
[Cancer Research 60, 553-559, February 1, 2000]
© 2000 American Association for Cancer Research
Regulation of Tumor Necrosis Factor-related Apoptosis-inducing Ligand Sensitivity in Primary and Transformed Human Keratinocytes1
Martin Leverkus2,
Manfred Neumann,
Thilo Mengling,
Charles T. Rauch,
Eva-Bettina Bröcker,
Peter H. Krammer and
Henning Walczak
Departments of Dermatology [M. L., T. M., E-B. B.] and Pathology [M. N.], University of Würzburg Medical School, 97080 Würzburg, Germany; Immunex Corporation, Department of Protein Chemistry, Seattle, Washington 98101 [C. T. R.]; and Deutsches Krebsforschungszentrum Tumor Immunology Program, German Cancer Research Center, 69120 Heidelberg, Germany [P. H. K., H. W.]
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ABSTRACT
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Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)
has been shown to exert potent cytotoxic activity against many tumor
cell lines but not against normal cells. It has been hypothesized that
this difference in TRAIL sensitivity between normal and transformed
cells might be due to the expression of the non-death-inducing TRAIL
receptors (TRAIL-R) TRAIL-R3 and TRAIL-R4, presumably by competition
for limited amounts of TRAIL. To assess the regulation of resistance
versus sensitivity to TRAIL in primary as well as
transformed keratinocytes, we examined TRAIL sensitivity, TRAIL
receptor expression, and intracellular signaling events induced by
TRAIL. Although TRAIL induced apoptosis in primary as well as
transformed keratinocytes, a marked difference in sensitivity could be
observed with primary keratinocytes (PK) being 5-fold less sensitive to
TRAIL than transformed keratinocytes (TK). Yet both cell types
exhibited similar TRAIL receptor surface expression, suggesting that
expression of TRAIL-R3 and TRAIL-R4 may not be the main regulator of
sensitivity to TRAIL. Biochemical analysis of the signaling events
induced by TRAIL revealed that PK could be sensitized for TRAIL and,
similarly, for TRAIL-R1- and TRAIL-R2-specific apoptosis by
pretreatment of the cells with cycloheximide (CHX). This sensitization
concomitantly resulted in processing of caspase-8, which did not occur
in TRAIL-resistant PK. These data indicate that an early block of
TRAIL-induced apoptosis was present in PK compared with TK or PK
treated with CHX. Interestingly, cellular FLICE inhibitory protein
(cFLIP) levels, high in PK and low in TK and several other
squamous cell carcinoma cell lines, decreased rapidly after treatment
of PK with CHX, correlating with the increase in TRAIL sensitivity and
caspase-8 processing. Furthermore, ectopic expression of cFLIP long
(cFLIPL) in TK by transfection with a
cFLIPL expression vector resulted in resistance to
TRAIL-mediated apoptosis of these cells. Thus, our results demonstrate
that TRAIL sensitivity in PK is primarily regulated at the
intracellular level rather than at the receptor level.
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Introduction
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Apoptotic cell death is a fundamentally important biological
process that is required to maintain the integrity and homeostasis of
multicellular organisms. Inappropriate or impaired apoptosis has been
implicated in the development of many human diseases, including cancer
(1)
. The death-inducing members of the
TNF3
family, TNF and CD95/APO-1/Fas ligand have been studied most
intensively leading to the elucidation of their role in
activation-induced cell death, autoimmune disorders, immune privilege,
and tumor evasion from the immune system (reviewed in Ref.
2
). TRAIL is a recently identified member of the TNF
family (3)
. TRAIL primarily kills tumor cells but not
normal cells (3)
. In addition, TRAIL exerts potent
antitumor activity in vivo without exhibiting systemic
toxicity (4)
. Five different cellular receptors for TRAIL,
all members of the TNF receptor family, have been identified thus far
(2)
. First, the two death receptors TRAIL-R1 and TRAIL-R2,
which are characterized by cytoplasmic death domains, were identified.
