Induced Apoptosis in Glioma Cells - Science Direct

Biochemical and Biophysical Research Communications 284, 1162–1167 (2001) doi:10.1006/bbrc.2001.5104, available online at http://www.idealibrary.com on

A Protective Role of PKC⑀ against TNF-Related Apoptosis-Inducing Ligand (TRAIL)-Induced Apoptosis in Glioma Cells Hisaaki Shinohara,* Nobuhiko Kayagaki,† Hideo Yagita,† Naoki Oyaizu,* Motoi Ohba,‡ Toshio Kuroki,‡ and Yoji Ikawa* ,§ ,1 *Department of Retroviral Regulation, Medical Research Division, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8519, Japan; †Department of Immunology, Juntendo University School of Medicine, Bunkyo-ku, Tokyo 113-8421, Japan; ‡Institute of Molecular Oncology, Showa University, Shinagawa-ku, Tokyo 142-8555, Japan; and §Ikawa Laboratory, Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-0198, Japan

Received May 16, 2001

To elucidate the molecular mechanism(s) involved in the TRAIL-induced apoptosis sensitivity, we conducted the following experiments utilizing TRAIL-sensitive and -resistant glioma cells. We examined the expression of TRAIL receptors mRNA, but no significant differences were detected in those cells. TRAIL-resistant cells were sensitized to TRAIL-induced apoptosis by staurosporine pretreatment and preferentially expressed PKC⑀. Since several lines of evidence suggest that PKC may play a protective role for apoptosis, we analyzed the involvement of PKC⑀ in TRAIL-induced apoptosis by an adenovirus vector expression system. We found that TRAIL susceptibility was augmented by the expression of a dominant negative PKC⑀ in TRAIL-resistant cells. Conversely, PKC⑀ introduction in TRAIL-sensitive cells resulted in the reduction of TRAIL-induced apoptosis. Taken together, these data suggest that PKC⑀ may be a regulator of susceptibility to TRAIL-induced apoptosis in gliomas and probably other malignancies. © 2001 Academic Press

Key Words: PKC; TRAIL; apoptosis; PKC inhibitor.

We have previously reported that glioma cells are resistant to Fas-induced cell death, instead it promotes cell cycle progression via ERK (1). As contrasted with Fas, TRAIL was reported to have a hopeful potential Abbreviations used: TNF, tumor necrosis factor; rhTRAIL, human recombinant TRAIL; PKC, protein kinase c; PI, propidium iodide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2, 5-dipheniltetrazonium bromide; ERK, extracellular signal regulated kinase; mAb, monoclonal antibody; M.O.I., multiplicity of infection; phosphoinositide 3-kinase, PI3-K. 1 To whom correspondence should be addressed at Ikawa Laboratory, Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. Fax: ⫹81 48 4679790. E-mail: [email protected]. 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

for the treatment of cancer without causing toxic effects to normal tissues in vivo (2, 3). However, some neoplasmic cells are resistant to cell death induced by TRAIL. We examined several glioma cell lines for their sensitivity to TRAIL-induced cell death and found that the presence of two types of glioma cells; TRAILinduced apoptosis sensitive and TRAIL-induced apoptosis-resistant. By comparison of the characters between these two types of glioma cell lines, we tried to elucidate molecular mechanisms, which may regulate TRAIL-induced apoptosis. TRAIL was identified as a type II membrane protein belonging to TNF family, which induces cell death in the tumor cells of various origin (4 – 6). At least four receptors of TRAIL (TRAIL-R1, -R2, -R3, -R4) have been discovered (4, 7–14). Both TRAIL-R1 and -R2 contain a death domain that possesses a cell killing potential. In contrast to theses proapoptotic receptors, TRAIL-R3 and -R4 do not transmit an apoptotic signal and have been proposed to confer protection from TRAIL-induced apoptosis (8, 10, 11, 14, 15). Thus the types of TRAIL-receptor used in the cells appear to be one of the key determinants for TRAIL-induced apoptosis sensitivity. PKCs play crucial roles in biological regulation such as cell proliferation, differentiation, apoptosis and neoplastic transformation. Especially, current reports have indicated that PKC activation can inhibit apoptosis. For example, a potent PKC activator, phorbol ester prevents apoptosis induced by Fas in Jurkat T cells (21, 22). PKC is a family of serine–threonine kinase and to date, 12 different isoforms have been identified and are classified into three major groups based on their structures and on their activation mechanisms. Conventional PKCs (␣, ␤ I, ␤ II, ␥) require phosphatidylserine (PS), diacyl glycerol (DAG) or phorbol esters and

