Calcium/Calmodulin-dependent Protein Kinase II Regulation of c-FLIP Expression and Phosphorylation in Modulation of Fas-mediated Signaling in Malignant Glioma Cells*

Fas, upon cross-linking with Fas ligand (FasL) or Fas agonistic antibody, transduces apoptotic yet also proliferative signals, which have been implicated in tumor pathogenesis. In this study, we investigated the molecular mechanisms that control Fas-mediated signaling in glioma cells. Fas agonistic antibody, CH-11, induced apoptosis in sensitive glioma cells through caspase-8 recruitment to the Fas-mediated death-inducing signaling complex (DISC) where caspase-8 was cleaved to initiate apoptosis through a systematic cleavage of downstream substrates. In contrast, CH-11 stimulated cell growth in resistant glioma cells through recruitment of c-FLIP (cellular Fas-associated death domain (FADD)-like interleukin-1β-converting enzyme (FLICE)-inhibitory protein) to the Fas-mediated DISC. Three isoforms of long form c-FLIP were detected in glioma cells, but only the phosphorylated isoform was recruited to and cleaved into a p43 intermediate form in the Fas-mediated DISC in resistant cells. Calcium/calmodulin-dependent protein kinase II (CaMK II) activity was up-regulated in resistant cells. Treatment of resistant cells with the CaMK II inhibitor KN-93 inhibited CaMK II activity, reduced c-FLIP expression, inhibited c-FLIP phosphorylation, and rescued CH-11 sensitivity. Transfection of CaMK II cDNA in sensitive cells rendered them resistant to CH-11. These results indicated that CaMK II regulates c-FLIP expression and phosphorylation, thus modulating Fas-mediated signaling in glioma cells.

that has multiple cysteine-rich repeats in the extracellular domain and an intracellular motif termed a death domain (DD). Binding of FasL or CH-11 to the Fas extracellular domain induces trimerization of Fas, resulting in the recruitment of the intracellular adapter FADD (5,6). FADD has a carboxylterminal DD and an amino-terminal death effector domain (DED). Through its DED, FADD recruits the DED-containing apoptosis-initiating proteases caspase-8 (7,8) and caspase-10 (9 -11) to the Fas receptor to assemble a DISC (12). In the DISC, caspase-8 is cleaved through autoproteolysis of caspase-8 molecules in close proximity (13). Active caspase-8 subunits are released into the cytoplasm to cleave downstream effector caspases such as caspase-3 (14), which subsequently cleaves its substrates such as DNA fragmentation factor 45 (DFF45) (15), to execute programmed cell death.
Recently, the recruitment of other DED-containing proteins to the Fas-mediated DISC, which has been described, modulates DISC functions. These include a family of virus-encoded proteins referred to as v-FLIP (16,17). v-FLIP contains two DEDs that can bind to the Fas/FADD complex to inhibit Fasmediated apoptosis by interfering with the recruitment of caspase-8 to the DISC. A mammalian cellular homolog of v-FLIP is termed c-FLIP (18), CASH (19), CASPER (20), CLARP (21), FLAME1 (22), I-FLICE (23), MRIT (24), and Usurpin (25). These studies, however, have generated controversy as to the functions of c-FLIP in apoptosis. Some groups have described it as pro-apoptotic (19 -21, 24), whereas others as anti-apoptotic (18,22,25). Recent analysis of Fas-mediated DISC in c-FLIPtransfected BJAB cells has shown that c-FLIP proteins are recruited to the Fas-mediated DISC to inhibit caspase-8 cleavage (26,27), which supports the role of c-FLIP as an antiapoptotic molecule.
