceptible to Fas ligand-induced apoptosis because of counteracting physiological cellular inhibitors. One of these regulators of death receptor-mediated apoptosis was termed FLIP1 (for FLICE inhibitory protein), which is predominantly expressed in lymphoid tissues (4, 5). Cross-linking of Fas-sensitive cells by the Fas ligand or an agonistic antibody induces apoptosis through procaspase-8 recruitment to the Fas-mediated death-inducing signaling complex (DISC), where procaspase-8 is cleaved to initiate apoptosis through a systematic cleavage of downstream substrates. The recruitment of the caspase-8 inhibitor cFLIP into the DISC prevents the cleavage of procaspase-8, resulting in concomitantly reduced apoptosis (6).

Multiple splice variants of cFLIP have been reported, but to date only a long and a short form, designated cFLIPL and cFLIPS, respectively, could be detected on a protein level. It has been shown that, in the presence of cFLIPS, procaspase-8 is recruited into the DISC but remains unprocessed (7). Thus FLIPS appears to be a good candidate to block apoptosis in death receptor-mediated caspase-8 dependent pathways.

In previous investigations it has been demonstrated that protein transduction is a powerful tool for introducing full-length proteins into cells without the help of viral or chemical transporters (8, 9). The principle of protein transduction originates from the biology of various viruses. In vivo analysis of the transduction properties of the HIV TAT domain demonstrated that almost all cells within the body, even those protected by the blood-brain barrier, were targeted by TAT fusion proteins after intraperitoneal application (10).

Transient blockage of apoptosis may be useful in clinical settings in which the initiation of death receptor-mediated apoptosis is the main pathogenic principle, such as certain forms of acute liver failure (11), reperfusion injury (12-14), or sepsis (15). In this context, the aim of this study was to transiently inhibit the Fas-mediated activation of initiator procaspase as a key regulator step in apoptosis by protein transduction of FLIPS and to restrain death receptor-mediated apoptosis in mice.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cloning and Expression of TAT-FLIPS—Total RNA was isolated from human peripheral blood lymphocytes by using RNeasy mini-columns (Qiagen, Hilden, Germany). The short form of human FLIPS cDNA was generated by using the ThermoScript reverse transcriptase PCR system (Invitrogen) with the forward (5'-CAAGCCATGGCTGCTGAAGTCATCCATCAGGTTGAA-3') and reverse (5'-CCGCTCGAGTCACATGGAACAATTTCCAAGAATTTTCA-3') primers. The pTAT vector (generous gift of S. F. Dowdy) has an N-terminal His6 leader followed by the 11-amino acid TAT protein transduction domain and a polylinker (16). The PCR product was cloned into NcoI/XhoI-cut pTAT vector.

A TAT construct containing the FLIP domain mutated within the death effector domain (named TAT-{Delta}FLIPS) served as a specific negative control that lacks the ability to protect against Fas-induced apoptosis. The plasmid was generated by site-directed mutagenesis within the first death effector domain of FLIP. Therefore, the amino acids RFD at the positions 64-66 of cFLIP (17) were replaced by AAA using the QuikChange mutagenesis kit (Stratagene). The complementary oligonucleotides 5'-CTCTACAGAGTGAGGGCGGCCGCCCTGCTCAAACGTATC-3' (forward) and 5'-GATACGTTTGAGCAGGGCGGCCGCCCTCACTCTGTAGAG-3' (reverse) were used for the desired mutation. The underlined sequences were the replaced amino acids.

The resulting constructs of a selected colony harboring the cDNAs of TAT-FLIPS as well as control plasmids TAT-{Delta}FLIPS and pTAT-{beta}-galactosidase (the latter one kindly provided by S. F. Dowdy) were transformed into competent Escherichia coli BL21 (DE3)pLysS bacteria, followed by the induction of expression in Luria-Bertani media with 500 µM isopropyl-{beta}-D-thiogalactopyranoside. After 3 h of incubation at 37 °C, cells were harvested by centrifugation (6,5000 x g for 10 min at 4 °C) followed by sonification in binding buffer (500 mM NaCl, 20 mM Tris-HCl, and 5 mM imidazole, pH 7.9). The suspensions were clarified by centrifugation (14,000 x g for 20 min at 4 °C), and the supernatants containing TAT proteins were purified under native conditions using pre-equilibrated nickel-nitrilotriacetic acid columns (Qiagen). To remove the high background of contaminating bacterial proteins, columns were washed by the stepwise addition of increasing imidazole concentrations. Finally, the target proteins were eluated with an elution buffer containing 500 mM NaCl, 20 mM Tris-HCl, and 100 mM imidazole (pH 7.9). The removal of salt was performed using a disposable PD-10 (Sephadex G-25) desalting column equilibrated in an RPMI medium. The fusion proteins were either used immediately after purification or stored at 4 or -80 °C.