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Ca ⫹⫹; novel PKCs (␦, ⑀, ␩, ␪) require only DAG and PS; and atypical PKCs (␨, ␫, ␭) are dependent on PS for activation (reviewed in Ref. 20). In glioma cells, PKC action is implicated as a key mediator of the proliferation of highly malignant gliomas (reviewed in Ref. 23). Besides its critical role in proliferation of glial cell tumors, PKC has been also implicated in the regulation of apoptosis in glioma cells. It is still unclear, however, which isoforms of PKC and which site of action are actually involved to block a death signal. We addressed these questions and show here that the TRAIL-resistant glioma cells preferentially expressed the PKC⑀ isoform. The expression of a dominant negative form of PKC⑀ (Ax-D/N⑀) using the adenovirus vector expression system led to upregulate TRAIL-induced apoptosis in the TRAIL-resistant glioma cells. These data suggest that PKC isoform ⑀ may act as protective machinery against TRAILinduced apoptosis. MATERIALS AND METHODS Cells and reagents. Human glioma cell lines (A-172, T98G, and YKG-1) were obtained from the Health Science Research Resources Bank (Osaka, Japan). The caspase colorimetric protease assay kits were purchased from MBL (Nagoya, Japan); a broad-spectrum PKC inhibitor, staurosporine, from Sigma (St. Louis, MO) and a PI3-K specific inhibitor, LY294002, from Calbiochem (La Jolla, CA). Caspase-3specific inhibitor, Ac-Asp-Met-Gln-Asp-CHO and caspase-8 inhibitor, Ac-Ile-Glu-Thr-Asp-CHO was purchased from Peptide Institute Inc., Osaka, Japan. The recombinant human TRAIL was prepared as described (26). Phorbol 12-myristate 13-acetate (PMA) and phosphatidylserine (PS) were from Sigma (St. Louis, MO). Estimation of cell viability. A WST-8 assay was applied for estimating cellular viability, utilizing a commercially available kit (Cell Counting Kit-8, Wako Pure Chemical, Osaka, Japan) as described previously (1). In brief, cells were seeded at 0.7 ⫻ 10 4 cells per well onto a 96-well culture plate and cultured over night to allow the cells adhere. The following day, or 24 h after adenovirus infection, rhTRAIL was added at given concentrations, and the 10 ␮l per well of the WST-8 was added 2 h before the end of culture. The cell-bound dye was measured by optical absorption at 450 nm using a microplate reader. Cell viability after rhTRAIL treatment was calculated as follows: % viability ⫽ 共 A 450/640 rhTRAIL-treated cells/A 450/640 untreated cells兲 ⫻ 100. RT-PCR for human TRAIL receptors. Total RNA was isolated from glioma cells and from Jurkat T cells using acid-guanidiniumthiocyanate-phenol-chloroform extraction method. RNA samples (1 ␮g each) were tested for DNA contamination by PCR with human GAPDH primers. After confirming no DNA contaminants, cDNA synthesis was performed by reverse transcription using a RNA PCR kit (Perkin–Elmer, Norwalk, CT) supplied with random hexamers according to the manufacturer’s instructions. The primer preparations for human TRAIL receptors (TRAIL-R1, -R2, -R3, -R4) were performed as described (5). Assay for caspase activity. Caspase-3 and -8 activity was determined by utilizing a commercially available caspase colorimetric protease assay kit, in which Asp-Glu-Val-Asp-P-nitroaniline and Ile-Glu-Thr-Asp-P-nitroaniline are used as the substrates of

caspase-3 and -8 respectively. Assaying was performed according to the manufacturer’s instructions. In brief, the caspase substrate was added to the cell lysate at a final concentration of 200 ␮M and incubated at 37°C for 2 h. The samples were read at 405 nm in a microplate reader. TRAIL-induced caspase activity was determined by comparing the results with the level of the untreated control cells as follows:

Caspase activity ⫽ 共 A 405/640 rhTRAIL-treated cells ⫺ background reading兲/ 共 A 405/640 untreated cells ⫺ background reading兲.

