Forum: Ligation of Fas and Cell Death


Moderator: David L. Vaux

The Walter and Eliza Hall Institute, Royal Parade, Parkville, Victoria 3052, Australia.


Opening Statement: Can Cell Death Induced by Fas Be Blocked by Bcl-2?

18 June 2000

David Vaux

With 250 papers appearing every week on apoptosis, it is not surprising that this relatively new field is subject to many areas of controversy. One area of contention concerns how some cell death triggers are wired to the caspases, the key cell death effector proteins. This Web discussion is about how ligation of the receptor Fas (also called CD95 or APO-1) leads to cell death, in particular whether Fas-triggered cell death can be signaled by pathways that can be blocked by Bcl-2 (or Bcl-xL), and, if so, the nature of the molecules that make this connection.

A very simplified diagram of the two pathways to cell death is shown in Fig. 1. Apoptosis induced in many ways, such as by removal of growth factors, activation of p53, or treatment of cells with steroids or chemotherapeutic drugs, can be inhibited by Bcl-2. Apoptosis induced by ligation of Fas causes assembly of a "death inducing signaling complex" (DISC) involving FADD and caspase 8, that then activates downstream caspases independently of the steps that Bcl-2 can inhibit. What is hotly debated is whether Fas is always able to activate enough caspase 8 to cross the apoptotic threshold, or whether, in addition, Fas signals via other molecules leading to activation of caspase 9, via steps that can be blocked by Bcl-2.



Fig. 1. A schematic of the two pathways leading to caspase activation and cell death.

A few of the reports in the literature that address whether Bcl-2 or Bcl-xL can inhibit Fas-induced apoptosis are listed below (1-35). Note that this list is far from complete and is organized according to references that support a Bcl-2 inhibitable pathway (1-25) and those that oppose this pathway (25-35) and have brief comments about the systems used for the experiments or significant contributions to the issue. The literature illustrates how difficult the issue has been to resolve. Several groups have published papers supporting both sides. Moreover, the experimental systems used can't explain the discrepancies, because evidence supporting both sides has come from cell-free systems, tissue culture systems, and in vivo systems.

Several candidate molecules have been proposed to participate in the connection between the "Bcl-2 inhibitable" and Fas, FADD, caspase 8 pathways. These include Daxx, which is reported to bind directly to the cytoplasmic domain of Fas and promote apoptosis via JNK; ceramide, which is generated by Fas-activated sphingomyelinases and affects the mitochondria; FLASH (36), which binds directly to Fas; and Bid, a pro-apoptotic "BH3-only" member of the Bcl-2 family (37) that when cleaved by caspase 8 can directly antagonize Bcl-2.

Despite the number of papers describing inhibition of Fas-induced apoptosis by Bcl-2, the controversy persists because in some cases there are other explanations for the observations made. For example, Jurkat T cells have often been used to demonstrate inhibition of Fas-induced apoptosis by Bcl-2, but it is very easy to select Jurkat cells that have lost the caspase 8 gene, so in some cases Fas-resistance may be due to this rather than expression of Bcl-2. In most experimental systems Fas is ligated by antibodies rather than its ligand. It is possible that the antibodies used to bind Fas also provide other signals, perhaps via their Fc domains. In many cases, Fas only efficiently induces apoptosis in the presence of cycloheximide. It has been proposed that inhibition of translation by cycloheximide can cause apoptosis by a Bcl-2-inhibitable pathway.

In 1998, Scaffidi et al. (25) proposed a model to explain the differences between cells in which Fas-induced apoptosis could be inhibited by Bcl-2 and those in which it couldn't. They suggested that in Type 1 cells high levels of caspase 8 were rapidly activated upon Fas ligation, and Bcl-2 expression could not affect apoptosis that followed. In Type II cells only low levels of caspase 8 became activated and were insufficient to activate downstream caspases directly. However, caspase 8 was somehow able to signal across to the Bcl-2-inhibitable pathway to ultimately cause apoptosis.

