High Cell Surface Death Receptor Expression Determines Type I Versus Type II Signaling*

Previous studies have suggested that there are two signaling pathways leading from ligation of the Fas receptor to induction of apoptosis. Type I signaling involves Fas ligand-induced recruitment of large amounts of FADD (FAS-associated death domain protein) and procaspase 8, leading to direct activation of caspase 3, whereas type II signaling involves Bid-mediated mitochondrial perturbation to amplify a more modest death receptor-initiated signal. The biochemical basis for this dichotomy has previously been unclear. Here we show that type I cells have a longer half-life for Fas message and express higher amounts of cell surface Fas, explaining the increased recruitment of FADD and subsequent signaling. Moreover, we demonstrate that cells with type II Fas signaling (Jurkat or HCT-15) can signal through a type I pathway upon forced receptor overexpression and that shRNA-mediated Fas down-regulation converts cells with type I signaling (A498) to type II signaling. Importantly, the same cells can exhibit type I signaling for Fas and type II signaling for TRAIL (TNF-α-related apoptosis-inducing ligand), indicating that the choice of signaling pathway is related to the specific receptor, not some other cellular feature. Additional experiments revealed that up-regulation of cell surface death receptor 5 levels by treatment with 7-ethyl-10-hydroxy-camptothecin converted TRAIL signaling in HCT116 cells from type II to type I. Collectively, these results suggest that the type I/type II dichotomy reflects differences in cell surface death receptor expression.

Previous studies have shown that signaling initiated at the Fas receptor differs among various cells (18). In particular, socalled type I cells generate large amounts of DISC, activate substantial caspase 8, and directly trigger the apoptotic pathway through caspase 8-mediated cleavage of procaspase 3. In contrast, type II cells generate smaller amounts of DISC, activate less caspase 8, and rely on caspase 8-mediated cleavage of Bid followed by subsequent signal amplification through the mitochondrial apoptotic pathway for sufficient caspase 3 activation to kill the cell (18 -20). As a consequence, death ligand signaling in type II cells (but not type I cells) is inhibited by overexpression of anti-apoptotic Bcl-2 family members and other changes that dampen activation of the mitochondrial pathway. In addition, death ligand-induced killing in type II cells, but not type I cells, is inhibited by treatment with phorbol esters (21), which serve as a surrogate for signaling pathways that activate protein kinase C isoforms. Thus, whether signaling proceeds through type I or type II signaling has important ramifications for potential mechanisms of resistance to death ligand-induced killing.
The biochemical basis for the dichotomy into type I versus type II cells is incompletely understood. Early studies suggested that Fas expression is similar between the two cell types (18). Subsequent publications reported that linkage of Fas to the actin cytoskeleton might be different between type I and type II cells (22). More recent studies have suggested that type II cells might have lower levels of the lipid phosphatase PTEN, which is thought to modulate Bcl-2 function (23), or differential sensi-tivity to the endogenous caspase inhibitor XIAP (24,25). It is not clear, however, how these differences explain the dichotomy in DISC formation and subsequent signaling in type I versus type II cells.
In the present work we report that type II cells have less Fas receptor on their surfaces than type I cells. Building on this observation, we show that type II cells can be converted to type I cells by forced overexpression of the Fas receptor and that type I cells can be converted to type II cells by Fas down-regulation. In addition, we demonstrate that the same cells can exhibit type I signaling for Fas and type II signaling for TRAIL receptorinduced cell death, suggesting that the choice of signaling pathway is not a cell-intrinsic feature. Collectively, these observations provide new insight into an important cell type-specific difference in death ligand signaling.
