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J Biol Chem, Vol. 275, Issue 12, 8610-8617, March 24, 2000

Tumor Necrosis Factor- and Fas Activate Complementary Fas-associated Death Domain-dependent Pathways That Enhance Apoptosis Induced by -Irradiation^*

Kotohiko Kimura and Edward P. Gelmann

From the Department of Oncology, Lombardi Cancer Center, Georgetown University, Washington, D.C. 20007-2197

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Activation of either tumor necrosis factor receptor 1 or Fas induces a low level of programmed cell death in LNCaP human prostate cancer cells. We have shown that LNCaP cells are entirely resistant to -radiation-induced apoptosis, but can be sensitized to irradiation by TNF-. Fas activation also sensitized LNCaP cells to irradiation, causing nearly 40% cell death 72 h after irradiation. Caspase-8 was cleaved and activated after exposure to tumor necrosis factor (TNF)-. However, after exposure to anti-Fas antibody caspase-8 cleavage occurred only between the 26-kDa N-terminal prodomain and the 28-kDa C-terminal region that contains the protease components. Although anti-Fas antibody plus irradiation induced apoptosis that could be blocked by the pancaspase inhibitor zVAD, there was no measurable caspase-8 activity after exposure to anti-Fas antibody. The effector caspases-6 and -7, and to a lesser extent caspase-3, were activated by TNF-, but not by anti-Fas antibody. Anti-Fas antibody, like TNF- also activated serine proteases that contributed to cell death. Exposure of LNCaP cells simultaneously to TNF- and anti-Fas antibody CH-11 resulted in marked enhancement of apoptosis that occurred very rapidly and was still further augmented by irradiation. Rapid apoptosis that ensued from combined treatment with TNF-, anti-Fas antibody, and irradiation was completely blocked either by zVAD or expression of dominant negative Fas-associated death domain. Our data shows that there are qualitative differences in caspase activation resulting from either TNF receptor 1 or Fas. Simultaneous activation of these receptors was synergistic and caused rapid epithelial cell apoptosis mediated by the caspase cascade.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Programmed cell death is critical for normal tissue homeostasis and control of cell growth. Genetic disruptions of cell death pathways can be oncogenic (1, 2). In addition, cancer cells develop resistance to programmed cell death as a mechanism of chemotherapy and radiation resistance. Strategies to restore the cell death response to chemotherapy or radiation may reverse treatment resistance in some cancers. The prostate cancer cell line LNCaP, like most clinical prostate cancers, is dependent on androgens for growth and, like tumor tissue synthesizes the tissue-specific prostate-specific antigen, a serine protease (3-6). The hormone dependence of LNCaP cells is reminiscent of early stage prostate cancer, but the cells share characteristics of advanced prostate cancers in that they do not undergo apoptosis in response to either androgen deprivation or -irradiation (7-10). We have shown that TNF-¹ or C₂-ceramide, at doses that, by themselves, induce little or no cell death, sensitize LNCaP cells to irradiation so that combined treatment with either TNF- and irradiation or C₂-ceramide and irradiation induces death of the cultures (11). We have now studied the effect of Fas activation on the sensitivity of LNCaP cells to radiation-induced cell death. We have also found that Fas and TNF- have complementary effects on the activation of cell death pathways in LNCaP and together induced a markedly accelerated cell death response compared with activation of either TNFR-1 or Fas alone.

TNF- is an inflammatory cytokine that can induce a diverse range of biological responses (12, 13). Signaling by TNF- is initiated by binding to TNFR-1, which causes the association of an adapter protein TRADD with the intracellular death domain of the TNFR-1 molecule (14). TRADD mediates the subsequent recruitment of an adapter protein FADD to form a death-inducing signaling complex, which initiates apoptosis through activation of caspase-8, a proximal element in the cascade of cysteine proteases (15-21). Caspase-8 in turn induces activation of caspase-3 and caspase-7, which cleave PARP, and of caspase-6 that cleaves lamin B thus contributing to dissolution of the nuclear envelope (22-25). Signaling by TNF- also activates anti-apoptotic cell signals via activation of the transcription factor NFB that may override the effects of apoptosis pathways in some cells (26-28).

Signaling through Fas is thought to be less complex than signaling though TNFR-1. Fas is a cell surface receptor that is activated by binding of Fas ligand or agonistic anti-Fas antibody (29). Fas receptor interacts with FADD to activate caspase-8 and, subsequently, the effector caspases-3, -7, and -6 (30, 31). Unlike TNF- receptor, Fas is not linked to activation of NFB, and therefore is a pure death receptor that lacks antagonistic anti-apoptotic signaling.