The mechanism of this "cross-talk" is also debated. A favorite candidate is Bid, which can be cleaved by caspase 8, and then antagonize Bcl-2. However, because Bid is not cleaved during Fas-induced hepatocyte apoptosis in caspase 3 knock out mice (38), caspase 3 rather than caspase 8 may be the main Bid-cleaving caspase. There are also many arguments for and against other molecules proposed to mediate the crosstalk, such as FLASH, ceramide, and Daxx.

The ultimate resolution of these controversies will depend on critical experiments. It is hoped that analysis and discussion in this forum of the data already published will clarify the discrepancies and help in the formulation of new experiments that may resolve the biologically relevant signaling mechanisms that control cell death.

Literature that supports Bcl-2/Bcl-xL inhibition of Fas-induced apoptosis Experiments with myeloid cells (FDC-P1) showing partial protection only (1) Experiments with Hela cells (2) Experiments with human breast carcinoma (MCF-7) (3, 17, 18) Experiments with mouse mastocytoma cells (P815) and ConA-activated mouse splenocytes (4) Experiments in cell-free systems (5, 6) Experiments with hepatic expression of a Bcl-2 transgene (7) Experiments with hepatic expression of a Bcl-2 transgene; the hepatocytes were protected but the mice died (8) Experiments with Jurkat T cells (9, 10, 13, 21) Experiments with mouse mastocytoma cells (P815) (11) Experiments with human tonsillar cells (12) Evidence that Bcl-2 inhibits Fas-induced apoptosis signaled via Daxx (14) Evidence that c-myc-induced apoptosis requires Fas-FasL interaction and is blocked by Bcl-2 (15) Evidence that Bcl-2 and SMN synergize to give greater protection from apoptosis (16) Evidence that Bcl-2 blockable Fas-triggered killing is mediated by Bid (19, 20) Evidence that Bcl-2 can inhibit FADD-independent activation of capsase 8 as well as apoptosis downstream of caspase 8 activation (22) Evidence that Bcl-xL inibits Fas-induced death upstream of caspase 8 activation (23) Experiments with hepatocytes from Bid-/- mice showing resistance to killing by anti-Fas antibodies (24) Evidence for Type II cells in which a low level of caspase 8 is activated and cells are protected by Bcl-2 (25)

Literature that opposes a role for Bcl-2/Bcl-xL in blocking Fas-induced apoptosis Evidence that Bcl-2 could protect against Fas-induced apoptosis only if Bag-1 was co-expressed in Jurkat T cells (26) Experiments with cytotoxic T cell targets (27) Experiments with mouse T hybridoma cells (2B4) (28) Experiments with mouse lymphoid cells (29) Experiments with T cell blasts (30) Experiments with mouse cell lines (31) Experiments in cell-free systems (32, 34) Evidence that Bcl-xL did not protect against death induced by a Fas-caspase 8 fusion construct (33) Evidence that Fas causes apoptosis and Bid cleavage and that Bcl-xL inhibits Bid-independent cytochrome c release but does not inhibit cell death (35) Evidence for Type I cells in which a high level of caspase 8 is activated and cells are not protected by Bcl-2 (25) REFERENCES


