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Killing tumour cells by alkylphosphocholines: Evidence for involvement of CD95
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European Journal of Cell Biology 80, 1 ± 10 (2001, January) ´ Urban & Fischer Verlag ´ Jena http://www.urbanfischer.de/journals/ejcb
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Killing tumour cells by alkylphosphocholines: Evidence for involvement of CD95 Astrid Matzkea, Ulrich Massingb, Harald F. Krug1)a a b
Forschungszentrum Karlsruhe, Institute for Toxicology and Genetics, Karlsruhe/Germany Tumour Biology Centre, Freiburg/Germany
Received July 25, 2000 Accepted August 18, 2000
Alkylphosphocholines ± BCL-2 ± caspases ± CD95 ± apoptosis Many lipids act as cellular messengers and lead to a variety of different cellular responses. Out of the group of these compounds the ceramides are able to induce apoptosis, and some synthetic lipids can mimic this effect. Apoptosis is an important mechanism whereby chemotherapeutics exhibit their anti-oncogenic activity. Although, some lipid analogues were used in clinical trials, they exert severe side effects and their mechanism of action is widely unknown. We present here a new class of synthetic alkylphosphocholines (APC) that induce programmed cell death in leukaemia cells. The signs of apoptosis arise after 1 h of incubation with these compounds as shown by phosphatidylserine externalisation followed by caspase activation and DNA fragmentation. We demonstrate that the molecular target of these lipids is upstream of caspases and Bcl-2. Experiments with FADD dominant negative cells reveal that induction of apoptosis occurs on the level of CD95 and that these compounds can now be optimised for their capacity to activate the apoptosis-inducing receptor CD95.
Introduction Phospholipid analogues represent a new class of antitumour agents targeted to the cell membrane. They are membraneaffecting antiproliferative drugs that exert a pronounced cytostatic action on different neoplastic cell lines in vitro and show antineoplastic activity in vivo (Berdel, 1991; Muschiol et al., 1988; Noseda et al., 1988). The first compounds of this group which were synthesised represented metabolically stabilised analogues of 2-lysophosphatidylcholine. Later, it has been shown that the glycerol moiety is not an essential structural element and APC exert similar antitumour effects
Dr. Harald F. Krug, Forschungszentrum Karlsruhe, Institute for Toxicology and Genetics, P.O. Box 3640, D-76021 Karlsruhe/Germany, e-mail: [email protected], Fax: 49 7247 823 557.
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(Hilgard et al., 1993). Further investigations of the structureactivity relationship among a variety of synthetic APC indicated that a long alkyl chain and a phosphocholine moiety are necessary for sufficient antineoplastic effects of the lipid analogues (Unger and Eibl, 1986). A prominent example of APC is 1-O-octadecyl-2-O-methylsn-glycero-3-phosphocholine (Edelfosine, Et-18-OCH3) which is experimentally used in purging leukaemic bone marrow prior to autologous bone marrow transplantation (Vogler and Berdel, 1993; Königsmann et al., 1996). Another encouraging example of APC is hexadecylphosphocholine (Miltefosine, HePC) (Unger et al., 1989). It is used in the topical treatment of skin metastases resulting of breast cancer and lymphomas (Unger et al., 1988). Systemic application of HePC is not possible due to its gastro-intestinal side effects. In contrast to naturally occurring phospholipids, HePC contains no ester, hydroxyl, amide or amino group adjacent to the phosphocholine head group. The application of APC in tumour therapy is made possible by the fact that APC are toxic preferentially against leukaemia cells but spare normal blood progenitor cells, possibly due to a different lipid metabolism in tumour cells (Andreesen et al., 1978; Vogler et al., 1987; Verdonck et al., 1990; Mollinedo et al., 1997). Furthermore, APC show only marginal substrate properties to phospholipid metabolising enzymes which increases their half-life and allows accumulation in tumour cells (Hoffman et al., 1986). Nevertheless, the mechanisms of tumouricidal action of APC remain largely unknown. Here we present 8 newly synthesised, enantiomerically pure APC compounds (Massing and Eibl, 1994) (Fig. 1). The objective was to develop APC with high antineoplastic activity and lower side effects as HePC. The new compounds are therefore structurally similar to HePC, but contain an additional functional group: acetic ester (R/S-O-acetyl), hydroxyl (R/S-OH), acetamide (R/S-N-acetyl), amino (R/S-NH2) adjacent to the phosphocholine moiety (2-position) and have been synthesised in the R- and S-configuration, respectively. In order to achieve the same effective length of the apolar hydrocarbon chain as HePC, the new compounds are based on an octadecyl chain instead of an hexadecyl chain.
0171-9335/01/80/01-1 $15.00/0
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A. Matzke, U. Massing, H. F. Krug
Hoechst 33342 staining
After incubation, 1 106 cells were incubated with a final concentration of 10 mM Hoechst 33342 (Sigma) for 10 min, washed with PBS to reduce background fluorescence and finally visualised with a fluorescence microscope (Axiovert S100, Zeiss, Jena). Microscopy was performed with excitation at 364 nm and emission at 460 nm. Pictures were taken with a Hamamatsu CCD 4880 ± 80 camera and further optimisation was performed by use of the Openlab 2.05 software (Improvision, UK).
Analysis of oligonucleosomal DNA fragments
Apoptotic DNA fragments were isolated according to Herrmann et al. (1994). The separation was carried out on 1.8% agarose gels. DNA fragments were stained with 0.5 mg/ml ethidium bromide and DNA was visualised by UV light.
Western blot
Fig. 1. Structures and abbreviations of synthetic alkylphosphocholines (APC). All compounds are enantiomerically pure R- and Sconfigurated. * indicates the chirality centre.
The action of these APC on human tumour cell lines was studied. Since promising results were obtained in the treatment of leukaemia by bone marrow purging (Vogler and Berdel, 1993; Königsmann et al., 1996), we chose for our investigations leukaemia cell lines.
Materials and methods Reagents
All cell culture reagents and media were from Life Technologies (Eggenstein, Germany). Caspase-inhibitors zVADfmk and zD-DCB and CD95 ligand were obtained from Alexis (Grünberg, Germany). Apoptosis-inducing anti-CD95 was obtained from Boehringer Mannheim. APC were synthesised by U. Massing, Tumour Biology Centre, Freiburg, Germany. Stock solutions were prepared in ethanol. The final concentration of the vehicles within the incubation mixture did not exceed 1%. All other chemicals were obtained from Sigma, Deisenhofen.
Cell culture and growth conditions
HL-60 cells were obtained from ATCC (Rockville, MD) and grown in suspension in a standard culture medium consisting of RPMI 1640 supplemented with 15% foetal calf serum (FCS). Jurkat neo and bcl-2 were cultivated as described elsewhere (Armstrong et al., 1996). SKW neo, bcl-2 and crmA were cultivated as described before (Strasser et al., 1995). BJAB wt and FADDdn cells were cultivated as described elsewhere (Chinnaiyan et al., 1996).
Cell proliferation and viability
The cell number was measured with a Coulter Counter (Coulter Electronics GmbH, Krefeld). Cell viability was checked by trypan blue dye exclusion and the number of viable cells was estimated using a Neubauer chamber.
Proteins were solubilised from control and stimulated cells by lysis in gel-sample buffer (0.16 M Tris-HCl, 4% SDS, 20% glycerol, 4% 2mercaptoethanol, 0.08% bromophenol blue), size fractionated on 10% or 12.5% SDS-polyacrylamide gels and then transferred to PVDF membranes by electroblotting. After blocking ( > 1 hour at room temperature in 10% skim milk in phosphate-buffered saline) membranes were incubated for > 1 hour at room temperature with the indicated antibodies, washed and incubated for 1 hour with the secondary horseradish peroxidase-conjugated antibody. Membranes were finally probed with enhanced chemoluminescence reagent (Amersham, Braunschweig) and exposed to X-ray films. Antibodies for Western blotting: anti-PARP (Boehringer Mannheim), anti-CPP32 (Transduction Laboratories, Mamhead, UK), anti-lamin B (Santa Cruz Biotechnology, Santa Cruz, USA).
Annexin-V-Fluos staining
Cells (1 106) were stained for 15 min in HEPES-buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, 5 mM CaCl2) containing 1 mg/ ml propidium iodide and 2% Annexin-V-Fluos solution (Boehringer Mannheim). After washing once with HEPES-buffer, Annexin-Vpositive cells were visualised using a fluorescence microscope (Axiovert S100, Zeiss, Jena). Colorisation and merging was done with the Openlab 2.05 software (Improvision, Coventry, UK).