Cell Culture and in Vitro Apoptosis Analysis by DNA Fragmentation Assay and Annexin V Staining—The human T lymphocytic Jurkat cells and the human B-cell line BJAB (American Type Culture Collection, Manassas, VA) were cultured in RPMI supplemented with 10% fetal calf serum and penicillin-streptomycin. For experiments, the cells were seeded at a density of 1.5 x 106 cells/ml. An investigation of anti-Fas-induced apoptosis was performed by preincubation of cells with a TAT fusion protein (500 nM) for 30 min prior to the addition of 100 ng/ml anti-Fas antibody (clone 7C11, Immunotech, Marseille, France) for the indicated times (Fig. 1).



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  		FIG. 1.
Transducibility of TAT-FLIPS and TAT-{Delta}FLIPS in vitro. A, 106 Jurkat cells were incubated with 500 nM TAT-FLIPS or 500 nM TAT-{Delta}FLIPS for the indicated times at 37 °C. Equal protein amounts (10 µg/lane) were resolved on a 15% SDS-polyacrylamide gel and transferred onto nitrocellulose membrane for immunoblotting. Transduced TAT fusion proteins were detected with an anti-FLIP antibody. B, 106 Jurkat cells were incubated with different amounts of TAT-FLIPS or TAT-{Delta}FLIPS, respectively, at 37 °C for 30 min and processed as described above.

 
Stimulations were terminated by aspirating the medium and washing five times with ice-cold phosphate-buffered saline. The rate of apoptosis was analyzed by a DNA fragmentation assay using the diphenylamine reagent. Briefly, cells were harvested by centrifugation, lysed in a buffer containing 0.5% Triton X-100, 5 mM Tris, pH 8, and 20 mM EDTA for 30 min on ice and centrifuged for 15 min at 14,000 x g to separate DNA fragments (supernatants) from intact chromatin (pellets). Subsequently, each DNA sample in the supernatant and the pellet was precipitated in 10% trichloroacetic acid at 4 °C overnight and quantified using a diphenylamine reagent after hydrolysis in 5% trichloroacetic acid at 95 °C for 15 min. The percentage of fragmented DNA in each sample was calculated as the amount of DNA in the supernatant divided by the total DNA for that sample (supernatant plus pellet). Significance was assessed by a two-way analysis of variance followed by a Fisher's protected least significant difference (PLSD) test and was assumed statistically significant for p values < 0.05.

In other experiments, apoptosis was quantified by Annexin V staining and fluorescence-activated cell sorter analysis. After 4 h of stimulation the cells were harvested, washed with RPMI supplemented with 2% fetal calf serum, and Annexin V staining (ApoAlert, Annexin VFITC, BD Biosciences) was performed according to the manufacturer's instructions. Fluorescence was analyzed by an EPICS XL® (Coulter, Krefeld, Germany). Flow cytometry data were analyzed using the EPICS System II software.

Cell Lysis and Immunoblotting—Cells were lysed in modified ice-cold Frackelton cell lysis buffer (18). Insoluble material was removed by centrifugation (14,000 x g for 10 min at 4 °C). Protein concentration was determined (Bradford method) according to the manufacturer's instructions (Bio-Rad). Equal protein amounts (20 µg/lane) were resolved on 15% SDS-PAGE gels and transferred to a nitrocellulose membrane (Amersham Biosciences) for immunoblotting. Western blots were performed using a polyclonal anti-FLIP NT antibody (Biomol, Hamburg, Germany), a polyclonal anti-cleaved caspase-3 antibody (Asp-175), or a monoclonal anti-caspase-8 antibody (1C12 [PDB] , both from BioLabs, Frankfurt, Germany), respectively, and horseradish peroxidase-linked anti-mouse or anti-rabbit antibody (both from Promega), respectively. Immune complexes were visualized by enhanced chemiluminescence (ECL, Amersham Biosciences).