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Chem. 273: 33099-33102.[Abstract/Full Text] Perez, D., and White, E. (1998) E1b 19k inhibits Fas-mediated apoptosis through FADD-dependent sequestration of FLICE. J. Cell Biol. 141: 1255-1266.[Abstract/Full Text] Yin, X.M., Wang, K., Gross, A., Zhao, Y.G., Zinkel, S., Klocke, B., Roth, K.A., and Korsmeyer, S.J. (1999) Bid-deficient mice are resistant to Fas-induced hepatocellular apoptosis. Nature 400: 886-891.[Medline] Scaffidi, C., Fulda, S., Srinivasan, A., Friesen, C., Li, F., Tomaselli, K.J., Debatin, K.M., Krammer, P.H., and Peter, M.E. (1998) Two CD95 (APO-1/Fas) signaling pathways. EMBO J. 17: 1675-1687.[Abstract/Full Text] Takayama, S., Sato, T., Krajewski, S., Kochel, K., Irie, S., Millan, J.A., and Reed, J.C. (1995) Cloning and functional analysis of BAG-1 - a novel Bcl-2-binding protein with anti-cell death activity. Cell 80: 279-284.[Medline] Chiu, V.K., Walsh, C.M., Liu, C.C., Reed, J.C., and Clark, W.R. (1995) Bcl-2 blocks degranulation but not fas-based cell-mediated cytotoxicity. J. Immunol. 154: 2023-2032.[Medline] Memon, S.A., Moreno, M.B., Petrak, D., and Zacharchuk, C.M. (1995) Bcl-2 blocks glucocorticoid- but not Fas- or activation-induced apoptosis in a T cell hybridoma. J. Immunol. 155: 4644-4652.[Medline] Strasser, A., Harris, A.W., Huang, D.C.S., Krammer, P.H., and Cory, S. (1995) Bcl-2 and Fas/APO-1 regulate distinct pathways to lymphocyte apoptosis. EMBO J. 14: 6136-6147.[Abstract] Moreno, M.B., Memon, S.A., and Zacharchuk, C.M. (1996) Apoptosis signaling pathways in normal T cells differential activity of Bcl-2 and IL-1beta-converting enzyme family protease inhibitors on glucocorticoid- and fas-mediated cytotoxicity. J. Immunol. 157: 3845-3849.[Medline] Huang, D., Cory, S., and Strasser, A. (1997) Bcl-2, Bcl-xL and adenovirus protein E1B19kD are functionally equivalent in their ability to inhibit cell death. 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(1999) Caspase cleaved BID targets mitochondria and is required for cytochrome c release, while BCL-XL prevents this release but not tumor necrosis factor-R1/Fas death. J. Biol. Chem. 274: 1156-1163.[Abstract/Full Text] Imai, Y., Kimura, T., Murakami, A., Yajima, N., Sakamaki, K., and Yonehara, S. (1999) The CED-4-homologous protein FLASH is involved in Fas-mediated activation of caspase-8 during apoptosis. Nature 398: 777-785.[Medline] Adams, J.M., and Cory, S. (1998) The Bcl-2 protein family: Arbiters of cell survival. Science 281: 1322-1326.[Abstract/Full Text] Woo, M., Hakem, A., Elia, A.J., Hakem, R., Duncan, G.S., Patterson, B.J., and Mak, T.W. (1999) In vivo evidence that capase-3 is required for Fas-mediate apoptosis of hepatocytes. J. Immunol. 163: 4909-4916.[Medline]

The Middle Ground

20 June 2000

Guy Salvesen

David Vaux raised an issue that has become religious in its intensity in the apoptosis field. People like to take sides, and as usual the answer is probably somewhere in the middle. I guess David’s question is "where is the middle"?

A contemporary view of apoptotic signaling defines the "Bcl-2- inhibitable pathway" as the "Intrinsic Apoptotic Pathway" and the "Fas- triggered pathway" as a subset of the death receptor, or "Extrinsic Apoptotic Pathway". I’m not sure when these definitions were suggested, but you can find them in (1).

Both pathways are thought to converge at the activation of pro- caspase-3. Therefore, it would seem that the activation of pro-caspase-3 by caspase-8 of the Extrinsic Pathway is more efficient than sending a signal through the mitochondrial Intrinsic route. Which is easier (more logical)? To cut a piece of string with some scissors that are already at hand; or to cut a rope binding someone so they can go to the hardware store to buy the scissors, get them home to you so you can cut the string?