Results APC are cytotoxic towards human leukaemia HL-60 cells
HL-60 cells were seeded at 5 105 per ml and incubated for 48 hours with increasing concentrations of APC. Cell number was measured and viability of the cells was estimated by trypan blue dye exclusion test. With increasing concentrations of APC, we could observe a growth-inhibitory and cytotoxic effect of all APC (Fig. 2). As shown in Table I, R-N-acetyl is the most toxic compound (LD50 4 mM). S-O-acetyl and S-Nacetyl were effective in the same concentration range, whereas R-O-acetyl (LD50 68 mM) and R-OH (LD50 30 mM) were the least toxic compounds. In general, the s-configurated isomers showed higher antitumoural activity than their rconfigurated counterparts. This is obvious when comparing the LD50 values of R-O-acetyl (LD50 68 mM) and S-O-acetyl (LD50 5 mM). HL-60 cells are affected by R-O-acetyl only in high concentrations, whereas S-O-acetyl is ten times as effective in the lower micromolar range. In our hands, the LD50 values of the most potent compounds R-N-acetyl, S-N-acetyl and S-O-acetyl bear comparison to those of Et-18-OCH3 (LD50 12 mM) and HePC (LD50
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Induction of apoptosis by alkylphosphocholines
Fig. 2. Dose-response curves of HL-60 cells after APC treatment. HL-60 cells were seeded with 500 000 cells/ml. Viability was determined by trypan blue dye exclusion test after an incubation time of 48 hours. Data represent the mean of 3 experiments. Error bars were omitted but the standard deviation was < 10%. Viability is presented as a percentage of total cells.
Tab. I. LD50 values of HL-60 cells. compound
LD50 [mM]
R-O-acetyl R-OH R-NH2 S-OH S-NH2 Et-18-OCH3 HePC S-O-acetyl S-N-acetyl R-N-acetyl
68 30 26 21 13 12 7.5 7 5 4
HL-60 cells were seeded at a concentration of 5 105 per ml and were treated with increasing concentrations of APC. LD50 values were calculated from dose-response curves after an incubation time of 48 hours.
7.5 mM), indicating that our lipids have similar or even better potency in killing leukaemia cells in vitro.
APC induce apoptosis in HL-60 cells
From light microscopy studies, evidence was given that APC might act via the induction of apoptosis. APC-treated cells produced blebbing of plasma membranes and segregation of apoptotic bodies. To clarify whether these morphological alterations and the cytotoxic and growth-inhibitory effect of APC might be indeed a result of induced apoptosis, the APC compounds were further tested for their ability to induce apoptosis in HL-60 cells. Since R-N-acetyl was the most potent compound further studies dealing with the mechanisms of action of APC were mainly carried out with this compound.
Externalisation of PS early in APC-induced apoptosis
One of the early events in apoptosis is the externalisation of phosphatidylserine (PS) to the outer leaflet of the plasma membrane (Martin et al., 1995). We have used a PS-binding protein (annexin-V-fluos) as a specific probe to detect apoptosis-dependent redistribution of this phospholipid, which is normally confined to the inner plasma membrane leaflet. Cells were counterstained with
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propidium iodide to distinguish apoptotic from necrotic cells. Cells were visualised using a fluorescence microscope. Fig. 3a shows light microscopy pictures of control cells (top line) and APC-treated cells (bottom lines). Fluorescent microscopy pictures of annexin-V-stained cells (green) are seen in Fig. 3b, and propidium iodide-stained cells (red) in Fig. 3c. Fig. 3d shows a merged image of the three previous pictures. Already after 1 hour, R-N-acetyl-treated cells were annexinV-positive. Green fluorescence indicated the binding of annexin-V to PS at the outer plasma membrane leaflet. Characteristic green fluorescent rings formed around the cells. At that time point, only a few necrotic cells could be seen indicated by the red fluorescence of propidium iodide, nor did the cells bleb. The externalisation of phosphatidylserine is the first apoptotic sign detectable upon APC treatment. Green fluorescence of annexin-V-positive cells appeared after 1 hour of treatment (Fig. 3b, middle line) and sustained several hours. After 16 hours, however, most of the cells were necrotic or late apoptotic and were annexin-V- as well as propidium iodidepositive (Fig. 3d, bottom line) indicating that the plasma membrane integrity has been lost.
APC induce plasma membrane blebbing and chromatin condensation
One of the characteristic and early described features of apoptosis is the blebbing of the plasma membrane of apoptotic cells. As can be seen in light microscopy this phenomenon could be observed in APC-treated cells (Fig. 3a). The formation of membrane protrusions and blebs is more prominent when depicted with a scanning electron microscope. Severe membrane blebbing started about 8 hours after APC treatment (data not shown) and was clearly obvious after 16 hours of treatment with 20 mM APC (Fig. 4b). Additionally, during apoptosis the structure of the cell nucleus changes and typical rearrangements of chromatin appear during cell death (Dini et al., 1996). DNA staining of APC-treated cells with the fluorescent dye Hoechst 33342 revealed condensed and fragmented nuclei. After 16 hours the chromatin of APC-treated cells was condensed and showed a much brighter and more intense fluorescence compared to untreated control cells as illustrated in Fig. 5.
APC induce DNA fragmentation and cleavage of the death substrate PARP
After having observed the morphological alterations in APCtreated cells, we investigated the biochemical changes that accompany apoptosis due to activation of caspases and endonucleases. All eight APC compounds were tested for their ability to induce apoptosis in HL-60 cells. As a marker for apoptosis we investigated the activation of endonucleases resulting in the formation of a DNA-ladder due to internucleosomal cleavage of the DNA (Wyllie, 1980). The characteristic 180 ± 200-bp DNA fragments were recognised as a DNA-ladder on conventional agarose gel electrophoresis. Moreover, we investigated the proteolytic cleavage of poly(ADP-ribose)polymerase (PARP) (Kaufmann et al., 1993) as a second marker for apoptosis. The native 116-kDa PARP is a substrate for caspase-3 (Cohen, 1997) and cleavage of PARP during apoptosis yields a 86-kDa product which can be detected by Western blotting using specific antibodies.
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Fig. 3. Annexin V-fluos staining of HL-60 cells. Control HL-60 cells and APC-treated cells were stained with the phosphatidylserinebinding protein annexin-V-fluos (green), and DNA was stained with propidium iodide (red) at a concentration of 0.1 mg/ml for 15 minutes.
Fig. 4. APC induce plasma membrane blebbing in HL-60 cells. Scanning electron microscopy pictures of control HL-60 cells (a) and a cell treated with 20 mM APC for 16 hours (b).
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Top line: untreated control cells; middle line: 20 mM R-N-acetyl, 1 hour; bottom line: 20 mM R-N-acetyl, 16 hours. (a) Light microscope picture, (b) annexin V-fluos staining, (c) propidium iodide staining, (d) merged image of the three previous pictures. Magnification: 328 .
All compounds except R-OH and R-O-acetyl were able to induce DNA fragmentation (Fig. 6b) and PARP cleavage (Fig. 6a) in HL-60 cells. Upon treatment with 20 mM R-OH or 20 mM R-O-acetyl, neither PARP cleavage (Fig. 6a, lanes 2 and 6) nor DNA fragmentation (Fig. 6b, lanes 3 and 7) could be detected. As shown in Fig. 2, R-OH and R-O-acetyl were the least toxic compounds. All other compounds were able to induce DNA fragmentation and PARP cleavage in HL-60 cells. In R-N-acetyl-treated cells, DNA fragmentation could first be monitored at about 5 hours after treatment with 20 mM of the synthetic lipid (Fig. 6c). Upon 16 hours treatment of cells with R-N-acetyl, the APC was able to induce DNA fragmentation at a concentration as low as 2.5 mM (Fig. 6d, lane 4).
Specific activation of caspases in APC-induced apoptosis
Fig. 5. Chromatin condensation induced by APC in HL-60 cells. Nuclei of HL-60 cells were stained with Hoechst 33342 dye in untreated control cells (a) and in cells treated with 20 mM APC for 16 hours (b). Magnification: 276 .
To point out specifically the importance of caspase proteases in APC-induced apoptosis, we analysed the activation of caspases at different time points during treatment. The processing of the two caspase substrates PARP and lamin b (Kaufmann, 1989; Kaufmann et al., 1993) was investigated by means of Western blotting using specific antibodies. Cleavage of PARP, as well as the degradation of lamin b started 5 hours after APC treatment and was clearly obvious after 10 hours treatment (Fig. 7a, b). Moreover, activation of caspases itself is dependent on proteolytic cleavage (Thornberry and Lazebnik, 1998). We therefore analysed the disappearance of the 32-kDa pro-
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Fig. 6. Induction of apoptosis by APC. The ability of all different APC to induce apoptosis was analysed using the cleavage of poly(ADPribose)polymerase and the formation of a DNA-ladder due to internucleosomal DNA cleavage as markers. HL-60 cells (1 106 per ml culture medium) were incubated with 20 mM of each APC for 16 hours. Cleavage of PARP was monitored in a Western blot using antiPARP antibodies (a). DNA fragments were visualised by agarose gel electrophoresis (b). Furthermore, HL-60 cells were treated with 20 mM of R-N-acetyl and DNA fragmentation was analysed after different incubation times (c). In (d), HL-60 cells were treated with increasing concentrations of R-N-acetyl for 16 hours and probed for DNA fragmentation. (M: DNA molecular weight marker)
caspase-3 (CPP32) by proteolytic cleavage (Erhard and Cooper, 1996; Liu et al., 1996). As shown in Fig. 7c processing of the pro-enzyme started after 7.5 hours and is almost complete at 10 hours of treatment with APC.
