1
Research Unit Molecular Oncology, Clinic for General Surgery and Thoracic
Surgery, Christian-Albrechts-University, 24105 Kiel, Germany
2
Department of Medicine, Christian-Albrechts-University, 24105 Kiel,
Germany
*
Author for correspondence (e-mail:
hkalthoff{at}email.uni-kiel.de
)
Accepted April 23, 2001
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SUMMARY |
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Key words: CD95-induced cell death, Pancreatic cancer, Human, Immune escape
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INTRODUCTION |
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Our laboratory has recently demonstrated that the majority of human
pancreatic adenocarcinoma cell lines is largely refractory to CD95-induced
apoptosis (Ungefroren et al.,
1998), an observation that has
been independently confirmed by others (von Bernstorff et al.,
1999
). Intriguingly,
resistance correlated with expression of FAP-1 in 6/6 pancreatic tumor cell
lines, as shown by reverse transcriptase-polymerase chain reaction (RT-PCR)
(Ungefroren et al., 1998
).
Prompted by the recent identification of a pancreatic carcinoma cell line
(Capan-1) that was sensitive to CD95-mediated apoptosis and totally lacked
FAP-1 expression, we set out to analyze in greater depth the putative
inhibitory role of FAP-1 on CD95-signal transduction in pancreatic tumor
cells. Our results show that FAP-1 provides these cells with (partial)
protection against CD95-mediated apoptosis via an as yet unrecognized
mechanism: sequestration of CD95 in the Golgi complex and prevention of CD95
transport to the cell surface.
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MATERIALS AND METHODS |
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Construction of a FAP-1 expression plasmid and generation of stable
transfectants of the Capan-1 cell line
The entire FAP-1/PTPL1 cDNA (nucleotides -77 to +7401) in the pSV7d vector
(kindly donated by Drs C.-H. Heldin and J. Saras, Uppsala, Sweden) was excised
with PstI and SalI and subcloned into the
PstI/SalI sites of pBSK plasmid (Stratagene, Heidelberg,
Germany). The FAP-1 cDNA was then released with NotI and
ApaI from the pBSK polylinker and inserted in sense orientation into
the NotI/ApaI sites of pcDNA3 (Invitrogen, San Diego, CA,
USA). The resulting plasmid was transfected as closed circular DNA into
Capan-1 cells using LipofectAmine (Life Technologies, Inc.). Control cells
received the empty pcDNA3 vector. 72 hours after transfection individual
G418-resistant clones were selected with geneticin, propagated and screened
for FAP-1 expression by RT-PCR analysis and immunoblotting. The clones with
the strongest FAP-1 expression were used for further analysis.
Inhibition of protein tyrosine phosphatases
Cells were incubated with various concentrations of the protein tyrosine
phosphatase inhibitor sodium orthovanadate (Calbiochem, Bad Soden, Germany),
alone or in combination with anti-CD95-antibody CH11 (Coulter Immunotech,
Hamburg, Germany), TNF- or 5-fluorouracil in normal growth medium for
24 hours. Subsequently, apoptosis was evaluated by the JAM assay (see
below).
Cytoplasmic microinjection
The microinjection experiments followed a previously published protocol
(Yanagisawa et al., 1997),
except that in our experiments apoptosis was detected by annexin V-FITC
instead of Hoechst 33342 staining. Briefly, 3x104 Panc89
human pancreatic adenocarcinoma cells were plated on CELLocate 175 µm
(Eppendorf, Hamburg, Germany) in a 35-mm plastic culture dish and grown for 24
hours. The microinjection experiments were performed with an automatic
microinjection system (Eppendorf transjector 5246, micromanipulator 5171 and
Femtotips (Eppendorf). Synthetic tripeptides (Ac-SLV and Ac-SLY, 100 mM in
K-PBS containing 0.1% (w/v) FITC-dextran (Sigma, Deisenhofen, Germany)) were
microinjected into the cytoplasm of Panc89 cells using 0.7 second injection
time and 100-120 hPa injection pressure. Successful miroinjection of each cell
was verified by cytoplasmic staining with FITC-dextran. Immediately after
microinjection, CD95 monoclonal antibody CH11 (500 ng/ml) was added to the
medium. 4 hours after injection the cells were washed in PBS and stained with
the Annexin V-FITC Apoptosis Detection Kit I (Pharmingen, Hamburg, Germany).
Apoptosis of microinjected cells was evaluated microscopically by counting
cells that displayed staining with annexin V-FITC (the concentration of the
microinjected FITC was so low that it did not interfere with the annexin
V-coupled FITC) or annexin V-FITC and propidium iodide, whereas viable cells
remained unstained. In all experiments approximately 100 cells were
microinjected with each peptide.
RNA isolation and RT-PCR analysis of FAP-1 and c-FLIP
Total RNA from Panc89, Jurkat, Capan-1 cells as well as various FAP-1 and
vector-transfected Capan-1 cells was isolated with RNA Clean (AGS, Heidelberg,
Germany) according to the manufacturer's instructions. The general RT-PCR
protocol as well as PCR oligonucleotide primer sequences for FAP-1 and GAPDH
was described in detail earlier (Ungefroren et al.,
1998), and was applied in this
study except that a different FAP-1 oligonucleotide sense primer
(5'-CATGGCAGCCCTTCCCCATCTG-3') was used, resulting in a specific
fragment of 945 bp. Amplification of c-FLIP mRNA used an identical protocol
except that the number of amplification cycles at 59°C was reduced to 20.
The oligonucleotide primer sequences for c-FLIP were as follows: c-FLIP-sense:
5'-ATTGGTGAGGATTTGGATAAATCTG-3', c-FLIP-antisense:
5'-GTGGGCGTTTTCTTTCTTGTCTC-3'.