Analysis of Cell Surface Fas, DR4, and DR5 Expression-Aliquots containing 2.5 ϫ 10 5 human cells were fixed with 4% (w/v) paraformaldehyde for 30 min. After 2 washes in PBS containing 2% (v/v) FBS (PBS/FBS), cells were incubated with saturating amounts (1.0 g) of APO-1-1 murine anti-human Fas antibody, DR4, or DR5 monoclonal antibodies on ice for 45 min, washed twice with PBS/FBS, incubated with PE-or APCconjugated anti-mouse IgG for an additional 30 min on ice, washed, and subjected to flow microfluorimetry. To quantitate cell surface antibody binding, beads conjugated with known amounts of PE (Quantibrite TM PE beads, BD Biosciences) were run in parallel, and fluorescence histograms were analyzed as suggested by the supplier.
To further assess cell surface Fas expression, 2 ϫ 10 6 cells were treated with cell-impermeable biotinylating reagents as described previously (28). Biotinylated cell surface proteins were pulled down with streptavidin-agarose, separated by SDS-PAGE, transferred to nitrocellulose, and probed with anti-Fas antibody .
Hepatocytes were also isolated from C57BL/6 mice by collagenase perfusion and purified by Percoll gradient centrifugation as previously described (29,30). Thymocytes were isolated by passage through wire mesh as described (31). After fixation with 4% paraformaldehyde for 30 min on ice, 2.5 ϫ 10 5 cells were stained with 1.0 g of FITC-conjugated Jo2 anti-mouse Fas antibody on ice for 45 min, washed, and analyzed by flow cytometry.
Plasmids-Plasmid encoding Histone H2B fused to the C terminus of enhanced green fluorescent protein (pEGFP-histone H2B) was a kind gift from J. van Deursen (Mayo Clinic, Rochester, MN). Plasmid encoding S peptide-tagged Bcl-x L was constructed by inserting cDNA encoding codons 2-234 of the Bcl-x L open reading frame into the AscI and EcoRI sites of pSPN (27). pCMS5A-EGFP-H2B, which encodes EGFP fused to histone H2B and also contains a multiple cloning site behind the CMV promoter, was described elsewhere (32). pCMS5A-EGFP-H2B encoding Bcl-2 was generated by ligating fulllength Bcl-2 variant 1 behind the CMV promoter (33). Human full-length Fas was cloned into the EcoRI and NotI sites of pEF1, which contains the eukaryotic initiation factor 1␣ promoter. All open reading frames were confirmed to be free of mutations by automated sequencing.
Human Fas Promoter Cloning and Luciferase Reporter Assay-The sequence corresponding to the FAS promoter region Ϫ1739/Ϫ19 (34) was PCR-amplified from human Jurkat genomic DNA with the following primers: 5-TTAAGGATC-CCTCATCTCACTGGG-3Ј (forward) and 5Ј-TGAGGGATC-CTCCGAAGTG-3Ј (reverse). The 1736-bp PCR product was digested with BamHI and inserted into BglII site of pGL3 basic vector (Promega). Reporter assays were performed using a dual luciferase strategy as recently described (35). 24 h after transfection, cell lysates were prepared, assayed for firefly luciferase activity using a Lumat LB 9501 luminometer (Berthold Technologies, Oak Ridge, TN), and normalized using Renilla luciferase activity (35).
Transfection-To assess the impact of experimental manipulations on Fas-mediated apoptosis, 1 ϫ 10 7 log phase cells growing in antibiotic-free medium were suspended in 400 l of medium containing one of the following sets of additives: (i) 4 g of pEGFP-histone H2B and 1000 nM Bid (or control) siRNA, (ii) 4 g of pEGFP-histone H2B and 30 g of pSPN-Bcl-x L (or pSPN empty vector), or (iii) 30 g of pCMS5A-EGFP-H2B-Bcl-2 (or pCMS5A-EGFP-H2B with an empty second multiple cloning site). After incubation for 5 min, cells were subjected to electroporation using a BTX 830 square wave electroporator (BTX, San Diego, CA) delivering a single 10-ms pulse at 240 V for leukemia lines or 300 V for solid tumor cell lines. Cells transfected with Bcl-x L (or Bcl-2) were incubated for 24 h before the addition of CH.11 or etoposide. To assess the effects of Fas up-regulation, cells were incubated for 16 h in the presence of 5 M Q-VD-OPh to minimize apoptosis induced by Fas overexpression itself, then transferred to Q-VD-OPh-free medium immediately before the addition of CH.11 or diluent.
