The tyrosine kinase Lck is involved in regulation of ... - Nature

Oncogene (2003) 22, 176–185

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The tyrosine kinase Lck is involved in regulation of mitochondrial apoptosis pathways Claus Belka*,1,3, Charlotte Gruber1,3, Verena Jendrossek1, Sebastian Wesselborg2 and Wilfried Budach1 1 Department of Radiation Oncology, University of Tu¨bingen, Hoppe Seyler Str. 3, D-72076 Tu¨bingen, Germany; 2Department of Internal Medicine I, University of Tu¨bingen, Otfried Mu¨ller Str. 10, D-72076 Tu¨bingen, Germany

The induction of apoptosis requires the activation of a highly coordinated signaling network ultimately leading to the activation of caspases. In previous experiments we and others have shown that the tyrosine kinase Lck is required for adequate apoptosis induction in response to ionizing radiation, ceramide incubation and overexpression of the HIV-TAT protein. However, the position of Lck within given apoptotic signaling cascades remains unclear. We therefore aimed to define the role of Lck during radiationinduced apoptosis. Apoptosis induction in response to ionizing radiation, CD95 or TRAIL receptor stimulation was determined in Jurkat T-cells, the Lck-deficient Jurkat clone JCaM1.6- and Lck-retransfected JCaM1.6/Lck. No apoptosis, release of cytochrome c, breakdown of the mitochondrial potential were detectable during the first 48 h after irradiation of JCaM1.6 cells. In parallel, no activation of caspase-9, -8 and -3 was detectable. Since mitochondrial apoptosis pathways act within a feedback mechanism during death-receptormediated apoptosis, the influence of the Lck defect on CD95/Fas/Apo-1-L or TRAIL-induced apoptosis was also tested. Both stimuli induced apoptosis in Lckdeficient cells. However, the kinetics of apoptosis induction determined by caspase-8, -9 and -3 activation as well as DWm breakdown was slowed. We conclude that the Lck deficiency influences early steps during radiation-induced mitochondrial alterations. Oncogene (2003) 22, 176–185. doi:10.1038/sj.onc.1206103 Introduction The apoptotic process is regulated by a wide array of diverse molecules. Depending on the stimulus applied two apoptotic cascades can be distinguished: whereas death receptor stimulation acts via a direct activation *Correspondence: C Belka, Department of Radiation Oncology, University of Tu¨bingen, Hoppe Seyler Str. 3, Tu¨bingen D-72076, Germany; E-mail: [email protected], http:www.radiation-research.de 3 These two authors share first authorship Received 8 July 2002; revised 30 September 2002; accepted 4 October 2002

of the caspase cascade at the receptor level, DNA damage and stress-mediated caspase activation and apoptosis are triggered mainly by mitochondrial alterations (Belka et al., 2000b, 2001; Newton and Strasser, 2000). Mitochondrial apoptosis is basically regulated by a complex interplay of pro- and antiapoptotic Bcl-2-like proteins. DNA damage or cellular stress triggers the transcriptional activation of the proapoptotic Bax-like molecules Bax, Bak and the BH3-only proteins Noxa and Puma. Although the interplay of Bax-like molecules and BH3-only proteins is not completely understood, it has been shown that an interaction of molecules belonging to either group is crucial for the subsequent release of cytochrome c (Cheng et al., 2001). The release of cytochrome c together with dATP and APAF-1 in turn triggers the autoproteolytic activation of caspases (reviewed in: Belka and Budach, 2002; Rudner et al., 2002). In this regard, the role of certain caspases was also found to be different. While caspase-8, which is directly activated at the receptor complex, was shown to be the key caspase for death-receptor-mediated apoptosis (Varfolomeev et al., 1998), caspase-9 is crucial for DNA-damage-related apoptosis and is activated following cytochrome c release from the mitochondrial intermembrane space (Hakem et al., 1998; Kuida et al., 1998; Soengas et al., 1999). In this context, we have shown that the apoptotic process triggered by ionizing radiation is delayed, but not abrogated in cells lacking caspase-8 (Belka et al., 2000b). Thus, two distinct pathways are responsible for the induction of apoptosis in response to death receptor ligation or DNA damage or stress (Belka et al., 2000b; Engels et al., 2000; Dunkern et al., 2001; Stepczynska et al., 2001; Tomicic et al., 2002; Wieder et al., 2001). In addition to the essential role of caspases, it was shown that the tyrosine kinase Lck is involved in the regulation of apoptosis induced by ionizing radiation (Belka et al., 1999), the HIV-TAT protein (Manna and Aggarwal, 2000) and ceramide (Manna et al., 2000). In this regard, it is important that the release of ceramides from cellular membranes is considered to be a key step during radiation-induced apoptosis in some (Santana et al., 1996; Pena et al., 2000) but not all cell models (Burek et al., 2001).