The c-FLIP gene is composed of 13 exons that are clustered within ϳ200 kilobases within the caspase-8 and caspase-10 genes on human chromosome 2q33 to 34 (25,28). c-FLIP is expressed as four main mRNA splice variants but only two forms of protein in human tissues (18,20). The short form protein (c-FLIP S , M r ϳ28) contains two DEDs and is structurally related to v-FLIP; the longer form (c-FLIP L , M r ϳ55) is structurally similar to caspase-8 and contains two DEDs and a caspase-like domain that lacks catalytic activity (18). c-FLIP L is expressed in many tissues, but c-FLIP S is found mainly in lymphatic tissue (18). Expression of c-FLIP mRNA and proteins is regulated by mitogen-activated protein kinase kinase in T lymphocytes (29) and by phosphatidylinositol 3-kinase in tumor cells (30). Here we show calcium/calmodulin-dependent protein kinase II (CaMK II) regulates c-FLIP expression and phosphorylation in malignant glioma cells.
Cell Death Assay and Cleavage of Caspases, DFF45, and c-FLIP-For cell death, cells were seeded in 96-well plates at 2ϫ10 4 cells/well and treated at 37°C for 24 h with 1 g/ml anti-Fas CH-11 or 100 M KN-93 alone or in combination. Cell death was determined by crystal violet assay (37). For cleavage of caspases, DFF45, and c-FLIP, subconfluent cells were treated with 1 g/ml CH-11 either in the presence or absence of 100 M KN-93 at 37°C for the times indicated. At each time point, cells were lysed in lysis buffer (1%Triton X-100, 150 mM NaCl, 10% glycerol, 20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, complete protease inhibitor mixture). After centrifugation at 16,000 ϫ g for 15 min at 4°C, supernatants were subjected to Western blotting.
DISC Analysis by Immunoprecipitation-Immunoprecipitation for DISC analysis was performed according to a modified protocol as we previously reported (38). 5 ϫ10 7 cells were stimulated with 1 g/ml CH-11 (mouse IgM) for 30 min at 37°C and then lysed for 30 min on ice with lysis buffer. In unstimulated controls, the cells were lysed, and 1 g of CH-11 was added to 1 ml of cell lysates to immunoprecipitate non-stimulated Fas receptors. The soluble fraction was immunoprecipitated with 20 l of goat anti-mouse IgM-agarose overnight at 4°C and analyzed by Western blotting and two-dimensional PAGE immunoblotting.
Two-dimensional-PAGE, 32 P-Labeling, and Dephosphorylation-For each sample, 10 7 cells were either treated with 100 M KN-93 for 24 h at 37°C or left untreated and lysed in 40 mM Tris-HCl, pH 8.0, 1% Triton X-100, 65 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, complete protease inhibitor mixture. After centrifugation, the supernatants were precipitated with acetone. The pellets were dissolved in 9.8 M urea, 2% CHAPS, 0.5% pH 3-10 non-linear IPG buffer (Amersham Biosciences) 65 mM dithiothreitol, applied by rehydration in 13-cm DryStrips (pH 3-10 non-linear), and electrofocused with IPGphor TM System following the manufacturer's protocol (Amersham Biosciences). The strips were equilibrated with 50 mM Tris-HCl, pH 8.8, 6 M urea, 30% glycerol, 2% SDS, 10 mg/ml dithiothreitol and subjected to SDS-PAGE followed by transfer to a nitrocellulose membrane (Bio-Rad) and immunoblotting. For labeling of cellular phosphoproteins, cells were incubated for 1 h in phosphate-free Dulbecco's modified Eagle's medium (DMEM) and then labeled for 4 h in phosphate-free DMEM supplemented with 0.5 mCi/ml 32 P. Cells were lysed, and cell extracts were subjected to two-dimensional-PAGE followed by autoradiography and immunoblotting, as described above. For dephosphorylation of cellular phosphoproteins, cell lysates obtained from cells were treated with 0.2 units/ml acid phosphatase for 3 h at 37°C and subsequently subjected to two-dimensional PAGE immunoblotting.