Generation of Stable Cell Lines-To down-regulate Fas expression, A498 renal cancer cells were transduced with lentiviral particles that were packaged in HEK293T cells transfected with the MISSION Lentiviral (Sigma) vector pLKO.1puro containing a hairpin shRNA against human Fas mRNA (GenBank TM NM_000043) TRCN0000038696 (R#6, CCG-GGTGCAGATGTAAACCAAACTTCTCGAGAAGTTTGG-TTTACATCTGCACTTTTTG), the packaging vector psPAX2 and envelope vector pMD2.G. Virus containing non-targeting shRNA (#SHC001V) was used to transduce A498 cells as a control. After viral infection on two successive days, cells were cultured in medium containing 2 g/ml puromycin for a week, stained with APO-1-1 anti-Fas antibody, and examined for cell surface Fas expression by flow microfluorimetry.
To derive cells deficient in both Bax and Bak, lentivirus encoding Bak shRNA (25) targeting nucleotides 1713-1731 of the Bak mRNA (GenBank TM NM_001188) was packaged and transduced into HCT116 BAX Ϫ/Ϫ cells (36), which were selected in puromycin as described above.
Assays for Apoptosis-After the indicated treatment, cells were stained with APC-conjugated annexin V in 140 mM NaCl, 2.5 mM CaCl 2 , and 10 mM HEPES (pH 7.4) as instructed by the supplier. After 20,000 events were collected on a BD Biosciences FACSCanto flow cytometer, data were analyzed by gating on EGFP-histone H2B-positive cells (typically 60 -70% of transfected Jurkat cells) and assessing APC-annexin V binding. Alternatively, treated cells were stained with propidium iodide and subjected to flow microfluorimetry (37,38) to detect cells with Ͻ2n DNA content.
DISC Analysis-The Fas DISC was immunoprecipitated as described previously (40). In brief, aliquots containing 5 ϫ 10 7 cells were treated with 500 ng/ml CH.11 anti-Fas antibody for the indicated periods of time. After cell lysates were prepared as described above, aliquots containing 4 mg of protein were precleared by sedimentation at 14,000 ϫ g for 5 min, supplemented with 10 g of rabbit anti-mouse IgM that was precoupled to protein A-and protein G-Sepharose using dimethyl pimelimidate, and incubated for 2 h at 4°C. As a control, 1 ml of cell lysate from 5 ϫ 10 7 untreated cells was precleared, supplemented with 2.0 g of CH.11 antibody, and treated with immobilized anti-mouse IgM in parallel with treated samples. At the end of the incubation, beads were sedimented at 14,000 ϫ g for 3 min and washed 5 times with cell lysis buffer. Immunoprecipitated complexes were released from the beads by boiling for 5 min in SDS sample buffer, subjected to SDS-PAGE, transferred to nitrocellulose, and sequentially probed with reagents that recognize FADD and caspase-8.

Expression of FADD, Procaspase 8, c-FLIP, and XIAP Is Similar in Cells with Type I and Type II Signaling-Our
initial experiments used a series of human lymphoid cell lines, many of which were previously employed in the original identification of type I versus type II signaling (18). To rigorously establish whether signaling was type I or type II, cells were transfected with empty vector or cDNA encoding Bcl-x L , treated with agonistic anti-Fas antibody, and examined for apoptosis. Consistent with previous reports, Bcl-x L diminished apoptosis induced by the agonistic anti-Fas antibody CH.11 (Fig. 1, A  Subsequent studies examined expression of components of the extrinsic pathway as well as XIAP. Immunoblotting demonstrated that levels of FADD, procaspase 8, c-Flip, and XIAP did not distinguish type I from type II cells (Fig. 1E). Additional studies failed to demonstrate a differential effect of the actin depolymerizing agent latrunculin A on CH.11-induced apoptosis in Jurkat and SKW6.4 cells (Fig. 1F), suggesting that involvement of the actin cytoskeleton is also not a critical difference between cells with the two types of signaling. These observations prompted us to examine other potential differences between cells that exhibit type I and type II Fas signaling.