In LNCaP cells TNF- induces activation of caspases-8, -6, and -7, appearance of classical DNA fragmentation ladders and 20% cell death within 72 h after treatment. This effect can be blocked by the pancaspase inhibitor zVAD. In the presence of TNF-, irradiation, which has no effect by itself, results in 60-70% cell death at 72 h after exposure. In the presence of TNF-, irradiation induces the activation of serine proteases that are inhibited by TLCK and slightly enhances caspase activation (11). Therefore, two separate proteolytic pathways are activated in response to the combined treatment of LNCaP cells with TNF- and irradiation. To further understand the cell death pathways activated by irradiation in sensitized LNCaP cells, we studied the response of LNCaP cells to agonistic Fas antibody combined with -irradiation. These experiments were undertaken to compare the effects of Fas activation with those of TNF- in order to elucidate the mechanism by which resistant cells can become sensitized to radiation-induced apoptosis. We found that Fas activation caused similar sensitization of the cells to irradiation as seen with TNF-. The cell death pathways induced by the two agonistic receptor/ligand interactions were not entirely redundant and could be combined to produce accelerated cell death when both TNFR-1 and Fas were activated simultaneously.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture-- Human prostate cancer cell line LNCaP was routinely cultured at 37 °C in improved minimal essential medium (Biofluids, Rockville, MD) supplemented with 5% fetal calf serum using standard cell culture procedures (3, 4). Twenty-four hours before exposure to TNF- (Roche Molecular Biochemicals, Indianapolis, IN) and/or cross-linking anti-Fas antibody (clone CH-11, Immunotech, Westbrook, ME) and/or irradiation, the medium was changed to improved minimal essential medium without phenol red (Biofluids) supplemented with 5% charcoal-stripped calf serum. Caspase inhibitors were added 1 h before treatment of cells with TNF-, anti-Fas antibody, or -irradiation. z-Val-Ala-Asp(OMe)-CH₂F(zVAD) were purchased from Enzyme Systems Products (Livermore, CA). TLCK was purchased from Sigma. Neutralization antibody for human Fas receptor clone ZB-4 (Immunotech, Westbrook, ME) was added 1 h before treatment of either TNF- or CH-11. For -irradiation we used a JL Shepherd Mark I Irradiator [¹³⁷Cs] source with a dose rate of 209 centigray/min.

Construction of FADD-dominant Negative Transformants-- The expression vector for FADD-DN transformants, a pEF vector containing an N-terminal 79-amino acid deletion mutant of human FADD, was a gift from Andreas Strasser, University of Cambridge (32). The vector was transfected into LNCaP cells using LipofectAMINE (Life Technologies, Grand Island, NY), and the transformants were selected by puromycin. The expression of FADD-DN was confirmed by the Western blotting using anti-FADD polyclonal antibody (Transduction Laboratories, Lexington, KY).

Apoptosis Assays-- In situ end labeling assay is routinely used in our laboratory for apoptosis determination and has been previously described (33). Briefly cells were cultured in 12-well plates. Both floating and adhesive cells were collected together and fixed with 10% formaldehyde for 30 min. After washing with phosphate-buffered saline, the cells were spread onto glass slides. The first reaction was performed by incubating the cells at 37 °C for 2 h in the reaction buffer (50 mM Tris-HCl, pH 7.5, 5 mM MgCl₂, 60 µM 2-mercaptoethanesulfonic acid, and 0.005% bovine serum albumin) supplemented with 1.25 units/ml Klenow fragment (Roche Molecular Biochemicals, Indianapolis, IN), 200 µM dATP, 200 µM dCTP, 200 µM dGTP, and 200 pM biotinylated dUTP (Roche Molecular Biochemicals). The second reaction was performed by incubating the cells at 25 °C for 1 h in the peroxidase-conjugated avidin-biotin complex solution (Vector Laboratories, Burlingame, CA). Peroxidase reaction was performed using VIP (Vector Laboratories) as a substrate. After counter staining with methyl green, the cells with purple-stained nuclei were counted as apoptotic cells. Bars in the figure designate standard deviations (34).

Western Blotting-- 100 µg of total cellular protein were resolved by electrophoresis on either 8 or 10-20% SDS-polyacrylamide gel electrophoresis gels and transferred onto nitrocellulose membranes (Trans-Blot transfer medium, Bio-Rad). After blocking with 5% milk in 10 mM Tris-HCl, pH 8.0, 150 mM NaCl with 0.05% Tween 20, membranes were probed with monoclonal antibody to PARP (Enzyme Systems Products, Dublin, CA), rabbit antiserum to caspase-3 (a gift from Kristine Kikly, SmithKline Beecham, King of Prussia, PA), rat antiserum to caspase-7 (35) (a gift from Jun Ying Yuan, Harvard University), mouse monoclonal antibody to caspase-8 (clone N2, C15, and C5) (36) (gifts from Peter Krammer, German Cancer Research Center, Heidelberg, Germany), mouse monoclonal antibody to p18 fragment of caspase-8 (Cell Diagnostica, Munster, Germany), rabbit antiserum to DFF45 (37) (a gift from Xiao Dong Wang, University of Texas Southwestern Medical Center, Dallas, TX), mouse monoclonal antibody to lamin B (Calbiochem, La Jolla, CA), or goat anti-TNFR-1 polyclonal antibody (R & D systems, Minneapolis, MN) and visualized with enhanced chemiluminescence detection (Pierce). Western blotting for Fas was performed with a rabbit antiserum obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Western blotting for Fas ligand was performed with mouse monoclonal antibody from Pharmingen (San Diego, Ca).