CaMK II Activity Assay and CaMK II cDNA Transfection-CaMK II activity was assayed in cell lysates using CaMK II assay kits following the manufacturer's protocol (Upstate Biotechnology). For transfection, CaMK II cDNA (Stratagene, La Jolla, CA) was subcloned into pcDNA3.1 expression vector using EcoR I and HindIII restriction sites. Transfection of the pcDNA3.1 expression vector containing CaMK II cDNA into glioma cells was accomplished using the LipofectAMINE method following the manufacturer's protocol (Invitrogen). After transfected for 48 h, the cells were subjected to cell death assay and Western blot analysis for cleavage of caspases and DFF45.

Caspase-8 Is Recruited to the Fas-mediated DISC to Initiate
Apoptosis-Malignant glioma cells express cell surface Fas and are susceptible to agonistic Fas antibody-induced apoptosis (31). To illustrate the signal events in Fas-mediated apoptosis, we first analyzed the Fas-mediated DISC in sensitive glioma cells. Three glioma cell lines (U343MG, LN-18, T98G) were selected in the study for their expression of cell surface Fas, as shown by flow cytometry, and sensitivity to agonistic Fas antibody CH-11, as determined by crystal violet assay (data not shown). We stimulated these cells with 1 g/ml CH-11 (mouse IgM) for 30 min at 37°C, and the Fas-mediated DISC was immunoprecipitated using goat anti-mouse IgM-agarose and examined by Western blotting to identify the proteins that are incorporated into the Fas-mediated DISC. In unstimulated controls, cells were lysed, and CH-11 was then added to the lysates to immunoprecipitate unstimulated Fas. Total cell protein extracts from unstimulated cells were also included in the Western blot analysis to examine endogenous expression of the proteins.
Western blots detected one p45 protein band of Fas, one p25 protein band of FADD, four protein bands of caspase-8, and four protein bands of caspase-10 in the Fas-mediated DISC in CH-11 stimulated cells (Fig. 1A). Two caspase-8 precursor proteins (p55 and p53) were detected in both the cell extracts and the DISC, but two caspase-8 first-step cleavage products (p43 and p41) were detected only in the DISC. Similarly, two caspase-10 precursor proteins (p59 and p54) and two caspase-10 first-step cleavage products (p47 and p43) were detected in the DISC. Next we examined the Fas-medicated DISC to look for the other two DED-containing proteins; c-FLIP and phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes-M r 15 (PED/PEA-15) (36). The long form p55 c-FLIP L and the short form p25 c-FLIP S were endogenously expressed in the cells as shown by Western blot anal-ysis of the cell extracts ( Fig. 1A), but they were not detected in the Fas-mediated DISC in these CH-11-sensitive cells (Fig.  1A). PED/PEA-15 was reported to inhibit Fas-mediated apoptosis (39,40). Western blots failed to detect PED/PEA-15 in the Fas-mediated DISC in these sensitive glioma cells (Fig. 1A).
Caspase-8 cleavage occurs in two consecutive steps in Fasmediated DISC, first-step cleavage to produce p12, p43, and p41 subunits and second-step cleavage to generate prodomain and active p18 and p10 subunits (41) to cleave downstream caspase-3 p32 precursors into large p20 and p17 and small p10 subunits (42). To examine this caspase-8-initiated cascade, we analyzed cell extracts collected from the cells exposed to 1 g/ml CH-11 to look for cleavage products. Western blots detected caspase-8 first-step cleavage p43 and p41 products within 30 min and second-step cleavage p18 proteins within 60 min after CH-11 stimulation (Fig. 1B). Western blots also detected caspase-3 cleavage p20 and p17 subunits after CH-11 stimulation (Fig. 1B). Finally, we examined the cell extracts for cleavage products of DFF45, a caspase-3 downstream substrate (15,43). Two forms of DFF45, the long form DFF45 and the short form DFF35, were endogenously expressed in the glioma cells and proteolytically cleaved upon CH-11 stimulation (Fig. 1B) 1. DISC analysis of CH-11-sensitive glioma cells. Each column represents one cell line as indicated above the panels. The proteins detected are indicated to the left, and Western blot (WB) antibodies are indicated to the right. A, DISC analysis. Anti-Fas CH-11 (1 g/ml) was added to the cells either after cell lysis in unstimulated cells (Ϫ) or for 30 min before cell lysis in stimulated cells (ϩ). The samples were immunoprecipitated with goat anti-mouse IgM-agarose and analyzed by WB. Cell extracts (EX) were also included in the Western blot analysis for the presence of the endogenous proteins in the cells. B, cleavage of caspases and DFF45. The cells were treated with 1 g/ml CH-11 for the times indicated, and cell lysates were examined by Western blot using anti-caspase-8, caspase-3, and DFF45 antibody, respectively.

