Protein tyrosine kinase p56lck-deficiency confers hypersusceptibility to q-fluorophenylalanine (pFPhe)-induced apoptosis by augmenting mitochondrial apoptotic pathway in human Jurkat T cells

Phenylalanine analog, q-fluorophenylalanine (pFPhe)-mediated cytotoxicity and several apoptotic events including mitochondrial cytochrome c release, activation of caspase-9, -3, and -8, Bid cleavage, degradation of PARP and PLCc-1, and DNA fragmentation were more significant in p56lck-deficient Jurkat T cells (JCaM1.6) than in wild-type Jurkat T cells (E6.1). The susceptibility of JCaM1.6 toward apoptogenic activity of pFPhe decreased after acquisition of p56lck by transfection. The p56lck kinase activity increased 1.6-fold at 15–30 min after pFPhe treatment. The pan-caspase inhibitor (z-VAD-fmk) completely blocked the pFPhe-mediated apoptotic changes except caspase-9 activation. The caspase-8 inhibitor (z-IETD-fmk), which failed to influ- ence pFPhe-induced caspase-9 activation, completely blocked caspase-8 activation and PLCc-1 degradation with a marked reduction in caspase-3 activation and PARP degradation, indicating pFPhe-induced caspase-8 activation as a downstream event of mitochondria-dependent activation of caspase-9. These results indi- cate that the deficiency of p56lck augments pFPhe-induced mitochondrial cytochrome c release and resultant apoptotic cell death in Jurkat T cells.

A protein tyrosine kinase (PTK) p56lck is a typical non-recep- tor TPK of src-family and is expressed almost exclusively in T cells [1]. The p56lck interacts with cytoplasmic regions of CD4 and CD8 coreceptor molecules, and thus plays an important role in relay- ing TCR-mediated activation signal. In addition, p56lck is known to relay signals from b chain of the IL-2 receptor, indicating critical roles of p56lck for T-cell activation and proliferation. The impor- tance of p56lck for T-cell propagation is initially indicated by vir- tue of its overexpression, resulting from retroviral insertion into the lck locus, in two Molony murine leukemia virus-induced lym- phoid tumors [2]. Besides the typical roles of p56lck directly asso- ciated with T-cell propagation, it has also been shown that p56lck is required for FasL expression during activation-induced T cell apoptosis [3]. Although these previous studies have indicated that p56lck is associated with activation-induced T-cell apoptosis mainly via upregulating FasL expression, several recent evidences have indicated a direct requirement of p56lck for certain types of apop- tosis induced by ionizing radiation, ceramide or rosmarinic acid, through modulating mitochondria-dependent apoptotic signaling pathway [4–6]. On the other hand, p56lck-deficient murine helper T cell clone resulting from p56lck-specific antisense RNA expres- sion was hypersusceptible to apoptosis when activated through TCR [7]. Although the previous data have suggested that the role of p56lck involved in T-cell apoptosis might be distinctive depend- ing on initial triggers provoking apoptosis, the precise mechanism remains obscure.

Several studies have reported that amino acid analogs possess growth inhibitory activity toward tumor cells in culture and exper- imental tumors in vivo, and are able to enhance chemo- or radio- sensitivity of tumors [8,9]. Induction of apoptosis has also been implicated in the inhibitory activity of amino acid analogs on the growth of tumor cells [10,11]. These previous results have proposed the possible application of amino acid analogs to pharmacological treatment of malignant conditions including cancers. A primary mechanism underlying these antitumor activities of amino acid analogs, leading to cell damage, is thought to be their incorpora- tion into cellular proteins in substitution for the intact forms and induction of structurally aberrant proteins with impaired function or degradation [12,13], which can be more significant in tumor cells than in normal cells due to the difference of their mitotic rates. However, the involvement of apoptotic cell death in the inhibitory activity of amino acid analogs against tumor cells, which is req- uisite for evaluating their potency as a chemotherapeutic agent, remains largely unknown.