Higher Levels of Cell Surface Fas in Type I Cells-Building on previous studies of Fas trafficking to the cell surface (28), we examined cell surface Fas in this panel of lymphoid cell lines using two different approaches. First, cell surface proteins were biotinylated using cell-impermeable reagents, pulled down from cell lysates using streptavidin-agarose, and probed with anti-Fas antibody. Results of this analysis ( Fig. 2A) suggested much higher levels of Fas on the surface of SKW6.4 and H9 cells (type I signaling) compared with Jurkat, CEM, and Molt-3 cells (type II signaling). To complement this approach, flow microfluorimetry was utilized to quantitate cell surface Fas levels on fixed but unpermeabilized cells. As indicated in Fig. 2B, the cells with type I signaling had 2-4-fold higher mean fluorescence intensity after staining with APO-1-1 anti-Fas antibody than cells with type II signaling. In further studies we estimated the absolute number of receptors using PE fluorescence calibration beads and approximated cell surface area based on measurements of cell diameter. Results of this analysis ( Fig. 2C and Table 1) confirmed that the density of cell surface Fas was higher in type I cells even after differences in size were taken into account.
To rule out the possibility that this difference in receptor number occurred only in tissue culture cell lines, cell surface Fas expression was examined on freshly isolated mouse thymocytes and hepatocytes, which exhibit type I and type II Fas signaling, respectively (25,41). As indicated in Fig. 2D, murine thymocytes had almost 10 times as much cell surface Fas receptor as hepatocytes despite the larger size of the latter cells.
Higher Fas mRNA in Type I Cells Reflects Increased mRNA Stability-To explore the basis for higher cell surface Fas expression in type I cells, we performed a series of experiments using the human leukemia cell lines.
First, cell lysates were examined for Fas expression. As shown in Fig. 3A, higher levels of Fas protein were detected by immunoblotting in whole cell lysates from type I cells compared with type II cells. Moreover, qRT-PCR using three different reference transcripts ( Fig. 3B and supplemental Fig. S2) showed that type I cell lines (SKW6.4 and H9) contain more Fas mRNA than type II cells (Jurkat, CEM, and Molt-3). Collectively, these results suggest that type I cells express more Fas message and protein rather than trafficking a disproportionate fraction of Fas protein to the cell surface.
It was reported previously that Fas expression is regulated at the transcriptional level (e.g. Refs. [42][43][44][45]. To investigate the possibility that transcription factors regulating the Fas promoter might be more highly activated in type I versus type II cells, we transfected cells with a reporter construct containing firefly luciferase behind the Fas promoter. Type II Jurkat cells had 3-fold higher luciferase activity than type I SKW6.4 cells (Fig. 3C), arguing against the possibility that differences in promoter activation account for the higher Fas mRNA in type I versus type II cells.
To assess the possibility that Fas mRNA might be more stable in type I versus type II cells, we treated cells with actinomycin D to block transcription (along with Q-VD-OPh to block actinomycin-induced apoptosis), then performed qRT-PCR for Fas mRNA. Results of these experiments (Fig. 3, D and E) indicated that the mean half-life of Fas mRNA is 2.9 -3.4 h in type I cells and 1.4 -1.5 h in type II cells, suggesting that the higher Fas mRNA in type I cells reflects greater Fas mRNA stability.     Fig. S3) protected Jurkat cells transfected with empty vector but not Jurkat cells transfected with Fas cDNA. In contrast, Bcl-x L continued to protect these cells from etoposide, a mitochondrial pathwaydependent stimulus (46 -48), independent of Fas expression (Fig. 4D), ruling out indiscriminant neutralization of the Bcl-x L in Fas-transfected cells. Consistent with type I behavior, CH.11 also induced more DISC formation and procaspase 8 cleavage in Fas-transfected Jurkat cells than in cells transfected with empty vector (Fig. 4E, DISC). As a result, cleaved caspase 3, which contributes to cellular disassembly (49), was more evident in Fas-transfected cells than in those transfected with empty vector (Fig. 4E, Extract), explaining how Fas transfection could lead to more cell death.
