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Ligation of Fas and Cell Death

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

18 June 2000

David Vaux

Opening Statement for the Discussion of Apoptosis Signaling: Can Cell Death Induced by Fas Be Blocked by Bcl-2?

Moderator: David L. Vaux

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

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)

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