Recently we have found that q-fluorophenylalanine (pFPhe), the phenylalanine analog, which possesses enough structural similarity to compete with phenylalanine for phenylalanyl-tRNA activation and for its incorporation into cellular proteins, can exert cytotoxicity through apoptotic cell death in human acute leukemia Jurkat T cells. In addition, we have found that the lack of p56lck in Jurkat T cells potentiate the pFPhe-induced apoptosis, suggesting an inhibitory effect of p56lck on pFPhe-induced apoptosis. In order to understand further not only the mechanism by which pFPhe induces apoptosis, but also the component of pFPhe-mediated apoptotic signaling pathway, which is augmented in the absence of p56lck, in the present study, we have investigated pFPhe-induced apoptotic signaling pathway and its modulation by p56lck using wild-type Jurkat T cells (clone E6.1), p56lck-deficient Jurkat T cells (clone JCaM1.6) and p56lck-transfected JCaM1.6 (clone JCaM1.6/lck). The results demonstrate that pFPhe induces apoptosis in Jurkat T cells via mitochondria-dependent death signaling pathway includ- ing mitochondrial cytochrome c release and resultant activation of caspase-9 and -3, which is negatively regulated by p56lck, suggest- ing that a novel suppressive effect of p56lck on apoptosis mediated by pFPhe in human acute leukemia Jurkat T cells.

Materials and methods

Reagents, antibodies, and cells. q-Fluorophenylalanine (pFPhe) was purchased from Sigma Chemical (St. Louis, MO). ECL Western blotting kit was from Amersham (Arlington Heights, IL). Anti-cyto- chrome c, anti-Fas and anti-FasL were purchased from Pharmingen (San Diego, CA). Anti-caspase-3, anti- poly (ADP-ribose) polymer- ase (PARP), anti-Bid, anti-PLCc-1, anti-p56lck, and anti-b-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti- caspase-8, anti-caspase-9, anti-caspase-7, and anti-Bid were from Cell Signaling Technology (Beverly, MA). A broad-range caspase inhibitor z-VAD-fmk and caspase-8 inhibitor z-IETD-fmk were obtained from Calbiochem (San Diego, CA). Annexin V-FITC apoptosis kit was from Clontech (Takara Bio Inc., Shiga, Japan). Human acute leukemia Jurkat T cell line E6.1, p56lck-deficient Jurkat T cell clone JCaM1.6, Jurkat T cell clone A3, and FADD-deficient Jurkat T cell clone I2.1 were purchased from ATCC (Manassas, VA). Stable transfectants of Jurkat JCaM1.6 (JCaM1.6/vector and JCaM1.6/lck) were supplied from Dr. Arthur Weiss (University of California, San Francisco, CA). Jurkat T cells were maintained in RPMI 1640 (Life Technologies, Gaithersburg, MD) containing 10% FBS, 20 mM HEPES (pH 7.0), 5 10¡5M b-mercaptoethanol, and 100 lg/ml gen- tamycin.

Cytotoxicity assay. The cytotoxic effect of pFPhe on Jurkat T cells was analyzed by MTT assay reflecting the cell viability. Briefly, Jurkat T cells (5 104) were added to the serial dilution of pFPhe in 96-well plates. At 20 h after incubation, 50 ll of MTT solution (1.1 mg/ml) was added to each well and incubated for an additional 4 h. After centrifugation, the supernatant was removed from each well and then 150 ll of DMSO was added to dissolve the colored formazan crystal produced from MTT. OD values of the solutions were measured at 540 nm by a plate reader.

DNA fragmentation analysis. Apoptotic DNA fragmentation induced in Jurkat T cells following pFPhe treatment was deter- mined by Triton X-100 lysis methods using 1.2% agarose gel elec- trophoresis as previously described [14]. Flow cytometric analysis. Cell cycle progression of Jurkat T cells following pFPhe treatment was analyzed by Flow cytometry as described elsewhere [15]. The extent of necrosis was detected with Annexin V-FITC apoptosis kit. The cells (1 106) were washed with 1X binding buffer and then incubated with Annexin V-FITC and PI for 15 min before being analyzed by flow cytometry according to the manufacturer’s instructions.