Key players in the Intrinsic Pathway are the apical caspase-9 whose activation is initiated by its co-factor Apaf-1, following release of cytochrome-C from mitochondria (2). Both Apaf-1 and cytochrome-C seem to be obligatory for the activation of caspase-9 (3, 4), so one can predict that mice ablated in these genes should not be able to support the Intrinsic Pathway. This is indeed the case. Though there are minor differences in apoptotic phenotype, caspase-9 knockouts (5, 6), the Apaf-1 knockout (7, 8), and the cytochrome-C knockout (9) are unable to support apoptosis triggered by Intrinsic Pathway stimuli such as ionizing radiation and cytotoxic drugs. On the other hand all of these knockouts show unimpaired apoptosis via Extrinsic Pathway triggers such as TNF. Indeed the TNF response of embryonic cells in the cytochrome-C knockout is reportedly enhanced (9)! The simplest explanation is that the Intrinsic Pathway is not required in these cases when the trigger is TNF: the Extrinsic Pathway does not need help from the Intrinsic.

Therefore, the apparent requirement of the Intrinsic Pathway in those several cellular and in vivo paradigms (alluded to in David’s post) where the initial trigger is through the Extrinsic Pathway is probably telling us that we are missing something. Something is binding the scissors. Something is putting the breaks on caspase-8, or more likely or caspase-3, in these cells. The "something" should be an additional pro-apoptotic factor that only the Intrinsic Pathway can elaborate, and it should take the breaks off. Since the major difference between the pathways is the requirement of protein flux from mitochondria, then this factor should reside there. Is it AIF (10), or does it await discovery?

The field has come a long way in revealing the key players: the framework of apoptosis. But it’s abundantly clear that many cell types respond differently to the same trigger. Therefore, placed on the framework is a level of control and sophistication that allows precise regulation of death decisions. This is the middle ground, and understanding the cell-specific controls is probably where the future of the field lies. For those of religious fervor on the Bcl- 2/Fas/Extrinsic/Intrinsic questions… Remember – "God is in the details".


1. Stennicke, H. R., and Salvesen, G. S. (2000) Caspases - controlling intracellular signals by protease zymogen activation. Biochim Biophys Acta 1477, 299-306. [Medline]

2. Li, P. et al. (1997) Cytochrome c and dATP-Dependent Formation of Apaf- 1/Caspase-9 Complex Initiates an Apoptotic Protease Cascade. Cell 91, 479- 489. [Medline]

3. Stennicke, H. R. et al. (1999) Caspase-9 can be activated without proteolytic processing. J Biol Chem 274, 8359-8362.[Abstract/Full Text]

4. Rodriguez, J., and Lazebnik, Y. (1999) Caspase-9 and APAF-1 form an active holoenzyme. Genes Dev 13, 3179-84.[Abstract/Full Text]

5. Kuida, K. et al. (1998) Reduced apoptosis and cytochrome c-mediated caspase activation in mice lacking caspase 9. Cell 94, 325-37. [Medline]

6. Hakem, R. et al. (1998) Differential requirement for caspase 9 in apoptotic pathways in vivo. Cell 94, 339-52. [Medline]

7. Yoshida, H. et al. (1998) Apaf1 is required for mitochondrial pathways of apoptosis and brain development. Cell 94, 739-50. [Medline]

8. Cecconi, F. et al. (1998) Apaf1 (CED-4 homolog) regulates programmed cell death in mammalian development. Cell 94, 727-37. [Medline]

9. Li, K. et al. (2000) Cytochrome c deficiency causes embryonic lethality and attenuates stress-induced apoptosis. Cell 101, 389-99. [Medline]

10. Susin, S. A. et al. (1999) Molecular characterization of mitochondrial apoptosis-inducing factor. Nature 397, 441-6. [Medline]