Lck and mitochondrial apaptosis pathways C Belka et al

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Lck belongs to the src-like tyrosine kinase family and is normally required for the adequate propagation of Tcell receptor signals. After stimulation of the T-cell receptor complex by the respective antigen in combination with an MHC molecule, Lck becomes activated and is involved in the phosphorylation of critical residues of the TCR complex. These phophorylations are essential for downstream signaling events including calcium influx, activation of the transcription factor NFAT and the mitogenic kinases Erk1 and Erk2 (Straus and Weiss, 1992; Kabouridis et al., 1997; Samelson, 2002). Using normal Jurkat Tcells, the Lck-deficient Jurkat subclone JCaM1.6 and Lck-transfected JCaM1.6/Lck cells, we could show previously that Lck is also important for the regulation of apoptosis in response to ionizing radiation (Belka et al., 1999). JCaM1.6 and JCaM1.6/Lck were initially employed to prove that Lck is involved in T-cell receptor signal transduction (Straus and Weiss, 1992). This finding is paralleled by similar results from B-cells, where the B-cell receptor associated src-like kinase BTK is essential for radiation-induced apoptosis (Uckun et al., 1996). In this context, the kinase domain of BTK was crucial for the apoptotic response to ionizing radiation. In addition to the findings on BTK, it was shown that activation of Lck by CD19 crosslinking promoted radiation-induced apoptosis in a radiation-resistant B-lymphoma line (Waddick et al., 1993). Similarly, the src-like kinase Lyn was suggested to be involved in apoptosis regulation in response to UV light in DT40 B-cells (Qin et al., 1997). Since Lck has been shown to be involved in apoptosis induction in response to ceramide, radiation and HIVTAT protein, we aimed to further define its role for

apoptotic signaling. To this end, we analyzed the key steps in apoptosis signaling cascade in Lck-deficient JCaM1.6 and Lck expressing JCaM1.6 cells upon death receptor ligation and irradiation.

Results Apoptosis induction, Lck function and expression In a first set of experiments apoptosis induction after irradiation of JCaM1.6 and JCaM1.6/Lck cells with 10 Gy was followed over 72 h. As shown in Figure 1a, the onset of apoptosis was abrogated in cells lacking Lck with no apoptosis being detectable prior to 72 h (23.9711% in Lck-negative cells vs 71.7712.4%). At 96 h apoptotic changes also increased in Lck-deficient cells (41.2722.5%). However, a strong difference between the Lck-deficient and the Lck-re-expressing cells (75.3711.6%) remained detectable. In parallel experiments, the expression levels of Lck and the function of Lck-mediated signaling pathways were tested in Jurkat, JCaM1.6 and JCaM1.6/Lck. Using an antibody against the C-terminus of Lck, the expression of Lck was absent in JCaM1.6 cells, while comparable levels of Lck were detectable in Jurkat and JCaM1.6/Lck cells (Figure 1b). The functional integrity of Lck-mediated signaling pathways in the JCaM1.6/ Lck cells was verified by analyzing the capability of the respective cell line to respond to CD3/T-cell receptor (TCR) stimulation with an activation of Erk1/2 kinases. The CD3/TCR complex was stimulated using phytohemagglutinin-L (PHA-L, 50 mg/ml) and activation of Erk1/2 was detected using activation specific antibodies

