Oncogene (2003) 22, 176–185 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc

The is involved in regulation of mitochondrial pathways

Claus Belka*,1,3, Charlotte Gruber1,3, Verena Jendrossek1, Sebastian Wesselborg2 and Wilfried Budach1

1Department 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 of the caspase cascade at the receptor level, DNA highly coordinated signaling network ultimately leading to damage and stress-mediated caspase activation and the activation of caspases. In previous experiments we and apoptosis are triggered mainly by mitochondrial alter- others have shown that the Lck is required ations (Belka et al., 2000b, 2001; Newton and Strasser, for adequate apoptosis induction in response to ionizing 2000). radiation, ceramide incubation and overexpression of the Mitochondrial apoptosis is basically regulated by a HIV-TAT . However, the position of Lck within complex interplay of pro- and antiapoptotic Bcl-2-like given apoptotic signaling cascades remains unclear. We . DNA damage or cellular stress triggers the therefore aimed to define the role of Lck during radiation- transcriptional activation of the proapoptotic Bax-like induced apoptosis. Apoptosis induction in response to molecules Bax, Bak and the BH3-only proteins Noxa and ionizing radiation, CD95 or TRAIL receptor stimulation Puma. Although the interplay of Bax-like molecules and was determined in Jurkat T-cells, the Lck-deficient BH3-only proteins is not completely understood, it has Jurkat clone JCaM1.6- and Lck-retransfected been shown that an interaction of molecules belonging to JCaM1.6/Lck. No apoptosis, release of cytochrome c, either group is crucial for the subsequent release of breakdown of the mitochondrial potential were detectable cytochrome c (Cheng et al., 2001). The release of during the first 48 h after irradiation of JCaM1.6 cells. In cytochrome c together with dATP and APAF-1 in turn parallel, no activation of caspase-9, -8 and -3 was triggers the autoproteolytic activation of caspases (re- detectable. Since mitochondrial apoptosis pathways act viewed in: Belka and Budach, 2002; Rudner et al., 2002). within a feedback mechanism during death-receptor- In this regard, the role of certain caspases was also mediated apoptosis, the influence of the Lck defect on found to be different. While caspase-8, which is directly CD95/Fas/Apo-1-L or TRAIL-induced apoptosis was activated at the receptor complex, was shown to be the also tested. Both stimuli induced apoptosis in Lck- key caspase for death-receptor-mediated apoptosis deficient cells. However, the kinetics of apoptosis induc- (Varfolomeev et al., 1998), caspase-9 is crucial for tion determined by caspase-8, -9 and -3 activation as well DNA-damage-related apoptosis and is activated follow- as DWm breakdown was slowed. We conclude that the Lck ing cytochrome c release from the mitochondrial deficiency influences early steps during radiation-induced intermembrane space (Hakem et al., 1998; Kuida et al., mitochondrial alterations. 1998; Soengas et al., 1999). In this context, we have shown that the apoptotic process triggered by ionizing Oncogene (2003) 22, 176–185. doi:10.1038/sj.onc.1206103 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 Introduction in response to death receptor ligation or DNA damage or stress (Belka et al., 2000b; Engels et al., 2000; The apoptotic process is regulated by a wide array of Dunkern et al., 2001; Stepczynska et al., 2001; Tomicic diverse molecules. Depending on the stimulus applied et al., 2002; Wieder et al., 2001). two apoptotic cascades can be distinguished: whereas In addition to the essential role of caspases, it was death receptor stimulation acts via a direct activation 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 *Correspondence: C Belka, Department of Radiation Oncology, Aggarwal, 2000)and ceramide (Manna et al., 2000). In University of Tu¨ bingen, Hoppe Seyler Str. 3, Tu¨ bingen D-72076, this regard, it is important that the release of ceramides Germany; from cellular membranes is considered to be a key step E-mail: [email protected], http:www.radiation-research.de during radiation-induced apoptosis in some (Santana 3These two authors share first authorship Received 8 July 2002; revised 30 September 2002; accepted 4 et al., 1996; Pena et al., 2000)but not all models October 2002 (Burek et al., 2001). Lck and mitochondrial apaptosis pathways C Belka et al 177 Lck belongs to the src-like tyrosine kinase family and apoptotic signaling. To this end, we analyzed the key is normally required for the adequate propagation of T- steps in apoptosis signaling cascade in Lck-deficient cell receptor signals. After stimulation of the T-cell JCaM1.6 and Lck expressing JCaM1.6 cells upon death receptor complex by the respective in combina- receptor ligation and irradiation. tion with an MHC molecule, Lck becomes activated and is involved in the of critical residues of the TCR complex. These phophorylations are essential Results for downstream signaling events including influx, activation of the factor NFAT Apoptosis induction, Lck function and expression and the mitogenic Erk1 and Erk2 (Straus and Weiss, 1992; Kabouridis et al., 1997; Samelson, 2002). In a first set of experiments apoptosis induction after Using normal Jurkat Tcells, the Lck-deficient Jurkat irradiation of JCaM1.6 and JCaM1.6/Lck cells with subclone JCaM1.6 and Lck-transfected JCaM1.6/Lck 10 Gy was followed over 72 h. As shown in Figure 1a, cells, we could show previously that Lck is also the onset of apoptosis was abrogated in cells lacking Lck important for the regulation of apoptosis in response with no apoptosis being detectable prior to 72 h to ionizing radiation (Belka et al., 1999). JCaM1.6 and (23.9711% in Lck-negative cells vs 71.7712.4%). At JCaM1.6/Lck were initially employed to prove that Lck 96 h apoptotic changes also increased in Lck-deficient is involved in T-cell