Immunoblot analysis, immunohistochemistry and immunofluorescence
For analysis of FAP-1, CD95, caspase-8 and ß-actin proteins by western
blotting, pancreatic tumor cells were lysed in RIPA buffer (0.1% SDS, 1% NP40,
and 0.5% sodium deoxycholate in PBS) for 20 minutes on ice followed by one
freeze-thaw cycle. For detection of PARP, Capan-1 cells were lysed in 62.5 mM
Tris/HCl, pH 6.8, 6 M urea, 5% ß-mercaptoethanol, sonicated for 15
seconds and incubated at 65°C for 15 minutes. In both cases lysates were
cleared by centrifugation and total cellular proteins separated on standard 5%
(FAP-1) or 10% (CD95, caspase-8, ß-actin, PARP) SDS-polyacrylamide gels.
The fractionated proteins were blotted onto PVDF membranes (Immobilon-P,
Millipore, Eschborn, Germany). Following blocking in 5% non-fat milk the
membrane was incubated with anti-FAP-1 (C-20, Santa Cruz Biotechnology, Santa
Cruz, CA, USA), anti-CD95 (C-20, Santa Cruz Biotechnology), anti-caspase-8
(Upstate Biotechnology, Lake Placid, NY, USA), anti-ß-actin (AC-15,
Sigma) or anti-poly(ADP-ribose) polymerase (PARP) (Ab-2, Calbiochem)
antiserum. After binding of the respective horse radish peroxidase
(HRP)-coupled secondary antibodies, antigens were visualized by enhanced
chemiluminescence (ECL-kit, Amersham-Buchler, Braunschweig, Germany). For
immunohistochemistry pancreatic tumor tissue was cryosectioned (5-10 µm),
fixed for 10 minutes in cold acetone and air-dried. Immunohistochemistry was
carried out with the Vectastain ABC kit (Biologo, Wettenberg, Germany)
according to the manufacturer's instructions and using the appropriate
secondary antibodies against goat immunoglobulin. In some experiments the
FAP-1 antiserum was preincubated with a tenfold (immunoblot) or 13-fold
(immunohistochemistry) molar excess of the corresponding blocking peptide
(C-20P, Santa Cruz Biotechnology) prior to incubation with immunoblots or
tissue sections.
For indirect immunofluorescence staining, cells grown on glass coverslips were fixed with ice-cold methanol for 10 minutes or 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 30 minutes at room temperature (RT) and subsequently washed with Tris-buffered saline (TBS). PFA-fixed cells were permeabilised with 0.02% saponin in TBS for 30 minutes at RT. Unspecific binding sites were blocked with TBS containing 0.1% BSA for 60 minutes at RT. Primary antibodies diluted in TBS were incubated overnight at 4°C. After washing in TBS, secondary fluorophore-labelled antibodies were incubated at 37°C for 60 minutes. After several washes in TBS, cells were mounted with slow fadeTM mounting medium (Molecular Probes, Eugene, OR, USA). Primary antibodies were goat anti-FAP-1, rabbit anti-CD95 antibody (both C-20, Santa Cruz Biotechnology) or mouse monoclonal anti-ß-COP (maD, Sigma, St Louis, MO, USA). Secondary antibodies were donkey anti-goat Alexa Probes 546 (Molecular Probes, Eugene, OR, USA), donkey anti-rabbit FITC and donkey anti-mouse FITC (both from Jackson ImmunoResearch Lab., West Grove, PA, USA). In some experiments cells were treated with brefeldin A (Biomol, Hamburg, Germany) at a concentration of 1 µg/ml for 60 minutes before fixation. For stimulation experiments cells on coverslips were incubated for various times with the anti-CD95 agonistic antibody APO1-3 (Alexis Biochemicals, San Diego, CA, USA) at a concentration of 2 µg/ml (alone or in combination with brefeldin A) or TRAIL (R&D Systems, Wiesbaden, Germany), and then fixed in ice-cold methanol. Confocal laser scanning analysis was carried out with a Zeiss LSM 510 laser scanning microscope (Carl Zeiss Jena, Jena, Germany). Double staining pictures represent optical slices of 0.5 µm taken from the middle of the cells. Original magnification was 400x.
FACS analysis
For FACS analysis, confluent cells were incubated for 10 minutes with
pre-warmed Trypsin-EDTA-solution (Life Technologies Inc.). Trypsin was
neutralised by addition of growth medium and cells were collected by
centrifugation for 10 minutes at 600 g. The cell pellet was
resuspended in at least 5 ml growth medium and incubated for 30-45 minutes
under standard culture conditions for recovery from trypsinisation. For
further experiments, cells were counted and about 500,000 cells/ml were
dispensed into 15 ml polypropylene tubes. The cells were stimulated with
anti-CD95 antibody CH11 (500 ng/ml) for the indicated time points under
standard culture conditions. After addition of PBS containing 0.1% sodium
azide (PBS-azide), cells were pelleted and washed twice in PBS-azide.
Apoptosis was measured by binding of FITC-labeled annexin V (Clontech).
Briefly, 105 cells were pelleted, resuspended in 0.2 ml of
Hepes-buffered saline, and 10 µl of FITC-labeled enhanced annexin V and 100
ng of propidium iodide (PI) were added. After incubation for 15 minutes at
room temperature in the dark, samples were brought to 0.5 ml with PBS.
Detection was done by fluorescence flow cytometry (Galaxy Argon Plus, Dako)
and results were analysed with the FLOMAX software (Dako). Those cells
exhibiting high staining with annexin V were regarded as being apoptotic.
