(Received for publication, May 18, 1995; and in revised form, July 14, 1995)
From the
Relatively little is known about oncogene involvement in the
regulation of Fas-mediated apoptosis. Inhibition of Fas-induced cell
death by the bcl-2 oncogene has been demonstrated to be only
partial. In light of a growing body of evidence for the Abl kinase as a
negative regulator of cell death, we sought to determine whether Abl
expression could protect against Fas-mediated cell death. To address
this question, we utilized two separate strategies. In the first, we
expressed human Fas in K562, a chronic myelogenous leukemia cell line,
which constitutively expresses bcr-abl and examined the
effects of Fas ligation in these cells. Fas-positive K562 transformants
(K562.Fas) were found to be protected against Fas-mediated cell death.
However, down-regulation of Bcr-Abl protein levels in K562.Fas cells
using antisense oligonucleotides targeted to bcr-abl mRNA
rendered these cells highly susceptible to Fas-induced death. In the
second approach we utilized a Fas-positive HL-60 cell line, which we
transfected with a temperature-sensitive mutant of v-Abl.
HL-60.v-Abl transfectants were found to be protected from
Fas-induced apoptosis at the permissive but not the restrictive
temperature for the Abl kinase. Taken together, these observations
identify the Abl kinase as a negative regulator of Fas-mediated cell
death. Since Abl was also found to block apoptosis mediated by
ceramide, a recently proposed downstream effector of the apoptotic
pathway initiated by Fas, we propose that Abl exerts its protective
effects downstream of the early Fas-initiated signaling events.
Fas (CD95), a 48-kDa cell surface protein belonging to the tumor
necrosis factor receptor family (Itoh et al., 1991; Oehm et al., 1992) is expressed in various tissues including
thymus, heart, lung, and liver (Watanabe-Fukunaga et al.,
1992). Ligation of Fas with anti-Fas antibody (Trauth et al.,
1989; McGahon et al., 1995) or its specific ligand (Suda et al., 1993) induces a death signal in many cell types of
different hematopoietic origin, including T and B cells (Trauth et
al., 1989; Owen-Schaub et al., 1992) and a variety of
hematopoietic and nonhematopoietic cell lines (McGahon et al.,
1995). Mutations in the fas gene or its specific ligand are
responsible for lymphoproliferative disorders in the lpr and gld mutant mouse, respectively (Watanabe-Fukunaga et
al., 1992; Takahashi et al., 1994). A role for Fas in
Ca-independent T cell-mediated cytotoxicity has also
recently been found (Rouvier et al., 1993). Fas-mediated
cytotoxic T lymphocyte killing has also been found as an alternative
lytic pathway in a perforin-deficient cytotoxic T lymphocyte hybridoma
(Walsh et al., 1994) and perforin null mice (Kagi et
al., 1994). Activation-induced cell death in T cell hybridomas has
recently been demonstrated to proceed via a cell autonomous Fas/Fas
ligand interaction (Brunner et al., 1995).
There is a
growing body of evidence that identifies the Abl kinase as a negative
regulator of apoptosis. Expression of v-abl can confer growth
factor independence on several cell types, including mast cells (Pierce et al., 1986) and lymphoid lines (Mathey et al.,
1986). Bcr-Abl expression can also render some myeloid lines
IL-3()-independent (Daley and Baltimore, 1988; Hariharan et al., 1988). More recent studies have shown that expression
of the Abl kinase can confer resistance to apoptosis in some cell
types. For example, activation of v-abl is associated with the
suppression of apoptosis in hematopoietic cells (Evans et al.,
1993) and constitutive expression of the p210 Bcr-Abl protein inhibits
apoptosis in CML progenitor lines upon IL-3 withdrawal (Bedi et
al., 1994). In addition, down-regulation of Bcr-Abl protein levels
by antisense oligonucleotides targeted to bcr-abl mRNA have
been shown to render K562 cells (an apoptosis-resistant CML cell line
that expresses a deregulated form of the abl oncogene: bcr-abl) susceptible to apoptosis (McGahon et al.,
1994). Moreover, transfection of a temperature-sensitive mutant of
v-Abl (Kipreos et al., 1987) into the HL-60 human
promyelocytic leukemia cell line also renders cells of this line
resistant to agents that are normally very effective inducers of
apoptosis in these cells, providing further evidence that deregulated
Abl kinase activity can confer resistance to apoptotic cell death.