Immunoblotting. Immunoblotting was performed as reported previously (1). Whole cell lysates were subjected to 12% polyacrylamide gel electrophoresis after boiling, and then electroblotted onto nitrocellulose membrane (Bio-Rad, Richmond, CA). The membrane was probed with the following antibodies: mAbs to PKC␣, PKC⑀, and PKC␫ from Transduction Laboratory (Santa Cruz, CA), and polyclonal abs to PKC␣, ⑀, and ERK from Santa Cruz (Santa Cruz, CA). The bound antibodies were visualized by using the Photope-Star Western Blot Detection kit (New England Biolabs, Schwalbach, Germany). Construction of adenovirus vector. The preparation and construction of adenovirus vector were performed as described previously (27). In brief, the cDNA coding for the rabbit ⑀ isoform of PKC (Ax-⑀), a dominant-negative mutant of the PKC⑀ (Ax-D/N⑀), and ␤-galactosidase (Ax-lacZ) were inserted into the SwaI site of a cosmid cassette pAxCAwt (37). By using these cosmids, recombinant adenoviruses containing each PKC gene were generated and were mixed with the EcoT22I-digested DNA terminal protein complex of Ad5dIX and transfected into 293 cells, in which a recombinant adenovirus vector was generated through homologous recombination. Highly concentrated virus was prepared, and the titer was determined by infection onto 293 cells. The Ax-D/N⑀ was generated by the substitution of arginine for lysine at the amino acid 436 residue within the ATP binding site of the catalytic domain (38). The dominant-negative nature of the ⑀ isoform was confirmed functionally by its incapability of phosphoryrating histon H1, as described below. Immunoprecipitation and in vitro kinase assay. At appropriate time points, cells were lysed by lysis buffer (30 mM Tris–HCl pH 7.5, 500 mM NaCl, 1.5 mM MgCl 2, 0.1% Triton X-100, 1 mM EDTA, 1 mM DTT, 0.1% aprotinin, 1 mM PMSF, 0.1 mM Na 3VO 4, 0.125 ␮M Okadic acid). Immunoprecipitation of PKC was performed by the addition of 2 ␮g of the respective antibody to the 200 ␮g of cell lysate protein for 1 h incubation, and Sepharose-protein A (Sigma) for 1 h at 4°C. The immunoprecipitates were washed with lysis buffer once and twice with kinase buffer (20 mM Tris, pH 7.4, 10 mM MgCl 2, and 1.2 mM CaCl 2), and resuspended in 40 ␮l of reaction buffer (kinase buffer containing 0.5 mg of H1 histon (Sigma), 10 ␮M ATP, 50 ␮g/ml PS, 50 ng/ml PMA, and 5 ␮Ci of [␥- 32P]ATP (6000 Ci/mM). Reaction was terminated by the addition of SDS sample buffer, boiled, and the reaction products were applied to a 12% SDS–polyacrylamide gel and the extent of histon H1 phosphorylation was determined by autoradiography. Measurement of DNA fragmentation. For the measurement of DNA fragmentation, we employed a technique of propidium iodide (PI) staining (31, 32) with miner modification. In brief, the cells cultured in 12-well dish were collected by trypsinization and washed by PBS. The cells were resuspended in 1.0 ml of PI-staining buffer (PBS containing 0.2% Triton X-100 and 0.2 mg/ml of RNase) and 0.5 ml of PI (0.1 mg/ml) was added. After 10 min incubation, PI stained samples were analyzed on a flow cytometry (FACS Calibur, Becton Dickinson, San Jose, CA) utilizing CELLQuest software (Becton Dickinson).