receptor signal transduction (Straus cells (41.2722.5%). However, a strong difference and Weiss, 1992). This finding is paralleled by similar between the Lck-deficient and the Lck-re-expressing results from B-cells, where the B-cell receptor associated cells (75.3711.6%)remained detectable. src-like kinase BTK is essential for radiation-induced In parallel experiments, the expression levels of Lck apoptosis (Uckun et al., 1996). In this context, the and the function of Lck-mediated signaling pathways kinase domain of BTK was crucial for the apoptotic were tested in Jurkat, JCaM1.6 and JCaM1.6/Lck. response to ionizing radiation. In addition to the Using an antibody against the C-terminus of Lck, the findings on BTK, it was shown that activation of Lck expression of Lck was absent in JCaM1.6 cells, while by CD19 crosslinking promoted radiation-induced comparable levels of Lck were detectable in Jurkat and apoptosis in a radiation-resistant B-lymphoma line JCaM1.6/Lck cells (Figure 1b). The functional integrity (Waddick et al., 1993). Similarly, the src-like kinase of Lck-mediated signaling pathways in the JCaM1.6/ Lyn was suggested to be involved in apoptosis regula- Lck cells was verified by analyzing the capability of the tion in response to UV light in DT40 B-cells (Qin et al., respective cell line to respond to CD3/T-cell receptor 1997). (TCR)stimulation with an activation of Erk1/2 kinases. Since Lck has been shown to be involved in apoptosis The CD3/TCR complex was stimulated using phytohe- induction in response to ceramide, radiation and HIV- magglutinin-L (PHA-L, 50 mg/ml)and activation of TAT protein, we aimed to further define its role for 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), 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 Lck and mitochondrial apaptosis pathways C Belka et al 178 directed against the active kinase (Figure 1c). After determined. As shown in Figure 2, cleavage of PARP by stimulation of normal Jurkat cells with PHA-L, Erk1/2 caspase-3 was not detectable in JCaM1.6 cells, whereas kinase was rapidly activated as indicated by the it clearly occurred in the Lck-expressing JCaM1.6 cells. detection of phosphorylated Erk1/2. No such activation Although caspase-8 is an activator caspase for was detectable in JCaM1.6 cells, whereas treatment of receptor-mediated apoptosis, recent work indicated that JCaM1.6/Lck cell with PHA-L clearly induced Erk1/2 it acts as executor caspase during radiation or drug- phosphorylation. Taken together, the data prove that induced apoptosis (Belka et al., 2000b; Engels et al., intact Lck is absent in JCaM1.6 cells that do not 2000; Wieder et al., 2001). In accordance with our undergo apoptosis in response to irradiation, whereas previous results no activation of caspase-8 was detect- normal Jurkat cells and retransfected JCaM1.6/Lck able in Lck-deficient cells (Figure 2). express functional active Lck. 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 Radiation-induced caspase activation course of caspase activation in our experiments. Caspase- Caspases are key regulators for the induction and 9 activation preceded activation of caspase-3 and -8. execution of apoptosis. Caspase-9 is considered to be Up to this point, the data show that none of the key the most apical caspase involved in DNA-damage- initiator and executor caspases are activated in response induced apoptosis. Procaspase-9 is activated after to ionizing radiation in Lck-deficient cells. binding to a complex composed of dATP, APAF-1 and cytochrome c that is released from the mitochon- Activation of mitochondrial apoptosis steps in response drium (Li et al., 1997). Thus, it is the initiator caspase to radiation for the execution of the mitochondrial apoptosis path- ways. Since the release of cytochrome c and the breakdown of In order to determine in how far the absence of Lck the mitochondrial membrane potential (DCm)is con- influences the activation of caspase-9, the time course of sidered to be the crucial step for the activation of the caspase-9 processing was determined using antibodies mitochondrial death pathway (Li et al., 1997; Soengas against the active sub-unit (Figure 2). As shown in et al., 1999; Susin et al., 1999), the role of Lck for the Figure 2, activation of caspase-9 was absent in Lck- release of cytochrome c and breakdown of the deficient cells whereas in Lck-expressing cells caspase-9 mitochondrial membrane potential was tested. As activation was detectable as early as 6 h after irradiation. shown in Figure 3a, the staining pattern of cytochrome Caspase-3 is activated by caspase-9 and functions as c was found to be focal in JCaM1.6 cells after key executor for the apoptotic process. Thus, caspase-3 irradiation, whereas in JCaM1.6/Lck cells a diffuse is responsible for many aspects of the apoptotic staining of cytochrome c occurred, which was indicative phenotype. Consistent with the finding of caspase-9 for the release of cytochrome c from the mitochondrial processing, no activation of caspase-3 was detectable in intermembrane space into the cytosol. Conversely, Lck-deficient cells. In order to further substantiate this almost no breakdown of the DCm was detectable in finding, cleavage of the caspase-3 substrate PARP was 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 Figure 2 Radiation-induced caspase activation in JCaM1.6 cells. Radiation-induced cleavage of caspase-9, -3 and -8 as well as expression level of either molecule was detectable in cleavage of the caspase-3 substrate PARP is absent in Lck-deficient Lck-deficient cells (data not shown). 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/ Influence of Lck on death receptor signaling Lck cells (right panel)10–72 h after irradiation with 10 Gy ionizing radiation. Data show one representative of three independent The strong effect of the Lck deficiency on DNA- experiments. damage-mediated apoptosis prompted us to test the

