Fas-Mediated Apoptosis Fas Aggregation Does Not Correlate with

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Fas Aggregation Does Not Correlate with Fas-Mediated Apoptosis Yang-ja Lee1 and Emily Shacter Cross-linking of cell surface Fas molecules by Fas ligand or by agonistic anti-Fas Abs induces cell death by apoptosis. We found that a serine protease inhibitor, N-tosyl-L-lysine chloromethyl ketone (TLCK), dramatically enhances Fas-mediated apoptosis in the human T cell line Jurkat and in various B cell lines resistant to Fas-mediated apoptosis. The enhancing effect of TLCK is specific to Fas-induced cell death, with no effect seen on TNF-␣ or TNF-related apoptosis-inducing ligand-induced apoptosis. TLCK treatment had no effect on Fas expression levels on the cell surface, and neither promoted death-inducing signaling complex formation nor decreased expression levels of cellular inhibitors of apoptosis (FLICE inhibitory protein, X chromosome-linked inhibitor of apoptosis, and Bcl-2). Activation of the Fas-mediated apoptotic pathway by anti-Fas Ab is accompanied by aggregation of Fas molecules to form oligomers that are stable to boiling in SDS and ␤-ME. Fas aggregation is often considered to be required for Fas-mediated apoptosis. However, sensitization of cells to Fas-mediated apoptosis by TLCK or other agents (cycloheximide, protein kinase C inhibitors) causes less Fas aggregation during the apoptotic process compared with that in nonsensitized cells. These results show that Fas aggregation and Fas-mediated apoptosis are not directly correlated and may even be inversely correlated. The Journal of Immunology, 2001, 167: 82– 89.


as (APO-1/CD95) is a transmembrane protein that belongs to the TNFR superfamily (1). Cross-linking of surface Fas molecules by the Fas ligand or agonistic anti-Fas Abs activates apoptotic death programs (1, 2). Defects in the expression of the Fas gene or impairments of Fas-linked signaling contribute to a variety of severe disorders associated with lymphoproliferation, inflammation, and autoimmunity (2, 3). The contribution of the Fas-mediated cell death pathway to controlling peripheral T cell immune responses is well characterized (3– 6). Fas is also involved in the elimination of self-Ag-activated B cells in the germinal center (7–9). Germline Fas mutations result in autoimmune lymphoproliferative disorders (10, 11). Fas activates apoptosis through a series of protein-protein interactions associated with the intracellular domain of the receptor. Upon Fas stimulation, a set of effector proteins is recruited, forming the death-inducing signaling complex (DISC)2 (12). This complex contains the Fas-associated death domain protein (FADD) (13, 14), which binds to Fas by interacting with a cytoplasmic portion of Fas known as the death domain (15). The complex also contains FLICE (FADD-like IL-1␤-convert-

Laboratory of Immunology, Division of Therapeutic Proteins, Food and Drug Administration, Center for Biologics and Evaluation and Research, Bethesda, MD 20892 Received for publication December 1, 2000. Accepted for publication April 16, 2001. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Address correspondence and reprint requests to Dr. Yang-ja Lee, Biochemistry Section, Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Building 10, Room 4N258, Bethesda, MD 20892. E-mail address: [email protected] 2 Abbreviations used in this paper: DISC, death-inducing signaling complex; TLCK, N-tosyl-L-lysine chloromethyl ketone; TPCK, N-tosyl-L-phenylalanine chloromethyl ketone; CHX, cycloheximide; VP-16, etoposide; FADD, Fas-associated death domain protein; FLICE, FADD-like IL-1␤-converting enzyme; PARP, poly(ADP-ribose) polymerase; TRAIL, TNF-related apoptosis-inducing ligand; FLIP, FLICE-inhibitory protein; IAP, inhibitor of apoptosis; XIAP, X chromosome-linked IAP; PKC, protein kinase C; PI, propidium iodide; NLVS, 4-hydroxy-5-iodo-3-nitrophenylacetyl-LeuLeu-Leu-vinylsulfone; BIM, bisindolylmaleimide; LLnL, N-acetyl-L-leucinal-L-leucinal-L-norleucinal; BD-FMK, Boc-Asp-fluoromethyl ketone.