TRAIL-R3 is a truncated receptor that is membrane anchored, whereas
TRAIL-R4 is a receptor with a cytoplasmic domain lacking a functional
death domain. Finally, OPG, in addition to binding to the TNF ligand
family member OPG ligand/receptor activator of nuclear factor kappa B
ligand, also interacts with TRAIL (5)
. The
identification of a whole family of TRAIL receptors shows the
complexity of this death system and is indicative of its important
physiological role. It has been suggested that the differential
expression of non-death receptors of TRAIL, particularly TRAIL-R3,
might be responsible for the difference in sensitivity to TRAIL-induced
apoptosis observed between normal and transformed cells by competing
with the TRAIL death receptors TRAIL-R1 and TRAIL-R2 for limited
amounts of the ligand (6
, 7)
. In addition to determining
the role of TRAIL in keratinocyte biology, we set out to address the
question of differential TRAIL sensitivity in human PK compared with
TK. We compared the two cell types in terms of TRAIL sensitivity,
receptor expression, and intracellular signaling events induced by
TRAIL. Our data clearly demonstrate that TRAIL resistance of human PK
is maintained by intracellular inhibition of the TRAIL death pathway
rather than by non-death receptors of TRAIL.
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Materials and Methods
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Materials.
The protease inhibitor ZVAD-fmk was obtained from Bachem (Heidelberg,
Germany). All other reagents, if not indicated otherwise, were of
reagent grade and were obtained from Serva (Heidelberg, Germany). The
following Ab were used: anti-PARP Ab (G. Poirier, Centre Hospitalier
Université Laval Research Center, Quebec, Canada),
anti-CPP32 polyclonal Ab (D. Nicholson, Merck Frosst Corp., Quebec,
Canada), Flice Ab (C-15; Ref. 8
), and cFlip Ab (NF-6; Ref.
9
). TRAIL-R1 (clone M 271), TRAIL-R2 (clone M 413),
TRAIL-R3 (clone M 430), and TRAIL-R4 (clone M 444) Ab for analysis of
TRAIL-R1 through TRAIL-R4 surface expression (10)
and
recombinant LZ-TRAIL were generated at Immunex Corp. (Seattle,
WA) (11)
. Horseradish peroxidase-tagged donkey anti-rabbit
Ab and Horseradish peroxidase- and FITC-tagged goat anti-mouse IgG were
from PharMingen (Hamburg, Germany).
Tissue Culture.
PK cultures were prepared from newborn foreskins as described
previously (12)
. Cells were kept in serum-free
keratinocyte growth medium (Cellsystems, St. Katharinen,
Germany) and used only up to passage 3 for all experiments. The
spontaneously TK line HacaT, SCL-1, and SCL-2 were kindly
provided by Dr. N. Fusenig (Deutsches Krebsforschungszentrum,
Heidelberg, Germany) and were cultured as described (13)
.
SCC12F cells were obtained and cultured as described elsewhere
(14)
.
Reverse Transcription-PCR.
RNA was prepared with Qiagen (Hilden, Germany) RnEasy Kit
according to the manufacturers recommendation. RNA was reverse
transcribed and the resulting cDNA was analyzed with 100 pmol of each
of the forward and reverse primer pairs (MWG Biotech, Munich, Germany)
for the presence of TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, and
ß-actin, yielding amplification products of 506, 502, 612, 453, and
219 bp, respectively. Primers and numbers of cycles used are described
in Table 1
. Samples in which reverse transcriptase was omitted were amplified for
40 cycles with ß-actin-specific primers to exclude genomic DNA
contamination. Setup experiments demonstrated exponential amplification
for cycle number and primer pairs used (data not shown).
PCR conditions [measured using a Perkin-Elmer (Norwalk, CT)
Gene Amp PCR System 2400] were as follows: denaturation for 5 min at
94°C and then the indicated number of cycles (Table 1)
with
denaturation (94°C, 1 min), primer annealing (55°C, 1 min), and DNA
polymerization (72°C, 1 min) and a final extension step of 7 min at
72°C. PCR products were electrophoresed on a 2.5% agarose gel,
stained with ethidium bromide, and photographed on an UV screen.
Western Blot Analysis.
Total cellular proteins were collected as described (15)
with the exception that Complete protease inhibitor cocktail
(Boehringer Mannheim, Mannheim, Germany) was used. A total of 2075
µg of protein were electrophoresed on SDS-PAGE gels and transferred
to nitrocellulose membranes. Membranes were blocked and hybridization
was performed with Ab to PARP, caspase 8, caspase 3, or cFLIP.