Preparation of cell lysate and Western blot analysis. Cellular lysates were prepared by suspending 5 £ 106 Jurkat T cells in 200 ll of lysis buffer (137 mM NaCl, 15 mM EGTA, 1 mM sodium ortho- vanadate, 15 mM MgCl2, 0.1% Triton X-100, 25 mM MOPS, 2.5 lg/ml proteinase inhibitor E-64, pH 7.2). The cells were disrupted by son- ication and extracted at 4 °C for 30 min. An equivalent amount of protein lysate (20 lg) was electrophoresed on 4–12% SDS gradient polyacrylamide gel with MOPS buffer and then electrotransferred to Immobilon-P membranes. Detection of each protein was per- formed using an ECL Western blotting kit according to the manu- facturer’s instructions.

Detection of mitochondrial cytochrome c in cytosolic protein extracts. To assess mitochondrial cytochrome c release in Jurkat T cells following pFPhe treatment, cytosolic protein extracts were obtained as described elsewhere [14]. The cytosolic extracts free of mitochondria were analyzed for cytochrome c by Western blotting.

Immunoprecipitation and kinase assay. For immunoprecipitation of p56lck, 70 lg of cellular lysate at 1 lg/ll were allowed to react with 2 lg of rabbit polyclonal anti-p56lck antibody at 4 °C for 2 h. The immune complexes were recovered by addition of 30 ll of pro- tein G-agarose. The a-casein kinase activity of immunoprecipitated p56lck was assayed as previously described [15]. Phosphorylation of a-casein was measured by incubating the protein G-agarose beads with 30 ll of kinase assay cocktail (15 lg of a-casein, 10 lCi [c-32P]ATP, 100 lM ATP, 1X kinase assay buffer) for 30 min at 30 °C. The reaction mixture was electrophoresed on an 11% SDS–poly-acrylamide gel and then electrotransferred to an Immobilon-P membrane. The membrane was blot-dried, and phosphorylation of the casein was detected by autoradiography and quantitated by a phosphoimage analyzer. For Western analysis of immuno- precipitated p56lck, the membrane was probed with monoclonal anti-p56lck antibody and detection was performed using an ECL Western blotting kit.

Results and discussion

Apoptogenic effect of q-fluorophenylalanine (pFPhe) on wild-type Jurkat T cells (clone E6.1) and p56lck-deficient Jurkat T cells (clone JCaM1.6)

To understand apoptogenic effect of q-fluorophenylalanine (pFPhe) as well as its modulation by a src-family protein tyrosine kinase (PTK) p56lck, the cytotoxic effect of pFPhe on p56lck-positive Jurkat T cells (clone E6.1) and p56lck-deficient Jurkat T cells (clone JCaM1.6) was compared by MTT assay. When the cells were treated with pFPhe at various concentrations of 0.63–2.5 mM for 20 h, the decline in the cell viability, which occurred in a dose-dependent manner, appeared to be more significant in JCaM1.6 than in E6.1 (Fig. 1A). Under the same conditions, the apoptotic DNA fragmen- tation as well as the ratio of sub-G1 phase representing apopto- tic cells was more apparent in JCaM1.6 than in E6.1 (Fig. 1B and C). These results indicate that the higher susceptibility of p56lck- deficient JCaM1.6, as compared with p56lck-positive E6.1, toward the cytotoxicity of pFPhe is attributable to induced apoptotic cell death that can be enhanced in the absence of p56lck.