RT-PCR-- TNF-, TNF-, TRAIL, and DR3mRNA were assayed by PCR amplification of cDNA isolated from LNCaP cells. The primer sequences for PCR amplification are as follows: TNF-, left, 5'-GGCTCCAGGCGGTGCTTGTTCC-3' and right, 5'-CAGGCTTGTCACTCGGGGTTCG-3'; TNF-, left, 5'-GGTCCAGCTCTTCTCCTCCCAGTA-3' and right, 5'-GCGAAGGCTCCAAAGAAGACAGTA-3'; TRAIL, left, 5'-GTGGCAACTCCGTCAGC-3' and right, 5'-GCCCAGAGCCTTTTCATT-3'; DR3, left, 5'-ATGGCGATGGCTGCGTGTCCT-3' and right, 5'-GGTGGCCGGTGGTGGGGTCAGAG-3'. PCR reactions were cycled at 94 °C for 1 min, 55 °C for 1 min and 72 °C for 30 s. For TNF- 32 cycles were completed, for TNF- 37 cycles, for TRAIL 35 cycles, and for DR3 33 cycles.

Caspase-8 Fluorimetric Assay-- Caspase-8 activity was measured by IETD-amc cleavage using the Apoalert caspase-8 assay kit (CLONTECH, Palo Alto, CA). For the assay, 3 × 10⁶ cells were washed with phosphate-buffered saline and suspended in 50 µl of lysis buffer provided by the manufacturer. The rest of the assay was done according to the manufacture's protocol. Fluorescence was measured using an F-4500 spectrophotometer (Hitachi Ltd., Japan) with 400 nm excitation and 505 nm emission filter.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

To demonstrate that Fas activation sensitized cells to irradiation we used CH-11, an anti-Fas antibody that cross-links Fas. Fig. 1 shows that CH-11-sensitized LNCaP cells to cell death induced by both 8 and 20 Gy -irradiation. This was somewhat in contrast to our previously published results with radiation and TNF- that did not result in more apoptosis at 20 Gy than at 8 Gy (11).

View larger version (12K): [in this window] [in a new window]   Fig. 1.   CH-11 sensitized LNCaP cells to radiation-mediated apoptosis. LNCaP cells were treated with 4 µg/ml CH-11 and/or 8 or 20 Gy -radiation. The percentage of apoptotic cells was determined by the in situ end labeling assay 72 h after exposure.

Since it was possible that either Fas or TNF- were sensitizing LNCaP cells to irradiation by increasing expression of each other or other death ligands or receptors, we analyzed the expression of death ligands and receptors during induction of apoptosis by irradiation and Fas or TNFR-1 activation. After exposure to either 8 Gy or TNF-, Fas expression in LNCaP cells was increased slightly at 6, 24, and 48 h after exposure and combined treatment with TNF- and irradiation generated slightly more Fas than either treatment alone (Fig. 2A). Since both TNF- and irradiation increased Fas expression to some degree, it was possible that Fas may have mediated TNF- sensitization of LNCaP cells to irradiation. To determine the significance of increased Fas expression on the effect of TNF- we used an anti-Fas neutralizing antibody ZB4. Fig. 2B shows that Fas neutralizing antibody blocked apoptosis induced by concomitant treatment with radiation and CH-11, but it had no effect on apoptosis induced by either TNF- or TNF- plus irradiation.

View larger version (45K): [in this window] [in a new window]   Fig. 2.   Effect of TNF- and CH-11 on expression of death ligands and receptors. A, Western blots of Fas and Fas ligand at 6, 24, and 48 h after exposure of LNCaP cells to 40 ng/ml TNF- and/or 20 Gy irradiation. As controls, extracts from LNCaP cells were treated with 100 µM etoposide were included. The anti-Fas antibody used for Western blotting detected both unglycosylated Fas lower band and glycosylated Fas upper band. B, LNCaP cells were treated with either 40 ng/ml TNF- or 4 µg/ml CH-11 and/or 20 Gy irradiation in the presence of 10 µg/ml neutralizing antibody to Fas receptor (clone ZB4) or a control antibody at the same concentration. The percentage of apoptotic cells was determined by the in situ end labeling assay 72 h after treatment. C, RT-PCR assay of TNF-, TNF-, TRAIL, and DR-3 mRNA expression at 6 and 24 h after treatment of LNCaP cells with 40 ng/ml TNF- and/or 4 µg/ml CH-11 and/or 20 Gy irradiation. As a control we used LNCaP cell treated with 30 nM okadaic acid for 48 h. D, Western blot of TNFRI in extracts of LNCaP cells 24 h after treatment with combinations of 40 ng/ml TNF-, 4 µg/ml CH-11, and 20 Gy irradiation. As a control, LNCaP cells were treated with 30 nM okadaic acid for 48 h.