APC-induced apoptosis can be prevented by the caspase inhibitors zVADfmk and zD-DCB as well as by overexpression of CrmA
To demonstrate that activation of caspases is functionally relevant in APC-induced apoptosis, the effect of the peptidebased caspase inhibitors zVADfmk and zD-DCB in APCtreated cells was investigated. zVADfmk, a fluoromethyl ketone inhibitor, is a competitive and irreversible inhibitor of caspases 1 ± 10 (Garcia-Calvo et al., 1998) and zD-DCB is a
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Fig. 7. Activation of Caspases is required for R-N-acetyl-induced apoptosis. The degradation of the death substrates PARP (a), and lamin b (b), as well as the processing of pro-caspase 3 (CPP32) (c) was observed in R-N-acetyl-treated HL-60 cells over a time period of 24 hours by means of Western blotting using specific monoclonal antibodies. Furthermore, the effect of the caspase inhibitors zVADfmk and zD-DCB was observed during R-N-acetyl-induced apoptosis. Cells were pre-treated with the indicated concentrations of inhibitor for 30 min and further treated with 30 mM of R-N-acetyl for 16 hours. Control cells were left untreated. Apoptosis was detected by DNA fragmentation (d) and PARP cleavage (e). SKW6 cells engineered to overexpress the viral caspase inhibitor CrmA (SKW6 crmA) and SKW6 cells transfected with an empty vector (SKW6 neo) were treated with increasing concentrations of R-N-acetyl or with 1 mg/ml apoptosisinducing anti-CD95 antibody (aCD95) for 16 hours and probed for DNA fragmentation (f) and PARP cleavage (g).
broad-range caspase inhibitor which can easily enter target cells due to a hydrophobic moiety (Henkart, 1996). APCinduced killing of HL-60 cells was almost completely prevented when cells were pre-incubated with 100 mM of inhibitors for 15 minutes (data not shown). Neither DNA fragmentation (Fig. 7d) nor PARP cleavage (Fig. 7e) could be observed when cells were pre-treated with zVADfmk or zD-DCB. In our test system, the inhibitor zD-DCB was more potent than zVADfmk since it blocked DNA fragmentation and PARP cleavage at a lower concentration (50 mM) than zVADfmk. To further stress the importance of caspases in APC-induced apoptosis, we used B lymphoblastoid SKW6 cells engineered to overexpress the cowpox virus CrmA protein, a direct and specific inhibitor of caspase proteases (Ray et al., 1992; Cohen, 1997). Overexpression of CrmA prevents cells from apoptosis induced by a variety of stimuli, such as CD95 ligand (Strasser
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et al.,1995) or UV radiation (Rehemtulla et al., 1997). Here we show that CrmA protects cells from APC-induced apoptosis. In APC-treated SKW6 crmA cells, cleavage of caspase substrate protein PARP (Fig. 7g) as well as the fragmentation of DNA (Fig. 7f) is totally prevented. CrmA is a highly specific inhibitor of group I and III caspases, with the exception of caspase-6 (Garcia-Calvo et al., 1998), indicating that activation of such caspases is crucial in APC-induced cell death.
Role of mitochondria and Bcl-2 in APC-induced apoptosis
There is evidence that Bcl-2 plays a role in preventing cells from apoptosis (Adams and Cory, 1998). We were interested whether Bcl-2 can rescue cells within which apoptosis had been induced by APC. Therefore, we examined the effect of Bcl-2 overexpression during APC-induced apoptosis in Jurkat T cells and in B lymphoblastoid SKW6 cells. Both cell lines were transfected with either Bcl-2 vector or empty vector (Strasser et al., 1995; Armstrong et al., 1996). Cells were incubated with increasing concentrations of APC and lysates were probed for PARP cleavage and DNA fragmentation. As controls, we used taxol and the topoisomerase inhibitor etoposide, which are well-known inducers of apoptosis (Kaufmann, 1989; Solary et al., 1994). Interestingly, we observed that overexpression of Bcl-2 protects only the Jurkat T cells from APC-induced apoptosis (Fig. 8a, b). Within Jurkat T cells, neither PARP cleavage nor DNA degradation could be detected in the Bcl-2 overexpressing cells, whereas the control cells were strongly affected by the alkylphosphocholines. On the other hand, Bcl2 overexpression within the lymphoblastoid B cells SKW6 could not rescue APC-treated cells. Within the SKW6 cell line, the Bcl-2 overexpressing cells were just as much affected by APC treatment as the control cells. Transfected SKW6 cells of each type, neo or bcl-2, showed similar patterns for DNA laddering as well as for PARP cleavage (Fig. 8c, d). To further investigate these obvious differences between the apoptotic pathways within the two cell lines, the action of APC was compared with the action of an apoptosis-inducing anti-CD95 antibody. Such an antibody can trigger apoptosis via the CD95 pathway by crosslinking CD95 receptor molecules (Oehm et al., 1992). Anti-CD95-treated cells showed a similar pattern of DNA fragmentation compared to APC-treated cells. Again, apoptosis was induced in both neo-type clones, and Bcl-2 acted protective only within Jurkat T cells (Fig. 8e, JB). These data demonstrate that Bcl-2 protects cells from the apoptotic action of APC, but this phenomenon is clearly restricted to specific cell types and not a common property.
CD95 is involved in APC-induced apoptosis
Searching for a more upstream target of APC, we investigated whether the CD95 (Fas/APO-1) receptor-mediated signalling pathway is involved in APC-induced apoptosis. We therefore used a cell line defective in the CD95 pathway, the B lymphoma cell line BJAB, which was transfected with a dominant negative expression construct of the Fas-associated death domain (FADDdn). Expression of such FADD dominant negative mutants abrogates CD95-induced apoptosis in these cells (Chinnaiyan et al., 1995, 1996). We investigated the action of APC on FADDdn BJAB cells, as well as on control BJAB cells which had been transfected with an empty vector (BJAB wt). The apoptotic nature of APC-treated cells was confirmed by assay of PARP cleavage and DNA fragmentation.
Fig. 8. Role of mitochondria and Bcl-2 in APC induced apoptosis. Two Bcl-2-overexpressing cell lines, Jurkat T cells and SKW6 cells were incubated with increasing concentrations of APC,and lysates were probed after 16 hours for internucleosomal DNA cleavage on agarose gel electrophoresis (a, c) and PARP cleavage by means of Western blotting (b, d). As a control (e), we used an anti-CD95 antibody (aFas, Boehringer) at a concentration of 1 mg/ml for 5 h to induce apoptosis by triggering the CD95 receptor pathway. JN: Jurkat neo, JB: Jurkat bcl-2, SN: SKW6 neo, SB: SKW6 bcl-2, Tax: Taxol 1 mM 16 hours, Et: Etoposide 64 mg/ml 16 hours. M: DNA molecular weight marker.
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After treatment of BJAB cells with APC, only the wild-type cells underwent apoptosis. Within the dominant negative FADD mutants, neither PARP cleavage nor the formation of a DNA ladder could be observed (Fig. 9a, b). This indicates strongly that caspases and endonucleases were not activated within these mutants. Even after treatment with high concentrations of APC up to 50 mM the cells remained intact and did not segregate apoptotic bodies (data not shown). As a control, the specific CD95 ligand (CD95L) (Nagata and Golstein, 1995) was used to check the activation of the CD95 pathway and its blockade in the dominant negative mutants. Treatment with CD95L resulted in apoptosis only within the wild-type cells but not within the FADDdn mutants (Fig. 9b).
Discussion The knowledge about signal transduction pathways leading to death or survival of a given cell is permanently increasing (Dragovich et al., 1998), and dysregulation or modulation of the apoptotic mechanisms seem to be involved in oncogenesis (Stambolic et al., 1999) or present a chance for new ways in tumour therapy (McGill and Fisher, 1997). For this purpose we designed 8 new alkylphosphocholine compounds. They are lysophosphatidylcholine analogues that were synthesised according to minimal structural requirements for antineoplastic activities, namely a long alkyl chain and a phosphocholine polar head group.
Fig. 9. APC fail to induce apoptosis in FADDdn cells. BJAB wildtype (wt) and BJAB engineered to express a dominant negative version of FADD (FADDdn) were used to investigate whether CD95 plays a role in APC-induced apoptosis. Both cell lines were incubated with increasing concentrations of R-N-acetyl for 16 hours and probed for internucleosomal DNA cleavage (a). Furthermore, cells were incubated with 30 mM R-N-acetyl for different time intervals and probed for PARP cleavage by means of Western blotting. As control, we used the CD95 ligand (CD95 lig, 300 ng/ml) to induce apoptosis via the CD95 pathway (b). M: DNA molecular weight marker.