Oncogene Lck and mitochondrial apaptosis pathways C Belka et al 179

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 means 7s.d. (n ¼ 3)( b).

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-receptor- mediated apoptosis. Therefore, we analyzed the activa- tion 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 Lck- deficient and Bcl-2-overexpressing cells. Similar data were revealed when PARP cleavage was analyzed. Figure 4 Influence of Bcl-2 on radiation-induced apoptosis and caspase activation. Radiation-induced apoptosis and activation of To further substantiate these findings, the release of caspases is abrogated in Bcl-2 overexpressing cells. Jurkat vector cells cytochrome c and breakdown of the mitochondrial and Bcl-2 overexpressing cells were exposed to 10 Gy ionizing membrane potential were determined. radiation. (a)Apoptosis induction was determined every 12 up to In accordance with the apoptosis data presented 72 h by flow cytometry. (b)Processing of caspase-3 and cleavage of PARP were analyzed by Western blotting. Data show means7s.d. above, the breakdown of DCm upon CD95 death (n ¼ 3)( a)or one representative of three independent experiments ( b). 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 influence of Lck on other apoptosis-inducing stimuli. breakdown of DCm was slowed but not abrogated Activation of the CD95 or DR4/DR5 TRAIL receptors (Figure 8b). Cytochrome c release indicated by is known to rapidly induce apoptosis. Thus, JCaM1.6, diffuse staining of the was detectable regard- JCaM1.6/Lck, Jurkat and Bcl-2 overexpressing Jurkat less of the Lck deficiency or Bcl-2 overexpression cells were treated with 100 ng/ml of an agonistic CD95 (Figure 8d). antibody or 100 ng/ml TRAIL (Figures 5a, b and 6). Taken together, Lck-deficient cells clearly undergo In contrast to the observed apoptosis resistance upon apoptosis in response to death receptor stimulation, irradiation, no apoptosis resistance towards TRAIL and while being resistant to radiation-induced apoptosis.