Figure 1 Radiation-induced apoptosis in Lck-deficient cells. JCaM1.6 are negative for Lck expression and function and do not undergo apoptosis in response to ionizing radiation, whereas Jurkat control cells and retransfected JCaM1.6/Lck cells express functional Lck and are sensitive to irradiation. (a) Jurkat cells, Lck negative JCaM1.6 and retransfected JCaM1.6/Lck cells were exposed to 10 Gy ionizing radiation and apoptosis induction was determined every 12 up to 72 h by flow cytometry. (b) Expression of Lck was verfied by Western blotting using an antibody against the C-terminus of Lck. (c) Downstream signaling of Lck was determined by Erk1/2 activation in response to phytohaemagglutinin stimulation. Data show means7s.d. (n ¼ 3) (a) or one representative of three independent experiments (b + c). Oncogene


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directed against the active kinase (Figure 1c). After stimulation of normal Jurkat cells with PHA-L, Erk1/2 kinase was rapidly activated as indicated by the detection of phosphorylated Erk1/2. No such activation was detectable in JCaM1.6 cells, whereas treatment of JCaM1.6/Lck cell with PHA-L clearly induced Erk1/2 phosphorylation. Taken together, the data prove that intact Lck is absent in JCaM1.6 cells that do not undergo apoptosis in response to irradiation, whereas normal Jurkat cells and retransfected JCaM1.6/Lck express functional active Lck. Radiation-induced caspase activation Caspases are key regulators for the induction and execution of apoptosis. Caspase-9 is considered to be the most apical caspase involved in DNA-damageinduced apoptosis. Procaspase-9 is activated after binding to a complex composed of dATP, APAF-1 and cytochrome c that is released from the mitochondrium (Li et al., 1997). Thus, it is the initiator caspase for the execution of the mitochondrial apoptosis pathways. In order to determine in how far the absence of Lck influences the activation of caspase-9, the time course of caspase-9 processing was determined using antibodies against the active sub-unit (Figure 2). As shown in Figure 2, activation of caspase-9 was absent in Lckdeficient cells whereas in Lck-expressing cells caspase-9 activation was detectable as early as 6 h after irradiation. Caspase-3 is activated by caspase-9 and functions as key executor for the apoptotic process. Thus, caspase-3 is responsible for many aspects of the apoptotic phenotype. Consistent with the finding of caspase-9 processing, no activation of caspase-3 was detectable in Lck-deficient cells. In order to further substantiate this finding, cleavage of the caspase-3 substrate PARP was

Figure 2 Radiation-induced caspase activation in JCaM1.6 cells. Radiation-induced cleavage of caspase-9, -3 and -8 as well as cleavage of the caspase-3 substrate PARP is absent in Lck-deficient JCaM1.6 cells, while it is readily detectable in Lck-retransfected cells. Caspase activation was determined by Western blot analysis of cytosolic extracts from JCaM1.6 cells (left panel) and JCaM1.6/ Lck cells (right panel) 10–72 h after irradiation with 10 Gy ionizing radiation. Data show one representative of three independent experiments. Oncogene