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FIG. 1. Apoptosis sensitivity induced by rhTRAIL in glioma cells. (A) Susceptibility to rhTRAIL-induced cell death was assessed by a WST-8 and indicated as relative to untreated controls at time 24 h after rhTRAIL treatment. Data represent mean ⫾ SD of three experiments. (B) TRAIL receptor mRNA expression in glioma cell lines and in Jurkat T cells was determined by RT-PCR as described under Materials and Methods. Sizes of PCR products for each primer pair are R-1, 506 bp; R-2, 502 bp; R-3; 612 bp; R-4, 453; and GAPDH, 226 bp, respectively. (C) Relative cell viability of was estimated by WST-8 method at time 24 h after rhTRAIL (50 ng/ml) treatment (filled bar). Results from DMSO-controls and inhibitors alone are indicated in open bar. LY294002 and staurosporine was added 1 h prior to the treatment of rhTRAIL. DMSO was used as controls at concentration 1 ␮g/ml. Data are expressed as mean ⫾ SD of three independent experiments. *P ⬍ 0.01 compared to the DMSO ⫹ rhTRAIL sample. (D) Effects of PMA or staurosporine on TRAIL-induced caspase-8 and caspase-3 activation. Specific inhibitor for caspase-8 and -3 was added 1 h prior to the treatment of rhTRAIL at concentration 100 ␮M. PMA (100 ng/ml) and staurosporine (30 nM) were added 30 min and 1 h before addition of rhTRAIL, respectively. Cells were lysed at 1 h for caspase-8 activity and 3 h treated for caspase-3 activity after treatment of rhTRAIL (50 ng/ml). The caspase activity was determined as described under Materials and Methods. Data represent mean ⫾ SD of three independent experiments. *P ⬍ 0.01 compared to the rhTRAIL-treated sample.

Statistical analysis. Statistical significance was assessed by Student’s t-test and P ⬍ 0.05 was considered as significant. Composite treatments were analyzed by Microsoft Excel Software.

RESULTS AND DISCUSSION We initially examined the cell death sensitivity against the rhTRAIL on three glioma cell lines; A-172, T98G, YKG-1 and Jurkat T cell line. The viability of the cells after 24-h culture with various doses of rhTRAIL was estimated by WST-8 assay. As shown in Fig. 1A, each glioma cell lines showed the varying degree of susceptibility against TRAIL; T98G showed highest cell death induced by rhTRAIL which was followed by A-172, whereas YKG-1 showed resistance to cell death as far as we examined at the concentration of

rhTRAIL up to 50 ng/ml. No viable cells of Jurkat, a known TRAIL-sensitive cell line (4), were observed at the concentration of 5 ng/ml of rhTRAIL. These observations may raise a possibility that different TRAIL receptor expression of either death inducing (TRAILR1, -R2) or nondeath inducing (TRAIL-R3, -R4) may determine the susceptibility/resistance for cell death by TRAIL. To examine this possibility, we conducted RT-PCR for the mRNA expression of TRAIL receptors in glioma cell lines and also in Jurkat cells (Fig. 1B). The Jurkat cells were found to express mRNAs of all TRAIL receptors and all glioma cell lines were positive for TRAIL-R2 and -R3 mRNA, while TRAIL-R1 mRNA expression was observed only in A-172. Notably, celldeath sensitive T98G and cell-death resistant YKG-1