To examine the potential effect of Abl on Fas-initiated cell
killing, we adopted a dual approach. First, we transfected K562, a
chronic myelogenous leukemia cell line that expresses bcr-abl with a cDNA encoding human fas. Transfected K562 clones
were found to be resistant to Fas-mediated cell death. However,
down-regulation of Bcr-Abl expression using antisense oligonucleotides
corresponding to bcr-abl mRNA rendered these cells susceptible
to Fas-induced death. In the second approach we transfected HL-60 (a
Fas-positive cell line) with a temperature-sensitive mutant of v-Abl
(v-Abl). HL-60.v-Abl
transfectants were found
to be protected from Fas-induced apoptosis at the permissive
temperature for the Abl kinase. Finally, we demonstrate that Abl can
block apoptosis induced by ceramide, a recently proposed downstream
effector of the apoptosis pathway initiated by Fas. Our observations
demonstrate that Abl can act as a negative regulator of Fas-induced
cell death and that it exerts its regulatory effects downstream of
Fas-initiated ceramide production.
For detection of Fas-positive
clones, cells (5 10
cells/well in a 96-well plate)
were washed in phosphate-buffered saline, fixed in 70% ethanol on ice
for 30 min, washed again, and preincubated in phosphate-buffered saline
containing 2% calf serum to block Fc receptors and minimize nonspecific
staining. Cells were stained using a 1:50 dilution of a mouse
anti-human Fas IgG monoclonal antibody (UB2, Kamiya Biomedical Co.)
followed by a goat anti-mouse FITC-conjugated F(ab`)
Ab at
a 1:50 dilution. Clones were analyzed on a FACScan flow cytometer.
Cell death was also quantitated by flow cytometry. The criteria used for assessment of cell death in this case were based on two parameters: changes in light scattering properties of dead cells due to cell shrinkage and increased granularity (Yamada and Ohyama, 1980; Wyllie and Morris, 1982; McGahon et al., 1995), and their permeability to the DNA binding dye propidium iodide (PI). Cells were incubated with 5 µg/ml PI at room temperature and analyzed immediately.
Figure 1:
A, expression of human Fas in
K562 cells. The parental K562 cells and their transfectants were
stained with an FITC-conjugated secondary antibody alone, GAM-FITC
(control), or with an anti-Fas Ab followed by a GAM-FITC Ab (anti-Fas),
and Fas expression was determined on a FACScan as described under
``Materials and Methods.'' B, resistance of K562.Fas
transformants to Fas-mediated cell death. K562 () and K562.Fas
(
) cells (10
/ml) were incubated for an 18-h period
with varying concentrations of anti-Fas Ab and apoptosis was
quantitated using morphological criteria as described under
``Materials and Methods.'' An independent measure of cell
viability as assessed by PI uptake was also performed. The ability of
the Fas-positive Jurkat (
) cell line to undergo Fas-mediated
killing under the same culture conditions was also assessed by both
morphological criteria and PI uptake. All data presented are averages
of triplicate experiments. C, expression of human Fas in
murine P815 cells. The parental P815 cells (P815) and their
transfectants (P815.Fas) were stained with an FITC-conjugated secondary
antibody alone, GAM-FITC, or with an anti-Fas Ab, followed by a
GAM-FITC Ab, and Fas expression was determined on a FACScan as
described under ``Materials and Methods.'' D,
dose-dependent killing of P815.Fas transfectants with anti-Fas Ab.