After incubation with appropriate secondary Ab, bands were visualized
using the enhanced chemiluminescence detection kit (Amersham, Arlington
Heights, IL). Blots were subsequently rehybridized with anti-tubulin Ab
(Sigma, St. Louis, MO) to show equal loading of the gel.
FACScan Analysis.
Subconfluent TK or PK were detached from the plates by EDTA treatment
before brief treatment with trypsin. A total of 5 x 105 cells were resuspended in ice-cold PBS
containing 1% BSA (PBS/BSA) and reacted in 5 µg/ml
monoclonal Ab or isotyped control IgG and then in FITC-conjugated
secondary Ab. A total of 105 cells were analyzed
by FACScan (Becton Dickinson and Co., San Jose, CA).
Induction of Cell Death by Recombinant Human TRAIL or TRAIL-R1-
and TRAIL-R2-Specific Monoclonal Ab.
Preconfluent TK or PK were washed once with PBS and 1 ng1 µg/ml
recombinant LZ-TRAIL or diluent alone was added. CHX and TRAIL were
added to the culture medium simultaneously. Cultures were maintained at
37°C until analysis. For induction of apoptosis with TRAIL-R1- and
TRAIL-R2-specific monoclonal Ab, 96-well plates were coated with the
indicated concentrations of the monoclonal Ab overnight at 4°C. Then
3 x 105 PK were added to wells
that were washed three times with PBS. After attachment for at
least 2 h, CHX (1 µg/ml) or diluent alone was added and cell
death was determined after a 16-h incubation.
Apoptosis and Cytotoxicity Assays.
Crystal violet staining of surviving attached cells was performed
1624 h after addition of LZ-TRAIL, as described (16)
.
Briefly, cells were seeded in 24-well plates (1 x 105 cells) or in 96-well plates (2 x 104 cells) and treated as described in
"Results." Absorbance of eluted crystal violet was measured in an
ELISA reader at 570 nm. Parallel plates were examined by propidium
iodide staining and flow cytometrical analysis of subdiploid DNA
content as described (17)
. The percentage of apoptotic
nuclei was always >95% for conditions where viability was determined
as background (0%) in the cytotoxicity assay. In some experiments
apoptosis was determined by detection of internucleosomal DNA
fragmentation by cell death detection ELISA (Cell Death Detection ELISA
Plus, Boehringer Mannheim) used according to the manufacturers
instructions. Analysis of half-maximal response to TRAIL-induced
apoptosis was determined by nonlinear regression analysis.
Transient Transfection Assay for cFLIP.
Sixteen to thirty-six hours before transfection with Fugene (Boehringer
Mannheim), 5 x 105 HacaT cells
were seeded per well in six-well plates. Cells were cotransfected with
0.5 µg of pGREEN LANTERN-1 (Life Technologies, Inc., Eggenstein,
Germany) GFP expression vector (pGL) and increasing
amounts of pcDNA3-cFLIP vector (up to 0.5 µg; Ref. 9
).
The total amount of transfected DNA was kept constant to 1 µg DNA by
adding the appropriate amount of pcDNA3 control vector. After
transfection, the cells were incubated 2436 h to permit recovery and
maximum expression (i.e., assessed by GFP expression). For
assessment of apoptosis in response to TRAIL, cultures were either
untreated or incubated with 50 ng/ml of LZ-TRAIL for an additional
16 h. Cell death was analyzed by determining the percentage of
propidium iodide-positive cells in the GFP-expressing
population.
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Results
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Higher Levels of TRAIL Are Necessary to Induce Apoptosis in PK
Compared with TK.
To determine the effect of TRAIL on human keratinocytes, we first
analyzed PK and TK in terms of TRAIL-induced cytotoxicity, apoptosis,
and cleavage of PARP. PARP is a known substrate of caspase 3 (CPP-32;
Ref. 18
) and specific cleavage of PARP is a hallmark of
cellular apoptosis. Cells were initially treated with 250 ng/ml of
recombinant human LZ-TRAIL. Cell death was visualized morphologically
by phase contrast microscopy (Fig. 1A)
and cellular apoptosis was assayed by determination of
internucleosomal DNA fragmentation 16 h (Fig. 1B)
or
PARP cleavage 4 h (Fig. 1C)
after addition of LZ-TRAIL.