FIG. 2. Fas-mediated DISC in CH-11-resistant glioma cells.
A, DISC analysis. The cell lines are indicated above the panels. Cell extracts (EX) and immunoprecipitated samples collected from unstimulated (Ϫ) and CH-11-stimulated (ϩ) cells were analyzed as described in Fig. 1A. B, cleavage of caspases and DFF45. Each column represents one cell line as indicated above in Fig. 2A. The cells were treated as described above in Fig. 1B and examined on Western blot (WB) for cleavage products of caspase-8, casapase-3, and DFF45. C, kinetic analysis of c-FLIP cleavage. The cell lines are indicated under the panels. The CH-11-sensitive (U343MG, LN-18, T98G) and CH-11-resistant (LN-215, LN-443, LN-464) cell lines were treated with 1 g/ml CH-11 for the times indicated, and c-FLIP proteins were examined on Western blot using anti-c-FLIP NF6 antibody.
precursors and p43 and p41 caspase-8 first-step cleavage products ( Fig. 2A). These findings indicated that FADD and caspase-8 are recruited to the Fas-medicated DISC, where caspase-8 completes its first-step cleavage. However, Western blot analysis failed to show caspase-8 second-step cleavage products in these resistant cells treated with CH-11 (Fig. 2B), indicating that caspase-8 second-step cleavage is inhibited in the DISC. Western blots also failed to detect caspase-3 and DFF45 cleavage products (Fig. 2B), which indicated that caspase-8-initiated cascade is inhibited as well in the resistant cells. Neither caspase-10 precursors nor its cleavage products was detected in the Fas-mediated DISC (data not shown) due to the lower levels of caspase-10 expression in these cells, as we previously reported (38).
We further examined the Fas-medicated DISC to look for c-FLIP and PED/PEA-15. The long form p55 c-FLIP L and the short form p25 c-FLIP S were endogenously expressed in these glioma cells (Fig. 2A). However, Western blot analysis of the DISC revealed three forms of c-FLIP proteins, a very weak band of p55 c-FLIP L , a new band of p43 c-FLIP, and a band of p25 c-FLIP S (Fig. 2A). These findings indicated that c-FLIP L is recruited to the DISC and cleaved into an intermediate p43 form, as previously reported in c-FLIP transfectants (18,26,27). Caspase-8 first-step cleavage occurred in both CH-11-sensitive and -resistant glioma cells (Figs. 1A and 2A), but it appeared that only resistant cells showed simultaneous recruitment of caspase-8 and c-FLIP L to the DISC, resulting in c-FLIP L cleavage into a p43 intermediate form ( Fig. 2A). To further confirm this, we examined c-FLIP L cleavage in the sensitive and resistant cell lines. Indeed, Western blots detected p43 intermediate products in the resistant cells (LN-215, LN-443, LN-464) but not in the sensitive cells (U343MG, LN-18, T98G) after exposure to 1 g/ml CH-11 (Fig. 2C). Western blots also detected PED/PEA-15 in the Fas-mediated DISC ( Fig. 2A). Simultaneous detection of caspase-8 first-step cleavage products, c-FLIP, and PED/PEA-15 suggested that both c-FLIP and PED/PEA-15 proteins may be responsible for inhibition of caspase-8 second-step cleavage in the Fas-mediated DISC in the CH-11-resistant glioma cells.