Apoptosis assays
Relative sensitivity to CD95-induced apoptosis of Panc89 and Capan-1 cells
was evaluated by annexin V-FITC staining (see Cytoplasmic Microinjection and
FACS analysis above), caspase-8 and PARP cleavage assays (see Immunoblot
analysis above) and the JAM DNA fragmentation assay (Matzinger et al.,
1991). The JAM assay was
performed as described previously (Ungefroren et al.,
1998
). Briefly,
1x104 pancreatic tumor cells were labeled with 0.5 µl
[3H]thymidine (370 kBq/µl; Amersham-Buchler) per well (100
µl) for 3 hours. Following removal of the label and one wash with PBS, the
labeled cells were incubated for 24 hours in normal growth medium containing
100 or 500 ng/ml anti-CD95 CH11 antibody. Control cells received medium
without CH11. Subsequently, cells were lysed in 0.1% SDS for 20 minutes at
37°C (to ensure complete release of nuclear DNA) and harvested on glass
fiber filters. The percentage of specific DNA fragmentation, being indicative
of apoptosis, was calculated as: % viability=(E/S)x100, where
E (experimental) is c.p.m. of retained DNA in the presence of CH11
antibody and S (spontaneous) is c.p.m. of retained DNA in the absence
of CH11 antibody.
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RESULTS |
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De novo expression of FAP-1 in the CD95-sensitive pancreatic
carcinoma cell line Capan-1 decreases sensitivity to CD95-mediated
apoptosis
The CD95-sensitive, FAP-1 negative cell line Capan-1 was stably transfected
with an expression plasmid carrying the entire approx. 7.7 kb FAP-1 cDNA and
the neomycin resistance gene. A total of 72 clones of FAP-1-transfected,
G418-resistant clones were obtained, of which 20 were positive for FAP-1 mRNA,
as revealed by RT-PCR. An assessment of relative FAP-1 expression using
semiquantitative RT-PCR revealed that FAP-1 expression in transfected Capan-1
clones was approximately 20-fold lower than in Panc89 cells (data not shown).
Immunoblot analysis of these transfectants confirmed that this mRNA was
translated into protein, as indicated by the approximately 250 kDa FAP-1
protein (Fig. 2A). However, due
to comparatively low expression, it was not possible to precisely quantify
protein levels. Several attempts to increase FAP-1 expression in the Capan-1
cell line, e.g. by stable transfection with a FAP-1 cDNA in which the
nucleotides flanking the ATG start codon had been mutated to a Kozak consensus
sequence, did not result in higher protein levels. Capan-1-FAP-1 clones were
then tested functionally for their susceptibility to undergo CD95-mediated
apoptosis by measuring anti-CD95-induced DNA fragmentation. Sensitivity to
CD95-mediated killing was greatly decreased in these clones (see
Fig. 2B). The JAM assay data
for Capan-1-FAP-1 clone 2 were corroborated by results from PARP cleavage
assays although PARP cleavage reflecting caspase-3 activity was not completely
inhibited relative to the vector control
(Fig. 2C). The observed
differences in CD95-sensitivity were not caused by changes in CD95 levels as
transfection with FAP-1 or empty vector did not affect CD95 expression
(Fig. 2A). In order to rule out
a general increase in apoptosis resistance (which might have developed during
the selection procedure) we included additional control experiments. (1) In
contrast to FAP-1 expressing clones, none of several clones containing the
empty vector (Fig. 2B) nor
G418-resistant clones that had been transfected with the FAP-1 expression
plasmid but lacked FAP-1 mRNA and protein (data not shown), ever showed
protection against anti-CD95-induced cell death. (2) As shown in
Fig. 2B, FAP-1 transfection did
not increase resistance towards TRAIL (Apo2-L)-induced apoptosis. (3) Cleavage
of caspase-8 in response to anti-CD95 but not TRAIL was inhibited in
Capan-1-FAP-1 cells, in contrast to the vector control
(Fig. 2D).
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Inhibition of protein tyrosine phosphatases increases sensitivity to
CD95-mediated apoptosis in Panc89 and Capan-1-FAP-1 cells
If resistance to CD95-mediated killing depended on the activity of cellular
protein tyrosine phosphatases, e.g. FAP-1, then treatment of cells with the
phosphatase inhibitor sodium orthovanadate would be expected to render
CD95-resistant cells more sensitive to CD95 killing. As shown in
Fig. 3, orthovanadate strongly
decreased viability of Panc89 and Capan-1-FAP-1 cells treated with anti-CD95
in a dose-dependent manner, while having a comparatively small effect on
parental and vector-transfected Capan-1 cells (data not shown). The
sensitization effect of orthovanadate was not due to increased CD95 expression
on the cell surface (data not shown). Moreover, it was specific for anti-CD95
since it was not observed for TNF- and 5-fluorouracil
(Fig. 3).
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Inhibition of the interaction between CD95 and FAP-1 increases
sensitivity to CD95-mediated apoptosis in Panc89 cells
From binding studies in vitro and in yeast it was known that the second and
fourth PDZ domains of FAP-1 interact with the three C-terminal amino acids
(SLV) of human CD95 and that this interaction is necessary and sufficient for
FAP-1 to exert its anti-apoptotic function in DLD-1 cells (Yanagisawa et al.,
1997). We therefore
specifically abrogated CD95-FAP-1 interactions by competitive inhibition with
Ac-SLV. As shown in Table 1,
direct cytoplasmic microinjection of this tripeptide into Panc89 cells
resulted in a mean 5.5-fold (±2.2, n=4) induction of
CH11-triggered apoptosis compared to cells that received the Ac-SLY control
peptide.