pFPhe-induced apoptotic signaling pathway in E6.1 and JCaM1.6

To examine the modulation mechanism of pFPhe-mediated apoptosis by p56lck in Jurkat T cells, we compared pFPhe-induced apoptotic signaling pathway between p56lck-positive E6.1 and p56lck-negative JCaM1.6. Since mitochondrial cytochrome c release plays an important role in the commitment of apoptosis provoked by many physiological and nonphysiological signals, and since the released cytochrome c together with apoptotic protease activat- ing factor-1 (Apaf-1) activates caspase-9 in the presence of dATP, and the latter then activates caspase-3 [16,17], we first examined, by Western blot analysis, that whether pFPhe-mediated apoptosis accompanied mitochondrial release of cytochrome c and this phe- nomenon occurred more significantly in JCaM1.6 than in E6.1. As shown in Fig. 2A, the level of the cytosolic cytochrome c increased in the presence of pFPhe (0.63–2.5 mM) in a dose-dependent man- ner, and the mitochondrial cytochrome c release was more appar- ent in JCaM1.6 than in E6.1. In addition, pFPhe-induced activation of caspase-9 and -3, and resultant degradation of PARP into two fragments appeared to be more dominant in JCaM1.6 than in E6.1 (Fig. 2B). The caspase-8 activation via the proteolytic degrada- tion of a 57-kDa proenzyme into 41-/43-kDa activated form was detected in both E6.1 and JCaM1.6 following treatment with pFPhe, and the level of 26-kDa Bid protein, which was previously cleaved by caspase-8 to produce the truncated Bid (tBid) causing the mito- chondrial cytochrome c release [18,19], appeared to decline in accordance with pFPhe-induced caspase-8 activation. Again, the pFPhe-induced caspase-8 activation as well as Bid cleavage was more significant in JCaM1.6 than in E6.1. As a potential mecha- nism underlying the apoptosis induced by antineoplastic drugs, upregulation of Fas ligand (FasL) and/or Fas expression has been implicated [20]. There was, however, no change in the level of FasL and Fas in both E6.1 and JCaM1.6 following exposure to 0.63–2.5 mM pFPhe, suggesting that Fas-death signaling might not be associated with pFPhe-induced apoptosis. To examine further an involvement of Fas/FasL system in pFPhe-mediated apoptosis, we compared cytotoxic effect of pFPhe on FADD-positive Jurkat T cells (clone A3) with that on FADD-deficient Jurkat T cells (clone I2.1), which was previously refractory to Fas-mediated apoptosis [21]. Irrespective of the FADD deficiency, both Jurkat T cell clones showed similar sensitivity to the cytotoxicity of pFPhe (data not shown), excluding the involvement of Fas/FasL system in the pFPhe-mediated apop- tosis. Since it has been reported that caspase-8 activation can play a role in drug-induced apoptotic signaling pathway either as an upstream event [14,19,22] or as a downstream event of mitochon- drial cytochrome c release [23], current data suggest that p56lck kinase might negatively influence either pFPhe-induced caspase-8 activation and subsequent generation of tBid triggering mitochon- drial cytochrome c release or pFPhe-induced mitochondrial cyto- chrome c release and resultant activation of caspase cascade.

Effect of ectopic expression of p56lck on hypersusceptibility of JCaM1.6 toward apoptogenic activity of pFPhe