Type I Versus Type II Signaling Is Death Receptor-dependent-If type I versus type II signaling were related to cell surface DR density, cells could potentially exhibit type I signaling for one death ligand and type II signaling for another. Consistent with this possibility, Fas transfection rendered Fas signaling in Jurkat cells resistant to Bcl-x L (Fig. 4C) but had no impact on ability of Bcl-x L to protect these cells from TRAIL (Fig. 4F). Instead, cells transfected with either empty vector or Fas cDNA were protected from TRAIL by Bcl-x L overexpression, suggesting that type I versus type II behavior is specific to the receptor rather than the cell line.
Threshold for Conversion of Type II to Type I Fas Signaling-To further explore the quantitative relationship between the amount of cell surface Fas and type II versus type I signaling, Jurkat cells were transfected with EGFP-H2B (to mark transfected cells) and various amounts of Fas plasmid without or with a fixed amount (30 g) of Bcl-x L plasmid. Increasing Fas plasmid led to increased cell surface Fas expression, which peaked at ϳ110,000 Fas molecules/cell when 4 -5 g of Fas-encoding plasmid was delivered to 10 7 cells (Fig. 5A and data  not shown). Notably, transfection with 1-2 g of Fas plasmid resulted in ϳ70,000 cell surface Fas molecules/cell, a level similar to type I SKW6.4 cells. Moreover, Bcl-x L expression did not affect Fas expression (inset, Fig. 5A), allowing us to examine the relationship between Fas expression and Bcl-x L protection.
Beginning 16 h after transfection, cells were treated with CH.11 and examined by annexin V staining for apoptosis in the EGFP-histone H2B ϩ population (supplemental Fig. S4). We then calculated the percent inhibition of apoptosis by Bcl-x L (Fig. 5B). In the face of constant Bcl-x L (inset, Fig. 5B), increasing Fas expression gradually decreased the protective effects of Bcl-x L . At a low CH.11 concentration (12.5 ng/ml), Bcl-x L inhibited Fas-mediated apoptosis up to 70% in Jurkat cells transfected with empty vector but only 20% in cells transfected with 4 -5 g of Fas plasmid. When cells that expressed the maximum amount of cell surface Fas were treated with a higher CH.11 concentration (100 ng/ml), Bcl-x L completely lost its inhibitory effect.
The results shown in Fig. 5, which demonstrate that increased Fas expression results in diminished Bcl-x L -induced protection from Fas-mediated apoptosis, are consistent with the hypothesis that high levels of Fas drive type I signaling. These results also suggest that, with the high level Bcl-x L expression achieved in this study, the threshold for type I signaling (defined as the inability of Bcl-x L to exert any detectable effect) was ϳ95,000 cell surface Fas molecules/Jurkat cell.
Conversion of Type I to Type II Signaling by Fas Downregulation-To rule out the possibility that the foregoing results were unique to lymphoid cells, we also examined solid tumor cell lines. Consistent with an earlier report that categorized lines based on sensitivity to soluble Fas ligand (50), we found that CH.11-induced apoptosis could be inhibited by Bcl-x L in HCT-15 cells (type II signaling) but not A498 cells (type I signaling; see below). As was the case with the lymphoid  OCTOBER 14, 2011 • VOLUME 286 • NUMBER 41 lines, A498 cells had 3-4-fold higher levels of cell surface Fas than HCT-15 cells (Fig. 6A). Further examination indicated that forced overexpression of Fas (Fig. 6, B and the inset in C) rendered Bcl-x L ineffective at inhibiting CH.11-induced apoptosis in HCT-15 cells (Fig. 6C), as was seen in Jurkat cells (Fig.  4C).