c-FLIP and CaMK II Are Up-regulated in Resistant Glioma Cells-The differential recruitment of c-FLIP and PED/PEA-15 to the Fas-mediated DISC suggests that c-FLIP and PED/ PEA-15 proteins may be up-regulated in the resistant glioma cells. Indeed, our earlier work has shown that CaMK II upregulates PED/PEA-15 expression and phosphorylation and modulates TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)-induced apoptosis in glioma cells (38). We speculated that a similar mechanism might exist in the glioma cells, which regulates c-FLIP expression and function. To test this hypothesis, we first investigated whether treatment of the resistant cells with KN-93, a CaMK II inhibitor, might restore CH-11 sensitivity. LN-215, LN-443, and LN-464 cell lines were treated with 1 g/ml CH-11 alone or in the presence of 100 M KN-93 for 24 h. Crystal violet analysis showed a consistent 20% cell growth in the CH-11-stimulated cells (Fig. 3A). These resistant cell lines, however, became sensitive to CH-11 in the presence of KN-93 (Fig. 3A). Kinetic analysis on Western blots showed caspase-8 first-step and second-step cleavage products in the cells exposed to CH-11 and KN-93 but not to KN-93 or CH-11 alone (Fig. 3B). Western blots also detected caspase-3 and DFF45 cleavage products (Fig. 3, C and D). These results indicated that the CaMK II inhibitor KN-93 rescues CH-11 sensitivity in resistant glioma cells and that CH-11-induced cell death in the resistant cells occurs through a caspase-8initiated caspase cascade, similar to the apoptotic signal pathway observed in the sensitive glioma cells (Fig. 1).
Given that caspase-8 is able to complete two-step cleavage in resistant cells after KN-93 treatment, we speculated that CaMK II might up-regulate c-FLIP expression and, thus, modulate Fas-mediated apoptosis in the resistant cells. To test this, we first compared the c-FLIP protein expression levels in the CH-11-sensitive and resistant glioma cell lines. Western blot analysis showed that both c-FLIP L and c-FLIP S were expressed at higher levels in the resistant (LN-215, LN-443, LN-464) than in the sensitive cell lines (U343MG, LN-18, T98G) (Fig.  4A) and that the c-FLIP L and c-FLIP S expression levels were markedly reduced in resistant cells after treatment with 100 M KN-93 for 24 h (Fig. 4A). Next, we examined CaMK II protein expression in the glioma cells and showed that CaMK II protein was highly expressed in CH-11-resistant cells as compared with the sensitive cells (Fig. 4A). Finally, we examined CaMK II activity to determine whether overexpressed CaMK II is biologically functional in glioma cells. CaMK II activity was measured in a total of 10 g proteins from each of the cell extracts using a specific kinase substrate and a mixture of kinase inhibitors to block the activity of other kinases. CaMK II activity was significantly higher in the resistant cell lines LN-215, LN-443, and LN-464 than in the sensitive cell lines U343MG, LN-18, and T98G (Fig. 4B). Treatment of the resistant cells with 100 M KN-93 for 24 h markedly reduced CaMK II activity in the resistant cells (Fig. 4B).