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FIG. 2. Expression of PKC in glioma cells. (A) Whole cell lysates were separated by SDS–PAGE and immunoblotting was performed PKC isoform-specific antibodies as indicated. ERK 1/2 is shown as a protein loading control. The results presented are representative of three experiments. (B) Time course of the PKC⑀ protein expression in Ax-PKC⑀-infected A-172 cell (M.O.I. ⫽ 8). The cells were lysed at indicated time after infection and were subjected to immunoblot analysis using anti-PKC⑀. The arrow indicates the position of the isoform of ⑀. (C) Kinase activity of PKC⑀ in adenovirus-infected glioma cells. Kinase activity was performed to phosphorylate histon H1. A-172 cells were infected with Ax-lacZ (lacZ; M.O.I. ⫽ 8), Ax-PKC⑀ (⑀; lane 2, M.O.I. ⫽ 4; ⑀; lane 3, M.O.I. ⫽ 8), and the dominant-negative mutant of PKC⑀ (D/N⑀; M.O.I. ⫽ 8), respectively for 24 h. The lysates were immunoprecipitated with antibody against the ⑀ isoform and reacted with [␥- 32P]ATP; the samples were then applied to SDS–PAGE and autoradiography. Ten micrograms per lane of histon H1 was subjected to gel. (D) Morphology of glioma cell (A-172) overexpressing PKC⑀ and PKC D/N⑀. Cells were infected with Ax-lacZ (lacZ), Ax-PKC⑀ (⑀), and Ax-PKC D/N⑀ (D/N⑀) at a M.O.I. of 11.6 and cultured for 24 h.

were found to present a similar mRNA pattern of TRAIL receptors, i.e., negative for TRAIL-R1 and positive for TRAIL-R2, -R3, and -R4. It was initially implicated that the expression of protective TRAIL receptors, -R3 and -R4 may render the cells resistant to TRAIL-induced cell death (8, 10, 14, 15). From RT-PCR study, however, we could not find any correlation between the sensitivity to TRAIL-inducing apoptosis and the mRNA expression of TRAIL receptors. In supporting this, further evidence suggests that the expression of TRAIL-R3 and -R4 is not different between the TRAIL-sensitive and -resistant cells (5, 6). In glioma cells, TRAIL-R3 and -R4 may also not be the main regulators of sensitivity in the TRAIL-induced apoptosis. Thus, the first possibility does not seem to take place in gliomas. The current accumulating evidence indicates a protective role of PKC against apoptosis. Activation of PKC induces a Fas-resistant phenotype in T cells and several kinds of cells (21, 22, 35, 36). As shown in Fig. 1C, we presented here that staurosporine, inhibitor of PKC, significantly and dose-dependently enhanced the

TRAIL-induced cell death at concentrations 3–30 nM in the TRAIL-resistant YKG-1 glioma cells. These data suggested that PKC may be involved in the protection of death signaling induced by TRAIL in glioma cells. Concurrently, the phosphoinositide 3-kinase (PI3-K)Akt/PKB pathway is reported to promote cell survival with the BCL-2 family member BAD or caspase-9 phosphorylation (16 –19, 28). The PI3-K specific inhibitor, LY294002 had no effects to TRAIL-induced apoptosis in the resistant cells. In this assay system, we could not find the involvement of PI3-K in TRAIL-inducing apoptosis. The link of PKC with death signal induced by TRAIL is still ambiguous at present time. The caspase-8 and caspase-3 have been shown to be critically involved in the induction of apoptosis by TRAIL (24, 25). We examined whether these caspases are differentially activated by TRAIL in TRAIL-resistant and -sensitive glioma cells (Fig. 1D, right). Staurosporine was found to increase the TRAIL-induced caspase-3 activation in YKG-1. Staurosporine itself had no effects on caspase-3 activation. We also asked whether PKC activation

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FIG. 3. Effects of Ax-PKC⑀ and Ax-PKC D/N⑀ infection on the viability and the caspase activity in rhTRAIL-treated glioma cells. (A) Ax-lacZ, Ax-PKC⑀, and Ax-PKC D/N⑀ were infected with T98G (upper) and YKG-1 (lower) 24 h prior to treatment of rhTRAIL. Cells were then cultured for 24 h in the absence (open bar) or presence (filled bar) of TRAIL (50 ng/ml). The viability was estimated by WST-8 Method. Data are expressed as mean ⫾ SD of three independent experiments. *P ⬍ 0.01 compared to the lacZ ⫹ rhTRAIL sample. (B) Ax-lacZ, Ax-PKC⑀, or Ax-PKC D/N⑀ as indicated were infected with T98G (left) or YKG-1 (right) cells 24 h prior to the treatment of rhTRAIL (50 ng/ml) at a M.O.I. of 11.6. Cells were lysed 3 h after the addition of rhTRAIL and caspase-3 activity was estimated as described under Materials and Methods. Forty micrograms of cell lysate was used in this experiment. Data represent mean ⫾ SD of triplicate samples. Similar results were obtained in two independent experiments. *P ⬍ 0.03 compared to lacZ ⫹ rhTRAIL sample.