Death Receptor Expression and Type I/II Signaling
To determine whether Fas down-regulation would convert signaling from type I to type II, A498 cells were transduced with Fas shRNA. After 1 week of selection, total (Fig. 6D, lower  panel) and cell surface Fas levels (Fig. 6D, upper panel) in A498 transductants were similar to those of HCT-15 cells. Fas shRNA-transduced A498 cells were less sensitive to CH.11 (Fig.  6E), consistent with lower cell surface Fas expression. Moreover, Bcl-x L now protected these cells from CH.11-induced apoptosis (Fig. 6E), consistent with conversion to type II signaling.
Cell Surface DR5 Up-regulation Converts TRAIL Signaling in HCT116 Cells from Type II to Type I-The preceding studies suggest that as little as a 3-fold change in cell surface Fas expression is sufficient to convert signaling from type II to type I. Additional experiments were performed to determine whether this was also the case with TRAIL signaling, where it was previously suggested that DR5 up-regulation makes TRAIL-induced killing of HCT116 cells Bax-independent (51,52).
Earlier studies suggested that TRAIL-induced killing in HCT116 colon cancer cells normally proceeds via a type II signaling pathway (51)(52)(53). Consistent with this conclusion, Bcl-x L protected cells from TRAIL-induced apoptosis (Fig. 7A).
To determine whether increased cell surface TRAIL receptor expression, like Fas expression, could convert cells from type II to type I signaling, we developed an HCT116 BAX Ϫ/Ϫ line (36) that also had Bak stably knocked down by shRNA (HCT116 BAX Ϫ/Ϫ /Bak shRNA, Fig. 7B). This line was resistant to induction of apoptosis by the topoisomerase II poison etoposide and the topoisomerase I poison SN-38 (Fig. 7C), two classes of agents that were previously shown to trigger apoptosis through the intrinsic pathway (46 -48, 54), confirming that the mitochondrial pathway was nonfunctional in these cells.
Because enforced overexpression of DR5 was extremely toxic to HCT116 cells (data not shown), we elected to up-regulate DR5 by treating cells with SN-38, the active metabolite of the drug irinotecan that was previously shown to enhance DR5 expression (55). We treated HCT116 BAX Ϫ/Ϫ /Bak shRNA cells with SN-38 for 48 h, then measured cell surface DR5 expression and TRAIL sensitivity. As shown in Fig. 7D, a 3-fold increase in cell surface DR5 was detected after a 48-h treatment with 6.25 nM SN-38. In the absence of SN-38 pretreatment, TRAIL did not induce apoptosis in these cells. However, after pretreat- ment with SN-38, TRAIL-induced apoptosis was observed (Fig.  7E). This apoptosis was not inhibited by Bcl-x L (Fig. 7F), demonstrating that TRAIL signaling had become type I when cell surface DR5 was up-regulated 3-fold.

DISCUSSION
Since its original description, the distinction between type I and type II death receptor signaling has been widely observed in vitro and in vivo. The biochemical basis for this dichotomy, however, has remained elusive. Results of the present study provide evidence that type I versus type II signaling is determined, at least in part, by the amount of cell surface receptor present. Because type II signaling is inhibited by changes that blunt signaling through the mitochondrial pathway (18) as well as protein kinase C activation (21,40), the present results suggest that changes in cell surface Fas expression can have implications for surfas sensitivity far beyond what would be expected by simple equilibrium binding calculations.