We previously showed that TNF- could induce the expression of its own mRNA (11). To determine whether CH-11 was affecting TNF- expression we analyzed TNF- mRNA in the cells treated with CH-11. Fig. 2C shows that anti-Fas antibody did not have any effect on the expression of TNF- mRNA. Moreover, anti-Fas antibody did not change the amount of mRNA for TNF-, TRAIL, or DR3. We also determined levels of TNFR-1 in LNCaP cells treated with TNF-, CH-11, and irradiation by Western blotting (Fig. 2D). There was no change in the levels of TNFRI in response to these exposures. In Fig. 2, C and D, okadaic acid treatment was included as a positive control since okadaic acid rapidly and efficiently induces caspase-dependent cell death in LNCaP cells. As a result of the experiments shown in Fig. 2 we do not believe that Fas was sensitizing LNCaP cells to irradiation by activating expression of other known death receptors or ligands.

Although both CH-11- and TNF--sensitized LNCaP cells to irradiation to a similar degree, we found that caspase activation differed between LNCaP cells treated with CH-11 and those treated with TNF-. Fig. 3A shows Western blots demonstrating activation of different components of the caspase cascade after either TNF- or CH-11 treatment. As we had previously shown, irradiation enhanced the low level of caspase-8 cleavage induced by TNF- in LNCaP cells (11). However, at 72 h after treatment there was substantially more caspase-8 cleavage induced by CH-11 than by TNF- as indicated by the presence of the p28 C-terminal cleavage product in the CH-11-treated cells. Note that both TNF- and okadaic acid appeared to cause both caspase-8 cleavage and an apparent increase in caspase-8 levels as indicated by the fact that the p55 caspase-8 precursor band was increased in intensity compared with control. The apparent increase in caspase-8 precursor may be similar to the increases in procaspases-3 and -7 that are essential for apoptosis in TSU-Pr1 prostate cancer cells treated with okadaic acid (38). Note that procaspase-3 is present at levels undetectable by Western blotting in untreated cells.

View larger version (36K): [in this window] [in a new window]   Fig. 3.   Caspase cleavage in LNCaP cells. A, LNCaP cells were treated with 40 ng/ml TNF-, 4 µg/ml CH-11, or 20 Gy irradiation. Caspase cleavage was determined by Western blotting 72 h after treatment. C15 antibody that recognizes the p18 peptide was used for detection of caspase-8. As a control, LNCaP cells were treated with 30 nM okadaic acid for 48 h. B, Western blots for caspase-8 and caspase-7 in cells treated with TNF- ± 20 Gy and various concentrations of TLCK shown at the bottom.

In contrast to the fact that more caspase-8 cleavage was seen after CH-11 exposure than after TNF- exposure, more cleavage of caspase-3, caspase-7, lamin B (a caspase-6 substrate) (39, 40), and PARP was seen after TNF- exposure. No caspase-10 cleavage was seen under any treatment conditions. Cleavage of DFF was slightly greater in the cells exposed to CH-11 than TNF-. Note that after exposure to okadaic acid we were able to detect both p18 and p10 terminal cleavage products of DFF45. We only detected a small amount of the 26-kDa partial cleavage product of DFF in LNCaP cells treated with either TNF- or CH-11. Although irradiation increased apoptosis induced by either TNF- or CH-11, irradiation had little, if any, effect on the cleavage of DFF caused by either TNF- or CH-11. This is consistent with the expectation that irradiation, in the presence of a death ligand, activates serine protease-dependent nucleolytic activity in these cells.²

Caspase activation is believed to be due predominantly to death ligand activation of the caspase cascade. However, we previously have shown that the serine protease inhibitor, TLCK, had a slight inhibitory effect on caspase activation in LNCaP cells treated with TNF- and irradiation. This observation was confirmed when we treated the cells with 40 µM TLCK, which is known to decrease apoptosis by half. A slight decrease in caspase-8, but not caspase-7 activation was seen (Fig. 3B).