Copyright © 2001 by The American Association of Immunologists

ing enzyme) (16, 17), which binds to FADD through its death effector domain (18). FLICE, also known as caspase-8, is activated in the DISC and serves as the initiator caspase for the proteolytic cascade that leads to the ultimate apoptotic dismantling of the cell (16 –18). Fas-mediated apoptosis is regulated in part by the levels of expression of Fas and Fas ligand (19 –21). Differences in cell susceptibilities to Fas-mediated apoptosis are also controlled by the regulation of signaling cascades, since not all Fas-positive cell types undergo apoptosis after stimulation of Fas (22–24). Activation of the serine/threonine protein kinase protein kinase C (PKC) by phorbol ester has been reported to repress apoptosis in several cell systems, which suggests that protein phosphorylation could also play a role in the regulation of Fas-induced apoptosis (25–28). In addition, the protein synthesis inhibitor cycloheximide (CHX) is known to sensitize cells to Fas-induced apoptosis (24, 29, 30), suggesting that resistance to Fasmediated cell death is mediated through short-lived proteins that may block Fas signaling pathways. One aspect of Fas signaling that has been less thoroughly studied is the possible role of aggregation of the receptor protein. In this report we show that N-tosyl-L-lysine chloromethyl ketone (TLCK), a serine protease inhibitor, sensitizes Jurkat T cells and various B cell lines to Fas-mediated cell death. The pro-apoptotic effect of TLCK is specific to Fas-induced cell death and is accompanied by modification of the Fas death domain. In untreated cells the Fas Ag exists as monomers, and upon receptor ligation Fas aggregates to form a high m.w. complex (12, 26, 31). We found that Ab-induced aggregation of Fas Ag was greatly reduced or unstable in the presence of TLCK despite the fact that a majority of cells underwent apoptosis. Other compounds, such as CHX and PKC inhibitors, which enhanced Fas-mediated apoptosis, also inhibited Fas aggregation. Even though receptor polymerization is believed to be necessary for the recruitment of signaling molecules (12, 31, 32), our finding suggests that death signals can be transduced with minimal or no aggregation of Fas receptor. 0022-1767/01/$02.00

The Journal of Immunology

Materials and Methods Cell lines and culture conditions The human leukemic T cell line Jurkat (clone E6-1) was obtained from the American Type Culture Collection (Manassas, VA). The Burkitt’s lymphoma cell lines JLP-119, ST-486, BL-41, and BJAB were provided by Dr. K. Bhatia (National Cancer Institute, National Institutes of Health, Bethesda, MD). The lymphoblastoid cell line VDSO was obtained from Dr. G. Tosato (Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD). All cell lines were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, and 50 ␮M 2-ME at 37°C in 5% CO2 in air.

Antibodies CH11, a mouse mAb (IgM) specific for an epitope on the extracellular domain of human Fas, was purchased from MBL International (Watertown, MA). PE-conjugated DX2, a mouse mAb (IgG1) specific for an epitope on the extracellular domain of human Fas, was obtained from PharMingen (San Diego, CA). 3D5, a mouse mAb (IgG1) specific for the intracellular death domain of human Fas, was purchased from Alexis Biochemicals (San Diego, CA). Fas/C20, rabbit antiserum recognizing the carboxyl-terminus of Fas (aa 300 –319) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-caspase-3 (mAb) and anti-X chromosome-linked IAP (anti-XIAP) mAb were obtained from Transduction Laboratories (Lexington, KY), anti-poly(ADP-ribose) polymerase (antiPARP) was purchased from Biomol (Plymouth Meeting, PA), anticaspase-8 mAb and anti-inhibitor of apoptosis (anti-IAP) mAb were obtained from PharMingen, and anti-FLICE inhibitory protein (anti-FLIP) mAb was purchased from Alexis Biochemicals. Anti-Bcl-2 (mAb) was obtained from Dako (Carpinteria, CA), and anti-Bcl-xL (polyclonal) was purchased from Santa Cruz Biotechnology. HRP- or PEconjugated secondary Abs were obtained from Southern Biotechnology (Birmingham, AL).

Morphological assessment of apoptosis using nuclear staining and fluorescence microscopy Cells were stained with Hoechst 33342 and propidium iodide (PI) and visualized using fluorescence microscopy as described previously (33, 34). A minimum of 200 cells were counted per sample and were classified as follows: 1) live cells (normal nuclei, blue chromatin with organized structure); 2) membrane-intact apoptotic cells (bright blue chromatin that is highly condensed, marginated, or fragmented); 3) necrotic cells (red, enlarged nuclei with smooth normal structure); and 4) membrane-permeable apoptotic cells (bright red chromatin, highly condensed or fragmented). The extent of apoptosis was calculated as the percentage of total apoptosis (classes 2 and 4) divided by the total number of cells counted.