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We tested these new compounds for their antineoplastic potency in cell culture systems of human leukaemia HL-60 cells. The APC showed a dose-dependent growth-inhibitory and cytotoxic effect on HL-60 cells. LD50 values were calculated from dose-response curves. The APC compounds showed different toxicity with regard to their chemical structure. In general, the S-configurated isomers were more toxic than their R-configurated counterparts. The compound bearing an Rconfigurated hydroxy or O-acetylic function were toxic only at very high concentrations. The low toxicity of R-OH and R-Oacetyl can be explained by the preferred degradation of these two R-(natural)-configurated compounds by phospholipidmodifying enzymes. Due to their chemical structure, Rconfigurated acetic ester or hydroxyl-function at the 2position, they represent good substrates for transacylases or reacylation and thus, can be quickly detoxified by the cells. It has been shown that R-OH and R-O-acetyl can be metabolised to an R-O-acyl compound which is similar to phosphatidylcholine in its chemical structure and can be tolerated by HL-60 cells (Hildenbrand et al., 1999). During the last years it has been discussed that lysophospholipids as well as analogues of lysophospholipids quickly disrupt plasma membrane integrity due to their detergent-like structure. Indeed, the plasma membrane becomes affected when exposing cells to high concentrations of APC. During the experiments shown here, APC were used in the lower micromolar range where they do not lyse cells (KaufmannKolle and Fleer, 1992). We show that the cytotoxicity of these APC is a result of induced apoptosis. Classical apoptotic features could be observed upon treatment with these substances. Shortly after APC addition, phosphatidylserine was transferred to the outer leaflet of the plasma membrane. This was the first detectable apoptotic event induced by these compounds. Phosphatidylserine externalisation occurred after only one hour of treatment when the plasma membrane integrity was still maintained. Fifteen hours later, however, most of the APC-treated cells were propidium iodide-positive indicating the disruption of the plasma membrane. At that time point, cells showed severe plasma membrane blebbing as could be seen in scanning electron microscope pictures. Comparable to CD95-induced apoptosis caspases and endonucleases were activated upon APC incubation. This was measured by analysis of degradation of two cellular substrates: poly(ADP-ribose)polymerase (PARP) and lamin b. Furthermore, activation of caspase-3 by proteolytic cleavage of the pro-enzyme could be observed. The importance of caspases in APC-induced apoptosis was stressed by the finding that the broad-range caspase inhibitors zVADfmk and zDDCB can almost completely suppress APC-induced apoptosis. Pre-treatment of HL-60 cells with these inhibitors prevented DNA fragmentation and PARP cleavage in APC-treated cells. To further stress the importance of caspases in APC-induced apoptosis, we used SKW6 cells engineered to overexpress the cowpox virus serpin-like caspase inhibitor CrmA. The CrmA protein is a potent (Ki < 20 nM) and selective inhibitor of group I caspases (caspase-1, -4, and -5) and most group III caspases (caspase-8, -9, -10). This cowpox virus is thought to facilitate infection through inhibition of apoptosis in host cells (Garcia-Calvo et al., 1998). In our test system, overexpression of CrmA resulted in increased survival compared to control cells which had been transfected with an empty vector. PARP cleavage and internucleosomal DNA cleavage was not de-
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tected after 16 hours treatment with up to 30 mM of APC in the crmA cells. On the same conditions, control cells were apoptotic. To examine whether Bcl-2 plays a role in APC-induced apoptosis, we used two Bcl-2-overexpressing cell lines, Jurkat T cells and the B lymphoblastoid cell line SKW6. We tested APC for their ability to induce apoptosis in these two cell systems. Interestingly, we found that Bcl-2 protects against APC-induced apoptosis only in Jurkat T cells. Neither PARP cleavage nor DNA fragmentation could be detected within APC-treated Bcl-2-overexpressing Jurkat cells in contrast to the control Jurkat cells. On the other hand, overexpression of Bcl-2 within the SKW6 cell line did not rescue APC-treated cells. Within control SKW6 cells as well as within the Bcl-2 overexpressing SKW6 cells PARP cleavage and DNA fragmentation could be monitored indicating the apoptotic nature of the cells. The same phenomenon was observed when incubating these two cell lines with an apoptosis-inducing antiCD95 antibody. Upon triggering of the CD95 pathway, Bcl-2 showed an inhibitory effect only within Jurkat T cells. An explanation can be given by recent findings of Scaffidi et al. (1998). According to their results, the pathway(s) downstream of CD95 can vary among different cell types. Two different pathways were investigated by M. Peter and co-workers and accordingly cell lines were separated into two groups: type I and type II cells. Within type I cells (e. g. SKW6) CD95 triggering induced a strong caspase-8 activation at the deathinducing signalling complex (DISC). In these B-cell type cells, mitochondria were bypassed and other caspases, such as caspase-3 were directly activated subsequently leading to apoptosis. In type II cells (e. g. Jurkat T), however, only a few DISCs were formed or DISC activity was weak leading to the activation of mitochondria. The release of cytochrome c from mitochondria in turn resulted in the cleavage of caspase-8 and caspase-3 downstream of mitochondria in these T-cell type cells. Upon triggering with CD95 ligand or an apoptosisinducing and receptor-crosslinking antibody, all mitochondrial apoptogenic activities were blocked by overexpression of Bcl-2 in both cell types (Scaffidi et al., 1998). However, blocking the activation of mitochondria by overexpression of Bcl-2 had an apoptosis-inhibiting effect only in type II cells or Jurkat T cells which use a mitochondria-dependent pathway, but not in type I (SKW6) cells. According to this point of view, our experiments show that induction of apoptosis by APC can only be abrogated by Bcl-2 at the level of mitochondria using cell lines within which mitochondria and the release of cytochrome c are of importance. However, most tumour cells are type I cells and thus, induction of apoptosis by activation of the CD95 pathway occurs independently of the amount of Bcl-2 expressed. Since almost 50% of tumours in humans express high levels of Bcl-2, it is of great interest for the use of APC in tumour therapy that induction of apoptosis by APC in these cells can occur despite high levels of Bcl-2. Searching for a more upstream target of APC, we investigated whether the CD95 (Fas/APO-1) receptor signalling pathway is directly involved in APC-induced apoptosis. We therefore used a cell line defective in the CD95 pathway, the B lymphoma cell line BJAB, which was transfected with a dominant negative expression construct of the Fas-associated death domain (FADDdn). Expression of such FADD dominant negative mutants abrogates CD95-induced apoptosis in these cells (Chinnaiyan et al., 1995, 1996). We investigated the
action of APC on FADDdn BJAB cells. In contrast to wildtype BJAB cells, apoptosis did not occur in APC-treated BJAB FADDdn cells. Cleavage of the death substrate PARP or DNA could not be observed. Thus, CD95 seems to play an essential role in APC-induced apoptosis. The molecular mechanism by which APC activate CD95 remains still unclear. Nevertheless, our findings present the first hint that APC act via highly specific mechanisms involving activation of receptor(s), e. g. CD95, and downstream signalling cascades. This is a strong hint that the antitumoural action of APC is not only based on unspecific membrane alterations but also involves (specific) activation of receptors at the plasma membrane of the target tumour cells. It is important to notice that the APC compounds presented in this paper were newly synthesised. The aim was to create new APC compounds with high antitumoural activity but lesser side effects than the more prominent members out of the growing group of APC, Et-18-OCH3 and HePC, and to investigate mechanisms of action of these new compounds. Our APC compounds are comparable to Et-18-OCH3 and HePC in cell culture models with regard to their potency to kill tumour cells. They act by highly specific mechanisms involving induction of apoptosis. Although HePC and Et-18-OCH3 are already used in the clinical treatment of leukaemia or skin metastases derived from breast cancer, their mechanisms of action remained largely unknown. Several effects have been proposed to be of importance in the cytotoxic action of APC. This includes disturbance of the phospholipid metabolism of the plasma membrane (Modolell et al., 1979; Herrmann and Neumann, 1986), inhibition of cellular transport systems (Hoffman et al., 1992), modification of protein kinase C activity (Helfman et al., 1983; Daniel et al., 1988; Geilen et al., 1991; Heesbeen et al., 1991) and, recently, induction of apoptosis (Mollinedo et al., 1997; Diomede et al., 1993). Nevertheless, no molecular target of APC compounds has been found so far. Identification of the CD95 pathway as an essential part in APC-induced apoptosis points out that further studies should focus on a possible molecular target of APC at the level of plasma membrane receptors. Identification of a molecular target of APC compounds could be of great benefit for specific design or chemical modifications of the APC compounds known to date and could make tumour therapy with APC even more effective. Acknowledgements. We are grateful to Dr. Andreas Strasser, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia for the Bcl-2- and CrmA-overexpressing SKW6 cells, to Prof. Stanley Korsmeyer, Washington University School of Medicine, St Louis, USA, for the Bcl-2-overexpressing Jurkat cells and to Dr. Markus Peter for his help with these cells, as well as to Prof. Vishva Dixit, University of Michigan, Ann Arbor, USA, for the FADDdn BJAB cells. Scanning electron microscope pictures were taken at the Institute of Transuranium Elements, EC, JRC, Karlsruhe. We would therefore like to thank Prof. J. Van Geel, Ian Ray and Hartmut Thiele for their kind help. ± Special thanks to Prof. Reinhard Jahn, MPI Göttingen, for critically reviewing the manuscript. ± This work has been supported by grants from the Forschungszentrum Karlsruhe.