could lead to inhibit TRAIL-mediated caspase activation. PMA, a potent PKC activator, inhibited TRAILinduced caspase-3 activation in TRAIL-sensitive T98G glioma cells (Fig. 1D, left). Regarding the caspase-8 activation, although it was activated in TRAILsensitive T98G, rhTRAIL failed to activate the caspase-8 in TRAIL-resistant YKG-1. These results suggested that PKC may be involved in another apical caspase activation such as caspase-10 and act upstream of caspase-3 to inhibit TRAIL-induced death signal in glioma cells. Although the involvement of PKC in gaining TRAILresistance gliomas, it is obscure as to which PKC isofoems involved in this process. The evidence of PKC specific isoforms, ␣, ⑀, and ␫ are involved in blocking apoptosis has currently emerged (29 –34). To address this point, we examined the endogenous protein expression of PKC isoform ␣, ⑀, and ␫ by immunoblotting (Fig. 2A). Although varying levels of PKC ␣ expression was observed in the cells examined, the correlation between the levels of protein expression and the sensitivity to TRAIL was not apparent. The PKC ␫ protein expression was not varying in all glioma cell lines. Of note, relatively high levels of PKC⑀ expression was detected only in TRAIL-resistant YKG-1 cells, as compared to other cell lines. These results prompted us to speculate that PKC⑀ may act a protective role to TRAIL-induced apoptosis in gliomas. To examine the role of PKC⑀ in TRAIL-induced apoptosis, we next conducted overexpression of PKC⑀ and its dominant negative mutant in glioma cells by

adenovirus vector which carry the cDNAs of intact PKC⑀ and kinase negative form of PKC⑀ (Ax-⑀ and Ax-D/N⑀ respectively). The Ax-lacZ, ␤-galactosidase gene had been employed in our previous experiment (27), was used for control. The favorable level of PKC⑀ transduced by adenovirus vector was confirmed by three parameters; i.e., immunoblotting, in vitro kinase assay and morphological change of the cells. In the immunoblotting, we could detect the high levels of PKC⑀ expression from 24 to 72 h after infection of Ax-PKC⑀ (Fig. 2B). The kinase activity of the ⑀ isoform introduced by Ax-PKC⑀ was determined by its ability to phosphorylate histon H1. As shown in Fig. 2C, the kinase activity of the ⑀ isoform was highly stimulated in the presence of PS plus PMA, but the lacZ control and Ax-PKC D/N⑀ failed to induce the substrate phosphorylation. The morphology of cells at 24 h after infection with adenovirus was markedly changed; Ax-PKC⑀ infected cells became enlarged and flatted and Ax-PKC D/N⑀ infected cells exhibited an elongated dendrite like morphology (Fig. 2D). Utilizing this adenovirus expression system, we examined the effects of PKC⑀ expression in rhTRAILinduced apoptosis (Fig. 3A). The Ax-PKC⑀ infected T98G, a TRAIL-sensitive cell line, showed suppression of the cell death susceptibly by rhTRAIL treatment with increasing M.O.I. In marked contrast, TRAILresistant YKG-1 cells gained cell death sensitivity to rhTRAIL by the infection of the Ax-PKC D/N⑀ with increasing M.O.I. The caspase-3 activation by rhTRAIL was decreased by Ax-PKC⑀ infection in