Western blot analysis

83 gand (TRAIL) was obtained from U.S. Biological (Swanpscott, MA), and GO6976 and 4-hydroxy-5-iodo-3-nitrophenylacetyl-Leu-Leu-Leu-vinylsulfone (NLVS) were obtained from Calbiochem (San Diego, CA). PMA, bisindolylmaleimide I (BIM I) and etoposide (VP-16) were obtained from Sigma (St. Louis, MO). BIM VIII and N-acetyl-L-leucinal-L-leucinal-Lnorleucinal (LLnL; calpain inhibitor I) were purchased from Alexis Biochemicals. Boc-Asp-fluoromethyl ketone (BD-FMK) was obtained from Enzyme Systems Products (Dublin, CA).

Results TLCK sensitizes Jurkat cells to Fas-mediated apoptosis The human T cell line Jurkat has been widely used as a model to study Fas-mediated apoptosis (25–27, 31, 36 – 40). When Jurkat cells are treated with an agonistic anti-Fas Ab (CH11), they undergo a moderate degree of apoptosis. CHX is known to enhance Fas- or TNFR-mediated cell death in a variety of cells, but the mechanism of this effect is not known (24, 29). In addition to CHX, we found that a serine protease inhibitor, TLCK, dramatically enhances Fas-mediated cell death in Jurkat cells. As shown in Fig. 1, the enhancing effect of TLCK is specific to Fas-mediated cell death; it does not affect TNF-␣- or TRAIL-induced cell death and protects cells from etoposide (VP-16)-induced apoptosis. Thus, TLCK differs from CHX, which enhances both anti-Fas and TNF-␣-induced apoptosis and moderately enhances TRAIL-induced cell death. TLCK greatly accelerates the time-frame of antiFas-induced apoptosis, and it renders Jurkat cells responsive to much lower concentrations of anti-Fas. As shown in Fig. 2A, after 5 h of treatment with 50 ng/ml anti-Fas Ab, 46% of Jurkat cells die by apoptosis, but 5 and 10 ng/ml anti-Fas Ab alone induce no apoptosis. In the presence of TLCK (50 ␮M), 60 – 80% of Jurkat cells undergo apoptosis in response to 5–10 ng/ml anti-Fas, and 100% of cells undergo apoptosis in response to 50 ng/ml anti-Fas. The data in Fig. 2, A and B, also show the effect of TLCK on the rate of apoptosis in response to anti-Fas; within 30 min of treatment with TLCK and 50 ng/ml anti-Fas, 20% of cells show apoptotic features (condensed or fragmented nuclei), and by 2 h ⬎90% of cells have undergone apoptosis (Fig. 2B). This is in contrast to the slow apoptosis induced by anti-Fas alone. The percentage of apoptosis in Fig. 2, A and B, is assessed by staining cells with the nuclear dyes Hoechst 33342 and PI and counting apoptotic nuclei by fluorescent microscopy as described previously (33,

Total cell lysates were prepared by heating cells at 100°C for 10 min (typically 5 ⫻ 106 cells in 50 ␮l Laemmli buffer (35) without ␤-ME. Protein concentrations of lysates were determined using Bio-Rad protein assay reagent (Hercules, CA). Before SDS-PAGE, 2-ME (5%, v/v) was added, and cell lysates were heated for 3 min at 75°C. The total cell lysates (20 ␮g/lane, unless stated otherwise) were subjected to SDS-PAGE (8 – 16% polyacrylamide, gels from NOVEX, San Diego, CA). Proteins were then transferred electrophoretically to a polyvinylidene difluoride membrane (Immobilon-P; Millipore, Bedford, MA). The membrane was blocked at room temperature for 1 h with TBS-T (20 mM Tris (pH 7.4), 150 mM NaCl, and 0.05% Tween 20) containing 5% dry milk, incubated with a primary Ab in TBS-T containing 5% dry milk for 1 h at room temperature or overnight at 4°C, and washed for 10 min with TBS-T three times. The membrane was then incubated with HRP-conjugated secondary Ab for 1 h at room temperature. Bands were visualized by chemiluminescence using the ECL kit from NEN (Boston, MA).

Flow cytometric analysis Control or TLCK (50 ␮M)-treated cells (1– 4 ⫻ 106 cells) were incubated with PE-conjugated DX2 (mouse mAb against human Fas, 20 ␮l/106 cells) for 30 min on ice. After fixation with 2.5% formaldehyde, cells were analyzed using FACScan flow cytometry equipped with a CellQuest data analysis program (BD Biosciences, San Jose, CA). PE-conjugated antimouse IgG1 was used as a control for nonspecific staining.