When compared with diluent-treated control cells, LZ-TRAIL-treated PK
as well as TK showed a marked reduction of survival, induction of
internucleosomal DNA fragmentation, and PARP cleavage, indicating that
TRAIL induces apoptosis in PK as well as TK cells. Reduction of
survival and induction of internucleosomal DNA fragmentation or PARP
cleavage was completely inhibited by ZVAD-fmk, which is known to block
the caspase family of proteases (19)
, suggesting that
TRAIL-mediated apoptosis in keratinocytes requires a
caspase-dependent signal transduction pathway (Fig. 1)
. However,
analysis of the dose response to TRAIL- induced apoptosis revealed
differential sensitivity of PK when compared with TK (Fig. 2)
. PK required 500 ng/ml of LZ-TRAIL for maximal killing, whereas TK
required only 100 ng/ml for the maximal cytotoxic effect. Therefore,
our data demonstrate that although at lower levels of TRAIL a
differential sensitivity between normal and transformed keratinocytes
can be detected, both cell types succumb to high levels of TRAIL.

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Fig. 1. TRAIL induces apoptosis in PK and TK. PK or TK were
treated with 250 ng/ml of human LZ-TRAIL. A, TRAIL leads
to caspase-dependent cytotoxicity in both cell types. The arrowsindicate cell surface blebbing typical for cellular apoptosis.
B, The conditions are the same as in
A, analysis of internucleosomal DNA cleavage by
ELISA 16 h after stimulation. TRAIL-treated PK or TK show
induction of internucleosomal DNA cleavage when compared with control
cultures or cultures treated with LZ-TRAIL in the presence of the
pancaspase inhibitor ZVAD-fmk. Shown are results of triplicate samples.
Experiments were repeated at least three times with similar results.
C, The conditions are the same as in
A, 4 h after stimulation with LZ-TRAIL.
LZ-TRAIL leads to specific and caspase-dependent PARP cleavage in PK
and TK.
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TRAIL Receptor Expression Is Not the Main Regulatory Mechanism of
Resistance to TRAIL in PK.
It has been hypothesized that a difference in TRAIL sensitivity
between normal and transformed cells might be due to the expression of
the non-death-inducing TRAIL-R3 and TRAIL-R4 (6
, 7)
.
Therefore, we analyzed TRAIL-R1TRAIL-R4 expression on mRNA as
well as protein level. In PK, specific transcripts were detectable for
TRAIL-R1, -R2, and -R4 and, to a much lesser extent, for TRAIL-R3.
Similar mRNA expression levels were detectable in TK, although no
TRAIL-R3-specific transcripts were detectable (Fig. 3A)
. On the protein level, whereas PK strongly expressed
TRAIL-R1 and -R2, little if any surface expression of TRAIL-R3 and no
surface expression of TRAIL-R4 was detected. On the surface of TK,
expression of TRAIL-R1 and -R2 was detected, albeit to a lesser extent
than on the surface of PK, and no staining for TRAIL-R3 or -R4 was
detectable (Fig. 3B)
. Therefore, our data demonstrate that
both cell types exhibit a similar TRAIL receptor surface expression. To
further confirm that TRAIL-R3, TRAIL-R4, or OPG expression is not the
main regulatory mechanism against TRAIL-induced apoptosis in PK, we
compared treatment of PK with LZ-TRAIL or a combination of TRAIL-R1-
and TRAIL-R2-specific agonistic monoclonal Ab in the presence and
absence of CHX. This experiment allows for the direct differentiation
between a potential protective role of the non-death-inducing TRAIL-R3,
-R4, and OPG on the one hand and, on the other hand, intracellular
antiapoptotic mechanisms. Interestingly, in PK TRAIL-R1 and -R2
triggering induced dose-dependent apoptosis similar to that induced by
recombinant LZ-TRAIL. However, 10-fold higher concentrations of
Ab for similar killing efficiency were required when compared with
LZ-TRAIL, a fact that can be explained by different agonistic
activities of the monoclonal Ab and LZ-TRAIL, respectively (Fig. 3C)
. More importantly, the protein synthesis inhibitor CHX,
which has been shown to increase sensitivity to death ligand-induced
apoptosis (20)
, increased sensitivity to LZ-TRAIL or
TRAIL-R1 and TRAIL-R2 triggering (Fig. 3C)
, suggesting that
TRAIL-R3, TRAIL-R4, and/or OPG expression may not be the main mechanism
regulating TRAIL sensitivity in PK. Yet, these data point toward an
important role for intracellular signaling events in the regulation of
keratinocyte sensitivity and resistance to TRAIL.