CaMK II Regulates c-FLIP L Phosphorylation and Recruitment to the DISC-CaMK II regulates c-FLIP expression to modulate Fas-mediated apoptosis in glioma cells. Here, we further proposed that CaMK II might regulate c-FLIP phosphorylation and recruitment to the Fas-mediated DISC. To test this, we first subjected the cell extracts to two-dimensional PAGE immunoblots to identify isoforms of the c-FLIP proteins. Two-dimensional PAGE immunoblots detected one c-FLIP S spot in either the sensitive or resistant tumor cells (Fig. 5A), indicating that there is only one isoform of c-FLIP S endogenously expressed in glioma cells. In contrast, however, twodimensional PAGE blots revealed three isoforms of c-FLIP L , indicated as c-FLIP La , c-FLIP Lb , and c-FLIP Lc (Fig. 5A). Two of the isoforms, c-FLIP La and c-FLIP Lb , were present in both sensitive (U343MG, LN-18) and resistant cell lines (LN-215, LN-443), but isoform c-FLIP Lc was detected only in the resistant cell lines (Fig. 5A).
Treatment of the resistant cells with 100 M KN-93 for 24 h inhibited CaMK II activity (Fig. 4B) and eliminated the expression of the isoform c-FLIP Lc (Fig. 5A); the results suggested that c-FLIP Lc might be a phosphorylated protein. To test this hypothesis, we first treated cell extracts of resistant cell lines LN-215 and LN-443 with acid phosphatase for 3 h at 37°C and then subjected the cell extracts to two-dimensional PAGE immunoblots. The results showed that acid phosphatase treatment eliminated the c-FLIP Lc isoform in the resistant cells (Fig. 5A). Next, we labeled the resistant cell lines LN-215 and LN-443 with [ 32 P]orthophosphate and subjected the cell extracts to two-dimensional PAGE that was examined first by autoradiography and then by immunoblotting with anti-c-FLIP antibody. Autoradiography of the two-dimensional PAGE membrane revealed one 32 P-labeled spot among the three c-FLIP L isoforms (Fig. 5B) and subsequent immunoblot analysis of the same membrane showed that the 32 P-labeled spot was identical to c-FLIP Lc (Fig. 5B). In contrast, similar analysis of autoradiography and immunoblots of two-dimensional PAGE failed to show any phosphorylated spot of the c-FLIP S form (Fig. 5B).
CaMK II is a S/T protein kinase (44). To further confirm c-FLIP Lc phosphorylation, we examined cell extracts from the sensitive U343MG and resistant LN-215 cells on two-dimensional PAGE immunoblots using anti-phosphothreonine antibody. The same membranes were then stripped of primary and secondary antibodies and analyzed using c-FLIP antibody. The c-FLIP Lc spot, detected using c-FLIP antibody, was also recognized by anti-phosphothreonine antibody (Fig. 6). Similar analysis showed an absence of c-FLIP Lc in CH-11-sensitive U343MG cells (Fig. 6). Taken together, these studies indicated that c-FLIP Lc is a phosphoprotein and is highly expressed in the resistant glioma cells.
We showed that c-FLIP L was recruited to the Fas-mediated DISC only in the resistant cells ( Fig. 2A), and of three isoforms of c-FLIP L , only c-FLIP Lc was expressed in the resistant cells (Figs. 5 and 6). These results suggested that c-FLIP Lc , but neither c-FLIP La nor c-FLIP Lb , is recruited to the Fas-mediated DISC. Western blot analysis showed that immediately upon recruitment, c-FLIP L proteins were cleaved into p43 intermediate forms in the DISC ( Fig. 2A), and thus, we were not surprised by the fact that two-dimensional PAGE immunoblots using anti-c-FLIP antibody detected p43 intermediate form protein but not c-FLIP L in the CH-11-induced DISC in LN-215 cells (Fig. 6). The same membrane was then analyzed using anti-phosphothreonine antibody, and the results showed that p43 intermediate form protein was phosphorylated (Fig. 6); the results suggested that p43 intermediate forms are generated from the phosphoprotein c-FLIP Lc but not from c-FLIP La or c-FLIP Lb .