Chemicals TLCK and tosyl-L-phenylalanine chloromethyl ketone (TPCK) were purchased from Roche (Indianapolis, IN). TNF-related apoptosis-inducing li-

FIGURE 1. TLCK sensitizes Jurkat cells to Fas-mediated, but not TNF␣-, TRAIL-, or VP-16-induced apoptosis. Jurkat cells (5 ⫻ 105/ml) were pretreated with TLCK (50 ␮M) or CHX (1 ␮g/ml) for 30 min, followed by addition of anti-Fas Ab (CH11, 100 ng/ml), TNF-␣ (80 ng/ml), TRAIL (100 ng/ml), or VP-16 (50 ␮g/ml). After incubation for 1.5 h with anti-Fas or TNF-␣ or for 4 h with TRAIL or VP-16 at 37°C, cell death was assessed by nuclear morphology using Hoechst/PI staining and fluorescence microscopy (mean ⫾ SD of three separate experiments).


Fas AGGREGATION DOES NOT CORRELATE WITH Fas-MEDIATED APOPTOSIS 34). This morphological evidence for apoptosis correlates with the activation of the caspase cascade (Fig. 2, C–E). Total cell lysates of Jurkat cells treated with various concentrations of anti-Fas in the presence or the absence of TLCK for 0.5– 4 h were analyzed by Western blotting using Abs raised against human caspase-8 (Fig. 2C), caspase-3 (Fig. 2D), and PARP (Fig. 2E). Anti-Fas Ab at 50 ng/ml alone causes a slow decrease in procaspase-8, but in the presence of TLCK even 10 ng/ml anti-Fas causes extensive cleavage, and with 50 ng/ml anti-Fas all the procaspase-8 is cleaved within 2 h (Fig. 2C). Activation of caspase-3 is measured by cleavage of procaspase-3 and PARP, a substrate for caspase-3. As shown in Fig. 2, D and E, cotreatment with TLCK and anti-Fas causes accelerated caspase-3 activation. TLCK alone causes no cleavage of these proteins (not shown). There is a difference in cleavage patterns of caspase-3 in cells treated with anti-Fas alone or with anti-Fas and TLCK; treatment with anti-Fas alone appears to proceed directly to formation of the active p17 subunit, but with anti-Fas plus TLCK, the p20 fragment of caspase-3 is formed first and then converted into the active p17 subunit. The significance of this difference will be discussed later. TLCK sensitizes B cells to Fas-mediated apoptosis

FIGURE 2. Time course of apoptosis in Jurkat cells induced by antiFas Ab with or without TLCK. Jurkat cells (5 ⫻ 105/ml) were treated with different concentrations of anti-Fas Ab (CH11; 0, 5, 10, and 50 ng/ml) with or without TLCK (50 ␮M) for 0.5– 4 h at 37°C. Cells were collected at the times indicated, and apoptosis was assessed by nuclear morphology (A and B; mean ⫾ SD of at least three experiments). Total cell lysates were also prepared and analyzed for activation (cleavage) of caspase-8 (C), caspase-3 (D), and PARP (E) by Western blot immunoassay. Data are from a representative study of three separate experiments. kD, Kilodaltons.

FIGURE 3. TLCK potentiates Fas-mediated apoptosis in B cell lines. Jurkat and B cells (BJAB, VDSO, JLP-119, ST-486, and BL-41; 5 ⫻ 105/ml) were treated with anti-Fas (100 ng/ml) in the presence or the absence of TLCK (50 ␮M). After 4 h (A) and 22 h (B) apoptosis was assessed by nuclear morphology. C, Cells (Jurkat, BJAB, and VDSO) were incubated with various concentrations of anti-Fas (0, 10, 50, 100, and 250 ng/ml) in the presence (F) or the absence (E) of TLCK for 4 h and analyzed for apoptosis by nuclear morphology. Results are the mean ⫾ SD of three separate experiments.

The sensitization of cells to Fas-mediated apoptosis by TLCK is also seen in cells of the B lymphocytic lineage. Several Burkitt’s lymphoma cell lines (JLP-119, ST-486, BL-41, and BJAB) and a B lymphoblastoid cell line (VDSO) were treated with 100 ng/ml anti-Fas Ab in the absence or the presence of TLCK (50 ␮M) for 4 or 22 h, and apoptosis was assessed by nuclear morphology. Cells incubated without anti-Fas Ab and without TLCK were used as controls. As shown in Fig. 3, all B cell lines examined were relatively unresponsive to apoptosis induced by anti-Fas alone (compared with Jurkat cells under the same conditions). In the presence of TLCK, however, anti-Fas induced significant apoptosis in all B cell lines. TLCK (50 ␮M) alone induced minimal cell