We further investigated the differences in caspase-8 cleavage and activation by TNF- and CH-11 with Western blotting using antibodies specific for different regions of the caspase-8 molecule (36). In the left half of Fig. 4A it can be seen that TNF- alone induced cleavage only of a p43 fragment that corresponded to the procaspase-8 less the C-terminal p10 peptide. There was also an increase in caspase-8 p55 precursor in TNF--treated cells and even a greater increase in caspase-8 p55 in cells exposed to TNF- + irradiation (right half of Fig. 4A). In contrast, CH-11 activated cleavage of the N-terminal death effector domain-containing prodomains from the C-terminal protease domains as seen by the appearance of p26 detected by N2 antibody and of p28 detected by both C5 and anti-p28 antibody. Irradiation enhanced CH-11-induced cleavage of caspase-8 (compare third and seventh lanes in Fig. 4A). Although Western blotting with peptide-specific antibodies was consistent with the interpretation of the data shown at the bottom of Fig. 4A, we were not able to detect caspase-8 p10 in any of our samples or positive controls. Because of differences in procaspase-8 cleavage seen with TNF- and CH-11 treatment, we assayed caspase-8 activity using a fluorescent substrate. Fig. 4B shows that TNF- generated active caspase-8, but CH-11 with or without irradiation yielded no detectable caspase-8 activity. Therefore, cleavage of caspase-8 into p26 and p28 fragments was abortive and did not activate caspase activity.

View larger version (36K): [in this window] [in a new window]   Fig. 4.   Caspase-8 cleavage and activity in LNCaP cells. A, LNCaP cells were treated with combinations of 40 ng/ml TNF-, 1.0 µg/ml CH-11, and 20 Gy irradiation. Samples were taken at 48 h after treatment for Western blotting. The specificity of monoclonal antibodies for different fragments of caspase-8 is shown in the diagram at the bottom that is adapted from Scaffidi et al. (36). N2 antibody recognizes the N-terminal domain that contains the death effector domain (DED) regions. C15 and a commercial antibody to p18 recognize the larger protease component. C5 antibody is specific for the C-terminal p10 peptide. Our results with the p18 antibody and C15 were identical and are represented by a blot with C15 antibody. "NS" in the panel showing the C5 antibody blot means "nonspecific." B, cleavage of IETD-amc measured fluorimetrically and plotted relative to background fluorescence of untreated cells.

One of the nucleases activated in these cell death experiments was DFF45 as shown by Western blotting and by DNA cleavage into low molecular weight fragments (not shown). We investigated whether cell death in our experiments also involved serine protease pathways that could lead to the activation of other nucleases. We had observed that apoptosis induced by TNF- + irradiation could be inhibited completely only by a combination of cysteine and serine protease inhibitors, suggesting that two separate protease cascades were activated by the combined exposure (11). This implied that TNF- + irradiation activated a separate serine protease pathway that was independent of caspase activation (11). In contrast, we found that inhibition of apoptosis induced by CH-11 + irradiation could be fully abrogated by zVAD (Fig. 5). Although TLCK inhibited about 50% of the apoptosis induced by CH-11 + irradiation, zVAD blocked both apoptosis induced by CH-11 alone and CH-11 + irradiation. This suggested that serine protease activation after exposure to CH-11 + irradiation contributed to apoptosis, but was dependent on caspase activation. Inhibition of apoptosis induced by CH-11 + irradiation by zVAD implied that caspases played a predominant role in apoptosis induced by CH-11 + irradiation. Since we were able to demonstrate only minimal caspase-6 activation (Fig. 3A), no caspase-8 activity (Fig. 4B), and no detectable caspase-3, -7, or -10 cleavage (Fig. 3A), we hypothesize that some other unidentified caspase activity was responsible for cell death induced by CH-11 + irradiation.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Inhibition of apoptosis mediated by Fas and irradiation by zVAD and TLCK. LNCaP cells were treated with 4 µg/ml CH-11 and/or 20 Gy irradiation in the presence or absence of 50 µM zVAD and/or 20 µM TLCK, and the fraction of apoptotic cells was determined by the in situ end labeling assay 72 h after treatment.

Because there were differences in caspase cleavage and activation between TNF- and CH-11 treatment of LNCaP cells, we investigated the effect of simultaneous activation of TNFR-1 and Fas. We also assayed the effect of the two ligands together on radiation-induced apoptosis. TNF- and CH-11 together caused a sensitization to radiation-induced apoptosis and rapid induction of cell death. Even at concentrations that were ineffective when the ligands were used alone, together they activated apoptosis rapidly. In order to show comparisons between treatment with TNF-, CH-11, and irradiation and treatment with any one or two of these stimuli, we had to measure apoptosis at 24 h, a time when either TNF- or CH-11 and irradiation were not seen to induce apoptosis (Fig. 6A). However, combined treatment with TNF- and CH-11 induced up to 40% cell death at 24 h and up to 90% in the presence of irradiation. In the presence of TNF- there was a dose-response relationship between the amount of CH-11 added up to 1.0 µg/ml and cell death. In the presence of TNF- and irradiation, all three concentrations of CH-11 used in Fig. 6A had equivalent effects. Noteworthy was the difference in induction of apoptosis between concentrations of TNF- 4 ng/ml and irradiation in the absence and presence of CH-11. We had seen that apoptosis after either TNF- + irradiation or CH-11 + irradiation was dependent on both caspases and serine proteases. However, in the case of the combination of all three treatments, zVAD completely blocked cell death at 20 and 48 h (not shown), but TLCK had no effect on cell death under these conditions (Fig. 6B).