CaMK II cDNA Transfection in Sensitive Glioma Cells Resulting in Cell Resistance-To further define the function of CaMK II in modulation of Fas-mediated apoptosis in tumor cells, we transiently transfected CaMK II cDNA in CH-11sensitive U343MG and LN-18 cell lines. Crystal violet analysis showed a near 100% cell death (Fig. 7A) in untransfected or vector transfected U343MG and LN-18 cell lines upon stimulation with 1 g/ml CH-11 for 24 h. Western analysis revealed cleavage of caspase-8, caspase-3, and DFF45 in these untransfected or vector-transfected cell lines upon stimulation with 1 g/ml CH-11 for 3 h (Fig. 7B). In contrast, transfection of these CH-11-sensitive cells with CaMK II cDNA resulted in significantly increased expression of CaMK II proteins, as demonstrated on Western blots (Fig. 7A), and remarkably decreased cell death, as shown by crystal violet analysis (Fig. 7A). Western blot analysis further showed a significantly decreased cleavage of caspase-8, caspase-3, and DFF45 (Fig. 7B). CaMK II cDNA-transfected cells were further exposed to 1 g/ml CH-11 in the presence of 100 M KN-93 to inhibit CaMK II activity. KN-93 treatment restored the sensitivity of CaMK II cDNA-transfected cells to CH-11-induced cell death (Fig. 7A) and cleavage of caspase-8, caspase-3, and DFF45 (Fig. 7B). Collectively, the results indicate that CaMK II plays a crucial regulatory role in Fas-mediated apoptosis in tumor cells.

DISCUSSION
The Fas/FasL-signaling pathway is an essential process in the regulation of programmed cell death and has been implicated in the pathogenesis of immune system diseases and various malignancies (45). Recent studies have provided several lines of evidence to support the role of the Fas-signaling pathway in tumorigenesis of malignant gliomas. Gliomas express both Fas and FasL, and glioma-derived FasL induces apoptosis of Fas-positive T cells that infiltrate tumors, thus facilitating tumor escape from the host immune system (46). FasL stimulates gliomas to produce chemokines that modulate immune responses (47). FasL or the Fas agonistic antibody CH-11 triggers apoptosis in some glioma cells (48) but transduces proliferative signals in others (49). In the present study, we analyzed the Fas-mediated DISC and elucidated the molecular mechanisms that regulate the biological functions of Fas-signaling pathways in human gliomas.
The signal transduction of Fas-mediated apoptosis has been well characterized in transfectants and lymphocytes (45) but remains to be established in non-transfected solid tumor cells.
Here, we showed that Fas-mediated apoptosis in sensitive glioma cells occurs through recruitment of apoptosis-initiating caspase-8 and caspase-10 to the Fas-mediated DISC. Caspase-8 completes its consecutive two-step cleavage in the DISC, resulting in release of its active subunits into the cytoplasm that initiate programmed cell death by subsequent cleavage of downstream effector caspases such as caspase-3 (14,42) and caspase-3 substrates such as DFF45 (15,43). However, many glioma cells are resistant to Fas-mediated apoptosis. Analysis of the Fas-mediated DISC provides a new molecular model for Fas-mediated resistance in tumor cells. Apoptosis-initiating caspase-8 is recruited to the Fas-mediated DISC and completes its first-step cleavage, but its second-step cleavage is inhibited in the DISC. Detection of c-FLIP and PED/PEA-15 molecules in the DISC suggests that these DEDcontaining proteins may inhibit the second-step cleavage of caspase-8 and, thus, prevent Fas-mediated apoptosis in tumor cells.