The Journal of Immunology death. JLP-119, ST-486, and BL-41 showed less TLCK sensitization than the other three cell lines in the 4-h incubation period. Much greater TLCK-enhanced apoptosis was seen in these three cell lines in an overnight (22-h) incubation (Fig. 3B), while apoptosis induced by anti-Fas Ab alone (100 ng/ml) occurred in ⬍10% of the cells. The amount of anti-Fas Ab (50 –100 ng/ml) was not a limiting factor for induction of apoptosis in these cell lines, since 250 ng/ml anti-Fas Ab did not induce more apoptosis than 100 ng/ml in any cell line tested (Fig. 3C; data for JLP-119, ST-486, and BL-41 not shown). Sensitivity to anti-Fas Ab does not correlate with levels of Fas Ag expression, and TLCK has no effect on Fas expression levels The amount of Fas Ag on the cell surface was measured by flow cytometry using an mAb that recognizes the extracellular domain of Fas (DX2). The total amount of Fas Ag in cells was assessed by Western blot immunoassay using two different Abs that recognize the cytoplasmic domain of Fas; one is an mAb (clone 3D5) that recognizes an epitope in the death domain of Fas (31), and the other is a rabbit polyclonal antiserum that recognizes the carboxylterminal 20 aa of Fas (Fas/C-20). As shown in Fig. 4, A and B, the relative levels of Fas Ag detected by flow cytometry closely parallel those detected by Western blotting. Note that 4 times more

85 protein was loaded for JLP-119, ST-486, and BL-41 than for Jurkat, BJAB, and VDSO. Fas Ag expression in the EBV-negative Burkitt’s lymphoma cells (JLP-119, ST-486, and BL-41) was low, and these cells were resistant to anti-Fas-induced apoptosis, but the sensitivity to anti-Fas did not strictly correlate with the expression level of Fas Ag. BJAB and VDSO expressed higher levels of Fas than Jurkat, but were less sensitive to Fas-mediated apoptosis than Jurkat. In addition, the Burkitt’s lymphoma cells that expressed low levels of Fas could undergo significant apoptosis upon exposure to anti-Fas Ab if TLCK was also present (see Fig. 3). Fig. 4A also shows that TLCK minimally affect Fas expression levels on the cell surface of these cell lines. Note that TLCK did not significantly affect levels of expression of the intracellular domain of Fas, as shown by reactivity with the polyclonal Ab C20 (Fig. 4C). However, in each of these cell lines, reactivity to mAb 3D5, directed against the death domain of Fas, was decreased after treatment with TLCK, raising the possibility that TLCK might be involved in modification of the death domain of Fas. TLCK neither promotes DISC formation nor decreases expression of cellular apoptosis inhibitors Jurkat is a type II cell that shows little or no DISC formation compared with type I cells (41). To determine whether TLCK acts by enhancing DISC formation, studies were conducted to measure possible increased formation of DISC complexes in Jurkat cells treated with anti-Fas in the presence or the absence of TLCK. Using the methods described previously (12, 41), we found no evidence of DISC complexes even in cells pretreated with TLCK, suggesting that TLCK did not act by promoting DISC formation. We also examined the possibility that TLCK acts by decreasing the expression of endogenous inhibitors of cell death. A variety of cellular inhibitors of apoptosis have been described, such as FLIP (42), IAP (43), and XIAP (44), in addition to Bcl-2 family members. We tested the effects of TLCK on expression levels of these inhibitors (FLIP, IAP, XIAP, Bcl-2, and Bcl-xL). Jurkat cells were treated with anti-Fas Ab (CH 11, 50 ng/ml) alone or with TLCK (50 ␮M) for 0, 1, 2, 3, and 4 h. At each time point, total cell lysates were prepared and analyzed by Western blot immunoassay. Bcl-xL and IAP protein levels were too low to be detected under any condition. Results for the other anti-apoptotic proteins are shown in Fig. 5. No significant effects of TLCK on expression levels of these proteins were observed, with the exception of XIAP, which was cleaved slightly more when cells were treated with TLCK plus anti-Fas compared with anti-Fas alone. The late appearance and low level of the cleaved forms of XIAP suggest that they are probably a consequence, and not a cause, of the increased apoptosis occurring in the cells. That is, XIAP inhibits caspase activity, but it is also cleaved by caspases (45). Treating cells with TLCK alone produced no detectable cleavage of XIAP (data not shown). Cumulatively, the data suggest that the mechanism by which TLCK sensitizes cells to Fas-mediated cell death is not through loss of, or decrease in, these endogenous inhibitors of apoptosis. TLCK decreases activation-induced aggregation of Fas Ag