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FAP-1 localizes to intracellular vesicles and the Golgi complex
FAP-1 has been shown to physically interact with the cytoplasmic C terminus
of human CD95 in vitro (Maekawa et al.,
1994; Yanagisawa et al.,
1997
; Li et al.,
2000
) and in vivo (Li et al.,
2000
) and tripeptide-mediated
inhibition of this interaction strongly relieved its anti-apoptotic activity
in viable cells (Yanagisawa et al.,
1997
;
Table 1). However, attempts to
co-immunoprecipitate both proteins from unstimulated Panc89 cells and
Capan-1-FAP-1 cells failed, in contrast to experiments carried out in COS
cells transiently transfected with both a CD95 and a FAP-1 expression plasmid
(data not shown). These results raised the question of whether there is a
permanent (and stable) interaction between FAP-1 and CD95. We therefore
analysed the subcellular distribution of FAP-1 and CD95 in Panc89 cells by
confocal laser microscopy after double-labeling with antisera against FAP-1
and CD95 (Fig. 4). Immunolocalisation of FAP-1 protein in Panc89 cells revealed strong
immunoreactivity, predominantly in a juxtanuclear position, reminiscent of
Golgi localisation (Fig. 4A-C,
arrows) and distinct vesicular staining in the cell periphery
(Fig. 4C, arrowheads). To prove
that FAP-1 resides in the Golgi, colocalization studies were carried out with
antibodies against ß-COP (Duden et al.,
1991
), a member of the
coatomer, which is found enriched in the Golgi complex. In
Fig. 4 colocalization is
visualized by yellow colouring of cisternae-like structures
(Fig. 4C, arrow), which are
positive for both ß-COP (Fig.
4A) and FAP-1 (Fig.
4B). To confirm cisternal Golgi staining, cells were treated with
brefeldin A, a drug that blocks Golgi-dependent protein secretion (Chardin and
McCormick, 1999
). Brefeldin A
led to dissociation of ß-COP from the Golgi membranes and distribution
throughout the cytoplasm. The effect of brefeldin A on FAP-1 and ß-COP
staining in Panc89 cells is depicted in
Fig. 4D-F. After 1 hour of
brefeldin A treatment (1 µg/ml), ß-COP was found diffusely all over
the cell (Fig. 4D). Likewise,
there was no visible cisternal staining of FAP-1, but immunoreactivity in
vesicular structures appeared to be unaffected
(Fig. 4E) and was clearly
separated from ß-COP immunoreactivity (see arrowheads in
Fig. 4F). Similar experiments
were carried out for the cis-Golgi network marker p58 with similar results
(data not shown), indicating that a considerable fraction of FAP-1 protein
localizes to the Golgi complex.
|
FAP-1 and CD95 intracellular colocalization in the Golgi complex is
induced upon CD95 stimulation and is inhibited by brefeldin A treatment
Indirect immunofluorescence staining of Panc89 cells under control
conditions (Fig. 5A,D,G,K)
revealed that CD95 (green colour) is predominantly intracellular and displayed
a punctate fluorescence, consistent with its compartmentation within
organelles (Fig. 5D). As
already shown in Fig. 4, FAP-1
staining was found mostly in juxtanuclear Golgi localisation (red colour,
arrowheads in Fig. 5A-C). There
also appeared to be some focal nuclear staining
(Fig. 5C,I), the significance
of which is unclear at present. Both proteins colocalized only sporadically,
which is indicated by the yellow fluorescence upon overlaying the two signals
(Fig. 5G). To analyse whether
treatment with anti-CD95 has an effect on the subcellular distribution of both
proteins, Panc89 cells were challenged for 5 and 15 minutes, respectively,
with APO1-3 antibody and processed as above. Plasma membrane fluorescence for
CD95 was increased after 5 minutes, demonstrating increased CD95 on the cell
surface, mostly at sites of intercellular contact
(Fig. 5E, arrows), whereas
staining patterns for FAP-1 (Fig.
5B) and colocalization of CD95 and FAP-1
(Fig. 5H) appeared almost
unchanged. However, a dramatic enhancement of CD95 intracellular staining was
visible after 15 minutes of stimulation
(Fig. 5F) and both proteins now
strongly colocalized in the perinuclear Golgi complex
(Fig. 5I, arrowhead).
Cell-surface staining of CD95 appeared reduced rather than enhanced (compare
Fig. 5E and F), an observation
that was confirmed by measuring cell-surface-associated CD95 by FACS analysis
(data not shown). When TRAIL was used as a stimulus under the same conditions
the intracellular staining pattern for CD95 did not change
(Fig. 5K-M). This also applies
to FAP-1-negative Capan-1 cells stimulated under the same conditions with
anti-CD95 (Fig. 6A-C).
Simultaneous treatment of Panc89 cells with anti-CD95 and brefeldin A for 1
hour abolished the colocalization of FAP-1 and CD95 and the decrease in cell
surface-associated CD95 (Fig.
7D) and was associated with an increase in apoptosis after 4
hours, as measured by annexin V assay (Fig.
7E). The distinct subcellular localization of FAP-1 and CD95 under
normal and anti-CD95-stimulated conditions suggest that the in vivo
interaction of both proteins is stimulus-dependent and temporally restricted.
Together with the results from the brefeldin A experiments, these data point
to a novel mechanism of anti-apoptosis by FAP-1: prevention of CD95
trafficking to the cell surface in response to anti-CD95.
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FAP-1 is strongly expressed in pancreatic adenocarcinoma tissue
To evaluate the physiological significance of FAP-1 expression in vivo,
pancreatic adenocarcinoma specimens were analyzed by immunohistochemistry.
FAP-1 was found to be expressed in 13/14 cases with a strong immunoreactivity
being present in five tumors. No or only very faint staining was observed in
pancreatic duct cells in tissues from a healthy individual
(Fig. 8). This observation
supports the assumption that FAP-1 upregulation represents a characteristic
feature of ductal pancreatic adenocarcinoma cells, which potentially enables
them to escape from CD95-mediated apoptosis.