P815.WT (
) or P815.Fas (
) transfectants
(10
/ml) were incubated in 96-well plates for an 18-h period
with varying concentrations of anti-Fas Ab, and cell viability was
assessed by PI uptake as described under ``Materials and
Methods.''
It remained a possibility that the expressed Fas was not capable of activating the apoptotic pathway in transfected cells. Therefore we examined the ability of murine P815 cells infected with the same Fas-expressing retrovirus (P815.Fas) to undergo apoptosis upon Fas ligation. These mastocytoma cells do not express Bcr-Abl protein and express the c-Abl protein at normal levels (data not shown). P815.Fas cells expressed cell surface human Fas at levels that were similar to those of K562.Fas cells (Fig. 1C) and were significantly susceptible to Fas-mediated cell killing under the same incubation conditions used for the K562.Fas transfectants (Fig. 1D).
Figure 2:
A, down-regulation of Bcr-Abl
protein levels in K562.Fas cells. K562.Fas cells were either left
untreated or incubated in the presence of either 10 µM
AS-bcr-abl (AS) or 10 µM NS-bcr-abl (NS) for a 48-h time period. Cell lysates were prepared
from the treated samples and Bcr-Abl protein levels examined by
immunoblot analysis using anti-Abl (8E9) and horseradish
peroxidase-conjugated anti-Mouse IgG, as described under
``Materials and Methods.'' The positions of Bcr-Abl protein
(p210) and c-Abl protein (p145) are marked. The above lysates were also
probed for actin protein levels, which acted as an internal control for
protein loadings. The position of actin protein (p45) is also
indicated. B, effect of AS-bcr-abl (AS) and
NS-bcr-abl (NS) treatment on Fas-mediated killing in
K562 and K562.Fas cells. Untreated (),
AS-bcr-abl-treated (
), and NS-bcr-abl-treated
(
) K-562 and K-562.Fas cells were incubated for 18 h in the
presence of various concentrations of anti-Fas, and apoptotic cell
death was determined as described in Fig. 1B. Cell
viability was assessed by flow cytometric criteria based on scatter
properties and PI permeability as described in Fig. 1B.
The effect of lowering Bcr-Abl protein levels in K562 and K562.Fas cells on susceptibility to Fas-mediated cell death was then examined. Untreated, AS-bcr-abl-treated, and NS-bcr-abl-treated K562 and K562.Fas cells were incubated overnight in varying concentrations of anti-Fas, and cell death was assessed as before. K562.Fas cells treated with AS-bcr-abl oligonucleotides underwent extensive cell death upon Fas ligation (Fig. 2B and 3B). Cell death under these conditions was confirmed to be apoptotic by examination of stained-cytospun preparations of the treated cells (Fig. 2B and 3A). In contrast, NS-bcr-abl-treated or untreated cells remained resistant to Fas-mediated apoptosis. AS or NS-bcr-abl treatment alone had no effect on cell viability (data not shown). Furthermore, AS or NS-bcr-abl treatment had no effect on the cell viability of K562 cells upon exposure to anti-Fas (Fig. 2B).
To rule out the possibility that AS or NS-bcr-abl treatment of the Fas transformants was modulating the levels of Fas expression in these cells, thereby affecting the susceptibility of these clones to Fas-mediated death, cells were examined for Fas expression after treatment with the various oligonucleotides. These experiments confirmed that Fas expression in the K562 and K562.Fas cells remained constant following AS or NS-bcr-abl pretreatment (data not shown).