determined. As shown in Figure 2, cleavage of PARP by caspase-3 was not detectable in JCaM1.6 cells, whereas it clearly occurred in the Lck-expressing JCaM1.6 cells. Although caspase-8 is an activator caspase for receptor-mediated apoptosis, recent work indicated that it acts as executor caspase during radiation or druginduced apoptosis (Belka et al., 2000b; Engels et al., 2000; Wieder et al., 2001). In accordance with our previous results no activation of caspase-8 was detectable in Lck-deficient cells (Figure 2). The interpretation that caspase-9 acts as apical caspase, followed by activation of executor caspases including caspase-8 and -3, is supported by the time course of caspase activation in our experiments. Caspase9 activation preceded activation of caspase-3 and -8. Up to this point, the data show that none of the key initiator and executor caspases are activated in response to ionizing radiation in Lck-deficient cells. Activation of mitochondrial apoptosis steps in response to radiation Since the release of cytochrome c and the breakdown of the mitochondrial membrane potential (DCm) is considered to be the crucial step for the activation of the mitochondrial death pathway (Li et al., 1997; Soengas et al., 1999; Susin et al., 1999), the role of Lck for the release of cytochrome c and breakdown of the mitochondrial membrane potential was tested. As shown in Figure 3a, the staining pattern of cytochrome c was found to be focal in JCaM1.6 cells after irradiation, whereas in JCaM1.6/Lck cells a diffuse staining of cytochrome c occurred, which was indicative for the release of cytochrome c from the mitochondrial intermembrane space into the cytosol. Conversely, almost no breakdown of the DCm was detectable in irradiated JCaM1.6 cells whereas radiation induced a rapid breakdown of DCm in Lck-positive cells (Figure 3b). At this stage our data indicated that Lck deficiency interferes with important mitochondrial alterations involved in radiation-induced apoptosis. Since Bcl-2 is a key protein inhibiting mitochondrial alterations in response to DNA damage (Rudner et al., 2001b), we compared the phenotype of the JCaM1.6 cells with the phenotype of cells overexpressing Bcl-2. As shown in Figure 4, Bcl-2 overexpressing Jurkat cells did not undergo apoptosis upon stimulation with 10 Gy (Figure 4a). In accordance, no activation of caspases or cleavage of PARP was detectable (Figure 4b). In order to rule out the possibility that Bcl-2 or related proteins are upregulated in JCaM1.6 cells, the expression of Bcl-2, Bcl-xL and MCL-1 was tested by Western blotting. No relevant increase in the basal expression level of either molecule was detectable in Lck-deficient cells (data not shown). Influence of Lck on death receptor signaling The strong effect of the Lck deficiency on DNAdamage-mediated apoptosis prompted us to test the


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Figure 3 Role of Lck for mitochondrial alterations upon irradiation. Radiation-induced release of cytochrome c and breakdown of the mitochondrial membrane potential is not detectable in JCaM1.6 cells. (a) JCaM1.6 and JCaM1.6/Lck were irradiated with 10 Gy. (a) Release of cyctochrome c was determined by confocal laser scanning microscopy after immunostaining of cytochrome c. (b) Integrity of the mitochondrial membrane potential was determined at the indicated intervals by FACS analysis of TMRE-stained cells. Data show one representative of three independent experiments (a) or means7s.d. (n ¼ 3) (b).

Figure 4 Influence of Bcl-2 on radiation-induced apoptosis and caspase activation. Radiation-induced apoptosis and activation of caspases is abrogated in Bcl-2 overexpressing cells. Jurkat vector cells and Bcl-2 overexpressing cells were exposed to 10 Gy ionizing radiation. (a) Apoptosis induction was determined every 12 up to 72 h by flow cytometry. (b) Processing of caspase-3 and cleavage of PARP were analyzed by Western blotting. Data show means7s.d. (n ¼ 3) (a) or one representative of three independent experiments (b).

influence of Lck on other apoptosis-inducing stimuli. Activation of the CD95 or DR4/DR5 TRAIL receptors is known to rapidly induce apoptosis. Thus, JCaM1.6, JCaM1.6/Lck, Jurkat and Bcl-2 overexpressing Jurkat cells were treated with 100 ng/ml of an agonistic CD95 antibody or 100 ng/ml TRAIL (Figures 5a, b and 6). In contrast to the observed apoptosis resistance upon irradiation, no apoptosis resistance towards TRAIL and