Oncogene Lck and mitochondrial apaptosis pathways C Belka et al 180

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 CD95- receptor 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 Lck and mitochondrial apaptosis pathways C Belka et al 181 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 recep- tors, which is mainly executed independently of mito- chondrial 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 over- Figure 6 Influence of Bcl-2 overexpression on death-receptor- lapping signaling cascades for DNA damage and induced apoptosis. Bcl-2 overexpression does not abrogate CD95- receptor-mediated death (Belka et al., 2000b; Engels induced apoptosis. Jurkat vector and Bcl-2-overexpressing Jurkat et al., 2000; Newton and Strasser, 2000), it has been cells were treated with 100 ng/ml activating CD95 antibody (clone suggested that DNA-damage-induced apoptosis may be CH11). Induction of apoptotic cell death was then determined by FACS analysis at the indicated time points. Data show means7s.d. dependent on activation of death receptors (Belka et al., (n ¼ 3). 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 death- receptor-induced apoptosis with mitochondrial path- ways (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 Lck- deficient cells could therefore be explained by inade- quate 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 Figure 7 Influence of Lck and Bcl-2 on death-receptor-induced activation of caspase-8 and PARP. CD95-induced activation of phosphorylation by Lck is involved in CD95-induced caspases is slowed in Lck-deficient and Bcl-2-overexpressing Jurkat apoptosis. The evident differences may be simply cells. JCaM1.6, JCaM1.6/Lck (upper panel), Jurkat vector cells and explained by the altered time course of death-receptor- Bcl-2-overexpressing cells (lower panel)were exposed to 100 ng/ml induced apoptosis in Lck-deficient cells. Whereas activating CD95 antibody (clone CH11)for up to 12 h. Processing of procaspase-8 and cleavage of PARP were determined by Schlottmann determined apoptosis after 4 h after Western blot analysis. Data show one representative of three stimulation, Schraven determined apoptosis after 18 h. independent experiments (b). Thus, both results can easily be integrated into our observations. Several other observations point to a role of Lck and Discussion related during apoptosis induction. In this regard, it has been shown that the addition of peptides In conclusion, our data indicate that the presence of the containing the SH2 domains of Lck, Src or Abl tyrosine kinase Lck is required for the adequate interfered with apoptosis induction in a cell-free system execution of early steps of the mitochondrial apoptosis (Farschon et al., 1997). Since it is known that the SH2 pathways with strong effects on radiation-induced domain of Lck is required for interaction of Lck with apoptosis and only very limited influences on CD95- tyrosine phosphorylated protein structures (Couture and TRAIL-induced apoptosis. This interpretation is et al., 1996), the addition of the soluble Lck SH2 based on the findings that neither a breakdown of the domain might act as a dominant negative regulating mitochondrial membrane potential nor release of protein, blocking the interactions of Lck with yet cytochrome c occurred after irradiation of Lck-deficient unknown target structures that are involved in the cells. regulation of apoptosis. JCaM1.6 cells are not totally

Oncogene Lck and mitochondrial apaptosis pathways C Belka et al 182

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 means 7s.d. (n ¼ 3)( a and c).