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DISCUSSION |
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The anti-apoptotic function of FAP-1 was originally evaluated in Jurkat
cells and is thought to be mediated via its carboxyl-terminal phosphatase
catalytic domain (Sato et al.,
1995), implying a role for
tyrosine (de)phosphorylation in CD95 signal transduction. Although this issue
remains unresolved at present (Cuppen et al.,
1997
), we observed rapid
phosphorylation of several cellular proteins upon stimulation of the CD95
receptor pathway in Panc89 cells (K. Klosa and H. Ungefroren, unpublished) as
did others in different cellular models (Eischen et al.,
1994
; Simon et al.,
1999
). More importantly,
inhibition of protein tyrosine phosphatases using sodium orthovanadate clearly
sensitized Panc89 and FAP-1-transfected Capan-1 cells to CD95-induced cell
death and the magnitude of this effect correlated with expression of FAP-1.
The anti-apoptotic function of FAP-1 in FAP-1-transfected Capan-1 cells was
specific for anti-CD95-induced cell death since we did not observe protection
from cell death induced by TRAIL, in accordance with the fact that protein
tyrosine (de)phosphorylation has so far not been implicated in TRAIL-mediated
apoptosis.
In several studies a correlation of FAP-1 expression with sensitivity to
CD95 cytotoxicity has been observed, and is implicated in the relative
sensitivity of various transformed (Sato et al.,
1995) and non-transformed
cells to the death-inducing effect of CD95. Higher FAP-1 levels may cause
preferential survival of T-helper cells Th2 over Th1 cells after activation of
the CD95 pathway (Zhang et al.,
1997
) and upregulation of
FAP-1 has been implicated in the escape of HTLV-1-infected T cells from
CD95-mediated immune surveillance (Arai et al.,
1998
). In turn, downregulation
of FAP-1 was implicated in acquiring sensitivity to CD95-triggered apoptosis
in IL-2 activated T cells (Zhou et al.,
1998
) and was proposed to
underlie the increased apoptotic death in hematopoietic cells from patients
with myelodysplastic syndrome (Mundle et al.,
1998
). Functional evidence for
a protective role of FAP-1 in non-immune cells has so far been limited to
TMK-1 gastric carcinoma (Li et al.,
2000
), DLD-1 colon carcinoma
(Yanagisawa et al., 1997
) and
AIDS Kaposi's sarcoma (Mori et al.,
1996
) cells, although in the
latter report data on specific inhibition of FAP-1 were not provided. In other
cellular systems no correlation of FAP-1 expression with apoptosis resistance
was found (Peter et al., 1996
;
Komada et al., 1997
; Houghton
et al., 1997
; Hedlund et al.,
1998
), which could be
explained by some degree of cell type-specificity.
The presence of a band 4.1 domain (also called ERM or membrane-binding
domain) at the N terminus of the FAP-1 protein (Sato et al.,
1995; Maekawa et al.,
1994
; Saras et al.,
1994
; Banville et al.,
1994
) suggests that this
protein resides at the interface between the cytoskeleton and the plasma
membrane. However, using confocal microscopy we have, for the first time,
obtained experimental evidence that the majority of FAP-1 immunostaining in
Panc89 cells is located in an intracellular juxtanuclear position. Double
staining with ß-COP, a protein associated with Golgi cisternae, showed
distinct cisternal localisation of FAP-1. Golgi localization of both proteins
was confirmed by results from experiments involving the Golgi-disrupting agent
brefeldin A.
Upon anti-CD95 treatment more cytoplasmic FAP-1, along with CD95 antigenic
sites, were exposed synchronously in a juxtanuclear position, with strong
colocalization. This was detectable as early as 5 minutes after anti-CD95
addition and was further enhanced after 15 minutes. At the same time only a
little CD95 was present on the cell surface, despite a transient increase
after 5 minutes. The enhanced fluorescence of FAP-1 and CD95 after 15 minutes
of anti-CD95 stimulation was probably a result of a conformational change
rather than de novo protein synthesis, as immunoblot analyses did not indicate
any changes in total CD95 protein levels in up to 1 hour of CD95 stimulation
(data not shown). It has been reported that CD95 is shuttled to the cell
surface in fibroblasts by a protein secretory pathway involving the Golgi
apparatus (Bennett et al.,
1998). CD95 translocation from
intracellular stores to the plasma membrane has been described to occur in
(FAP-1 negative) hepatocytes during bile salt-mediated apoptosis by a Golgi-
and microtubule-dependent pathway (Sodemann et al., 2000). The data presented
here suggest that anti-CD95 treatment itself delivers a signal that induces
recruitment of cytoplasmic CD95 to the cell surface. Based on the distinct
spatio-temporal relationship between CD95 and FAP-1 in response to CD95
stimulation, in conjunction with a lack of cell surface sequestering of CD95,
we propose that FAP-1 negatively regulates CD95-induced apoptosis,
predominantly by interfering with its translocation from intracellular stores
to the cell surface in response to anti-CD95 and probably other agents that
induce apoptosis by a CD95-dependent mechanism. This process apparently
requires an intact Golgi complex, as disruption of Golgi anterograde transport
by brefeldin A abolished both the anti-CD95-induced colocalization of FAP-1
and CD95 and caused a decrease in plasma membrane staining for CD95, which
correlated with an increase in apoptosis. Inhibition of caspase-8 activation
which we (Fig. 2D) and others
(Li et al., 2000
) observed in
FAP-1 transfected cells is thought to be crucial for blocking the transmission
and execution of the CD95 death signal. However, although we cannot rule out
that a small fraction of FAP-1 present in the DISC directly interferes with
caspase-8 activation, it is more likely that the absence of caspase-8 cleavage
reflected the absence of efficient anti-CD95-induced receptor trimerization,
which in turn is a consequence of low receptor density on the cell surface.