Figure 4:
A, expression of human Fas in
HL-60.vector and HL-60.v-Abl transfectants. HL-60.vector
and HL-60.v-Abl
transfectants were incubated for 18 h at
the permissive (32 °C) and non-permissive (39 °C) temperatures
for the Abl kinase and Fas expression was monitored as described in Fig. 1A. B, effect of Abl kinase activity on
Fas-mediated cell killing in HL-60 vector and HL-60.v-abl
transfectants. HL-60.vector and HL-60. v-Abl
transfectants were treated with various concentrations of
anti-Fas Ab and incubated for an 18-h time period at either 32 °C
(
) or 39 °C (
) as indicated. Cell viability was
assessed by either analysis of the scatter properties of the treated
cells or as a measure of PI permeability. C, inhibition of
Fas-mediated DNA fragmentation in HL-60.v-Ablts transfectants at the
permissive temperature for the v-Abl kinase. Cells (0.5
10
/ml) were incubated under the conditions described in B, and fragmentation of the chromosomal DNA was assessed by
electrophoresis on a 1.5% agarose gel.
As expected, the v-Abl kinase also
protected against DNA fragmentation induced by Fas-mediated apoptosis
at the permissive temperature (Fig. 4C). Both vector
only and HL-60.v-Abl transformants that were maintained at
the non-permissive temperature for the kinase showed significant DNA
fragmentation upon Fas ligation (Fig. 4C). Incubation
temperature differences alone had no effect on Fas expression in these
cell types, as was confirmed by staining for Fas expression in cells
maintained at the permissive versus non-permissive temperature (Fig. 4A).
Figure 5:
A, down-regulation of Bcr-Abl
proteins levels renders K562 cells susceptible to ceramide-induced cell
death. Untreated (), AS-bcr-abl-treated (
), and
NS-bcr-abl-treated (
) K562 cells were incubated for 18 h
in the presence of varying concentrations of C
-ceramide.
Cell death was determined by morphological examination of cytospun
samples as described in Fig. 1B. B, inhibition
of ceramide-induced cell death by the Abl kinase. HL-60.vector and
HL-60.v-Abl
transfectants were incubated for 18 h in the
presence of varying concentrations of C
-ceramide at the
permissive (32 °C,
) versus non-permissive (39
°C,
) temperature for the Abl kinase. Cell death was
asessed by both analysis of the scatter properties of the treated cells
and PI uptake as described in Fig. 1B.
NS-bcr-abl treatment did not
increase the susceptibility of K562 cells to ceramide-induced apoptosis (Fig. 5A). To further confirm that Abl protected against
ceramide-induced death, we tested whether Abl kinase activity in the
HL-60 v-Abl transfectants could protect against apoptosis
induced by ceramide. Therefore, HL-60.v-Abl
cells were
incubated with varying concentrations of C
-ceramide at
permissive and nonpermissive temperatures for the Abl kinase. As shown
in Fig. 5B, the active Abl kinase provided a partial
protection against ceramide-induced apoptosis in HL-60.v-Abl
transfectants at the permissive temperature. In contrast,
HL-60.vector transfectants were found to be extremely susceptible to
apoptosis induced by ceramide at both permissive and non-permissive
temperatures (Fig. 5B).
In the present study we examined the effect of the abl oncogene on Fas-mediated apoptosis. K562.Fas transfectants were highly resistant to Fas-induced apoptosis, in contrast to murine P815 cells, which were infected with the identical fas construct. Using antisense oligonucleotides targeted to the mRNA of bcr-abl, we have found that down-regulation of Bcr-Abl protein levels in K-562.Fas cells renders these cells highly susceptible to Fas-mediated apoptosis. This suggests that bcr-abl is responsible for the resistance of these cells to Fas-mediated apoptosis. The concept of bcr-abl as a negative regulator of apoptosis is relatively new. Although this oncogene has been demonstrated to prolong cell survival in various growth factor-dependent myeloid cell lines (Daley and Baltimore, 1988; Hariharan et al., 1988), it was not until quite recently that these observations were extended to the study of various apoptosis-inducing stimuli. We have recently shown that bcr-abl is responsible for the resistance of K562 cells to apoptosis induced by a wide variety of chemotherapeutic agents (McGahon et al., 1994). Using a similar antisense strategy Bedi et al. (1994) also demonstrated that constitutive expression of bcr-abl could inhibit the apoptotic death seen in CML myeloid progenitor lines upon IL-3 withdrawal.