devoid of Lck expression, but were shown to express a the control of apoptosis (Han et al., 1997; Jimenez et al., kinase dead mutated Lck protein that retains the 1995). expression of the critical SH2 domain (Straus and Another important function of Lck is the regulation Weiss, 1992). Thus, a similar dominant negative of several ion transport systems. After activation of the mechanism may be operative in the JCaM1.6 cells. TCR, Lck triggers the generation of inositoltripho- In previous studies, it was shown that Lck phosphor- sphate, which in turn mediates the release of calcium ylates the phosphatidylinositol 3 kinase (PI3K)SH2 from endoplasmatic stores. Human T cells deficient for domain. The phosphorylation of the PI3K SH2 domain the inositoltriphosphate receptor-1, a functional down- induced dissociation from upstream signaling molecules, stream target of Lck, are highly resistant to ionizing for example, the (von Willebrand et al., radiation (Jayaraman and Marks, 1997). This suggests 1998). Furthermore, it was shown that Lck phosphor- that the modulation of calcium signals might be ylates and binds to tyrosine-phosphorylated paxillin, involved in the initial apoptotic phase. In addition, thereby influencing the cytoskeleton (Ostergaard et al., other ion fluxes were shown to be regulated by Lck in 1998). Lck is also required for the activation of GTPases response to ceramide and CD95 crosslinking apoptotic of the Rho family, which were shown to be involved in stimuli (Gulbins et al., 1997; Szabo et al., 1998).

Oncogene Lck and mitochondrial apaptosis pathways C Belka et al 183 The absence of Lck interferes with both, radiation flow cytometer (Becton Dickinson, Heidelberg, Ger- and ceramide-induced apoptosis (Belka et al., 1999; many). When indicated, apoptosis was also quantified Manna et al., 2000). In parallel, both stimuli depend on after staining of the cells with Hoechst 33342 and the presence of Bax (Wei et al., 2001; von Haefen et al., subsequent fluorescence microscopy. In brief, cells were 2002). Therefore, it is tempting to speculate whether Lck incubated with Hoechst 33342 at a final concentration of is required for the activation and conformational 1.5 mM for 15 min. Cell morphology was then deter- changes of Bax and subsequent cytochrome c release. mined by fluorescence microscopy (Zeiss Axiovert 135, In this regard, it has been shown that the agonistic Carl Zeiss, Jena, Germany)using an excitation wave- Leu3a antibody, which induces apoptosis via the CD4 length filter of 380 nm. A total of 250 cells were counted receptor complex, leads to a rapid activation of Lck and cells with intense chromatin clumping were con- followed by Bax transcription and activation of sidered as apoptotic. mitochondrial apoptosis (Tuosto et al., 2002). Taken together, we suppose that the influence of Lck Immunoblotting on the cytoskeleton architecture, ion fluxes or activation 5 of proapoptotic Bcl-2-like proteins might contribute to Cells (1 Â 10 )were lysed in a lysis buffer containing m its role for the proper activation of mitochondrial 25 m HEPES, 0.1% SDS, 0.5% deoxycholate, 1% m m m apoptosis signaling pathways in response to ionizing Triton X-100, 10 m EDTA, 10 m NaF and 125 m radiation. NaCI. After removing insoluble material by centrifuga- tion for 10 min at 13 000 r.p.m., 20 mg lysate was separated by SDS–PAGE. Blotting was performed Material and methods employing a tank blotting apparatus (Biorad, Munich, Germany)onto Hybond C membranes (Amersham, All biochemicals were from Sigma chemicals (Deisen- Braunschweig, Germany). Equal protein loading was hofen, Germany)unless otherwise specified. Hoechst confirmed by Ponceau S staining (Sigma). Blots were 33342 was purchased from Calbiochem. The proton blocked in TBS buffer containing 0.05% Tween 20 and shuttle carbonylcyanide-m-chlorphenylhydrazone (CCCP) 5% fetal calf serum at room temperature for 15 min. was purchased from Sigma and dissolved in DMSO. Caspase-9, -3 and PARP were detected using cleavage Phytohemagglutinin L was from Sigma (Deisenhofen, specific antibodies from Cell Signalling (Frankfurt, Germany). 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 Jurkat T-lymphoma cells were from ATCC (Bethesda, was tested using a Bcl-2 antibody (Santa-Cruz, Heidel- MD, USA). Cells were grown in RPMI 1640 medium berg, Germany). Lck expression was detected using an (Gibco Life Technologies, Eggenstein, Germany)and antibody against the C-terminus of Lck (BD Transduc- maintained in a humidified incubator at 371C and 5% tion-Labs, Heidelberg, Germany). Activation of Erk1/2 CO2. The Lck-deficient Jurkat clone (JCaM1.6)and the was determined using an activation specific antibody as Lck cDNA-expressing JCaM1.6/Lck+ clone were a kind described previously (Belka et al., 2000a). gift from A Weiss (University of California, San After repeated washings with TBS/Tween 20 (0.05%), Francisco, USA). The Bcl-2-overexpressing Jurkat cells the membrane was incubated with the secondary anti- and the respective vector cells were used as already body (alkaline phosphatase coupled anti-IgG 1 : 20 000, described (Belka et al., 2000b; Rudner et al., 2001a, b). Santa-Cruz-Biotech, Heidelberg, Germany)in TBS/ Tween for 3 h at room temperature and washed three Radiation times with TBS/Tween. The detection of antibody binding was performed by enhanced chemoluminescence. Cells were irradiated with 6 MV photons from a linear accelerator (LINAC SL25 Phillips)with a dose rate of Determination of the mitochondrial membrane potential 4 Gy/min at room temperature. The mitochondrial transmembrane potential (DCm)was Stimulation of the CD95/TRAIL death receptors analyzed using the DCm specific stain TMRE (Mole- cular probes, Mobitech, Go¨ ttingen Germany). In brief, CD95 stimulation was performed using the agonistic IgM at the indicated time points, 105 cells were stained in a antibody CH11 (100 ng/ml; UBI Biomol, Hamburg, solution containing 25 nm TMRE for 30 min. Staining Germany). TRAIL receptors were stimulated using re- was quantified by FL2 and scatter characteristics combinant human Flag-tagged TRAIL and a crosslinking employing a FACS Calibur flow cytometer (Becton antibody (Alexis Biochemicals, Gru¨ nberg, Germany). Dickinson, Heidelberg, Germany).