FIGURE 4. Effects of TLCK on Fas expression in Jurkat cells and different B cell lines. A, FACS analysis of Fas expression using PE-conjugated anti-extracellular domain Ab (DX2). Filled, Fas expression in resting (untreated) cells; solid line, Fas expression in cells treated with TLCK (50 ␮M) for 3 h; dotted line, background staining with PE-conjugated antimouse IgG. Scales are the same in all panels, and the mean anti-Fas fluorescence intensity is given in each panel. B, Western blot analysis of Fas expression in resting cells using anti-cytoplasmic domain Ab (3D5). C, Western blot analysis of Fas expression in control or TLCK-treated cells using anti-cytoplasmic domain Abs (mAb 3D5 and polyclonal Ab C20).

It has been reported that the Fas Ag, which exists as monomers in resting cells, aggregates to form high Mr complexes upon receptor ligation (12, 31). The appearance of Fas aggregates seems to coincide with the formation of the signaling complex (DISC), but the physiological relevance of Fas aggregates has not been demonstrated clearly. To determine whether TLCK enhances apoptosis through an effect on Fas aggregation, the following studies were conducted with Jurkat cells. Cells were activated with anti-Fas Ab as described for Fig. 2, and at various times after activation cell lysates were prepared. The anti-cytoplasmic domain mAb (3D5)



FIGURE 5. Effect of TLCK on expression of cellular inhibitors of apoptosis. Jurkat cells (5 ⫻ 105/ml) were incubated with anti-Fas Ab (CH 11, 50 ng/ml) alone or with TLCK (50 ␮M) for 0, 1, 2, 3, and 4 h. At each time point, total cell lysates from 5 ⫻ 106 cells were prepared and analyzed for XIAP (A), FLIPs/L (B), and Bcl-2 (C) by Western blot immunoassay. Data are from a representative study that was reproduced at least twice. kD, Kilodaltons.

was used for detection of Fas protein. As shown in Fig. 6A, stimulation of Jurkat cells with anti-Fas Ab (CH11, 50 ng/ml) induced the formation of SDS- and 2-ME-stable, high Mr aggregates (⬎200 kDa) of Fas Ag, as reported by others (26, 31). Formation of the high Mr Fas aggregates increased with time and required ligation of the Fas Ag (no aggregation in control cells). In the presence of TLCK, anti-Fas-induced receptor aggregation was significantly reduced even in the face of extensive apoptosis (90% in 2 h; see Fig. 2). In the absence of TLCK, the high Mr Fas aggregates formed by ligation of Fas Ag were stable (persisted for 4 h), but in the presence of TLCK, the few aggregates that were formed were processed into smaller species (Fig. 6A). A similar result was obtained using a polyclonal Ab to screen for Fas aggregates (Fig. 6B). The inhibition of activation-induced Fas aggregation by TLCK is not unique to Jurkat T cells. As shown in Fig. 7A, treatment of BJAB cells with anti-Fas Ab alone did not induce any significant apoptosis over 6 h, yet extensive Fas aggregation was observed (Fig. 7B). Cotreatment of cells with anti-Fas Ab and TLCK induced apoptosis in 60% of cells after a 4-h incubation, but in this case relatively little aggregation of Fas was seen (Fig. 7B). These results suggest that extensive Fas aggregation is not required for transduction of death signals from Fas receptor ligation. Note that there was an overall decrease in binding to the death domainspecific mAb 3D5 in TLCK-treated cells (Fig. 7B, top). That the total amount of Fas was unaltered is shown using the polyclonal Ab C20 (Fig. 7B, bottom). Other agents that enhance Fas-mediated apoptosis also inhibit activation-induced Fas aggregation CHX sensitizes a variety of cells to Fas- and TNFR-mediated cell death (24, 29, 30) (Fig. 1). On the other hand, PKC activation is suggested to inhibit Fas-mediated cell death, and some PKC inhibitors have been reported to enhance Fas-mediated cell killing (25–28). Experiments were conducted to determine whether these agents may also act by modulating Fas aggregation. The agents tested included CHX; TPCK (serine protease inhibitor); the PKC inhibitors bisindolylmaleimide (BIM) I, BIM VIII, and GO6976;

FIGURE 6. Time course of activation-induced aggregation of the Fas-Ag and the effect of TLCK on the formation of aggregates in Jurkat cells. Jurkat cells (5 ⫻ 105/ml) were incubated with anti-Fas Ab (CH 11, 10 or 50 ng/ml) alone or with TLCK (50 ␮M) for 0, 1, 2, 3, and 4 h. At each time point total cell lysates from 5 ⫻ 106 cells were prepared and analyzed for Fas Ag by Western blot immunoassay using mAb 3D5 (A) or polyclonal Ab C20 (B). Data are from a representative study that was repeated at least three times.