Details of the intrinsic pathway

21 June 2000

David Vaux

Guy Salvesen is right - God IS in the details (or maybe it's the Devil…). As he (Guy, not the other two) mentioned, caspase 3, caspase 9 and Apaf-1 knock out mice have very similar phenotypes, mainly featuring excess neurons. Recently, however, caspase 3 knockouts and Apaf-1 knockouts have been bred onto a pure C57BL/6 background (1,2). In both cases several animals have survived to adulthood, and even produced live offspring. Clearly, in the mouse, neither Apaf-1 nor caspase 3 are essential for developmental cell deaths or those that maintain constancy of cell number. Does this mean that the "intrinsic" or "cytochrome c / Apaf-1 dependent" apoptosis pathway is of minor significance? Because many of the early experiments were of necessity carried out on mouse embryo fibroblasts or ES cells, it will be very interesting to see which other cell types (such as CSF-dependent hemopoietic cells) will still be able to undergo apoptosis in response to growth factor starvation or Fas ligation. REFERENCES


1. Woo, M., Hakem, A., Elia, A. J., Hakem, R., Duncan, G. S., Patterson, B. J. & Mak, T. W. (1999) In vivo evidence that capase-3 is required for Fas-mediate apoptosis of hepatocytes. Journal of Immunology 163 4909-4916.[Medline]

2. Honarpour, N., Du, C. Y., Richardson, J. A., Hammer, R. E., Wang, X. D. & Herz, J. (2000) Adult Apaf-1-deficient mice exhibit male infertility. Developmental Biology 218 248-258. [Medline]

The TNF response, NF-kappa B and Bcl-2

28 June 2000

Tomas Jelinek

Homologous to the Fas pathway is the tumor necrosis factor (TNF)-alpha pathway, using many of the same intracellular effectors that are used by Fas. Yet the work of Baltimore, and Baldwin, suggests that what determines whether TNF- stimulated cells undergo apoptosis or proliferation depends on NF-kappa B, a transcription factor. Any effects of NF-kappa B are of necessity delayed by an hour or so, until transcription and translation of target genes can occur. Among those targets are anti-apoptotic Bcl-family members.

Figuring out how this works mechanistically and kinetically should shed light on the question of Bcl-2 inhibition of the Caspase-8 signal. Specific questions:

1. What is the time-course of activation of the various caspases following Fas (or TNF) administration?

2. When NF-kappa B is involved, what is the time-to-appearance of elevated anti-apoptotic proteins on the mitochondria? Is this time shorter than what is seen for Caspase 9 activation in the absence of NF-kappa B?

The TNF plot thickens

3 July 2000

David Vaux

The signal transduction pathways activated by TNF-alpha are more complicated than those activated by Fas ligand (FasL) for a number of reasons. First, there are of course two receptors for TNF rather than one. Addition of TNF usually activates NF-kappa B (requiring the adapter protein RIP) and Jun [requiring other adapter proteins, the TRAFs (tumor necrosis factor receptor-associated factors)], whereas activation of these pathways is not always seen after Fas ligation. Fas can directly associate with the Fas-associated death domain protein (FADD), leading to activation of caspase 8, whereas TNF receptor type 1 (TNF-R1) seems to associate with FADD only indirectly, via TRADD (TNF-R1-associated death domain protein). Activated NF-kappa B has been shown to transactivate a number of anti- apoptotic genes, including Bcl-xL, the Bcl-2 family member A1, and the inhibitor-of-apoptosis proteins IAP1 and IAP2.

It has been proposed that because TNF induces expression of cell death inhibitors (via NF-kappa B) as well as caspase activation (via FADD), many cells don't die when their TNFRs are ligated (1,2). This model does explain why cycloheximide together with TNF is a much more potent inducer of cell death, and why cells lacking components of NF-kappa B are more likely to die when exposed to TNF, but there are still some problems. One is the difficulty of explaining how a pathway requiring transcription and translation manages to beat a short pathway involving direct association of TRADD, FADD, and caspase 8.