CD95 was observed. However, apoptosis upon CD95 receptor stimulation or incubation with TRAIL was slowed in Bcl-2 overexpressing and also in Lck-deficient cells (Figure 5, not shown for TRAIL). In parallel, the expression levels of key molecules (CD95, DR5, FADD, caspase-8, -3) involved in death receptor signaling were tested by FACS and Western blotting. In this regard, no differences in the basal expression levels were detectable (Figure 5c and d). Caspase-8 is the initiator caspase for death-receptormediated apoptosis. Therefore, we analyzed the activation of caspase-8 in JCaM1.6, JCaM1.6/Lck, Jurkat vector and Bcl-2 overexpressing cells. As shown in Figure 7 activation of caspase-8 in response to CD95 stimulation was clearly detectable in all cell lines tested. However, the time course of activation differed. The lowest activation of caspase-8 was measured in Lckdeficient and Bcl-2-overexpressing cells. Similar data were revealed when PARP cleavage was analyzed. To further substantiate these findings, the release of cytochrome c and breakdown of the mitochondrial membrane potential were determined. In accordance with the apoptosis data presented above, the breakdown of DCm upon CD95 death receptor stimulation was slowed in Lck-deficient cells and to a lower extent in Bcl-2-overexpressing cells (Figure 8a and c). Similarly, TRAIL-induced breakdown of DCm was slowed but not abrogated (Figure 8b). Cytochrome c release indicated by diffuse staining of the cytoplasm was detectable regardless of the Lck deficiency or Bcl-2 overexpression (Figure 8d). Taken together, Lck-deficient cells clearly undergo apoptosis in response to death receptor stimulation, while being resistant to radiation-induced apoptosis. Oncogene


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Figure 5 Influence of Lck deficency on death-receptor-induced apoptosis. Jurkat cells express CD95 and TRAIL receptors and undergo death-receptor-induced apoptosis. Lck deficiency does not abrogate CD95- and TRAIL-induced apoptosis. (a) JCaM1.6, JCaM1.6/Lck cells were treated with 100 ng/ml activating CD95 antibody (clone CH11) (b) JCaM1.6 and JCaM1.6/Lck were treated with 100 ng/ml TRAIL for 48 h. Induction of apoptotic cell death was then determined by FACS analysis. (c) Expression of CD95receptor and TRAIL receptor of Jurkat Vector, JCaM1.6 and JCaM1.6/Lck-cells were determined by FACS analysis of immunostained cells (bold line: anti-receptor-antibody; thin line: unspecific control). (d) Expression of caspase-8, -3 and FADD in Jurkat Vector, Bcl-2 overexpressing Jurkat, JCaM1.6 and JCaM1.6/Lck-cells was analyzed by Western Blot analysis. Data show means7s.d. (n ¼ 3) (a) or one representative of three independent experiments (b–d). Oncogene


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Figure 6 Influence of Bcl-2 overexpression on death-receptorinduced apoptosis. Bcl-2 overexpression does not abrogate CD95induced apoptosis. Jurkat vector and Bcl-2-overexpressing Jurkat cells were treated with 100 ng/ml activating CD95 antibody (clone CH11). Induction of apoptotic cell death was then determined by FACS analysis at the indicated time points. Data show means7s.d. (n ¼ 3).

Figure 7 Influence of Lck and Bcl-2 on death-receptor-induced activation of caspase-8 and PARP. CD95-induced activation of caspases is slowed in Lck-deficient and Bcl-2-overexpressing Jurkat cells. JCaM1.6, JCaM1.6/Lck (upper panel), Jurkat vector cells and Bcl-2-overexpressing cells (lower panel) were exposed to 100 ng/ml activating CD95 antibody (clone CH11) for up to 12 h. Processing of procaspase-8 and cleavage of PARP were determined by Western blot analysis. Data show one representative of three independent experiments (b).

Discussion In conclusion, our data indicate that the presence of the tyrosine kinase Lck is required for the adequate execution of early steps of the mitochondrial apoptosis pathways with strong effects on radiation-induced apoptosis and only very limited influences on CD95and TRAIL-induced apoptosis. This interpretation is based on the findings that neither a breakdown of the mitochondrial membrane potential nor release of cytochrome c occurred after irradiation of Lck-deficient cells.