A number of explanations for the differences between type I and type II signaling have been previously advanced. Several studies have suggested that cells with type II signaling have elevated sensitivity to the endogenous caspase inhibitor XIAP (24,25). In the present study, however, immunoblotting failed to demonstrate a reproducible dichotomy in XIAP expression between cells with type I and type II signaling (Fig. 1E). Moreover, it has been unclear how elevated XIAP sensitivity could cause the observed difference in DISC formation between cells with type I and type II signaling (18). Instead, it has been reported that the ability of XIAP knockdown to specifically enhance death ligand-induced apoptosis in type II cells can be explained by increased sensitivity of effector caspases to activation (56), presumably reflecting removal of the block to caspase activation that results when XIAP is bound to the apoptosome (57).
While the present work was in progress, Peacock et al. (23) reported that differences between type I and type II signaling correlate with differences in expression of the lipid phosphatase PTEN, which is thought to modulate Bcl-2 function as well as a variety of additional Akt-mediated anti-apoptotic functions (58,59). Although treatment of Jurkat cells with the phosphoinositide 3-kinase inhibitor LY294002 was shown to diminish the unrestrained Akt signaling and increase the pace of Fasmediated apoptosis in PTEN-deficient cells (23), the previous study did not establish whether LY294002 altered either DISC formation or the requirement for Bid during Fas-mediated killing of these cells.
In the present study we determined whether signaling was through a type I or type II pathway by directly examining the effects of Bid knockdown or Bcl-x L /Bcl-2 overexpression. Using this original criterion for type I versus type II signaling, our results demonstrate that cells with type I signaling have a higher density of cell surface Fas than cells with type II signaling ( Table 1, Figs. 2 and 6). In particular, we observed high cell surface Fas expression on type I cells using nonpermeable biotinylating agents as well as flow cytometry after staining with anti-Fas antibody. Moreover, Fas down-regulation in cells with high cell surface death receptor converted type I signaling to type II as manifested by enhanced Bcl-x L sensitivity (Fig. 6E). Conversely, forced overexpression of Fas converted type II sig-  OCTOBER 14, 2011 • VOLUME 286 • NUMBER 41 naling to type I, as manifested by both increased DISC formation (Fig. 4E) and diminished Bcl-x L sensitivity (Fig. 4C). Importantly, Fas overexpression had no impact on type II signaling by TRAIL receptors in the same cells (Fig. 4F), suggesting that the type I/type II dichotomy is not an intrinsic feature of the cell, but is instead related to the death receptor itself.

Death Receptor Expression and Type I/II Signaling
These observations provide a new conceptual framework for understanding additional differences in cells that signal through type I versus type II pathways after death ligand exposure. It has previously been reported that phorbol esters inhibit Fas-mediated apoptosis in cells with type II but not type I signaling (21). Our recent studies indicated that phorbol esters diminish ligand-induced accumulation of Fas on the cell surface (28). The present demonstration that type I cells have more cell surface Fas before ligation provides a potential explanation for successful death induction in type I cells even when phorbol esters inhibit the accumulation of additional Fas at the cell surface.
In addition to providing new insight into the action of death ligands, the present results also have potential therapeutic implications. Previous studies have shown that the mitochondrial apoptotic pathway is commonly disabled in human cancers (60 -62). To the extent that death ligands such as TRAIL induce type II signaling, as appears to be the case in many cell types (52,(63)(64)(65), cancer cells would be expected to be resistant as a consequence of blocks to the mitochondrial pathway. On the other hand, additional observations have indicated that Fas and TRAIL receptors can be up-regulated by a variety of treatments, including DNA damaging agents (66 -68), the spindle poison paclitaxel (69), the proteasome inhibitor bortezomib (70 -72), histone deacetylase inhibitors (70,73), farnesyltransferase inhibitors (74), and cytokines such as interferon-␥ and tumor necrosis factor-␣ (75). These observations, coupled with our demonstration that an increase in Fas or DR expression as small as 3-fold can convert cells from type II to type I signaling, raise the possibility that appropriately chosen combinations of agents might be used to increase cell surface DR expression and overcome the mitochondrial block that prevents death ligandinduced apoptosis in cells with type II signaling.