the proteasome inhibitors LLnL and NLVS; and the caspase inhibitor, BD-FMK. The phorbol ester PMA, a PKC activator, was also tested. Each agent was used at a concentration that induced no cell death by itself (not shown). The effects of these agents on Fas-mediated apoptosis are shown in Fig. 8. The broad spectrum caspase inhibitor, BD-FMK, reduced Fas-mediated cell death to background levels, and PMA inhibited Fas-mediated cell death by 50%. CHX was comparable in magnitude to TLCK in its ability to sensitize cells to Fas-induced cell death. The PKC inhibitors, BIM I, BIM VIII, and GO6976 (not shown), enhanced Fas-mediated cell death. TPCK also enhanced cell death induced by anti-Fas Ab, while the proteasome inhibitors, LLnL and NLVS (not shown), had no effect on Fas-induced cell death. As shown in Fig. 8B, the results of morphological assay for apoptosis correlate with the degree of cleavage of caspase-8, caspase-3, and PARP. To determine whether there is any correlation between Fas aggregation and sensitivity to Fas-induced apoptosis, the aggregation of Fas Ag after stimulation by anti-Fas Ab in the presence of these agents was examined (Fig. 9). We found that all the agents that enhanced Fas-mediated apoptosis (e.g., CHX, TPCK, BIM I, BIM VIII, and GO6976) inhibited activation-induced aggregation of Fas Ag, while the agents that had no effect (e.g., NLVS and LLnL) or had an inhibitory effect (BD-FMK and PMA) on Fas-mediated apoptosis had no effect on activation-induced aggregation of Fas Ag. Quantification of the aggregates was accomplished by densitometry and is shown in Fig. 9B.

The Journal of Immunology

FIGURE 7. Effect of TLCK on activation-induced cell death and aggregation of the Fas-Ag in BJAB cells. BJAB cells (5 ⫻ 105/ml) were incubated with anti-Fas Ab (50 ng/ml) in the presence or the absence of TLCK (50 ␮M) for 0.5– 6 h. At the indicated time points, apoptosis was assessed by nuclear morphology (A) (average of two separate experiments), or total cell lysates were prepared and the activation-induced changes in Fas Ag were analyzed by Western blotting using mAb 3D5 or polyclonal Ab C20 (B). Equal amounts of total protein (20 ␮g) were loaded in each lane. Data are from a representative study that was repeated twice.

Discussion Fas-mediated apoptosis can be regulated by the levels of expression of Fas or Fas ligand, but there is not always a good correlation between the presence of Fas and apoptosis in response to Fas ligand (Refs. 22–24 and this study), indicating that intracellular signaling pathways associated with Fas-mediated apoptosis are essential for determining whether cells undergo apoptosis after activation of Fas. In this report cells resistant to Fas-mediated apoptosis were rendered sensitive by pretreating them with TLCK, which not only overcame the almost complete resistance to Fasmediated apoptosis found in Burkitt’s lymphoma and B lymphoblastoid cells, but also potentiated weak to moderate apoptotic signals generated by a low dose of anti-Fas Ab in Jurkat T cells. TLCK seems to facilitate intracellular signaling initiated by activation of Fas without changing the level of Fas expression, and this facilitation occurs in cells with a wide range of sensitivities to Fas-mediated apoptosis. After cross-linking with an agonistic Ab, SDS- and 2-ME-stable high molecular mass Fas aggregates (⬎200 kDa) are detected by Abs against either the death domain (3D5) or the carboxyl terminus (C20) of the Fas protein. SDS-stable forms of aggregated Fas have also been reported by other groups (12, 26, 31). The formation of these aggregates by ligation of Fas receptor increases with time, and in the absence of TLCK, the aggregates are stable. In the presence of TLCK, however, the formation of Fas aggregates is


FIGURE 8. Effects of various modulators on Fas-mediated apoptosis in Jurkat cells. Jurkat cells (5 ⫻ 105/ml) were preincubated with BD-FMK (50 ␮M), CHX (1 ␮M), TLCK (50 ␮M), TPCK (2 ␮M), LLnL (50 ␮M), PMA (50 nM), BIM I (10 ␮M), or BIM VIII (1 ␮M) for 30 min before anti-Fas (CH11) was added (50 ng/ml). After a 3-h incubation with antiFas, cell death was assessed by nuclear morphology (A; mean ⫾ SD of at least three separate experiments), and total cell lysates were prepared and analyzed for activation (cleavage) of caspase-8, caspase-3, and PARP by Western blot immunoassay (B). Data are from a representative study that was repeated at least three times. kD, Kilodaltons.