Since Bcl-2 is the key protein interfering with mitochondrial apoptosis induction (Rudner et al., 2001b), our conclusion that the presence of Lck is mandatory for proper functioning of the mitochondrial pathways is further substantiated by the observation that the phenotype of Lck-deficient cells strongly resembles that of Bcl-2-overexpressing cells. In accordance, apoptosis induction by death receptors, which is mainly executed independently of mitochondrial apoptosis pathways (Belka et al., 2000b; Engels et al., 2000; Newton and Strasser, 2000), is only partially attenuated in Lck-deficient cells. Although our own observations and data published by others suggest the presence of distinct albeit overlapping signaling cascades for DNA damage and receptor-mediated death (Belka et al., 2000b; Engels et al., 2000; Newton and Strasser, 2000), it has been suggested that DNA-damage-induced apoptosis may be dependent on activation of death receptors (Belka et al., 1998; Friesen et al., 1996). The induction of apoptosis by death ligands is mediated by a direct activation of caspase-8 via FADD (Muzio et al., 1996). The signal is strongly amplified by caspase-8-mediated activation of the BH3 only protein BID, which connects deathreceptor-induced apoptosis with mitochondrial pathways (Li et al., 1998). The data provided in the present study strongly support the assumption of distinct death pathways, since Lck deficiency only abrogates radiation, but not death-receptor-induced apoptosis. The altered time course of death-receptor-induced apoptosis in Lckdeficient cells could therefore be explained by inadequate activation of the mitochondrial amplification loop. The role of tyrosine kinase activation for apoptosis induction by death receptors has been a matter of debate since conflicting results have been published. Schraven (Schraven and Peter, 1995) showed that Lck is not involved in CD95-induced apoptosis. In contrast, Schlottmann et al. (1996) suggested that tyrosine phosphorylation by Lck is involved in CD95-induced apoptosis. The evident differences may be simply explained by the altered time course of death-receptorinduced apoptosis in Lck-deficient cells. Whereas Schlottmann determined apoptosis after 4 h after stimulation, Schraven determined apoptosis after 18 h. Thus, both results can easily be integrated into our observations. Several other observations point to a role of Lck and related enzymes during apoptosis induction. In this regard, it has been shown that the addition of peptides containing the SH2 domains of Lck, Src or Abl interfered with apoptosis induction in a cell-free system (Farschon et al., 1997). Since it is known that the SH2 domain of Lck is required for interaction of Lck with tyrosine phosphorylated protein structures (Couture et al., 1996), the addition of the soluble Lck SH2 domain might act as a dominant negative regulating protein, blocking the interactions of Lck with yet unknown target structures that are involved in the regulation of apoptosis. JCaM1.6 cells are not totally Oncogene


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Figure 8 Influence of Lck or Bcl-2 on death-receptor-induced activation of the mitochondria. (a) Integrity of the mitochondrial membrane potential was determined every 12 up to 48 h by FACS analysis of TMRE-stained cells in Lck-deficient JCaM1.6, JCaM1.6/ Lck after exposure to 100 ng/ml activating CD95 antibody (clone CH11). (b) JCaM1.6 and JCaM1.6/Lck were treated with 100 ng/ml TRAIL for 48 h, and the integrity of the mitochondrial potential was determined accordingly. (c) Jurkat vector cells and Bcl-2 overexpressing cells were exposed to 100 ng/ml activating CD95 antibody (clone CH11) for up to 48 h. Integrity of the mitochondrial membrane potential was determined every 12 up to 48 h by FACS analysis of TMRE-stained cells. (d) Release of cytochrome c was determined by confocal laser scanning microscopy after immunostaining of cytochrome c. Data show one representative of three independent experiments (b and d) or means7s.d. (n ¼ 3) (a and c).