Induction and Analysis of Anti-Fas-mediated Apoptosis in Vivo— Female Balb/c mice were obtained from Charles River (Sulzfeld, Germany) and used for experiments at 6-8 weeks of age. Apoptosis was induced by intraperitoneal injection of the monoclonal agonistic anti-Fas antibody Jo-2 (1 µg/g body weight; BD Biosciences). For survival studies, different groups of mice (five animals per group) were composed and received vehicle, TAT-{Delta}FLIPS alone, anti-Fas mAb Jo-2 alone, or in combination with either TAT-FLIPS or TAT-{Delta}FLIPS. Injection of 100 µg TAT-FLIPS or vehicle was performed intraperitoneally 1 h prior to and 4 h after application of the Jo-2 mAb. Experiments were performed at least in duplicate. A part of the mice were sacrificed 8 h after the application of Jo-2 mAb to excise the liver and small intestine for histological and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) analyses to determine the extent of apoptosis.

For an investigation of histology, hematoxylin-stained cryosections (5 µm) were used. Sections of liver and small intestine were acetone-fixed, stained for 7 s with hematoxylin, and examined by light microscopy at 200x magnification for liver sections and 100x magnification for small intestine sections by an experienced pathologist.

Apoptotic cells were identified in acetone-fixed liver sections (5 µm) using TUNEL analysis (TMR red in situ cell death detection kit; Roche Applied Science). Staining was performed according to the manufacturer's instructions. TUNEL-positive cells were then imaged by fluorescent microscopy at 400x magnification. Nuclei were counterstained with Hoechst (Hoechst number 33258; Sigma).

Statistics—Statistical evaluation of actual survival of mice was performed using the log rank test. The SPSS program version 11.0.1 for MS Windows was used.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
To inhibit Fas-mediated apoptosis by recombinant FLIP we generated a protein containing FLIPS fused to the TAT domain of the human immunodeficiency virus. cDNA coding for the short form (splice variant) of FLIP, designated FLIPS, was cloned into a pTAT bacterial expression vector that contains a N-terminal TAT protein transduction domain leader followed by a polylinker (19-21). To demonstrate the biological effect specifically mediated by the FLIP domain of TAT-FLIP, we used a TAT fusion protein containing a mutated form of FLIP that lacks the binding properties of the death effector domain 1 as a further control (TAT-{Delta}FLIPS) (17).

TAT-FLIPS Is Readily Transduced into Lymphocytic Cell Lines—To address the transduction efficiency of the recombinant protein, we incubated lymphocytic cell lines with TAT-FLIPS or TAT-{Delta}FLIPS, respectively. Fig. 1A shows that the recombinant TAT-FLIPS is readily detectable in the cells as shown by Western blot analysis of cell lysates from Jurkat cells. Within 5 min of incubation TAT-FLIPS could be detected in whole cell lysates by immunoblotting using an anti-FLIP antibody. After 2 h the concentration of intracellular TAT-FLIPS decreased, and after 4 h intracellular TAT-FLIPS was hardly detectable. There was substantially no difference between TAT-FLIPS and TAT-{Delta}FLIPS.

These data are consistent with the kinetics of TAT-{beta}-galactosidase and other TAT proteins published (22-24). Endogenous FLIP could not be detected either in Jurkat or in BJAB cells (data not shown) using Western blot analysis, indicating that the concentration of both intracellular TAT-FLIPS and TAT-{Delta}FLIPS exceeds the endogenous FLIP multiple.

Fig. 1B demonstrates that the protein transduction of TAT-FLIPS is concentration-dependent. TAT-FLIPS could be detected weakly in a Western blot if used in a final concentration of ~25 nM and reached a plateau at concentrations of 500 nM. Control fusion protein TAT-{Delta}FLIPS showed equal concentration dependence. For the following experiments we therefore chose 500 nM as a suitable concentration and 30 min of incubation time to deliver sufficient amounts into the cells in order to observe potential anti-apoptotic effects.