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TRAIL-sensitive T98G cells (Fig. 3B, upper) and conversely enhanced by Ax-PKC D/N⑀ infection in TRAILresistant YKG-1 cells (Fig. 3B, lower). Regarding the TRAIL-induced caspase-8 activation, overexpression of PKC⑀ or its dominant negative form did not affect caspase-8 activation (data not shown). Based on these observations, it is suggested that the site of PKC⑀ protection may locate upstream of caspase-3 activity in glioma cells and may not be involved in the caspase-8 activation. The activities of caspases were lower than those expected which may be partly due to the sensitivity of assaying system. Otherwise, in these glioma cells, PKC may contribute to the unknown cell deathsignaling cascade induced by TRAIL. Since these gliomas are resistance to Fas-mediated cell death (1), it is possible that caspase-8 homologues such as caspase-10 may mediate the other death-inducing pathway by TRAIL (13). We have thus shown that the TRAILsusceptibility was gained by staurosporine treatment and partially by the introduction of Ax-D/N⑀ in TRAILresistant glioma cells. These observations are implying the existence of a novel anti-apoptotic signaling pathway involving PKC against TRAIL-induced apoptosis. REFERENCES 1. Shinohara, H., Yagita, H., Ikawa, Y., and Oyaizu, N. (2000) Cancer Res. 60, 1766 –1772. 2. Ashkenazi, A., Pai, R. C., Fong, S., Leung, S., Lawrence, D. A., Marsters, S. A., Blackie, C., Chang, L., McMurtrey, A. E., Hebert, A., DeForge, L., Koumenis, I. L., Lewis, D., Harris, L., Bussiere, J., Koeppen, H., Shahrokh, Z., and Schwall, R. H. (1999) J. Clin. Invest. 104, 155–162. 3. Walczak, H., Miller, R. E., Ariail, K., Gliniak, B., Griffith, T. S., Kubin, M., Chin, W., Jones, J., Woodward, A., Le, T., Smith, C., Smolak, P., Goodwin, R. G., Rauch, C. T., Schuh, J. C. L., and Lynch, D. H. (1999) Nat. Med. 5, 157–163. 4. Wiley, S. R., Schooley, K., Smolak, P. J., Din, W. S., Huang, C.-P., Nicholl, J. K., Sutherland, G. R., Smith, T. D., Rauch, C., Smith, C. A., and Goodwin, R. G. (1995) Immunity 3, 673– 682. 5. Griffith, T. S., Chin, W. A., Jackson, G. C., Lynch, D. H., and Kubin, M. Z. (1998) J. Immunol. 161, 2833–2840. 6. Leverkus, M., Neumann, M., Mengling, T., Rauch, C. T., Brocker, E.-B., Krammer, P. H., and Walczak, H. (2000) Cancer Res. 60, 553–559. 7. Pan, G., O’Rourke, K., Chinnaiyan, A. M., Gentz, R., Ebner, R., Ni, J., and Dixit, V. M. (1997) Science 276, 111–113. 8. Pan, G., Ni, J., Wei, Y.-F., Yu, G.-L., Gentz, R., and Dixit, V. M. (1997) Science 277, 815– 818. 9. Walczak, H., Degli-Esposti, M. A., Johnson, R. S., Smolak, P. J., Waugh, J. Y., Boiani, N., Timour, M. S., Gerhart, M. J., Schooley, K. A., Smith, C. A., Goodwin, R. G., and Rauch, C. T. (1997) EMBO J. 16, 5386 –5397. 10. Degli-Esposti, M. A., Smolak, P. J., Walczak, H., Waugh, J., Huang, C.-P., DuBose, R. F., Goodwin, R. G., and Smith, C. A. (1997) J. Exp. Med. 186, 1165–1170. 11. Degli-Esposti, M. A., Dougall, W. C., Smolak, P. J., Waugh, J. Y., Smith, C. A., and Goodwin, R. G. (1997) Immunity 7, 813– 820. 12. Chaudhary, P. M., Eby, M., Jasmin, A., Bookwalter, A., Murray, J., and Hood, L. (1997) Immunity 7, 821– 830.

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