greatly diminished, and the aggregates are not stable, i.e., they degrade into smaller species. Yet, these cells with fewer aggregates undergo massive apoptosis. Receptor oligomerization or aggregation has been thought to be necessary for transmission of apoptotic signals (12, 31, 32, 46, 47). However, our results suggest that intensive aggregation is not required, and may even interfere with transduction of death signals caused by Fas receptor ligation. While the molecular structure of the aggregates is not well understood, our study clearly shows that there is an inverse correlation between the amount of aggregate formation and the level of cell death: more aggregate formation is associated with less cell death. This raises the intriguing possibility that the formation of aggregates might be a defense system that prevents excess death in response to Fas ligation. The mechanism by which TLCK decreases Fas aggregation and enhances Fas-mediated apoptosis is not known, but the decreased binding of an mAb (3D5) directed against an epitope in the death domain of Fas suggests that TLCK might act by modifying the death domain of Fas. Involvement of PKC in Fas signaling has been suggested (25– 28); activation of PKC antagonizes Fas-mediated apoptosis and inhibitors of PKC enhance the susceptibility of cells to Fas-mediated apoptosis. We thought that TLCK may sensitize cells to Fasmediated apoptosis through inhibition of PKC. In fact, one report has shown that TLCK can inhibit PKC (48). However, unlike authentic PKC inhibitors (BIM I and GO6976), the enhancing effect of TLCK on Fas-mediated apoptosis is not prevented by cotreatment with the PKC activator PMA (data not shown). In addition,


Fas AGGREGATION DOES NOT CORRELATE WITH Fas-MEDIATED APOPTOSIS that TLCK acts by converting cells from a type II to a type I apoptotic signaling pathway. Additional experiments are required to verify that this is, in fact, the case.

Acknowledgments We are grateful to Giovanna Tosato, Joy Williams, Richard Youle, Howard Anderson, Caroline Maylock, and Steven Wood for careful reading of the manuscript and for many useful suggestions.


FIGURE 9. Effects of various modulators on activation-induced aggregation of Fas Ag in Jurkat cells. Jurkat cells were treated as described in Fig. 7, and the same total cell lysates were used for analysis of Fas proteins. A, Western blot immunoassay for Fas-Ag using mAb 3D5. B, Intensities of monomers and aggregates in each lane were analyzed using the Macintosh densitometry program IMAGE (National Institutes of Health), and the percentage of polymerization was calculated as Fas aggregates/total Fas (monomers ⫹ aggregates). Data are from a representative study that was repeated twice.

we found no evidence for inhibition of PKC activity by TLCK under our experimental conditions (data not shown). Two different signaling pathways downstream of Fas have been proposed (41). As has been discussed, T cells can die by different routes, and the shift from resistance to sensitivity to apoptosis coincides with a shift from a type II to a type I pathway of apoptosis (3). In type I signaling, the death signal is propagated by a caspase cascade initiated by the activation of caspase-8 at the DISC. This is followed by a rapid cleavage of caspase-3 and other caspases, which, in turn, cleave vital substrates in the cell. In type II signaling, very little DISC is formed, and the caspase cascade is, instead, amplified by the mitochondria. Jurkat cells are considered to be type II cells in which DISC formation is low (41). What prevents FADD from associating with activated Fas in type II cells is currently unknown. One possibility is the presence of a blocking protein that binds to Fas only in type II cells, such as the 120-kDa protein described by Scaffidi et al. (41). Does TLCK treatment convert type II cells (like Jurkat) to type I cells? Several pieces of data suggest that this may be the case. The rates of caspase-8 and caspase-3 activation are accelerated by TLCK, and the cleavage pattern of the caspase-3 is changed (Fig. 2). In the presence of TLCK, the initial cleavage of procaspase-3 generates a p20 subunit that is slowly converted into the active p17 subunit, typical of type I cells, while without TLCK the active subunit p17 is formed without the p20 intermediate, typical of type II cells. In addition, TLCK enhances Fas-mediated apoptosis, but inhibits VP-16-induced apoptosis (Fig. 1), which proceeds solely through the mitochondrial pathway (38 – 40). Thus, it is unlikely that TLCK is simply increasing the upstream part of the Fas pathway, since this would be neutralized by its inhibitory effect on the mitochondrial amplification step. Taken together, these findings support the hypothesis

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