TAT-FLIPS Provides Protection from Fas-mediated Apoptosis in Vitro—To analyze the biological potency of transduced recombinant TAT-FLIPS, the inhibition of apoptosis in Jurkat and BJAB cell lines induced by the anti-Fas antibody was examined after incubation with TAT-FLIPS as well as with the control proteins TAT-{Delta}FLIPS or TAT-{beta}-galactosidase (Fig. 2).



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  		FIG. 2.
Reduction of Fas-mediated cell death by TAT-FLIPS in vitro. A, Jurkat cells were incubated either with 100 ng/ml {alpha}-Fas for 4 h at 37 °C only or for 30 min with 500 nM TAT-FLIPS or 500 nM TAT-{Delta}FLIPS followed by the addition of {alpha}-Fas. Untreated cells were incubated with vehicle. Detection of apoptosis was performed with a fluorescence-activated cell sorter using Annexin V-fluorescein isothiocyanate (FITC) antibody. The graphs show the result of a representative experiment. B, Jurkat or BJAB cells were analyzed regarding DNA fragmentation using a diphenylamine assay. Cells were incubated for 16 h at 37 °C either in untreated medium, medium containing 100 ng/ml {alpha}-Fas antibody, medium containing 500 nM TAT-FLIPS and 100 ng/ml {alpha}-Fas antibody, medium containing 500 nM TAT-{Delta}FLIPS and 100 ng/ml {alpha}-Fas antibody, or medium containing 500 nM TAT-{beta}-galactosidase and 100 ng/ml {alpha}-Fas antibody, respectively. DNA-fragmentation was analyzed photometrically after lysis and staining with diphenylamine (see "Experimental Procedures"). Means and S.D. of three independent experiments are shown. C, protection of {alpha}-Fas induced apoptosis in Jurkat cells by different concentrations of TAT-FLIPS. Cells were incubated for 16 h at 37 °C in medium containing 100 ng/ml {alpha}-Fas antibody and concentrations of TAT-FLIPS ranging up to 750 nM. Graph shows DNA fragmentation of cells using a diphenylamine assay. Significance (p < 0.05) is depicted by asterisks.

 
After 4 h of treatment of Jurkat cells with an agonistic anti-Fas mAb, the rate of apoptotic cells was significantly reduced from 35.1 ± 8.2% (anti-Fas mAb only) to 4.5 ± 3.2% if the cells were preincubated with TAT-FLIPS and reached levels of spontaneously occurring apoptosis in untreated cells (2.5 ± 0.8%) as determined by Annexin V staining. In cells pretreated with TAT-{Delta}FLIPS, a protection against {alpha}-Fas-induced apoptosis was not present (36.2 ± 6.8%) (Fig. 2A).

Fig. 2B confirms the significant reduction of apoptosis in both Jurkat and BJAB cells using a diphenylamine assay if the cells were treated with TAT-FLIPS before the induction of apoptosis with an agonistic {alpha}-Fas mAb (7C11). The control proteins TAT-{Delta}FLIPS or TAT-{beta}-galactosidase exhibited no protective effect on anti-Fas antibody-induced apoptosis. Fig. 2C shows that the anti-apoptotic effect of TAT-FLIPS is concentration-dependent, reaching significance if used at 100 nM or higher in a diphenylamine assay.

Anti-apoptotic Effect of TAT-FLIPS Is Mediated by the Inhibition of Caspase Activation—To determine which part of signal transduction is blocked by TAT-FLIPS in Fas-mediated apoptosis, we analyzed events downstream in death receptor-mediated apoptosis.

Caspases form a family of proteases that are necessary for the execution of apoptosis. They are synthesized as precursors and then activated by proteolytic cleavage. It has been shown that FLIP acts as an inhibitor of procaspase-8 activation (25).

Fig. 3A shows a time course of cleavage/activation of caspase-8 in Jurkat cells induced by an anti-Fas antibody. Cleavage of procaspase-8 can be demonstrated within 3 h after the induction of apoptosis. An increased concentration of the cleaved subunits of caspase-8 (p43/41 and p18) is visible in Western blot analysis 5 and 7 h after induction. If cells were preincubated with TAT-FLIPS, the cleavage of procaspase-8 could be completely blocked for at least 5 h. In cells that were exposed to anti-Fas mAb for 7 h in the presence of TAT-FLIPS, cleavage of procaspase-8 was still partially inhibited.



