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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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Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Materials and Methods |
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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 manufacturer’s 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).
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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 20–75
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 ng–1 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
16–24 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 manufacturer’s
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 24–36 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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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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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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. 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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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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. 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 45–90 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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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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REFERENCES |
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and by ultraviolet B radiation. J. Clin. Invest., 92: 462-470, 1993.
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B and protects against TRAIL-mediated apoptosis, yet retains an incomplete death domain. Immunity, 7: 813-820, 1997.[Medline]
B. Immunity, 7: 831-836, 1997.[Medline]
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) or TK (
) were treated for 24 h with increasing
concentrations of LZ-TRAIL in the absence (open symbols) or
presence (filled symbols) of 1 µg/ml CHX and subsequently
were analyzed by crystal violet staining. Mean and SD of four
independent experiments are shown as a percentage of control. Surviving
cells are dose dependently decreased in PK as well as in TK.
Half-maximal responses to TRAIL were determined by nonlinear regression
analysis. PK require 143 ng/ml LZ-TRAIL, whereas TK cells require 32
ng/ml LZ-TRAIL for half-maximal cytotoxicity. Upon addition of CHX,
dose-response curves in PK as well as in TK are shifted to lower
concentrations of TRAIL with half-maximal cytotoxicity reached at 14
ng/ml LZ-TRAIL in PK and 7 ng/ml in TK, respectively.