The small degree of colocalization of FAP-1 and CD95 at the plasma membrane
under normal conditions and the observation that FAP-1 colocalization (and
binding to) CD95 are stimulus-dependent might explain, at least in part, why
attempts to co-immunoprecipitate endogenous FAP-1 with CD95 or with other
constituents of the DISC have remained unsuccessful so far. The
intracellular/juxtanuclear localization of FAP-1 also explains the interaction
of FAP-1/PTP-BAS with I
B
(Maekawa et al.,
1999
). Since NF-
B can
protect cells from CD95-induced apoptosis in several cellular systems
(Wallach, 1997
; Zong et al.,
1998
; Dudley et al.,
1999
), regulation of
NF-
B activity via I
B
(Maekawa et al.,
1999
) or p75(NTR) (Irie et
al., 1999
) may thus represent
another mechanism of FAP-1 anti-apoptosis. How exactly FAP-1 interferes with
plasma membrane cycling of CD95 and whether this process involves
(de)phosphorylation events is currently under investigation in our laboratory
and will be the topic of a separate publication.
We have recently hypothesized that FAP-1 synthesis may be one mechanism by
which tumor cells protect themselves from killing by CD95 ligand. This
hypothesis is supported by the observation that FAP-1 is overexpressed in
pancreatic adenocarcinomas. A similar scenario is conceivable for other
gastrointestinal tumors; many colon carcinoma cells, despite expression of
CD95, are resistant to anti-CD95-induced apoptosis (von Reyher et al.,
1998), which in some cases may
be the consequence of FAP-1 synthesis (Yanagisawa et al.,
1997
). As in pancreatic
carcinoma, most human hepatoblastoma cell lines express CD95 on their surface
but are largely refractory to anti-CD95 treatment (Natoli et al.,
1995
), and notably, in a
recent immunohistochemical study 78% of hepatoblastomas were found to be FAP-1
positive (Lee et al., 1999
).
It is interesting to note that normal pancreas, liver and colon, three tissues
for which evidence for an apoptosis-suppressing role of FAP-1 in the
corresponding neoplastic cells has so far been obtained, have low or no
constitutive FAP-1 expression (Maekawa et al.,
1994
; Saras et al.,
1994
; Banville et al.,
1994
). The high metabolic
turnover in these tissues may depend critically on sufficient physiological
cell death regulated by the CD95 system (French et al.,
1996
; Krammer et al.,
1998
). It is thus conceivable
that tumor cells, through only moderate upregulation of FAP-1, partially
escape CD95-mediated apoptosis and thereby gain a selection advantage.
However, the large amount of FAP-1 present in many (pancreatic) CD95-resistant
tumor cells suggests that it may also serve other functions, e.g. interfering
with the phosphorylation-dependent vesicular transport of other
cell-surface-associated proteins.
In this report we show that in pancreatic adenocarcinoma cell lines FAP-1
negatively regulates CD95-mediated cell death. Undoubtedly, other proteins in
the CD95 signaling pathway may act in concert with FAP-1 to determine the
overall threshold of pancreatic tumor cells for resisting CD95-mediated
apoptosis. These include c-FLIP (Irmler et al.,
1997) and Bcl-XL
(Hinz et al., 2000
), an
anti-apoptotic member of the Bcl-2 family, which is highly expressed in
pancreatic carcinomas (Miyamoto et al.,
1999
; Hinz et al.,
2000
). In conjunction with the
observation that FAP-1 is overexpressed in pancreatic tumor tissue, these
findings suggest that pancreatic adenocarcinomas in vivo might escape
CD95-induced apoptosis, at least in part, through the expression of FAP-1. The
modulation of its expression or enzymatic activity by pharmacological or gene
therapeutial interventions may be an option for successful treatment of
patients with pancreatic carcinoma.
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ACKNOWLEDGMENTS |
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REFERENCES |
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Arai, M., Kannagi, M., Matsuoka, M., Sato, T., Yamamoto, N. and Fuji, M. (1998). Expression of FAP-1 (Fas-associated phosphatase) and resistance to Fas-mediated apoptosis in T cell lines derived from human T cell leukemia virus type 1-associated myelopathy/tropical spastic paraparesis patients. AIDS Res. Hum. Retroviruses 14,261 -267.[Medline]
Banville, D., Ahmad, S., Stocco, R. and Shen, S. H.
(1994). A novel protein-tyrosine phosphatase with homology to
both the cytoskeletal proteins of the band 4.1 family and the
junction-associated guanylate kinases. J. Biol. Chem.
269,22320
-22327.
Bennett, M., MacDonald, K., Chan, S. W., Luzio, J. P., Simari,
R. and Weissberg, P. (1998). Cell surface trafficking of Fas:
a rapid mechanism of p53-mediated apoptosis. Science
282,290
-293.
Chardin, P. and McCormick, F. (1999). Brefeldin A: the advantage of being uncompetitive. Cell 97,153 -155.[Medline]
Cuppen, E., Nagata, S., Wieringa, B. and Hendriks, W.
(1997). No evidence for involvement of mouse protein-tyrosine
phosphatase-BAS-like Fas-associated phosphatase-1 in Fas-mediated apoptosis.
J. Biol. Chem. 272,30215
-30220.
Duden, R., Griffith, G., Frank, R. and Kreis, T. E. (1991). ß-COP, a 110 kD protein associated with non-clathrin-coated vesicles and the Golgi complex, shows homology to ß-adaptin. Cell 64,649 -665.[Medline]
Dudley, E., Hornung, F., Zheng, L., Scherer, D., Ballard, D. and Lenardo, M. (1999). NF-kappaB regulates Fas/APO-1/CD95- and TCR-mediated apoptosis of T lymphocytes. Eur. J. Immunol. 29,878 -886.[Medline]
Eischen, C. M., Dick, C. J. and Leibson, P. J.