The viral homolog of the
c-abl gene is a 160-kDa tyrosine kinase, v-Abl (p160) (Abelson
and Rabstein, 1970). This v-Abl kinase can also function as a negative
regulator of apoptosis. Infection of early myeloid, granulocyte, or
primitive lymphoid lines with A-MuLV results in the generation of
IL-3-independent lines (Mathey et al., 1986; Pierce et
al., 1986; Rovera et al., 1987). Using a
temperature-sensitive mutant of v-Abl in which the kinase activity is
greatly reduced at 39 °C, Kipreos and Wang (Kipreos et
al., 1987) demonstrated that the kinase activity of v-Abl is
essential for the maintenance of factor-independent proliferation in an
IL-3-dependent line. Using the same v-Abl temperature-sensitive mutant,
we have demonstrated in this study that HL-60.v-Abl transfectants are resistant to Fas-mediated apoptosis at the
permissive temperature for the Abl kinase. Moreover, the differential
sensitivity to Fas-mediated apoptosis was not due to differential Fas
expression at the different temperatures, as was demonstrated by
anti-Fas staining. These data provide further evidence that the
resistance to Fas-mediated apoptosis is due to Abl kinase activity.
These results are of particular interest given the lack of
information concerning oncogene regulation of Fas-mediated apoptosis.
To date, Bcl-2 has been the only oncogene implicated in the regulation
of Fas-induced apoptosis. Itoh et al.(1993) demonstrated a
partial inhibition of Fas-mediated apoptosis in both an IL-3-dependent
FDC-PI line and WR9L cells transfected with Bcl-2. We have also
observed that the Abl kinase can protect against a wide variety of
apoptosis-inducing agents. ()Whether these stimuli share a
similar signal transduction pathway in the generation of apoptosis is,
however, unclear. Since both the Bcl-2 and Abl protein products have
been demonstrated to protect against Fas-mediated death, one obvious
possibility is that Abl may be exerting its anti-apoptotic activity
through the induction of Bcl-2. However, recent experiments by our
group suggest that the anti-apoptotic activity of the v-Abl kinase is
independent of Bcl-2 activity.
These observations may
explain the differences in inhibition of Fas-mediated apoptosis induced
by Abl and Bcl-2, as both repressors are working independently of each
other. One other possibility is that Abl and Bcl-2 may be acting at
different stages of the Fas-mediated signaling pathway that leads to
apoptosis.
Elucidation of the signal transduction events triggered by Fas ligation will provide a greater understanding of how Abl may exert its anti-apoptotic effects on Fas-mediated apoptosis. Recent investigations demonstrate that apoptotic signaling through Fas activates an acidic sphingomyelinase (Cifone et al., 1994), which results in sphingomyelin hydrolysis and the generation of ceramide. Ceramide can serve as a second messenger, which in turn can lead to the activation of Ras (Gulbins et al., 1995). Ceramide has been demonstrated to be a potent inducer of apoptosis in many cell types, including U937 cells (Jarvis et al., 1994) and HL-60 cells (Obeid et al., 1993). In the present study we found that both the Bcr-Abl and v-Abl tyrosine kinases inhibit ceramide-induced cell death. Hence, it is likely that Abl is acting downstream of ceramide-signaling events to regulate Fas-induced apoptosis.