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Effect of combined TNF- and CH-11 treatment on LNCaP cells. A, LNCaP cells were treated with various concentrations of TNF- and/or CH-11. The dose of CH-11 increased within each group of four bars as indicated by the triangles. Within each group the CH-11 doses were 0, 0.4, 1.0, and 4.0 µg/ml. Half of the samples were treated concomitantly with 20 Gy irradiation. Twenty-four hours later, the percentage of apoptotic cells was determined by the in situ end labeling assay. B, effect of protease inhibitors on apoptosis induced by TNF- plus CH-11 with or without irradiation. LNCaP cells were treated with different concentrations of TNF- and CH-11 shown in the figure with or without 20 Gy irradiation. 50 µM zVAD and 20 or 50 µM TLCK were used as shown. The percentage of apoptotic cells was determined 20 h after treatment by the in situ end labeling assay.

The radiation sensitization by TNF- + CH-11 was mediated entirely through the respective cell death receptor interaction with FADD since the expression of a FADD-DN abrogated the sensitization of LNCaP cells by TNF-, CH-11, or both (Fig. 7). Therefore combined activation of TNFR-1 and Fas in the presence of irradiation resulted in rapid activation of cell death mediated entirely by caspase pathways. Activation of either receptor alone in the presence of irradiation required a longer time period for cell death and the activation of serine as well as cysteine proteases. Since FADD-DN caused near total inhibition of apoptosis induced by TNF- + irradiation, we concluded that FADD was upstream of both caspase and serine protease activation in LNCaP cells.

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Effect of combined TNF- and CH-11 treatment on LNCaP cells and FADD-DN transformants. Parental LNCaP cells, vector-transfected LNCaP cells, and LNCaP cells transfected with FADD-DN were treated with 40 ng/ml TNF- and/or 1 µg/ml CH-11 and/or 20 Gy irradiation. The percentage of apoptotic cells was determined by the in situ end labeling assay 24 h after treatment.

Since FADD-DN blocked sensitization to radiation-induced apoptosis by TNF- + CH-11, we expected the combined effect of TNF- and CH-11 to be mediated through caspase activation and therefore expected to see substantial increases in caspase activation after simultaneous treatment with the two ligands. Fig. 8 shows Western blots on extracts 24 h after treatment. There was a substantial increase in caspase-8 and -7 activation after exposure to TNF-, CH-11, and irradiation. At the same time, the cleavage of lamin B, PARP, and DFF45 was increased by TNF- and CH-11. The degree of caspase cleavage at 24 h shown in Fig. 8 equaled or exceeded caspase activation at 72 h seen with either ligand alone + irradiation. Slight differences in caspase activation between Figs. 8 and 3A occur in our experiments and reflect some variability in results when very low levels of caspase-3, -6, and -7 are detected.

View larger version (41K): [in this window] [in a new window]   Fig. 8.   Caspase activation in LNCaP cells after treatment with TNF- and CH-11 with or without irradiation. LNCaP cells were treated with 40 ng/ml TNF- and/or 1 µg/ml CH-11 and/or 20 Gy irradiation. Cell extracts were prepared at 24 h for Western blotting. As a control, LNCaP cells were treated with 30 nM okadaic acid for 48 h. C15 monoclonal antibody was used for detection of caspase-8.