(1994). Tyrosine kinase activation provides an early and
requisite signal for Fas-induced apoptosis. J.
Immunol. 153,1947
-1954.
French, L. E. and Tschopp, J. (1996). Constitutive CD95 ligand expression in several non-lymphoid mouse tissues: implications for immune-protection and cell turnover. Behring Inst. Mitt. Oct(97),156 -160.
Hedlund, T. E., Duke, R. C., Schleicher, M. S. and Miller, G. J. (1998). Fas-mediated apoptosis in seven human prostate cancer cell lines: correlation with tumor stage. Prostate 36,92 -101.[Medline]
Hinz, S., Trauzold, A., Boenicke, L., Sandberg, C., Beckmann, S., Walczak, H., Bayer, E., Kalthoff, H. and Ungefroren, H. (2000). Bcl-XL protects pancreatic adenocarcinoma cells against CD95- and TRAIL-receptor-mediated apoptosis. Oncogene 19,5477 -5486.[Medline]
Houghton, J. A., Harwood, F. G., Gibson, A. A. and Tillman, D. M. (1997). The Fas signaling pathway is functional in colon carcinoma cells and induces apoptosis. Clin. Cancer Res. 3,2205 -2209.[Abstract]
Inazawa, J., Ariyama, T., Abe, T., Druck, T., Ohta, M., Huebner, K., Yanagisawa, J., Reed, J. C. and Sato, T. (1996). PTPN13, a Fas-associated protein tyrosine phosphatase, is located on the long arm of chromosome 4 at band q21.3. Genomics 31,240 -242.[Medline]
Irie, S., Hachiya, T., Rabizadeh, S., Maruyama, W., Mukai, J., Li, Y., Reed, J. C., Bredesen, D. E. and Sato, T. A. (1999). Functional interaction of Fas-associated phosphatase-1 (FAP-1) with p75(NTR) and their effect on NF-kappaB activation. FEBS Lett. 460, 191-8.[Medline]
Irmler, M., Thome, M., Hahne, M., Schneider, P., Hofmann, K., Steiner, V., Bodmer, J. L., Schroeter, M., Burns, K., Mattmann, C., Rimoldi, D., French, L. E. and Tschopp, J. (1997). Inhibition of death receptor signals by cellular FLIP. Nature 388,190 -195.[Medline]
Komada, Y., Inaba, H., Zhou, Y. W., Zhang, X. L., Tanaka, S., Azuma, E. and Sakurai, M. (1997). mRNA expression of Fas receptor (CD95)-associated proteins (Fas-associated phosphatase-1/FAP-1, Fas-associating protein with death domain/FADD, and receptor-interacting protein/RIP) in human leukaemia/lymphoma cell lines. Br. J. Haematol. 99,325 -330.[Medline]
Krammer, P. H. (1999). CD95(APO-1/Fas)-mediated apoptosis: live and let die. Adv. Immunol. 71,163 -210.[Medline]
Krammer, P. H., Galle, P. R., Moeller, P. and Debatin, K. M. (1998). CD95(APO-1/Fas)-mediated apoptosis in normal and malignant liver, colon, and hematopoietic cells. Adv. Cancer Res. 75,251 -273.[Medline]
Lee, S. H., Shin, M. S., Lee, J. Y., Park, W. S., Kim, S. Y., Jang, J. J., Dong, S. M., Na, E. Y., Kim, C. S., Kim, S. H. and Yoo, N. J. (1999). In vivo expression of soluble Fas and FAP-1: Possible mechanisms of Fas resistance in human hepatoblastomas. J. Pathol. 188,207 -212.[Medline]
Leithauser, F., Dhein, J., Mechtersheimer, G., Koretz, K., Bruderlein, S., Henne, C., Schmidt, A., Debatin, K. M., Krammer, P. H. and Moller, P. (1993). Constitutive and induced expression of APO-1, a new member of the nerve growth factor/tumor necrosis factor receptor superfamily, in normal and neoplastic cells. Lab. Invest. 69,415 -429.[Medline]
Li, Y., Kanki, H., Hachiya, T., Ohyama, T., Irie, S., Tang, G., Mukai, J. and Sato, T. (2000). Negative regulation of Fas-mediated apoptosis by FAP-1 in human cancer cells. Int. J. Cancer 87,473 -479.[Medline]
Maekawa, K., Imagawa, N., Nagamatsu, M. and Harada, S. (1994). Molecular cloning of a novel protein-tyrosine phosphatase containing a membrane-binding domain and GLGF repeats. FEBS Lett. 337,200 -206.[Medline]
Maekawa, K., Imagawa, N., Naito, A., Harada, S., Yoshie, O. and Takagi, S. (1999). Association of protein-tyrosine phosphatase PTP-BAS with the transcription-factor-inhibitory protein IkappaBalpha through interaction between the PDZ1 domain and ankyrin repeats. Biochem. J. 337,179 -184.[Medline]
Matzinger, P. (1991). The JAM test: a simple assay for DNA fragmentation and cell death. J. Immunol. Methods 145,185 -192.[Medline]
Miyamoto, Y., Hosotani, R., Wada, M., Lee, J. U., Koshiba, T., Fujimoto, K., Tsuji, S., Nakajima, S., Doi, R., Kato, M., Shimada, Y. and Imamura, M. (1999). Immunohistochemical analysis of Bcl-2, Bax, Bcl-X, and Mcl-1 expression in pancreatic cancers. Oncology 56,73 -82.[Medline]
Mori, S., Murakami-Mori, K., Jewett, A., Nakamura, S. and Bonavida, B. (1996). Resistance of AIDS-associated Kaposi's sarcoma cells to Fas-mediated apoptosis. Cancer Res. 56,1874 -1879.[Abstract]
Mundle, S., Mativi, B. Y., Bagai, K., Feldman, G., Cheema, P., Gautam, U., Reza, S., Cartlidge, J. D., Venugopal, P., Shetty, V., Gregory, S. A., Robin, E., Rifkin, S., Shah, R. and Raza, A. (1998). Spontaneous down-regulation of Fas-associated phosphatase-1 may contribute to excessive apoptosis in myelodysplastic marrows. Int. J. Hematol. 70,83 -90.