devoid of Lck expression, but were shown to express a kinase dead mutated Lck protein that retains the expression of the critical SH2 domain (Straus and Weiss, 1992). Thus, a similar dominant negative mechanism may be operative in the JCaM1.6 cells. In previous studies, it was shown that Lck phosphorylates the phosphatidylinositol 3 kinase (PI3K) SH2 domain. The phosphorylation of the PI3K SH2 domain induced dissociation from upstream signaling molecules, for example, the insulin receptor (von Willebrand et al., 1998). Furthermore, it was shown that Lck phosphorylates and binds to tyrosine-phosphorylated paxillin, thereby influencing the cytoskeleton (Ostergaard et al., 1998). Lck is also required for the activation of GTPases of the Rho family, which were shown to be involved in Oncogene

the control of apoptosis (Han et al., 1997; Jimenez et al., 1995). Another important function of Lck is the regulation of several ion transport systems. After activation of the TCR, Lck triggers the generation of inositoltriphosphate, which in turn mediates the release of calcium from endoplasmatic stores. Human T cells deficient for the inositoltriphosphate receptor-1, a functional downstream target of Lck, are highly resistant to ionizing radiation (Jayaraman and Marks, 1997). This suggests that the modulation of calcium signals might be involved in the initial apoptotic phase. In addition, other ion fluxes were shown to be regulated by Lck in response to ceramide and CD95 crosslinking apoptotic stimuli (Gulbins et al., 1997; Szabo et al., 1998).


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The absence of Lck interferes with both, radiation and ceramide-induced apoptosis (Belka et al., 1999; Manna et al., 2000). In parallel, both stimuli depend on the presence of Bax (Wei et al., 2001; von Haefen et al., 2002). Therefore, it is tempting to speculate whether Lck is required for the activation and conformational changes of Bax and subsequent cytochrome c release. In this regard, it has been shown that the agonistic Leu3a antibody, which induces apoptosis via the CD4 receptor complex, leads to a rapid activation of Lck followed by Bax transcription and activation of mitochondrial apoptosis (Tuosto et al., 2002). Taken together, we suppose that the influence of Lck on the cytoskeleton architecture, ion fluxes or activation of proapoptotic Bcl-2-like proteins might contribute to its role for the proper activation of mitochondrial apoptosis signaling pathways in response to ionizing radiation. Material and methods All biochemicals were from Sigma chemicals (Deisenhofen, Germany) unless otherwise specified. Hoechst 33342 was purchased from Calbiochem. The proton shuttle carbonylcyanide-m-chlorphenylhydrazone (CCCP) was purchased from Sigma and dissolved in DMSO. Phytohemagglutinin L was from Sigma (Deisenhofen, Germany). Cell culture Jurkat T-lymphoma cells were from ATCC (Bethesda, MD, USA). Cells were grown in RPMI 1640 medium (Gibco Life Technologies, Eggenstein, Germany) and maintained in a humidified incubator at 371C and 5% CO2. The Lck-deficient Jurkat clone (JCaM1.6) and the Lck cDNA-expressing JCaM1.6/Lck+ clone were a kind gift from A Weiss (University of California, San Francisco, USA). The Bcl-2-overexpressing Jurkat cells and the respective vector cells were used as already described (Belka et al., 2000b; Rudner et al., 2001a, b). Radiation Cells were irradiated with 6 MV photons from a linear accelerator (LINAC SL25 Phillips) with a dose rate of 4 Gy/min at room temperature.