Getting more to the point of the questions raised by Dr Jelinek, in most cases addition of TNF does not cause activation of any caspases, unless cycloheximide is also present. When apoptosis is induced by TNF it can usually be inhibited by CrmA (cowpox virus cytokine response modifier A), an inhibitor of caspases 1 and 8, but rarely by Bcl-2 or Bcl-xL.

Point 2 is an excellent one. (I wish I knew the answer!) I don't see how NF-kappa B-transactivated proteins could get to the mitochondria in time, so I would have to guess that maybe other proteins or pathways are involved. For example, maybe the low, steady-state levels of NF-kappa B are required for production of a short lived protein that can act in a rapid post- transcriptional pathway to inhibit caspase activation when TNF receptors are ligated (3). What might this protein be?

On a different (but related) note, a recent paper may help answer the question of why TNF alone rarely causes apoptosis in vitro, and why it causes necrosis rather than apoptosis when used in vivo. This paper (4) shows that in vivo, TNF causes death of tumor cells indirectly, by acting on the blood vessels that feed them. The tumor cells die a necrotic death due to loss of blood supply, rather than apoptosing cell-autonomously.

1 Beg, A.A. and Baltimore, D. (1996). An essential role for NF-kappa -B in preventing TNF-alpha-induced cell death. Science 274, 782-784.[Medline]

2 Wang, C.Y., Mayo, M.W. and Baldwin, A.S. (1996). TNF- and cancer therapy-induced apoptosis - potentiation by inhibition of NF-kappa-B. Science 274, 784-787.[Medline]

3 Kajino, S., Suganuma, M., Teranishi, F., Takahashi, N., Tetsuka, T., Ohara, H., Itoh, M. and Okamoto, T. (2000). Evidence that de novo protein synthesis is dispensable for anti-apoptotic effects of NF-kappa B. Oncogene 19, 2233-2239.[Medline]

4 Stoelcker, B., Ruhland, B., Hehlgans, T., Bluethmann, H., Luther, T. and Mannel, D.N. (2000). Tumor necrosis factor induces tumor necrosis via tumor necrosis factor receptor type 1-expressing endothelial cells of the tumor vasculature. Am. J. Pathol. 156, 1171-1176.[Medline]

Apoptosis in Neuronal and Cognitive Development?

19 March 2004

Rainer Spiegel

I wonder whether there is a particular reason for apoptosis other than the removal of damaged cells. From an evolutionary point of view, the removal of damaged cells would provide an advantage, as those individuals who have this mechanism would be more likely to survive and reproduce. However, does the removal of neurons (=neuronal apoptosis) in the central nervous system also help to form neuronal pathways that would otherwise not have formed, as they would have been inhibited by superfluous neurons? If these neuronal pathways are responsible for the acquisition of certain skills, then there might be a point to argue that neuronal and cognitive development benefit from apoptosis. I would be grateful for any expert advice or recommendations in this regard.

Apoptosis in Neuronal and Cognitive Development?

22 March 2004

Liz Adler

The elimination of numerous superfluous cells through apoptosis is critical to the proper development of the nervous system. Neuronal apoptosis during development can stem from lack of access to various neurotrophic factors (which suppress cell death pathways) or from activation of apoptotic pathways by death signals.

You may be interested in taking a look at the Granule Cell Survival Pathway and its associated i>Science Viewpoint by Vaudry et al., Regulators of Cerebellar Granule Cell Development Act Through Specific Signaling Pathways for a discussion of some signaling pathways involved in the survival of cerebellar granule cells.

Although not specific to neurons, the Perspective by Chao, Dependence Receptors: What Is the Mechanism? provides insight into the mechanism by which dependence receptors (which promote apoptosis in the absence of ligand) work.

General information on the extent and role of neuronal apoptosis during development can be found in many neuroscience texts.

Neuronal Apoptosis during Development

23 March 2004

Rainer Spiegel

Dear Professor Adler,

Thanks very much for your helpful comments about neuronal apoptosis during development. Your advice and the references you recommended are very much appreciated.

Yours sincerely,
Rainer Spiegel