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Fig. 3. TRAIL-R expression and monoclonal Ab-mediated cell death
in PK and TK. A, TRAIL-R mRNA expression. At the mRNA
level PK express TRAIL-R1 through TRAIL-R4, whereas TK express only
TRAIL-R1, -R2, and -R4. mRNA expression was determined by reverse
transcription-PCR. B, FACS analysis of TRAIL-R protein
expression. PK strongly express TRAIL-R1 and TRAIL-R2 on the surface,
whereas only minimal staining for TRAIL-R3 and no staining for TRAIL-R4
is detectable. TK express TRAIL-R1 and TRAIL-R2 weaker than PK but do
not express TRAIL-R3 or TRAIL-R4 on the cell surface. C,
TRAIL-R1 and -R2 agonistic monoclonal Ab induce cell death similar to
TRAIL in PK, and cells can be sensitized by CHX. Cells were incubated
with TRAIL-R1- and TRAIL-R2-specific monoclonal Ab as described in
"Materials and Methods." After 16 h, cell survival was
analyzed by crystal violet staining. Data are shown as a percentage of
control with SD (four wells for each condition).
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Caspase 8 Processing Is Inhibited in PK.
To understand the mechanism of intracellular regulation of TRAIL
sensitivity, we explored the intracellular events leading to
TRAIL-induced apoptosis of keratinocytes. For other death-inducing
ligands like CD95/APO-1/Fas ligand, it has been shown that caspase 8
and caspase 3 are critically involved in the induction of apoptosis
(21
, 22) . Therefore, we examined whether these caspases
are differentially activated by TRAIL in PK versus TK.
Indeed, LZ-TRAIL treatment led to rapid activation of caspase 8 and
caspase 3 in TK (Fig. 4A)
but not in PK (Fig. 4B)
. However, when PK were
treated with LZ-TRAIL in the presence of CHX, caspase 8 and caspase 3
were strongly activated (Fig. 4B)
, which was similar to the
activation observed in TK. Concomitantly, in the presence of CHX, PK
were as sensitive as TK to LZ-TRAIL-induced apoptosis (Fig. 2)
. These
data were indicative of an early apical block of TRAIL-induced
apoptosis in PK compared with TK and led us to examine proteins known
to interfere with caspase 8 activation at the DISC level.

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Fig. 4. TRAIL-dependent caspase activation and cFlipL
expression in PK and TK. A, TK were treated for the
indicated intervals with 100 ng/ml recombinant human LZ-TRAIL. Cell
lysates were prepared and analyzed by Western blotting with Ab to
caspase 8 and caspase 3. Detection of full-length caspase 8 or caspase
3 protein is decreased as early as 4590 min after stimulation. Note
appearance of specific caspase 8 (p43, p41, upper arrow) or
caspase 3 cleavage product p17 (lower arrow).
Rehybridization of the membrane with an anti-tubulin Ab demonstrated
comparable loading of protein. B, PK were treated in the
presence or absence of 1 µg/ml CHX with 100 ng/ml TRAIL. In the
absence of CHX, activation of caspase 8 or caspase 3 is inhibited. Upon
treatment with CHX, markedly increased cleavage of caspase 8 and
caspase 3 [determined by detection of cleavage products of caspase 8
(p43, p41, upper arrow), p18 (middle arrow), or
caspase 3 (p20, p17, lower arrows)] is detectable with
similar kinetics as demonstrated in A for TK. Inclusion
of 40 µM zVAD-fmk completely abrogates caspase 8 and
caspase 3 cleavage in PK, TK, or PK treated with CHX. C,
cFlipL is expressed in PK but not in TK.