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  		FIG. 3.
Cleavage of caspase-3 and caspase-8 is inhibited by TAT-FLIPS. 106 Jurkat cells were incubated with 100 ng/ml {alpha}-Fas antibody for the indicated times with or without 500 nM TAT-FLIPS or 500 nM TAT-{Delta}FLIPS, respectively, added 30 min before the addition of {alpha}-Fas. After the indicated times, cells were harvested. A, the Western blot analysis shows caspase-8 subunits using an antibody that recognizes full-length (57 kDa) as well as the cleaved intermediate p43/41 and the caspase-8 active subunit p18. B, membrane was stripped of bound antibody and reprobed with an antibody (Asp-175) that detects endogenous levels of the large fragment (19/17 kDa) of cleaved caspase-3. This antibody does not recognize full-length caspas-3 or other cleaved caspases. C, 106 Jurkat cells were incubated with or without TAT-FLIPS (500 nM), TAT-{Delta}FLIPS (500 nM), TAT-{beta}-galactosidase (500 nM), and/or {alpha}-Fas antibody (100 ng/ml) as indicated. Incubation with TAT fusion proteins was started 30 min before the addition of {alpha}-Fas. After 4 h cells were lysed, and a Western blotting of 10 µg whole lysate was performed using an {alpha}-caspase-8 antibody.

 
Fig. 3B shows the time course of cleavage of downstream caspase-3 using an antibody that specifically recognizes cleaved forms of 19 and 17 kDa. In the same experimental situation, caspase-3 activation is comparably inhibited, and the time course is similar to that of caspase-8 inhibition. In accordance with the lack of protection against apoptosis shown in Fig. 2, TAT-{Delta}FLIPS did not block {alpha}-Fas-induced cleavage of caspase-8 and caspase-3 (Fig. 3, A and B).

The inhibition of anti-Fas-mediated activation of caspase-8 by TAT-FLIPS is specific (Fig. 3C). Equimolar amounts of control fusion proteins exerted no inhibitory effect, indicating that the genuine FLIP domain itself is responsible for the demonstrated anti-apoptotic effects.

TAT-FLIPS Improves the Survival of Mice in Fas-mediated Multi-organ Failure—We wanted to further test the potential anti-apoptotic properties of TAT-FLIPS in a well characterized mouse model in which induction of Fas-mediated apoptosis leads to multi-organ failure and death within hours (11). TAT-FLIPS was administered intraperitoneally into Balb/c mice 1 h before and 4 h after the injection of 1 µg/g body weight of anti-Fas mAb (Jo-2). The actual cumulative survival of mice receiving anti-Fas antibodies plus TAT-FLIPS was significantly better compared with mice receiving anti-Fas mAb alone (p < 0.01) (Fig. 4). 9 of 25 mice (36% ± 8.9%) receiving anti-Fas mAb and TAT-FLIPS survived >4 weeks, whereas all mice of the anti-Fas group died during the first 2 days. The mean survival time of mice treated with an anti-Fas mAb alone was significantly shorter compared with mice receiving additional TAT-FLIPS (mean survival 10 ± 3.4 h versus 19.2 ± 5.7 h; p < 0.01). However, a complete inhibition of apoptosis during the peak effect of TAT-FLIPS in the first 8 h after administration could not be achieved. None of the mice treated with TAT-FLIPS or TAT-{Delta}FLIPS alone showed any toxic effects.



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  		FIG. 4.
TAT-FLIPS prolongs survival in Fas-mediated death in Balb/c mice. Balb/c mice were treated with 1 µg/g body weight {alpha}-Fas antibody (Jo-2) alone, in combination with either TAT-FLIPS or TAT-{Delta}FLIPS, or with TAT-{Delta}FLIPS alone. 10 milligrams of fusion protein per kilogram of body weight per dose was administered intraperitoneally 1 h before and 4 h after the injection of {alpha}-Fas. The graph shows the cumulative survival obtained from five independent experiments. Three of these experiments consisted of two groups ({alpha}-Fas alone and TAT-FLIPS plus {alpha}-Fas) with five animals each. In two of these experiments, two additional groups of treatment were included (TAT-{Delta}FLIPS and TAT-{Delta}FLIPS plus {alpha}-Fas). The resulting cumulative number of animals is depicted in the graph.