Nagata, S. (1997). Apoptosis by death factor. Cell 88,355 -365.[Medline]
Natoli, G., Ianni, A., Costanzo, A., De Petrillo, G., Ilari, I., Chirillo, P., Balsano, C. and Levrero, M. (1995). Resistance to Fas-mediated apoptosis in human hepatoma cells. Oncogene 11,1157 -1164.[Medline]
Okura, T., Gong, L., Kamitani, T., Wada, T., Okura, I., Wei, C. F., Chang, H. M. and Yeh, E. T. (1996). Protection against CD95/APO-1- and tumor necrosis factor-mediated cell death by a novel protein, sentrin. J. Immunol. 157,4277 -4281.[Abstract]
Peter, M. E., Kischkel, F. C., Hellbardt, S., Chinnaiyan, A. M., Krammer, P. H. and Dixit, V. M. (1996). CD95 (APO-1/Fas)-associating signalling proteins. Cell Death Differ. 3,161 -170.
Reed, J. C. (1997). Double identity for proteins of the Bcl-2 family. Nature 387,773 -776.[Medline]
Saras, J., Claesson-Welsh, L., Heldin, C. H. and Gonez, L.
J. (1994). Cloning and characterization of PTPL1, a protein
tyrosine phosphatase with similarities to cytoskeletal-associated proteins.
J. Biol. Chem. 269,24082
-24089.
Saras, J., Engstroem, U., Gonez, L. J. and Heldin, C. H.
(1997). Characterization of the interactions between PDZ domains
of the protein-tyrosine phosphatase PTPL1 and the carboxyl-terminal tail of
Fas. J. Biol. Chem. 272,20979
-20981.
Sato, T., Irie, S., Kitada, S. and Reed, J. C. (1995). FAP-1: a protein tyrosine phosphatase that associates with Fas. Science 268,411 -415.[Medline]
Simon, H.U., Yousefi, S., Dibbert, B., Hebestreit, H., Weber,
M., Branch, D. R., Blaser, K., Levi-Schaffer, F. and Anderson, G. P.
(1999). Role for tyrosine phosphorylation and Lyn tyrosine kinase
in fas receptor-mediated apoptosis in eosinophils.
Blood 92,547
-557.
Sodeman, T., Bronk, S. F., Roberts, P. J., Miyoshi, H. and Gores, G. J. (2000). Bile salts mediate hepatocyte apoptosis by increasing cell surface trafficking of Fas. Am. J. Physiol. Gastrointest. Liver Physiol. 278,992 -999.
Ungefroren, H., Voss, M., Jansen, M., Roeder, C., Henne-Bruns, D., Kremer, B. and Kalthoff, H. (1998). Human pancreatic adenocarcinomas express Fas and Fas ligand yet are resistant to Fas-mediated apoptosis. Cancer Res. 58,1741 -1749.[Abstract]
von Bernstorff, W., Spanjaard, R. A., Chan, A. K., Lockhart, D. C., Sadanaga, N., Wood, I., Peiper, M., Goedegebuure, P. S. and Eberlein, T. J. (1999). Pancreatic cancer cells can evade immune surveillance via nonfunctional Fas (APO-1/CD95) receptors and aberrant expression of functional Fas ligand. Surgery 125, 73-84.[Medline]
von Reyher, U., Strater, J., Kittstein, W., Gschwendt, M., Krammer, P. H. and Moeller, P. (1998). Colon carcinoma cells use different mechanisms to escape CD95-mediated apoptosis. Cancer Res. 58,526 -534.[Abstract]
Wallach, D. (1997). Cell death induction by TNF: a matter of self control. Trends Biochem. Sci. 22,107 -109.[Medline]
Yanagisawa, J., Takahashi, M., Kanki, H., Yano-Yanagisawa, H.,
Tazunoki, T., Sawa, E., Nishitoba, T., Kamishohara, M., Kobayashi, E.,
Kataoka, S. and Sato, T. (1997). The molecular interaction of
Fas and FAP-1. J. Biol. Chem.
272,8539
-8545.
Zhang, B. X., Brunner, T., Carter, L., Dutton, R. W., Rogers,
P., Bradley, L., Sato, T., Reed, J. C., Green, D. and Swain, S. L.
(1997). Unequal death in T helper cell (Th)1 and Th2 effectors:
Th1, but not Th2, effectors undergo rapid Fas/FasL-mediated apoptosis.
J. Exp. Med. 185,1837
-1849.
Zhou, Y. W., Komada, Y., Inaba, H., Azuma, E. and Sakurai, M. (1998). Down-regulation of fas-associated phosphatase-1 (FAP-1) in interleukin-2-activated T cells. Cell. Immunol. 186,103 -110.[Medline]
Zong, W. X., Bash, J. and Gelinas, C. (1998). Rel blocks both anti-Fas- and TNF alpha-induced apoptosis and an intact Rel transactivation domain is essential for this effect. Cell Death Differ. 5,963 -972.[Medline]