Left, Cell lysates from PK or TK were prepared and 50
µg of protein were analyzed by Western blotting with a cFLIP-specific
monoclonal Ab. A 55-kDa protein representing cFLIPL is
detectable in PK but not in TK. Right, PK were treated
for the indicated time intervals with CHX (1 µg/ml). A total of 75
µg of protein was analyzed by Western blotting with cFLIP-specific
Ab. Detectable levels of cFLIPL are reduced within 4590
min and are barely detectable within 5 h after treatment with CHX.
Inclusion of 40 µM zVAD-fmk does not prevent decay of
cFLIPL after CHX treatment. D, cFLIP
expression in different squamous cell carcinoma lines. PK (Lane
1) show strong expression of cFLIP, whereas squamous cell
carcinoma lines SCL-1 (Lane 2), SCL-2 (Lane
3), SCC12F (Lane 4), and TK (Lane
5) show weak or absent expression of cFLIP upon overexposure of
the blot. E, cFLIP overexpression confers resistance
against TRAIL-induced apoptosis. Twenty-four to thirty-six hours before
treatment with 50 ng/ml LZ-TRAIL, TK were transfected with the
indicated amounts of pcDNA3-cFLIP together with 0.5 µg of pGREEN
LANTERN-1 (Life Technologies, Inc.) GFP expression vector (pGL) and the
appropriate amount of pcDNA3 control vector necessary to keep the total
amount of transfected DNA constant to 1 µg DNA. Sixteen hours after
the beginning of treatment, cells were detached from the culture plates
and cell death was assessed by determining the percentage of propidium
iodide-positive cells in the GFP-positive population. Results
are shown as the percentage of maximal cell death determined in
control-transfected cultures (i.e., 0 µg cFLIP
expression vector). The results shown are averaged from three
independent experiments with positive control cell death ranging from
65 to 80%.
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Sensitization to TRAIL Correlates with cFLIP Expression in
Keratinocytes.
A cellular protein known to inhibit caspase 8 at the DISC is
cFLIP (9
, 23)
. Therefore, we analyzed cFLIP expression in
PK and TK. Western blot analysis showed that
cFLIPL (55 kDa) was present in PK. In contrast,
cFLIPL was virtually absent from TK (Fig. 4C)
. To examine the influence of CHX treatment on
cFLIPL expression in PK, we analyzed PK treated
for different lengths of time with CHX for cFLIPL
expression. Interestingly, cFLIPL levels were
markedly reduced within 4590 min and were barely detectable after
5 h, indicating that PK required active protein synthesis to
maintain cFLIPL protein levels (Fig. 4C)
. Moreover, three different squamous cell carcinoma lines
(SCC12F, SCL-1, and SCL-2) exhibited sensitivity to LZ-TRAIL-induced
apoptosis comparable with that of TK (data not shown) and contained
only low levels of cFLIPL as demonstrated by
Western blot analysis (Fig. 4D)
. Thus, our data indicate
that cFLIPL may act as an important regulator of
TRAIL-induced apoptosis in primary and malignant keratinocytes.
Overexpression of cFLIPL Decreases Sensitivity to
TRAIL.
To assess whether cFLIP may act as a regulator of sensitivity to
TRAIL-induced apoptosis in keratinocytes, we tested whether the ectopic
expression of cFLIPL influenced TRAIL sensitivity
of TK. As shown in Fig. 4E
, TK transfected with high or
intermediate amounts of cFLIP demonstrated a substantial reduction of
cell death when compared with vector-transfected controls. These
results indicate that cFLIP represents an important intracellular
regulator of resistance to TRAIL in keratinocytes (Fig. 4E)
.