Combined treatment of LNCaP cells with TNF- and CH-11 resulted in more rapid and extensive apoptosis. To determine if the time course of caspase activation was consistent with the timing of apoptosis after treatment with either ligand plus irradiation or both ligands together, we analyzed caspase-8 and caspase-7 cleavage at different time points after treatment with TNF-, CH-11, and irradiation. Fig. 9 shows a time course of caspase-8 activation after different treatments of LNCaP cells. Activation of caspase-8 after TNF- + irradiation was seen at 24 h and is consistent with initiation of apoptosis 24-48 h after exposure. Caspase-7 cleavage was seen in this experiment at 36 and 48 h at times when procaspase-7 levels were still increasing. Procaspases-8 also increased after exposure to TNF- + irradiation. In contrast, although CH-11 and irradiation induced apoptosis in the same time frame as did TNF- + irradiation, cleavage of caspase-8 to p26 and p28 occurred within the first 3 h after exposure. No caspase-7 cleavage was seen. In addition, procaspase-8 was depleted during the 48-h period and amounts of procaspase-7 did not change. Therefore it appears that blockage of caspase-8 activation in LNCaP cells occurred due to abortive cleavage and depletion of procaspase-8. Although p28 generation was also seen after treatment of LNCaP cells with CH-11 and TNF-, LNCaP cells treated with TNF- + CH-11 + irradiation showed activation of caspase-8 beginning 3-6 h after exposure. Procaspase-8 was not depleted until 48 h after exposure. Interestingly cleavage of some caspase-8 to p28 was also seen. Caspase-7 cleavage after TNF- + CH-11 + irradiation was seen at 18 h after exposure, consistent with the conclusion that caspase-7 was activated by caspase-8 in these cells. Moreover, procaspase-7 was increased between 3 and 15 h, but decreased after 15 h probably due to the combined effects of ongoing cell death and processing to active caspase-7 p20.

View larger version (46K): [in this window] [in a new window]   Fig. 9.   Time course of caspase activation in LNCaP cells. LNCaP cells were treated as shown on the left side of the figure. Protein extracts were made at the indicated times and processed for Western blotting for caspase-8 (left) and caspase-7 (right).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this paper we showed that a Fas ligand can cause radiation sensitization in a cell that is entirely resistant to radiation-induced apoptosis, similar to our previous findings with TNF-. Fas activation is an important element in cellular response to irradiation. For example, in lpr and gld mice the Fas/Fas ligand system is essential for radiation-mediated apoptosis (41). Our results show that levels of activation of death receptors can play an important role in radiation-induced death of cancer cells and can augment sensitivity to radiation. The levels of radiation used in this study were chosen to obtain a biological effect and are in excess of commonly used single-fraction doses used in therapeutic radiation.

Activation of TNFRI and Fas pathways each induce a low level of cell death in LNCaP cells. We observed differences between radiosensitization caused by TNF- and CH-11. For example, whereas there was no increment in cell death in the presence of TNF- when the dose of irradiation was increased from 8 to 20 Gy, cell death was dose dependent in the presence of CH-11. Also, there were differences in caspase activation downstream from TNFR-1 and Fas. TNF- induced an increase in procaspase-8 p55, but we were able to detect very little caspase-8 cleavage. CH-11 caused depletion of procaspase-8 and the appearance of caspase-8 cleavage products very early after treatment. However, caspase-8 was not activated. As a result, downstream effector caspases-3, -7, and -6 were activated to a greater degree after TNF- exposure than after CH-11. This implies that a very low level of caspase-8 activity was present and activated caspases-6 and -7 or that another upstream signaling caspase, but not caspase-10, transmitted death signals from the TNFR-1 to the downstream caspases. Little, if any difference was seen in the appearance of DFF cleavage products after exposure to TNF- compared with CH-11, although TNF- alone induced more LNCaP cell death than CH-11. Activation of caspase-8 and greater cleavage of effector caspases-3 and -7 and activation of caspase-6 correlated with the greater degree of cell death induced by TNF-.

We were able to show that the death inducing effects of CH-11 and TNF- were complementary. At doses that had no effect when either agent was used alone, the two agents together induced very rapid cell death. The effect of combining TNF- and CH-11 may have been primarily mediated at the level of caspase-8 since within 6 h after cells were exposed to 40 ng/ml TNF- and 1 µg/ml CH-11. There was an elevation of procaspase-8 p55 and appearance of activated caspase-8 cleavage peptides p43 and p18. Other reports of the interaction between TNF- and Fas ligand have suggested that each may augment cell death induced by the other through enhancement of death ligand expression (42, 43). For example, Spanaus et al. (42) suggested that TNF- induced increased expression of Fas, thereby sensitizing cells to Fas ligand. Based on the data in Fig. 2 we do not believe this to be the explanation for the cooperative effect of TNF- and CH-11 in LNCaP cells. Irradiation increased Fas expression in LNCaP cells to a much greater degree than did TNF- (Fig. 2A), yet irradiation enhanced TNF--induced apoptosis to a lesser degree than did CH-11. Moreover, blocking antibody to Fas did not affect apoptosis induced by TNF- with or without irradiation. For these reasons we do not believe that the synergy between CH-11 and TNF- can be attributed to increased signal transduction through Fas. Rather, we believe that the increased level and accelerated pace of apoptosis we observed after TNF- and CH-11 treatment of LNCaP cells is due to enhanced caspase-8 activation. This is similar to caspase-8 induction seen in synovial cells after exposure to TNF- and Fas ligand (44). Caspase-8 cleavage induced in LNCaP cells by CH-11 differed from the cleavage pattern described by Scaffidi et al. (36). After exposure to CH-11 LNCaP cells appeared to generate the residual p43 fragment that remains after cleavage of p10 from p55 procaspase-8. It is possible that the differences in caspase-8 cleavage contributed to the synergy seen in the induction of cell death after treatment with both TNF- and CH-11.