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European Journal of Cell Biology 80, 1 ± 10 (2001, January) ´ Urban & Fischer Verlag ´ Jena http://www.urbanfischer.de/journals/ejcb
1
Killing tumour cells by alkylphosphocholines: Evidence for involvement of CD95 Astrid Matzkea, Ulrich Massingb, Harald F. Krug1)a a b
Forschungszentrum Karlsruhe, Institute for Toxicology and Genetics, Karlsruhe/Germany Tumour Biology Centre, Freiburg/Germany
Received July 25, 2000 Accepted August 18, 2000
Alkylphosphocholines ± BCL-2 ± caspases ± CD95 ± apoptosis Many lipids act as cellular messengers and lead to a variety of different cellular responses. Out of the group of these compounds the ceramides are able to induce apoptosis, and some synthetic lipids can mimic this effect. Apoptosis is an important mechanism whereby chemotherapeutics exhibit their anti-oncogenic activity. Although, some lipid analogues were used in clinical trials, they exert severe side effects and their mechanism of action is widely unknown. We present here a new class of synthetic alkylphosphocholines (APC) that induce programmed cell death in leukaemia cells. The signs of apoptosis arise after 1 h of incubation with these compounds as shown by phosphatidylserine externalisation followed by caspase activation and DNA fragmentation. We demonstrate that the molecular target of these lipids is upstream of caspases and Bcl-2. Experiments with FADD dominant negative cells reveal that induction of apoptosis occurs on the level of CD95 and that these compounds can now be optimised for their capacity to activate the apoptosis-inducing receptor CD95.
Introduction Phospholipid analogues represent a new class of antitumour agents targeted to the cell membrane. They are membraneaffecting antiproliferative drugs that exert a pronounced cytostatic action on different neoplastic cell lines in vitro and show antineoplastic activity in vivo (Berdel, 1991; Muschiol et al., 1988; Noseda et al., 1988). The first compounds of this group which were synthesised represented metabolically stabilised analogues of 2-lysophosphatidylcholine. Later, it has been shown that the glycerol moiety is not an essential structural element and APC exert similar antitumour effects
Dr. Harald F. Krug, Forschungszentrum Karlsruhe, Institute for Toxicology and Genetics, P.O. Box 3640, D-76021 Karlsruhe/Germany, e-mail: [email protected], Fax: 49 7247 823 557.
1)
(Hilgard et al., 1993). Further investigations of the structureactivity relationship among a variety of synthetic APC indicated that a long alkyl chain and a phosphocholine moiety are necessary for sufficient antineoplastic effects of the lipid analogues (Unger and Eibl, 1986). A prominent example of APC is 1-O-octadecyl-2-O-methylsn-glycero-3-phosphocholine (Edelfosine, Et-18-OCH3) which is experimentally used in purging leukaemic bone marrow prior to autologous bone marrow transplantation (Vogler and Berdel, 1993; Königsmann et al., 1996). Another encouraging example of APC is hexadecylphosphocholine (Miltefosine, HePC) (Unger et al., 1989). It is used in the topical treatment of skin metastases resulting of breast cancer and lymphomas (Unger et al., 1988). Systemic application of HePC is not possible due to its gastro-intestinal side effects. In contrast to naturally occurring phospholipids, HePC contains no ester, hydroxyl, amide or amino group adjacent to the phosphocholine head group. The application of APC in tumour therapy is made possible by the fact that APC are toxic preferentially against leukaemia cells but spare normal blood progenitor cells, possibly due to a different lipid metabolism in tumour cells (Andreesen et al., 1978; Vogler et al., 1987; Verdonck et al., 1990; Mollinedo et al., 1997). Furthermore, APC show only marginal substrate properties to phospholipid metabolising enzymes which increases their half-life and allows accumulation in tumour cells (Hoffman et al., 1986). Nevertheless, the mechanisms of tumouricidal action of APC remain largely unknown. Here we present 8 newly synthesised, enantiomerically pure APC compounds (Massing and Eibl, 1994) (Fig. 1). The objective was to develop APC with high antineoplastic activity and lower side effects as HePC. The new compounds are therefore structurally similar to HePC, but contain an additional functional group: acetic ester (R/S-O-acetyl), hydroxyl (R/S-OH), acetamide (R/S-N-acetyl), amino (R/S-NH2) adjacent to the phosphocholine moiety (2-position) and have been synthesised in the R- and S-configuration, respectively. In order to achieve the same effective length of the apolar hydrocarbon chain as HePC, the new compounds are based on an octadecyl chain instead of an hexadecyl chain.
0171-9335/01/80/01-1 $15.00/0
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A. Matzke, U. Massing, H. F. Krug
Hoechst 33342 staining
After incubation, 1 106 cells were incubated with a final concentration of 10 mM Hoechst 33342 (Sigma) for 10 min, washed with PBS to reduce background fluorescence and finally visualised with a fluorescence microscope (Axiovert S100, Zeiss, Jena). Microscopy was performed with excitation at 364 nm and emission at 460 nm. Pictures were taken with a Hamamatsu CCD 4880 ± 80 camera and further optimisation was performed by use of the Openlab 2.05 software (Improvision, UK).
Analysis of oligonucleosomal DNA fragments
Apoptotic DNA fragments were isolated according to Herrmann et al. (1994). The separation was carried out on 1.8% agarose gels. DNA fragments were stained with 0.5 mg/ml ethidium bromide and DNA was visualised by UV light.
Western blot
Fig. 1. Structures and abbreviations of synthetic alkylphosphocholines (APC). All compounds are enantiomerically pure R- and Sconfigurated. * indicates the chirality centre.
The action of these APC on human tumour cell lines was studied. Since promising results were obtained in the treatment of leukaemia by bone marrow purging (Vogler and Berdel, 1993; Königsmann et al., 1996), we chose for our investigations leukaemia cell lines.
Materials and methods Reagents
All cell culture reagents and media were from Life Technologies (Eggenstein, Germany). Caspase-inhibitors zVADfmk and zD-DCB and CD95 ligand were obtained from Alexis (Grünberg, Germany). Apoptosis-inducing anti-CD95 was obtained from Boehringer Mannheim. APC were synthesised by U. Massing, Tumour Biology Centre, Freiburg, Germany. Stock solutions were prepared in ethanol. The final concentration of the vehicles within the incubation mixture did not exceed 1%. All other chemicals were obtained from Sigma, Deisenhofen.
Cell culture and growth conditions
HL-60 cells were obtained from ATCC (Rockville, MD) and grown in suspension in a standard culture medium consisting of RPMI 1640 supplemented with 15% foetal calf serum (FCS). Jurkat neo and bcl-2 were cultivated as described elsewhere (Armstrong et al., 1996). SKW neo, bcl-2 and crmA were cultivated as described before (Strasser et al., 1995). BJAB wt and FADDdn cells were cultivated as described elsewhere (Chinnaiyan et al., 1996).
Cell proliferation and viability
The cell number was measured with a Coulter Counter (Coulter Electronics GmbH, Krefeld). Cell viability was checked by trypan blue dye exclusion and the number of viable cells was estimated using a Neubauer chamber.
Proteins were solubilised from control and stimulated cells by lysis in gel-sample buffer (0.16 M Tris-HCl, 4% SDS, 20% glycerol, 4% 2mercaptoethanol, 0.08% bromophenol blue), size fractionated on 10% or 12.5% SDS-polyacrylamide gels and then transferred to PVDF membranes by electroblotting. After blocking ( > 1 hour at room temperature in 10% skim milk in phosphate-buffered saline) membranes were incubated for > 1 hour at room temperature with the indicated antibodies, washed and incubated for 1 hour with the secondary horseradish peroxidase-conjugated antibody. Membranes were finally probed with enhanced chemoluminescence reagent (Amersham, Braunschweig) and exposed to X-ray films. Antibodies for Western blotting: anti-PARP (Boehringer Mannheim), anti-CPP32 (Transduction Laboratories, Mamhead, UK), anti-lamin B (Santa Cruz Biotechnology, Santa Cruz, USA).
Annexin-V-Fluos staining
Cells (1 106) were stained for 15 min in HEPES-buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, 5 mM CaCl2) containing 1 mg/ ml propidium iodide and 2% Annexin-V-Fluos solution (Boehringer Mannheim). After washing once with HEPES-buffer, Annexin-Vpositive cells were visualised using a fluorescence microscope (Axiovert S100, Zeiss, Jena). Colorisation and merging was done with the Openlab 2.05 software (Improvision, Coventry, UK).