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Discussion
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TRAIL, a recently identified apoptosis-inducing member of the TNF
family, has been shown to exert potent cytotoxic activity against many
tumor cell lines (3)
. We were interested in examining the
effect of TRAIL on PK compared with TK because it has been suggested
that transformed cells but not primary cells are selectively killed by
TRAIL (3)
. In this study, we show that TRAIL can induce
apoptosis in TK and PK in a dose-dependent manner as shown by
morphological analysis, internucleosomal DNA fragmentation, and PARP
cleavage. Yet, PK and TK exhibit a marked difference in sensitivity to
TRAIL because 5-fold higher concentrations of TRAIL were required to
kill PK than were required to kill TK. Thus, our results are consistent
with the observation that at low concentrations of TRAIL PK are not
killed (24)
, yet in addition we show that PK are not
completely TRAIL-resistant but are only less sensitive than TK. Thus,
our data show that PK contain all necessary signaling components
required for TRAIL-induced apoptosis but that they nevertheless require
higher concentrations of TRAIL than TK do for efficient killing.
Due to the fact that TRAIL-R3, TRAIL-R4, and OPG do not signal for cell
death, it was proposed that these receptors might provide resistance to
TRAIL (6
, 7
, 25)
. Yet, this proposed function is rather
speculative because thus far it has never been tested in nontransfected
cells. When comparing the surface expression levels of the different
TRAIL receptors on PK and TK, we found, albeit at low levels, TRAIL-R3
on the surface of PK but not on the surface of TK, whereas TRAIL-R4 was
absent from the surface of both cell types despite strong mRNA
expression. Another difference between the two cell types was higher
expression of TRAIL-R1 and TRAIL-R2 on PK compared with expression on
TK. It is currently not known how the different TRAIL-R interact with
each other on the cell surface, nor is it known how much TRAIL-R3,
TRAIL-R4, or OPG is needed under physiological conditions to
potentially disrupt TRAIL-induced DISC formation. Although the stronger
expression of TRAIL-R1 and -R2 on PK would rather lead to the
prediction of higher sensitivity to TRAIL in these primary cells, we
could not exclude that low expression of TRAIL-R3 on their surface
might be responsible for mediating the observed relative resistance to
TRAIL. To address this question in detail, we performed experiments
that bypassed the potential effects of TRAIL-R3, TRAIL-R4, or OPG in PK
by induction of apoptosis with specific monoclonal Ab to TRAIL-R1 and
-R2. If the non-death-inducing TRAIL receptors played a role in
mediating resistance to TRAIL in the primary cells, then TRAIL-R1- and
TRAIL-R2-specific Ab should be able to induce apoptosis although TRAIL
would not. Yet, dose dependency of PK apoptosis was similar between
recombinant TRAIL and a mix of TRAIL-R1- and TRAIL-R2-specific Ab.
Similarly, in PK the CHX-mediated sensitization to apoptosis was
virtually identical for TRAIL-, TRAIL-R1-, and TRAIL-R2-induced cell
death. These data clearly indicate that mainly intracellular regulators
are responsible for the observed relative resistance of PK to TRAIL. In
addition, we can conclude that the extracellular regulation of TRAIL
sensitivity by TRAIL-R3, TRAIL-R4, or OPG plays a less important role,
at least in keratinocytes.
To date, little is known about the signaling events associated with
TRAIL receptor ligation. It is currently not known which adapter
proteins are recruited to the TRAIL-R DISC under physiological
conditions. However, overexpression of dominant negative forms of
Fas-associated death domain (FADD) and TNF-associated death domain
(TRADD) inhibited TRAIL-induced apoptosis (11
, 16
, 26)
. Our data confirm previous findings in myeloma and melanoma
cells and expand them to keratinocytes in as much as caspase 8 and
caspase 3 are necessary for induction of TRAIL-induced apoptosis
because inhibition of caspases completely abrogated their processing
and TRAIL-induced apoptosis in keratinocytes (27
, 28)
.