Recent studies of c-FLIP overexpression have provided two models for c-FLIP modulation of the Fas-mediated DISC (26,27). In the first model, caspase-8 and c-FLIP L are simultaneously recruited to the DISC, where caspase-8 completes its first-step cleavage, whereas c-FLIP L is cleaved into a p43 intermediate form. The p43 intermediate c-FLIP L remains in the DISC to interrupt caspase-8 second-step cleavage, and, thus inhibit caspase-8-initiated apoptosis. In contrast in another model, c-FLIP S is recruited to the DISC to inhibit first-step cleavage of caspase-8. Here, we demonstrated the p43 intermediate form in the Fas-mediated DISC, which supports the c-FLIP L -mediated model of regulation of Fas-mediated signaling. However, simultaneous detection of c-FLIP S and caspase-8 first-step cleavage in the DISC raises a question about the role of c-FLIP S in Fas signaling in glioma cells. It also remains to be clarified why both c-FLIP and PED are simultaneously recruited to the Fas-mediated DISC in glioma cells.
There is now increasing evidence that Fas transduces proliferative signals, but the signaling pathways remain poorly defined (50). Overexpression of c-FLIP L has been reported to activate nuclear factor kappa B (NF-B) through unknown signal pathways (51). On the other hand, c-FLIP S transfection up-regulates the proto-oncogene c-Fos in FADD-and caspase-8-dependent manner (52); the results suggest that c-FLIP Smediated cell proliferation occurs through the DISC. Here, we showed that glioma cells proliferate in response to CH-11 stimulation. Further detection of c-FLIP L and c-FLIP S in the Fasmediated DISC enhances the concept that c-FLIP proteins are recruited to the DISC to activate its downstream proliferative signals. PED/PEA-15 has also been shown to interfere with FADD and caspase-8 interactions and, thus, prevents DISC formation and subsequent apoptosis induced by FasL (39,40). PED/PEA-15 is recently reported to regulate Ras-initiated mitogen-activated protein kinase pathways through its DED (53) and, thus, modulate integrin-mediated cell growth, migration, and tumor metastasis (54,55), but the molecular events that connect the PED/PEA-15-mediated apoptotic signaling pathway to the Ras-initiated mitogen-activated protein kinase pathway remain elusive.
We have further identified three isoforms of c-FLIP L in glioma cells. The three isoforms were separated by the difference in their molecular weights and isoelectric points on two-dimensional PAGE. A combined two-dimensional PAGE 32 P autoradiography and immunoblot analysis further revealed that one of the three isoforms was phosphorylated. The phosphorylated c-FLIP L was expressed only in resistant cells, and inhibition of CaMK II activity in the resistant cells eliminated this phosphorylated isoform of c-FLIP L and rendered the cells sensitive to CH-11-induced apoptosis. Furthermore, we showed that the p43 intermediate form of c-FLIP L was phosphorylated, which suggested that the phosphorylated c-FLIP L isoform is recruited to and cleaved into a p43 intermediate form in the Fas-mediated DISC. The p43 form of c-FLIP L remains bound in the DISC to inhibit caspase-8-initiated apoptosis and, thus, switch the glioma cells from apoptosis to proliferation.
CaMK II has recently been implicated in the modulation of microcystin-induced apoptosis (56). Our earlier studies showed that CaMK II regulates PED/PEA-15 phosphorylation and, thus, modulates TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)-induced apoptosis in glioma cells (38). We now provide several lines of evidence that CaMK II up-regulates c-FLIP expression and phosphorylation to modulate Fasmediated signaling in the same tumor cells. Both c-FLIP L and c-FLIP S were highly expressed in resistant glioma cells, and treatment of these cells with the CaMK II inhibitor KN-93 reduced the expression of both long and short forms of c-FLIP and rescued the cell sensitivity to Fas-mediated apoptosis. CaMK II protein and biological activity were up-regulated, and KN-93 treatment eliminated the phosphorylated c-FLIP L in the resistant cells, suggesting that CaMK II may regulate c-FLIP L phosphorylation. Other kinases such as mitogen-activated protein kinase kinase, phosphatidylinositol 3-kinase, and protein kinase C have also been reported to regulate c-FLIP and PED/PEA-15 to modulate apoptosis (29,30,37). Therefore, further studies of these kinase-mediated signaling pathways will shed some light on the complex regulatory mechanisms that control tumor cell death and proliferation.