Results APC are cytotoxic towards human leukaemia HL-60 cells
HL-60 cells were seeded at 5 105 per ml and incubated for 48 hours with increasing concentrations of APC. Cell number was measured and viability of the cells was estimated by trypan blue dye exclusion test. With increasing concentrations of APC, we could observe a growth-inhibitory and cytotoxic effect of all APC (Fig. 2). As shown in Table I, R-N-acetyl is the most toxic compound (LD50 4 mM). S-O-acetyl and S-Nacetyl were effective in the same concentration range, whereas R-O-acetyl (LD50 68 mM) and R-OH (LD50 30 mM) were the least toxic compounds. In general, the s-configurated isomers showed higher antitumoural activity than their rconfigurated counterparts. This is obvious when comparing the LD50 values of R-O-acetyl (LD50 68 mM) and S-O-acetyl (LD50 5 mM). HL-60 cells are affected by R-O-acetyl only in high concentrations, whereas S-O-acetyl is ten times as effective in the lower micromolar range. In our hands, the LD50 values of the most potent compounds R-N-acetyl, S-N-acetyl and S-O-acetyl bear comparison to those of Et-18-OCH3 (LD50 12 mM) and HePC (LD50
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Induction of apoptosis by alkylphosphocholines
Fig. 2. Dose-response curves of HL-60 cells after APC treatment. HL-60 cells were seeded with 500 000 cells/ml. Viability was determined by trypan blue dye exclusion test after an incubation time of 48 hours. Data represent the mean of 3 experiments. Error bars were omitted but the standard deviation was < 10%. Viability is presented as a percentage of total cells.
Tab. I. LD50 values of HL-60 cells. compound
LD50 [mM]
R-O-acetyl R-OH R-NH2 S-OH S-NH2 Et-18-OCH3 HePC S-O-acetyl S-N-acetyl R-N-acetyl
68 30 26 21 13 12 7.5 7 5 4
HL-60 cells were seeded at a concentration of 5 105 per ml and were treated with increasing concentrations of APC. LD50 values were calculated from dose-response curves after an incubation time of 48 hours.
7.5 mM), indicating that our lipids have similar or even better potency in killing leukaemia cells in vitro.
APC induce apoptosis in HL-60 cells
From light microscopy studies, evidence was given that APC might act via the induction of apoptosis. APC-treated cells produced blebbing of plasma membranes and segregation of apoptotic bodies. To clarify whether these morphological alterations and the cytotoxic and growth-inhibitory effect of APC might be indeed a result of induced apoptosis, the APC compounds were further tested for their ability to induce apoptosis in HL-60 cells. Since R-N-acetyl was the most potent compound further studies dealing with the mechanisms of action of APC were mainly carried out with this compound.
Externalisation of PS early in APC-induced apoptosis
One of the early events in apoptosis is the externalisation of phosphatidylserine (PS) to the outer leaflet of the plasma membrane (Martin et al., 1995). We have used a PS-binding protein (annexin-V-fluos) as a specific probe to detect apoptosis-dependent redistribution of this phospholipid, which is normally confined to the inner plasma membrane leaflet. Cells were counterstained with
3
propidium iodide to distinguish apoptotic from necrotic cells. Cells were visualised using a fluorescence microscope. Fig. 3a shows light microscopy pictures of control cells (top line) and APC-treated cells (bottom lines). Fluorescent microscopy pictures of annexin-V-stained cells (green) are seen in Fig. 3b, and propidium iodide-stained cells (red) in Fig. 3c. Fig. 3d shows a merged image of the three previous pictures. Already after 1 hour, R-N-acetyl-treated cells were annexinV-positive. Green fluorescence indicated the binding of annexin-V to PS at the outer plasma membrane leaflet. Characteristic green fluorescent rings formed around the cells. At that time point, only a few necrotic cells could be seen indicated by the red fluorescence of propidium iodide, nor did the cells bleb. The externalisation of phosphatidylserine is the first apoptotic sign detectable upon APC treatment. Green fluorescence of annexin-V-positive cells appeared after 1 hour of treatment (Fig. 3b, middle line) and sustained several hours. After 16 hours, however, most of the cells were necrotic or late apoptotic and were annexin-V- as well as propidium iodidepositive (Fig. 3d, bottom line) indicating that the plasma membrane integrity has been lost.
APC induce plasma membrane blebbing and chromatin condensation
One of the characteristic and early described features of apoptosis is the blebbing of the plasma membrane of apoptotic cells. As can be seen in light microscopy this phenomenon could be observed in APC-treated cells (Fig. 3a). The formation of membrane protrusions and blebs is more prominent when depicted with a scanning electron microscope. Severe membrane blebbing started about 8 hours after APC treatment (data not shown) and was clearly obvious after 16 hours of treatment with 20 mM APC (Fig. 4b). Additionally, during apoptosis the structure of the cell nucleus changes and typical rearrangements of chromatin appear during cell death (Dini et al., 1996). DNA staining of APC-treated cells with the fluorescent dye Hoechst 33342 revealed condensed and fragmented nuclei. After 16 hours the chromatin of APC-treated cells was condensed and showed a much brighter and more intense fluorescence compared to untreated control cells as illustrated in Fig. 5.
APC induce DNA fragmentation and cleavage of the death substrate PARP
After having observed the morphological alterations in APCtreated cells, we investigated the biochemical changes that accompany apoptosis due to activation of caspases and endonucleases. All eight APC compounds were tested for their ability to induce apoptosis in HL-60 cells. As a marker for apoptosis we investigated the activation of endonucleases resulting in the formation of a DNA-ladder due to internucleosomal cleavage of the DNA (Wyllie, 1980). The characteristic 180 ± 200-bp DNA fragments were recognised as a DNA-ladder on conventional agarose gel electrophoresis. Moreover, we investigated the proteolytic cleavage of poly(ADP-ribose)polymerase (PARP) (Kaufmann et al., 1993) as a second marker for apoptosis. The native 116-kDa PARP is a substrate for caspase-3 (Cohen, 1997) and cleavage of PARP during apoptosis yields a 86-kDa product which can be detected by Western blotting using specific antibodies.
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Fig. 3. Annexin V-fluos staining of HL-60 cells. Control HL-60 cells and APC-treated cells were stained with the phosphatidylserinebinding protein annexin-V-fluos (green), and DNA was stained with propidium iodide (red) at a concentration of 0.1 mg/ml for 15 minutes.
Fig. 4. APC induce plasma membrane blebbing in HL-60 cells. Scanning electron microscopy pictures of control HL-60 cells (a) and a cell treated with 20 mM APC for 16 hours (b).
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Top line: untreated control cells; middle line: 20 mM R-N-acetyl, 1 hour; bottom line: 20 mM R-N-acetyl, 16 hours. (a) Light microscope picture, (b) annexin V-fluos staining, (c) propidium iodide staining, (d) merged image of the three previous pictures. Magnification: 328 .
All compounds except R-OH and R-O-acetyl were able to induce DNA fragmentation (Fig. 6b) and PARP cleavage (Fig. 6a) in HL-60 cells. Upon treatment with 20 mM R-OH or 20 mM R-O-acetyl, neither PARP cleavage (Fig. 6a, lanes 2 and 6) nor DNA fragmentation (Fig. 6b, lanes 3 and 7) could be detected. As shown in Fig. 2, R-OH and R-O-acetyl were the least toxic compounds. All other compounds were able to induce DNA fragmentation and PARP cleavage in HL-60 cells. In R-N-acetyl-treated cells, DNA fragmentation could first be monitored at about 5 hours after treatment with 20 mM of the synthetic lipid (Fig. 6c). Upon 16 hours treatment of cells with R-N-acetyl, the APC was able to induce DNA fragmentation at a concentration as low as 2.5 mM (Fig. 6d, lane 4).
Specific activation of caspases in APC-induced apoptosis
Fig. 5. Chromatin condensation induced by APC in HL-60 cells. Nuclei of HL-60 cells were stained with Hoechst 33342 dye in untreated control cells (a) and in cells treated with 20 mM APC for 16 hours (b). Magnification: 276 .
To point out specifically the importance of caspase proteases in APC-induced apoptosis, we analysed the activation of caspases at different time points during treatment. The processing of the two caspase substrates PARP and lamin b (Kaufmann, 1989; Kaufmann et al., 1993) was investigated by means of Western blotting using specific antibodies. Cleavage of PARP, as well as the degradation of lamin b started 5 hours after APC treatment and was clearly obvious after 10 hours treatment (Fig. 7a, b). Moreover, activation of caspases itself is dependent on proteolytic cleavage (Thornberry and Lazebnik, 1998). We therefore analysed the disappearance of the 32-kDa pro-
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Fig. 6. Induction of apoptosis by APC. The ability of all different APC to induce apoptosis was analysed using the cleavage of poly(ADPribose)polymerase and the formation of a DNA-ladder due to internucleosomal DNA cleavage as markers. HL-60 cells (1 106 per ml culture medium) were incubated with 20 mM of each APC for 16 hours. Cleavage of PARP was monitored in a Western blot using antiPARP antibodies (a). DNA fragments were visualised by agarose gel electrophoresis (b). Furthermore, HL-60 cells were treated with 20 mM of R-N-acetyl and DNA fragmentation was analysed after different incubation times (c). In (d), HL-60 cells were treated with increasing concentrations of R-N-acetyl for 16 hours and probed for DNA fragmentation. (M: DNA molecular weight marker)
caspase-3 (CPP32) by proteolytic cleavage (Erhard and Cooper, 1996; Liu et al., 1996). As shown in Fig. 7c processing of the pro-enzyme started after 7.5 hours and is almost complete at 10 hours of treatment with APC.