Upon treatment with TRAIL in the presence of CHX, PK were as sensitive
as TK. Concomitantly, TRAIL treatment led to similar activation of
caspase 8 in both cell types, suggesting that in the absence of the
protein synthesis inhibitor CHX an unidentified protein inhibits the
cytotoxic TRAIL signal at a very early stage, namely prior to caspase 8
activation. cFLIPL is a protein known to
interfere with the proximal death receptor-mediated cascade and has
been shown to suppress CD95-, TNF-, and TRAIL-mediated apoptosis
(23)
. Furthermore, it was recently shown that
cFLIPL inhibits caspase 8 activation at the CD95
DISC level (9)
. Our data clearly show that
cFLIPL is strongly expressed in PK and is
undetectable in TK. Upon CHX treatment, cFLIPL
levels decreased rapidly in PK, correlating with an increase in
sensitivity to TRAIL. In addition, transient transfection of
TRAIL-sensitive TK with cFLIPL rendered these
cells resistant to TRAIL-mediated apoptosis. Therefore, it is tempting
to speculate that the relative resistance of PK to TRAIL-induced
apoptosis is conferred by cFLIPL. In fact, the
analysis of a number of squamous cell carcinoma cell lines indicated
that this phenomenon may be of fundamental importance because low
levels of cFLIPL expression correlated well with
TRAIL sensitivity in all cell lines tested.
If nonmelanoma skin cancer is not treated by surgery, it often leads to
death due to metastasis. Common treatment regimens only have limited
success for metastatic disease. Therefore, effective new therapeutic
agents would be beneficial for the treatment of this disease. Indeed,
induction of apoptosis via death receptors has been shown to represent
one possible mechanism by which chemotherapeutic agents act on tumor
cells (29)
. Therefore, an attractive treatment option for
cancer might be by direct activation of TRAIL receptors. Interestingly,
it was recently shown that TRAIL exerts a potent antitumor activity
in vivo without exhibiting any systemic toxicity
(4)
. Our data suggest that TRAIL may represent a useful
option for the treatment of nonmelanoma skin cancer.
In summary, we first demonstrate a relative resistance to TRAIL-induced
apoptosis of PK compared with TK. Then we show that TRAIL sensitivity
is mainly regulated by intracellular factors protecting PK against the
cytotoxic signal induced by TRAIL. Furthermore, high
cFLIPL levels in PK correlated with inhibition of
caspase 8 activation and decreased sensitivity to TRAIL, whereas low
cFLIPL levels correlated with efficient caspase 8
activation and high TRAIL sensitivity in TK and squamous cell
carcinomas. Additionally, ectopic expression of
cFLIPL leads to TRAIL resistance of TK. Taken
together, these data suggest an important role for
cFLIPL in determining differential sensitivity of
PK versus TK. This mechanism of resistance to TRAIL-mediated
apoptosis indeed may be important in many different cellular
systems.
 |
ACKNOWLEDGMENTS
|
|---|
We thank Ingo Schmitz for purified anti-cFLIP
monoclonal Ab; D. Nicholson for antiserum to caspase 3; Eckhart
Kämpgen and Peter Friedl for helpful suggestions and critical
reading of the manuscript; Marc Schmidt for his help in setting up
transfection experiments; and Evi Horn, Eva Rieser, and Heiko Stahl for
excellent technical assistance.
 |
FOOTNOTES
|
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported in part by grants from
Erweiterte Forschungsfoerderung des Freistaates Bayerm (Nr. 6 a) and a
grant from Interdisziplinaerem Zentrum fuer Klinische Forschung
Wuerzburg (IZKF 01 KS 9603, Z 4-14) (to M. L.). H. W. was supported
by the AIDS Stipend Program of the Bundesministerium für Bildung
und Forschung. 
2 To whom requests for reprints should be
addressed, at University of Würzburg Medical School, Department
of Dermatology, Josef-Schneider-Strasse 2, 97080 Würzburg,
Germany. Phone: 0931-201-2710; Fax: 0931-201-2700. 
3 The abbreviations used are: TNF, tumor necrosis
factor; TRAIL, TNF-related apoptosis-inducing ligand; TRAIL-R, TRAIL
receptors; OPG, osteoprotegerin; PK, primary keratinocytes; TK,
transformed keratinocytes; ZVAD-fmk, z-Val-Ala-Asp-fluoromethyl ketone;
Ab, antibody; LZ-TRAIL, leucine zipper-TRAIL; PARP,
poly-ADP-ribose-polymerase; CHX, cycloheximide; DISC, death-inducing
signaling complex; cFLIPL, long form of cFLIP; GFP, green
fluorescence protein. 
Received 9/17/99.
Accepted 12/13/99.
 |
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