 
Because the hallmark of the model used is multi-organ failure, mice from each treatment group were sacrificed 8 h after the induction of apoptosis. Macroscopically obvious differences were found predominantly in the gross appearance of livers and intestine (Fig. 5A). Livers of mice that were treated with anti-Fas mAb showed a dark red-brown color with a rather deliquescent organ texture, whereas the intestine appeared red and contracted with small hemorrhages. In contrast, those changes were scarcely visible in mice that had received both TAT-FLIPS and anti-Fas mAb or were not detectable in untreated control mice. Mice treated with TAT-{Delta}FLIPS and anti-Fas mAb showed the same intra-abdominal appearance as anti-Fas mAb-treated mice. Other organs like heart, spleen, kidneys, lungs, and brain showed no gross difference (data not shown).



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  		FIG. 5.
TAT-FLIPS reduces liver apoptosis and intestinal degradation. A, the abdominal situs of representative mice from the experiment described in Fig. 4 is shown. Mice were sacrificed 8 h after injection of an {alpha}-Fas antibody. B, TUNEL assay of liver cryosections of sacrificed mice. The upper parts of the panel shows the nuclear staining with Hoechst, and the lower parts of the panel shows the TUNEL staining of the corresponding visual fields, magnification 400x. C, representative cryosections of small intestine of sacrificed mice with hematoxylin staining; magnification 100x.

 
Histological examinations of liver sections correlated well with these observations. TUNEL staining revealed a decrease in the number and intensity of apoptotic cells in animals that received both TAT-FLIPS and anti-Fas mAb when compared with anti-Fas mAb-treated animals. However, the number of TUNEL-positive cells was still lowest in mice that have received the vehicle only. Thus, TUNEL staining confirmed a partial inhibition of anti-Fas-induced liver cell apoptosis by TAT-FLIPS (Fig. 5B). Fig. 5C shows representative sections of small intestine stained with hematoxylin. Areas with the pronounced macroscopic differences described above were dissected showing a marked degradation of the luminal ends of villi detectable only in anti-Fas-treated mice.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Inhibition of apoptosis prevents organ failure in a variety of pathological conditions associated with increased apoptosis. Sensitivity toward apoptosis can be modulated at different levels in the signaling pathway. Initiation of apoptosis is partially inhibited in mice deficient for the intact Fas receptor or its ligand. Those mice are protected from ischemia-reperfusion injuries to some extent (12-14). Among the most potent inhibitors of caspase activation in the signaling cascade are endogenous proteins like Bcl-2, or Bcl-xL (26). Different approaches to the modulation of caspase activation have been investigated with the application of silencing RNA (27), chemical compounds, or retroviral gene transfer. Each approach exhibited advantages and limitations. Therapeutic inhibition of apoptosis has to be transient, because long term inhibition of apoptosis by overexpression of inhibitory proteins was associated with the risk of malignancies, predominantly lymphoma (28), as well as autoimmune diseases and other disorders (29). To use an endogenous proteinergic inhibitor as a therapeutic tool, this inhibitor has to be under regulatory control and has to be introducible into a cell to act intracellularly.

In the present study, recombinant TAT-FLIPS proteins inhibited Fas-mediated activation of caspases and apoptotic cell death in vitro and in mice. FLIP was chosen because, in contrast to other inhibitors, it particularly provides the advantages of proximal action in the signal cascade. FLIP is recruited into the DISC by binding to the Fas-associated death domain protein (FADD) and prevents the cleavage of procaspase-8 (4), which therefore may not initiate the caspase cascade. Of the two isoforms of FLIP that are known we used the short splicing variant FLIPS because the effect on apoptosis is contrarily discussed for the long form (30). FLIPS exerts only anti-apoptotic effects (31).