APC-induced apoptosis can be prevented by the caspase inhibitors zVADfmk and zD-DCB as well as by overexpression of CrmA
To demonstrate that activation of caspases is functionally relevant in APC-induced apoptosis, the effect of the peptidebased caspase inhibitors zVADfmk and zD-DCB in APCtreated cells was investigated. zVADfmk, a fluoromethyl ketone inhibitor, is a competitive and irreversible inhibitor of caspases 1 ± 10 (Garcia-Calvo et al., 1998) and zD-DCB is a
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Fig. 7. Activation of Caspases is required for R-N-acetyl-induced apoptosis. The degradation of the death substrates PARP (a), and lamin b (b), as well as the processing of pro-caspase 3 (CPP32) (c) was observed in R-N-acetyl-treated HL-60 cells over a time period of 24 hours by means of Western blotting using specific monoclonal antibodies. Furthermore, the effect of the caspase inhibitors zVADfmk and zD-DCB was observed during R-N-acetyl-induced apoptosis. Cells were pre-treated with the indicated concentrations of inhibitor for 30 min and further treated with 30 mM of R-N-acetyl for 16 hours. Control cells were left untreated. Apoptosis was detected by DNA fragmentation (d) and PARP cleavage (e). SKW6 cells engineered to overexpress the viral caspase inhibitor CrmA (SKW6 crmA) and SKW6 cells transfected with an empty vector (SKW6 neo) were treated with increasing concentrations of R-N-acetyl or with 1 mg/ml apoptosisinducing anti-CD95 antibody (aCD95) for 16 hours and probed for DNA fragmentation (f) and PARP cleavage (g).
broad-range caspase inhibitor which can easily enter target cells due to a hydrophobic moiety (Henkart, 1996). APCinduced killing of HL-60 cells was almost completely prevented when cells were pre-incubated with 100 mM of inhibitors for 15 minutes (data not shown). Neither DNA fragmentation (Fig. 7d) nor PARP cleavage (Fig. 7e) could be observed when cells were pre-treated with zVADfmk or zD-DCB. In our test system, the inhibitor zD-DCB was more potent than zVADfmk since it blocked DNA fragmentation and PARP cleavage at a lower concentration (50 mM) than zVADfmk. To further stress the importance of caspases in APC-induced apoptosis, we used B lymphoblastoid SKW6 cells engineered to overexpress the cowpox virus CrmA protein, a direct and specific inhibitor of caspase proteases (Ray et al., 1992; Cohen, 1997). Overexpression of CrmA prevents cells from apoptosis induced by a variety of stimuli, such as CD95 ligand (Strasser
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A. Matzke, U. Massing, H. F. Krug
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et al.,1995) or UV radiation (Rehemtulla et al., 1997). Here we show that CrmA protects cells from APC-induced apoptosis. In APC-treated SKW6 crmA cells, cleavage of caspase substrate protein PARP (Fig. 7g) as well as the fragmentation of DNA (Fig. 7f) is totally prevented. CrmA is a highly specific inhibitor of group I and III caspases, with the exception of caspase-6 (Garcia-Calvo et al., 1998), indicating that activation of such caspases is crucial in APC-induced cell death.
Role of mitochondria and Bcl-2 in APC-induced apoptosis
There is evidence that Bcl-2 plays a role in preventing cells from apoptosis (Adams and Cory, 1998). We were interested whether Bcl-2 can rescue cells within which apoptosis had been induced by APC. Therefore, we examined the effect of Bcl-2 overexpression during APC-induced apoptosis in Jurkat T cells and in B lymphoblastoid SKW6 cells. Both cell lines were transfected with either Bcl-2 vector or empty vector (Strasser et al., 1995; Armstrong et al., 1996). Cells were incubated with increasing concentrations of APC and lysates were probed for PARP cleavage and DNA fragmentation. As controls, we used taxol and the topoisomerase inhibitor etoposide, which are well-known inducers of apoptosis (Kaufmann, 1989; Solary et al., 1994). Interestingly, we observed that overexpression of Bcl-2 protects only the Jurkat T cells from APC-induced apoptosis (Fig. 8a, b). Within Jurkat T cells, neither PARP cleavage nor DNA degradation could be detected in the Bcl-2 overexpressing cells, whereas the control cells were strongly affected by the alkylphosphocholines. On the other hand, Bcl2 overexpression within the lymphoblastoid B cells SKW6 could not rescue APC-treated cells. Within the SKW6 cell line, the Bcl-2 overexpressing cells were just as much affected by APC treatment as the control cells. Transfected SKW6 cells of each type, neo or bcl-2, showed similar patterns for DNA laddering as well as for PARP cleavage (Fig. 8c, d). To further investigate these obvious differences between the apoptotic pathways within the two cell lines, the action of APC was compared with the action of an apoptosis-inducing anti-CD95 antibody. Such an antibody can trigger apoptosis via the CD95 pathway by crosslinking CD95 receptor molecules (Oehm et al., 1992). Anti-CD95-treated cells showed a similar pattern of DNA fragmentation compared to APC-treated cells. Again, apoptosis was induced in both neo-type clones, and Bcl-2 acted protective only within Jurkat T cells (Fig. 8e, JB). These data demonstrate that Bcl-2 protects cells from the apoptotic action of APC, but this phenomenon is clearly restricted to specific cell types and not a common property.
CD95 is involved in APC-induced apoptosis
Searching for a more upstream target of APC, we investigated whether the CD95 (Fas/APO-1) receptor-mediated signalling pathway is involved in APC-induced apoptosis. We therefore used a cell line defective in the CD95 pathway, the B lymphoma cell line BJAB, which was transfected with a dominant negative expression construct of the Fas-associated death domain (FADDdn). Expression of such FADD dominant negative mutants abrogates CD95-induced apoptosis in these cells (Chinnaiyan et al., 1995, 1996). We investigated the action of APC on FADDdn BJAB cells, as well as on control BJAB cells which had been transfected with an empty vector (BJAB wt). The apoptotic nature of APC-treated cells was confirmed by assay of PARP cleavage and DNA fragmentation.
Fig. 8. Role of mitochondria and Bcl-2 in APC induced apoptosis. Two Bcl-2-overexpressing cell lines, Jurkat T cells and SKW6 cells were incubated with increasing concentrations of APC,and lysates were probed after 16 hours for internucleosomal DNA cleavage on agarose gel electrophoresis (a, c) and PARP cleavage by means of Western blotting (b, d). As a control (e), we used an anti-CD95 antibody (aFas, Boehringer) at a concentration of 1 mg/ml for 5 h to induce apoptosis by triggering the CD95 receptor pathway. JN: Jurkat neo, JB: Jurkat bcl-2, SN: SKW6 neo, SB: SKW6 bcl-2, Tax: Taxol 1 mM 16 hours, Et: Etoposide 64 mg/ml 16 hours. M: DNA molecular weight marker.
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After treatment of BJAB cells with APC, only the wild-type cells underwent apoptosis. Within the dominant negative FADD mutants, neither PARP cleavage nor the formation of a DNA ladder could be observed (Fig. 9a, b). This indicates strongly that caspases and endonucleases were not activated within these mutants. Even after treatment with high concentrations of APC up to 50 mM the cells remained intact and did not segregate apoptotic bodies (data not shown). As a control, the specific CD95 ligand (CD95L) (Nagata and Golstein, 1995) was used to check the activation of the CD95 pathway and its blockade in the dominant negative mutants. Treatment with CD95L resulted in apoptosis only within the wild-type cells but not within the FADDdn mutants (Fig. 9b).
Discussion The knowledge about signal transduction pathways leading to death or survival of a given cell is permanently increasing (Dragovich et al., 1998), and dysregulation or modulation of the apoptotic mechanisms seem to be involved in oncogenesis (Stambolic et al., 1999) or present a chance for new ways in tumour therapy (McGill and Fisher, 1997). For this purpose we designed 8 new alkylphosphocholine compounds. They are lysophosphatidylcholine analogues that were synthesised according to minimal structural requirements for antineoplastic activities, namely a long alkyl chain and a phosphocholine polar head group.
Fig. 9. APC fail to induce apoptosis in FADDdn cells. BJAB wildtype (wt) and BJAB engineered to express a dominant negative version of FADD (FADDdn) were used to investigate whether CD95 plays a role in APC-induced apoptosis. Both cell lines were incubated with increasing concentrations of R-N-acetyl for 16 hours and probed for internucleosomal DNA cleavage (a). Furthermore, cells were incubated with 30 mM R-N-acetyl for different time intervals and probed for PARP cleavage by means of Western blotting. As control, we used the CD95 ligand (CD95 lig, 300 ng/ml) to induce apoptosis via the CD95 pathway (b). M: DNA molecular weight marker.