These observations lead us to consider how Abl may be signaling to prevent Fas-mediated apoptosis. One possibility arises from studies which indicate a role for protein kinase C activation in v-Abl-mediated abrogation of IL-3 dependence (Owen et al., 1993). Activation of the v-Abl tyrosine kinase stimulates phopholipase C-mediated breakdown of phosphatidylcholine to generate diacylglycerol, which may then activate protein kinase C. Protein kinase C activation by phorbol esters abolishes apoptosis in response to some stimuli (Tomei et al., 1988; Rajotte et al., 1992). More recently protein kinase C activation has been demonstrated to block ceramide production and ceramide-induced apoptosis (Hainovitz-Friedman et al., 1994). It is possible that Abl may activate protein kinase C to block ceramide-induced apoptosis, lending support to our hypothesis that Abl functions as a downstream regulator of Fas-initiated apoptosis.
Ras activation has also recently been found to be a critical component of the Fas-mediated signaling pathway (Gulbins et al., 1995). Ras is involved in the signaling events during both v-Abl- and Bcr-Abl-mediated transformation (Stacey et al., 1991; Pendergast et al., 1993). Expression of constitutively activated Ras in some cell types can prevent death after withdrawal of trophic support (Guerrero et al., 1986; Rukenstein et al., 1991). Transformation of murine myeloid cells to IL-3 independence by Bcr-Abl is dependent of the activation of p21 Ras (Mandanas et al., 1993). Thus a second possibility is that Abl may activate Ras via an alternative pathway and thereby prevent Fas-mediated Ras activation, thus preventing apoptosis. Bcr-Abl-mediated Ras activation can be achieved by direct interaction with the SH2 domain of the GRB-2 adaptor protein (Stacey et al., 1991; Pendergast et al., 1993), whereas activation of Ras by v-Abl may be achieved by phosphorylation of the SH2-containing protein SHC (Rozakis-Adcock et al., 1992). Experiments involving GRB-2 binding mutants of Abl further demonstrate that Ras activation is necessary step for transformation (Pendergast et al., 1993). Whether Ras activation by Abl is a necessary step in the prevention of Fas-mediated apoptosis remains unclear. In addition, since Abl is a tyrosine kinase and its signal transduction events involve the phosphorylation of many intracellular substrates, including the recently identified CRKL protein (ten Hoeve et al., 1994; Nichols et al., 1994), examination of the intracellular substrates necessary for Abl signaling may yield useful insight into how it is working as a negative regulator of apoptosis.
Finally, our observations lead us to consider the potential role of abl-mediated resistance to Fas-induced apoptosis, in the control of hematopoiesis. Bone marrow transplantation experiments indicate that the turnover of hematopoietic precursor cells are dependent upon Fas/Fas ligand interactions such that Fas-negative hematopoietic cells have a growth advantage (Ettinger et al., 1994). These studies suggest that resistance to Fas-mediated apoptosis may be an important element in proliferation control. The c-Abl kinase has also been implicated in the control of hematopoietic cell growth. Evidence that c-Abl function is necessary for normal hematopoiesis includes the demonstrations that homozygous null mutation of murine c-Abl results in lymphopenia and neonatal mortality (Schwartzberg et al., 1991; Tybulewicz et al., 1991). Thus a potential, yet unexplored mechanism for c-Abl-mediated control of hematopoiesis, is that Abl-mediated resistance to Fas-induced apoptosis may serve as a negative regulator of proliferation in hematopoietic cells. Recent studies (Owen-Schaub et al., 1995) have demonstrated that Fas/APO-1 is a target gene for transcriptional activation by p53. K562 cells transfected with a temperature-sensitive p53 mutant show a dramatic up-regulation of Fas at the permissive temperature only. These cells remain resistant to Fas-induced apoptosis, however, lending support to our current observations.
In summary, we have identified the Abl kinase as a negative regulator of Fas-mediated apoptosis. Inhibition of Fas-mediated apoptosis by Abl appears to be downstream of early signaling events mediated by Fas ligation. Knowledge of how the Abl kinase may signal in the prevention of apoptosis remains unclear. Further elucidation of the signaling events involved in Abl-mediated suppression of apoptosis will yield valuable insight into its regulation of Fas-mediated apoptosis.