Because cell death in LNCaP after exposure to TNF- + CH-11 was abrogated by zVAD and FADD-DN, but not affected by TLCK, it appears that an effect of simultaneous activation of TNFR-1 and Fas is rapid and efficient caspase activation and cell death within 24 h. Even though irradiation in addition to TNF- and CH-11 increased apoptosis compared with the two ligands alone, no sensitivity to TLCK was observed as with apoptosis induced by TNF- + irradiation (11) or CH-11 + irradiation, both of which took 72 h to evolve. This data suggests that cells can be sensitized to irradiation by very high levels of caspase activation and that under these conditions irradiation enhances caspase activation even further and apoptosis occurs within 24 h. However, under conditions where caspase activation is suboptimal, even though irradiation enhanced caspase activation over TNF- and Ch-11 alone, cell death required the activation of noncaspase proteases. Under the latter conditions, activation of noncaspase proteases occurred after 24 h, resulting in cell death at 72 h. The doses of irradiation used in these experiments were chosen for their biological effect and are in excess of doses that can be given in a single fraction of therapeutic irradiation.

The rate at which cells undergo apoptosis and the role of caspases and noncaspase proteases may depend on the magnitude of initial caspase activation. The role of different proteases may also be cell type specific. For example, lymphocytes are highly sensitive to induction of apoptosis by activation of Fas which results in caspase activation and rapid cell death (45, 46). In contrast epithelial cells may not have the same degree of caspase activation either because of reduced response to death ligands, or because of a lower level of procaspases. In fact, epithelial cells may have to synthesize procaspases in response to a death signal in order to sustain a death response (38). The involvement of serine proteases has been described in many cell types including epithelial cells and hepatoma cells (47-50). Recently Samejima et al. (51) showed that chicken hepatoma cells first activated caspases in response to a death signal, but were not fully committed to the point of no return in cell death until noncaspase proteases were also activated. Our observations that zVAD and TLCK both have a role in blocking apoptosis in LNCaP cells treated with either TNF- + irradiation or CH-11 + irradiation are consistent with the involvement of two classes of proteases in cell death. Although the data in this paper are consistent with the notion that caspase activation preceded serine protease activation, previously we found that in cells treated with TNF- and irradiation, caspases, and serine proteases had equal and independent contributions to cell death (11). Moreover, since TNF- + CH-11 + irradiation induced rapid cell death that was entirely caspase-dependent, we believe that early overwhelming caspase activation can achieve cell death in epithelial cells as in lymphocytes.

In these experiments we were able in induce a low, but reproducible level of LNCaP cell death with CH-11 antibody. Rokhlin et al. (52) demonstrated that a cross-linking of Fas receptor by anti-Fas antibody did not induce apoptosis in LNCaP cells and thus conclude that this cell line was resistant to Fas-mediated apoptosis. There are several differences in the experiments that may explain the discrepancy between their data and ours regarding sensitivity of LNCaP cells to Fas-mediated apoptosis. They used a strain of LNCaP cells that they termed LNCaP.FGC and used IPO-4 anti-Fas antibody. More importantly, Rokhlin et al. (52) treated LNCaP cells with anti-Fas antibody in the presence of 10% fetal calf serum. Our experiments are done in charcoal-stripped calf serum. The differences in the content of survival factors between the two types of serum probably explains the differences in LNCaP cell sensitivity to anti-Fas antibodies seen by the two groups. In fact, we agree that LNCaP cells are relatively resistant to Fas-mediated apoptosis, for instance, as compared with TNF--mediated apoptosis.

	ACKNOWLEDGEMENTS

We are grateful to our colleagues Kristine Kikly, Jun Ying Yuan, Peter Krammer, Xiao Dong Wang, and Andreas Strasser for providing invaluable reagents. We also thank Sarah Spiegel for helpful discussions.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant CA79912.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 202-687-2207; Fax: 202-784-1229; E-mail: Gelmanne@gunet.georgetown.edu.

² K. Kimura and E. P. Gelmann, unpublished data.

	ABBREVIATIONS

The abbreviations used are: TNF-, tumor necrosis factor ; PARP, poly(ADP-ribose) polymerase; z-VAD, z-Val-Ala-Asp(OMe)-CH₂F; TLCK, N-p-tosyl-L-lysine-chloromethyl ketone; PCR, polymerase chain reaction; RT, reverse transcriptase; TNFR-1, tumor necrosis factor receptor 1; TRADD, TNF receptor-associated death domain; FADD, Fas-associated death domain; FADD-DN, dominant negative mutant of FADD; DFF, DNA fragmentation factor; Gy, gray.

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