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We tested these new compounds for their antineoplastic potency in cell culture systems of human leukaemia HL-60 cells. The APC showed a dose-dependent growth-inhibitory and cytotoxic effect on HL-60 cells. LD50 values were calculated from dose-response curves. The APC compounds showed different toxicity with regard to their chemical structure. In general, the S-configurated isomers were more toxic than their R-configurated counterparts. The compound bearing an Rconfigurated hydroxy or O-acetylic function were toxic only at very high concentrations. The low toxicity of R-OH and R-Oacetyl can be explained by the preferred degradation of these two R-(natural)-configurated compounds by phospholipidmodifying enzymes. Due to their chemical structure, Rconfigurated acetic ester or hydroxyl-function at the 2position, they represent good substrates for transacylases or reacylation and thus, can be quickly detoxified by the cells. It has been shown that R-OH and R-O-acetyl can be metabolised to an R-O-acyl compound which is similar to phosphatidylcholine in its chemical structure and can be tolerated by HL-60 cells (Hildenbrand et al., 1999). During the last years it has been discussed that lysophospholipids as well as analogues of lysophospholipids quickly disrupt plasma membrane integrity due to their detergent-like structure. Indeed, the plasma membrane becomes affected when exposing cells to high concentrations of APC. During the experiments shown here, APC were used in the lower micromolar range where they do not lyse cells (KaufmannKolle and Fleer, 1992). We show that the cytotoxicity of these APC is a result of induced apoptosis. Classical apoptotic features could be observed upon treatment with these substances. Shortly after APC addition, phosphatidylserine was transferred to the outer leaflet of the plasma membrane. This was the first detectable apoptotic event induced by these compounds. Phosphatidylserine externalisation occurred after only one hour of treatment when the plasma membrane integrity was still maintained. Fifteen hours later, however, most of the APC-treated cells were propidium iodide-positive indicating the disruption of the plasma membrane. At that time point, cells showed severe plasma membrane blebbing as could be seen in scanning electron microscope pictures. Comparable to CD95-induced apoptosis caspases and endonucleases were activated upon APC incubation. This was measured by analysis of degradation of two cellular substrates: poly(ADP-ribose)polymerase (PARP) and lamin b. Furthermore, activation of caspase-3 by proteolytic cleavage of the pro-enzyme could be observed. The importance of caspases in APC-induced apoptosis was stressed by the finding that the broad-range caspase inhibitors zVADfmk and zDDCB can almost completely suppress APC-induced apoptosis. Pre-treatment of HL-60 cells with these inhibitors prevented DNA fragmentation and PARP cleavage in APC-treated cells. To further stress the importance of caspases in APC-induced apoptosis, we used SKW6 cells engineered to overexpress the cowpox virus serpin-like caspase inhibitor CrmA. The CrmA protein is a potent (Ki < 20 nM) and selective inhibitor of group I caspases (caspase-1, -4, and -5) and most group III caspases (caspase-8, -9, -10). This cowpox virus is thought to facilitate infection through inhibition of apoptosis in host cells (Garcia-Calvo et al., 1998). In our test system, overexpression of CrmA resulted in increased survival compared to control cells which had been transfected with an empty vector. PARP cleavage and internucleosomal DNA cleavage was not de-
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tected after 16 hours treatment with up to 30 mM of APC in the crmA cells. On the same conditions, control cells were apoptotic. To examine whether Bcl-2 plays a role in APC-induced apoptosis, we used two Bcl-2-overexpressing cell lines, Jurkat T cells and the B lymphoblastoid cell line SKW6. We tested APC for their ability to induce apoptosis in these two cell systems. Interestingly, we found that Bcl-2 protects against APC-induced apoptosis only in Jurkat T cells. Neither PARP cleavage nor DNA fragmentation could be detected within APC-treated Bcl-2-overexpressing Jurkat cells in contrast to the control Jurkat cells. On the other hand, overexpression of Bcl-2 within the SKW6 cell line did not rescue APC-treated cells. Within control SKW6 cells as well as within the Bcl-2 overexpressing SKW6 cells PARP cleavage and DNA fragmentation could be monitored indicating the apoptotic nature of the cells. The same phenomenon was observed when incubating these two cell lines with an apoptosis-inducing antiCD95 antibody. Upon triggering of the CD95 pathway, Bcl-2 showed an inhibitory effect only within Jurkat T cells. An explanation can be given by recent findings of Scaffidi et al. (1998). According to their results, the pathway(s) downstream of CD95 can vary among different cell types. Two different pathways were investigated by M. Peter and co-workers and accordingly cell lines were separated into two groups: type I and type II cells. Within type I cells (e. g. SKW6) CD95 triggering induced a strong caspase-8 activation at the deathinducing signalling complex (DISC). In these B-cell type cells, mitochondria were bypassed and other caspases, such as caspase-3 were directly activated subsequently leading to apoptosis. In type II cells (e. g. Jurkat T), however, only a few DISCs were formed or DISC activity was weak leading to the activation of mitochondria. The release of cytochrome c from mitochondria in turn resulted in the cleavage of caspase-8 and caspase-3 downstream of mitochondria in these T-cell type cells. Upon triggering with CD95 ligand or an apoptosisinducing and receptor-crosslinking antibody, all mitochondrial apoptogenic activities were blocked by overexpression of Bcl-2 in both cell types (Scaffidi et al., 1998). However, blocking the activation of mitochondria by overexpression of Bcl-2 had an apoptosis-inhibiting effect only in type II cells or Jurkat T cells which use a mitochondria-dependent pathway, but not in type I (SKW6) cells. According to this point of view, our experiments show that induction of apoptosis by APC can only be abrogated by Bcl-2 at the level of mitochondria using cell lines within which mitochondria and the release of cytochrome c are of importance. However, most tumour cells are type I cells and thus, induction of apoptosis by activation of the CD95 pathway occurs independently of the amount of Bcl-2 expressed. Since almost 50% of tumours in humans express high levels of Bcl-2, it is of great interest for the use of APC in tumour therapy that induction of apoptosis by APC in these cells can occur despite high levels of Bcl-2. Searching for a more upstream target of APC, we investigated whether the CD95 (Fas/APO-1) receptor signalling pathway is directly involved in APC-induced apoptosis. We therefore used a cell line defective in the CD95 pathway, the B lymphoma cell line BJAB, which was transfected with a dominant negative expression construct of the Fas-associated death domain (FADDdn). Expression of such FADD dominant negative mutants abrogates CD95-induced apoptosis in these cells (Chinnaiyan et al., 1995, 1996). We investigated the
action of APC on FADDdn BJAB cells. In contrast to wildtype BJAB cells, apoptosis did not occur in APC-treated BJAB FADDdn cells. Cleavage of the death substrate PARP or DNA could not be observed. Thus, CD95 seems to play an essential role in APC-induced apoptosis. The molecular mechanism by which APC activate CD95 remains still unclear. Nevertheless, our findings present the first hint that APC act via highly specific mechanisms involving activation of receptor(s), e. g. CD95, and downstream signalling cascades. This is a strong hint that the antitumoural action of APC is not only based on unspecific membrane alterations but also involves (specific) activation of receptors at the plasma membrane of the target tumour cells. It is important to notice that the APC compounds presented in this paper were newly synthesised. The aim was to create new APC compounds with high antitumoural activity but lesser side effects than the more prominent members out of the growing group of APC, Et-18-OCH3 and HePC, and to investigate mechanisms of action of these new compounds. Our APC compounds are comparable to Et-18-OCH3 and HePC in cell culture models with regard to their potency to kill tumour cells. They act by highly specific mechanisms involving induction of apoptosis. Although HePC and Et-18-OCH3 are already used in the clinical treatment of leukaemia or skin metastases derived from breast cancer, their mechanisms of action remained largely unknown. Several effects have been proposed to be of importance in the cytotoxic action of APC. This includes disturbance of the phospholipid metabolism of the plasma membrane (Modolell et al., 1979; Herrmann and Neumann, 1986), inhibition of cellular transport systems (Hoffman et al., 1992), modification of protein kinase C activity (Helfman et al., 1983; Daniel et al., 1988; Geilen et al., 1991; Heesbeen et al., 1991) and, recently, induction of apoptosis (Mollinedo et al., 1997; Diomede et al., 1993). Nevertheless, no molecular target of APC compounds has been found so far. Identification of the CD95 pathway as an essential part in APC-induced apoptosis points out that further studies should focus on a possible molecular target of APC at the level of plasma membrane receptors. Identification of a molecular target of APC compounds could be of great benefit for specific design or chemical modifications of the APC compounds known to date and could make tumour therapy with APC even more effective. Acknowledgements. We are grateful to Dr. Andreas Strasser, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia for the Bcl-2- and CrmA-overexpressing SKW6 cells, to Prof. Stanley Korsmeyer, Washington University School of Medicine, St Louis, USA, for the Bcl-2-overexpressing Jurkat cells and to Dr. Markus Peter for his help with these cells, as well as to Prof. Vishva Dixit, University of Michigan, Ann Arbor, USA, for the FADDdn BJAB cells. Scanning electron microscope pictures were taken at the Institute of Transuranium Elements, EC, JRC, Karlsruhe. We would therefore like to thank Prof. J. Van Geel, Ian Ray and Hartmut Thiele for their kind help. ± Special thanks to Prof. Reinhard Jahn, MPI Göttingen, for critically reviewing the manuscript. ± This work has been supported by grants from the Forschungszentrum Karlsruhe.
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