flow cytometer (Becton Dickinson, Heidelberg, Germany). When indicated, apoptosis was also quantified after staining of the cells with Hoechst 33342 and subsequent fluorescence microscopy. In brief, cells were incubated with Hoechst 33342 at a final concentration of 1.5 mM for 15 min. Cell morphology was then determined by fluorescence microscopy (Zeiss Axiovert 135, Carl Zeiss, Jena, Germany) using an excitation wavelength filter of 380 nm. A total of 250 cells were counted and cells with intense chromatin clumping were considered as apoptotic. Immunoblotting Cells (1 105) were lysed in a lysis buffer containing 25 mm HEPES, 0.1% SDS, 0.5% deoxycholate, 1% Triton X-100, 10 mm EDTA, 10 mm NaF and 125 mm NaCI. After removing insoluble material by centrifugation for 10 min at 13 000 r.p.m., 20 mg lysate was separated by SDS–PAGE. Blotting was performed employing a tank blotting apparatus (Biorad, Munich, Germany) onto Hybond C membranes (Amersham, Braunschweig, Germany). Equal protein loading was confirmed by Ponceau S staining (Sigma). Blots were blocked in TBS buffer containing 0.05% Tween 20 and 5% fetal calf serum at room temperature for 15 min. Caspase-9, -3 and PARP were detected using cleavage specific antibodies from Cell Signalling (Frankfurt, Germany). Caspase-8 was detected using a mouse monoclonal antibody directed against the p18 as described previously (Belka et al., 2000b) and was obtained from BioCheck (Mu¨nster). Bcl-2 expression was tested using a Bcl-2 antibody (Santa-Cruz, Heidelberg, Germany). Lck expression was detected using an antibody against the C-terminus of Lck (BD Transduction-Labs, Heidelberg, Germany). Activation of Erk1/2 was determined using an activation specific antibody as described previously (Belka et al., 2000a). After repeated washings with TBS/Tween 20 (0.05%), the membrane was incubated with the secondary antibody (alkaline phosphatase coupled anti-IgG 1 : 20 000, Santa-Cruz-Biotech, Heidelberg, Germany) in TBS/ Tween for 3 h at room temperature and washed three times with TBS/Tween. The detection of antibody binding was performed by enhanced chemoluminescence. Determination of the mitochondrial membrane potential

CD95 stimulation was performed using the agonistic IgM antibody CH11 (100 ng/ml; UBI Biomol, Hamburg, Germany). TRAIL receptors were stimulated using recombinant human Flag-tagged TRAIL and a crosslinking antibody (Alexis Biochemicals, Gru¨nberg, Germany).

The mitochondrial transmembrane potential (DCm) was analyzed using the DCm specific stain TMRE (Molecular probes, Mobitech, Go¨ttingen Germany). In brief, at the indicated time points, 105 cells were stained in a solution containing 25 nm TMRE for 30 min. Staining was quantified by FL2 and scatter characteristics employing a FACS Calibur flow cytometer (Becton Dickinson, Heidelberg, Germany).

Determination of apoptosis

Cytochrome c release

Cell death was quantified by FACS using scatter characteristics and propidium iodide (PI) exclusion (staining with 0.1 mg/ml PI) employing a FACS Calibur

The release of cytochrome c from the mitochondria was determined by confocal microcopy using a Leica TCS NT microscope. In brief, cells were immobilized on

Stimulation of the CD95/TRAIL death receptors

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cover slips with 0.1% poly-l-lysine, fixed with 2.5% parafomnaldehyde and permeabilized with 0.1% Triton X-100. After blocking with 10% fetal calf serum, cells were stained with primary antibodies for 1 h at room temperature. Cells were washed several times and incubated with secondary antibodies for 30 min. Finally, the cover slips were mounted with Mowiol (Sigma). Cytochrome c staining was performed with a mouse anti-cytochrome c antibody (PharMingen, Hamburg, Germany). As secondary antibody, a Alexa Fluortconjugated anti-mouse (Molecular Probes, Mobitech, Go¨ttingen, Germany) was employed. CD95 and DR5 receptor expression Surface expression of the CD95 and the DR5 receptor on Jurkat cells was determined by flow cytometry after

immuno staining. In brief, cells were incubated with 100 ng/ml CH11 (anti-CD95) or 10 mg/ml of an DR5 receptor antibody (Alexis, Gru¨nberg Germany) for 30 min at 371C. Afterwards, the cell suspension was washed in PBS for three times and cell were incubated with the secondary antibody for 30 min on ice. FACS analysis was then performed after three additional washes.

Acknowledgments The authors gratefully acknowledge the technical assistance of H Faltin. This work was supported by a grant of the Federal Ministry of Education and Research (Fo¨. 01KS9602) and the Interdisciplinary Center of Clinical research (IZKF) Tu¨bingen to CB and by the Deutsche Krebshilfe/Mildred Scheel Stiftung (10-1825-Be2) to CB and VJ.

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