To confirm the involvement of the p56lck in pFPhe-induced apop- tosis of Jurkat T cells as a negative modulator, we decided to exam- ine whether the hypersusceptibility of p56lck-deficient JCaM1.6 to the apoptogenic activity of pFPhe is reduced by ectopic expression of p56lck in JCaM1.6. As shown in Fig. 3A, Western blot analysis dem- onstrated that the stable transfectants of JCaM1.6 with the p56lck gene was able to express the p56lck protein. When the induced apoptotic sub-G1 peak after treatment with 2.5 mM pFPhe for 20 h was compared between p56lck-stable transfectants JCaM1.6/lck and p56lck-deficient JCaM1.6/vector by flow cytometry, the sub-G1 peak was more obvious in JCaM1.6/vector than in JCaM1.6/lck (Fig. 3B). In order to examine whether necrosis was accompanied by the apoptogenic activity of pFPhe as well as its modulation by the pres- ence of p56lck, JCaM1.6/lck and JCaM1.6/vector, which were treated with 2.5 mM pFPhe for 20 h, were analyzed by Annexin V staining. As shown in Fig. 3C, the treatment of JCaM1.6/lck and JCaM1.6/vec- tor with pFPhe caused an enhancement in the levels of early apop- totic cells stained only with Annexin V-FITC, and late apoptotic cells stained with both Annexin V-FITC and PI, and these apoptotic changes appeared to be more dominant in JCaM1.6/vector than in JCaM1.6/lck. Under these conditions, however, the necrotic cells stained only with PI were barely detected. These results confirmed that although the p56lck was not a prerequisite for pFPhe-mediated apoptotic signaling pathway, it could contribute to suppressing the pFPhe-induced apoptosis. Since current data showed that the presence of p56lck reduced the susceptibility to pFPhe-mediated apoptotic cell death of Jurkat T cells, we tested if the kinase activ- ity of p56lck in JCaM1.6/lck was altered after exposure to pFPhe. When the kinetic alteration of p56lck activity in JCaM1.6/lck follow- ing treatment with 2.5 mM pFPhe was assessed by in vitro kinase assay using a-casein as the substrate, the kinase activity of p56lck appeared to increase by approximately 1.6-fold at 15–30 min, and sustained to 1.2-fold-increased level at 1–6 h after treatment with pFPhe (Fig. 3D). At the same time, there was no significant change in the level of p56lck protein. These results demonstrated that the kinase activity of p56lck was enhanced following treatment with pFPhe in Jurkat T cells, supporting the involvement of 56lck kinase in the pFPhe-induced apoptotic signaling pathway as a negative modulator.

Effect of pan-caspase inhibitor z-VAD-fmk or caspase-8 inhibitor z-IETD-fmk on pFPhe-induced death signaling in JCaM1.6/lck

To elucidate further the death signaling pathway for pFPhe- induced apoptosis, which can be modulated by p56lck, we exam- ined the effect of the pan-caspase inhibitor (z-VAD-fmk), which is known to inhibit broad-range caspases [24], and the caspase-8 inhibitor (z-IETD-fmk) [25] on pFPhe-induced apoptotic events in JCaM1.6/lck. After cells were pretreated either with each caspase inhibitor or with both caspase inhibitors for 1 h, the cells were exposed to 2.5 mM pFPhe for 24 h. Although continuously growing JCaM1.6/lck showed a barely detectable apoptotic sub-G1 peak, it increased to the level of 47.2% in the presence of 2.5 mM pFPhe for 24 h (Fig. 4A). The pFPhe-induced sub-G1 peak was reduced to the basal level by pretreatment with z-VAD-fmk or with both z-VAD-fmk and z-IETD-fmk, whereas the sub-G1 peak was partially reduced to the level of 15.9% by pretreatment of z-IETD-fmk. Under the same conditions, Western blot analy- sis revealed that pFPhe-induced apoptotic events such as activation of caspase-8, -3 and -7, Bid cleavage, and cleavage of PARP and PLCc-1 were completely abrogated by z-VAD-fmk, whereas caspase-9 activation was sustained (Fig. 4B). In the presence of z-IETD-fmk, pFPhe-mediated caspase-9 activation was not influ- enced, but caspase-8 activation and PLCc-1 degradation were completely abrogated with a partial reduction in the activation of caspase-3 and -7, and PARP cleavage. These results indicated that pFPhe-induced caspase-8 activation were a downstream event of caspase-9 activation, and suggested that caspase-8 together with caspase-9 might contribute to activation of caspase-3 and -7, leading to cleavage of PLCc-1 and PARP.

In summary, these results demonstrate that the pFPhe-induced apoptotic signaling pathway leading to apoptotic DNA fragmen- tation was provoked by mitochondrial cytochrome c release and resultant activation of caspase cascade, which could be nega- tively regulated by the protein tyrosine kinase p56lck in Jurkat T cells. These results first provide the apoptogenic mechanism of an amino acid analog, q-fluorophenylalanine (pFPhe), along with its suppressive modulation by the protein tyrosine kinase p56lck in human acute leukemia Jurkat T cells.