To introduce FLIP into the cells we fused FLIPS genetically to the N-terminal protein transduction domain of human immunodeficiency virus TAT. The TAT domain allows proteins to pass the intact cell membrane, a method that was demonstrated as offering rapid introduction of a variety of full-length proteins into primary and transformed cells in vitro and in vivo (20, 32). In this study we show that Fas-induced apoptosis was inhibited in a time- and dose-dependent manner by TAT-FLIPS. This effect could be addressed to the inhibition of caspase-8 activation during the first 5 h after the induction of apoptosis, and it could be demonstrated to be specifically mediated by the FLIP domain because the control TAT fusion proteins did not provide protection against apoptosis. The observed decrease in inhibitory activity is due to rapid intracellular degradation of TAT-FLIPS. Intracellular degradation has been shown for other TAT fusion proteins with some variation. For example, TAT-FNK, is intracellularly active for 2 h (33), whereas galactosidase activity could be detected for >20 h after the application of TAT-{beta}-galactosidase (22). After fusing an active protein domain with a TAT-domain, the specific activity on a molar basis may not be comparable with the parental protein. Concentrations of other apoptosis-inhibitory TAT fusion proteins are reported to range from 0.3 pM TAT-FNK (33) to 50 µM TAT-BH4 (34) to warrant inhibitory effects in vitro. In our study, the sufficient concentration of TAT-FLIPS for anti-apoptotic effects was 500 nM. Because of the specific activity in vitro and the lack of visible side effects of TAT-FLIPS, we used up to 10 mg/kg body weight fusion protein for the experiments in mice. So, taken together, the stability and the dose effects of our TAT-FLIPS were within the previously published range of other TAT fusion proteins.

Although the principle of TAT-mediated protein transfer was discovered more than 15 years ago (35), there is only a very limited number of investigations in which TAT-proteins were used in vivo. Schwarze et al., was the first to demonstrate in mice that intraperitoneally applied denatured TAT-{beta}-galactosidase is taken up in a biologically active form by nearly all cells and organs and may even cross the blood-brain barrier (10). These experiments proved the principle of a new form for applying drugs, active proteins, or DNA into nearly all organs (36-38). However, only recently a few reports exploited protein transduction for therapeutic use in clinical relevant disease models in mice. Some groups reported the partial protection of local cerebral ischemia by the anti-apoptotic protein TATBcl-xL and a mutated variant of it, TAT-FNK, respectively (23, 33, 39). Similar results could be shown when using a different fusion protein (TAT-GDNF) in a similar mouse model for local cerebral ischemia-reperfusion injury (40). Myou et al., used protein transduction technology for a different clinical topic(41). They transduced a dominant negative Ras in a mouse asthma model, which resulted in inhibition of the airway inflammatory response by cytokine blockade (41).

We used a mouse model in which all of the mice died after antibody-mediated activation of Fas due to multi-organ failure. TAT-FLIPS protected 36% of the mice from death and delayed lethal multiple organ failure in all others. In addition, in the liver and the gut the protective effect of TAT-FLIPS has been demonstrated histologically. The effect was dose-dependent and time-limited. As mentioned, Fas-mediated apoptosis contributes to organ failure not only during ischemia-reperfusion injuries but also after certain infections like fulminant hepatitis B or intoxication and liver failure due to acute alcohol hepatitis or paracetamol intoxication (42-44). Inhibition of apoptosis has not been included in the therapeutic repertoire of those diseases yet. Probably the most effective way to introduce inhibition of apoptosis into the clinic is to use more than one principle and more than one inhibitor, of which TAT-FLIPS could be one.


   FOOTNOTES
 
* This work was supported by Deutsche Forschungsgemeinschaft (Bonn, Germany) Grant KU 760/6-1 (to U. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Both of these authors contributed equally to this work. Back

§ To whom correspondence should be addressed: University of Schleswig-Holstein, Campus Kiel, Dept. of Nephrology and Hypertension, Schittenhelmstr. 12, 24105 Kiel, Germany. Tel.: 49-431-597-1338; Fax: 49-431-597-1337; E-mail: kunzendorf{at}nephro.uni-kiel.de.

1 The abbreviations used are: FLIP, FLICE inhibitory protein; cFLIP, cellular FLIP; FLIPS, short form (splice variant) of FLIP; DISC, death-inducing signaling complex; mAb, monoclonal antibody; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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