Determination of apoptosis Cytochrome c release Cell death was quantified by FACS using scatter The release of cytochrome c from the mitochondria was characteristics and propidium iodide (PI)exclusion determined by confocal microcopy using a Leica TCS (staining with 0.1 mg/ml PI)employing a FACS Calibur NT microscope. In brief, cells were immobilized on

Oncogene Lck and mitochondrial apaptosis pathways C Belka et al 184 cover slips with 0.1% poly-l-lysine, fixed with 2.5% immuno staining. In brief, cells were incubated with parafomnaldehyde and permeabilized with 0.1% Triton 100 ng/ml CH11 (anti-CD95)or 10 mg/ml of an DR5 X-100. After blocking with 10% fetal calf serum, cells receptor antibody (Alexis, Gru¨ nberg Germany)for were stained with primary antibodies for 1 h at room 30 min at 371C. Afterwards, the cell suspension was temperature. Cells were washed several times and washed in PBS for three times and cell were incubated incubated with secondary antibodies for 30 min. Finally, with the secondary antibody for 30 min on ice. FACS the cover slips were mounted with Mowiol (Sigma). analysis was then performed after three additional Cytochrome c staining was performed with a mouse washes. anti-cytochrome c antibody (PharMingen, Hamburg, Germany). As secondary antibody, a Alexa Fluort- conjugated anti-mouse (Molecular Probes, Mobitech, Acknowledgments Go¨ ttingen, Germany)was employed. The authors gratefully acknowledge the technical assistance of H Faltin. This work was supported by a grant of the Federal CD95 and DR5 receptor expression Ministry of Education and Research (Fo¨ . 01KS9602)and the Interdisciplinary Center of Clinical research (IZKF)Tu¨ bingen Surface expression of the CD95 and the DR5 receptor to CB and by the Deutsche Krebshilfe/Mildred Scheel Stiftung on Jurkat cells was determined by flow cytometry after (10-1825-Be2)to CB and VJ.

References

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