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Advances in Brief |
Laboratory of Molecular Oncology and Cell Cycle Regulation, Howard Hughes Medical Institute, Departments of Medicine, Pharmacology, Genetics, Cancer Center, and the Institute for Human Gene Therapy, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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2;
P < 0.01). FLIP expression appeared elevated in
five of six (83%) TRAIL-resistant cell lines and only one of five
(20%) TRAIL-sensitive cells (2; P < 0.05). Two TRAIL-resistant lines that expressed DR4 contained an
A-to-G alteration in the death domain encoding arginine instead of
lysine at codon 441. The K441R polymorphism is present in 20% of the
normal population and can inhibit DR4-mediated cell killing in a
dominant-negative fashion. The expression level of KILLER/DR5, TRID,
TRUNDD or TRID, and TRUNDD did not correlate with TRAIL sensitivity
(P > 0.05). These results suggest that the major
determinants for TRAIL sensitivity may be the expression level of DR4
and FLIP. TRAIL-resistant cells became susceptible to TRAIL-mediated
apoptosis in the presence of doxorubicin. In TRAIL-sensitive cells,
caspases 8, 9, and 3 were activated after TRAIL treatment, but in
TRAIL-resistant cells, they were activated only by the combination of
TRAIL and doxorubicin. Our results suggest: (a)
evaluation of tumor DR4 and FLIP expression and host DR4 codon 441
status could be potentially useful predictors of TRAIL sensitivity, and
(b) doxorubicin, in combination with TRAIL, may
effectively promote caspase activation in TRAIL-resistant tumors. |
Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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, and lymphotoxin (1)
. TRAIL is
a potent inducer of apoptosis in a variety of transformed or cancer
cells of human and mouse origin but not normal cells (1
, 2)
.
The therapeutic use of the Fas/FasL or the TNF-/TNFR1
system in cancer treatment has been hampered by severe side effects
(3)
. The systemic administration of TNF causes a septic
shock-like response possibly mediated by nuclear factor-
B
activation, and the injection of agonist Ab to Fas can be lethal
(3
, 4)
. Compared to TNF- or Fas, TRAIL may be a safer
alternative because normal cells appear to be resistant, and it
activates nuclear factor-B only weakly (5)
.
Recently, evidence for the safety and potential efficacy of TRAIL
therapy against breast and colon cancer was obtained in a severe
combined immunodeficiency mouse model (6
, 7) .
Additionally, in cell culture, the human leucine zipper (LZ)-TRAIL had
no cytotoxic effects on normal cells, including human mammary
epithelial cells, human renal proximal tubule epithelial cells, human
lung fibroblasts, and human skeletal muscle cells but was toxic toward
mammary adenocarcinoma cells (6)
. The in vivo
experiments showed that the systemic administration of LZ-TRAIL into
mice inoculated with breast cancer cells prolonged survival. These
studies suggest that TRAIL may have a potential use for cancer
treatment.
TRAIL can modulate an apoptotic response by binding to one of four cell-surface receptors: Death receptor (DR) 4 (TRAIL-R1; Ref. 8 ), KILLER/DR5 (TRAIL-R2, TRICK2; Refs. 9, 10, 11, 12 ), TRID (DcR1, TRAIL-R3, or LIT; Refs. 5 , 10 , 13 , and 14 ), and TRUNDD (DcR2 or TRAIL-R4; Refs. 15, 16, 17 ). DR4 and KILLER/DR5 have two cysteine-rich extracellular ligand-binding domains and a cytoplasmic death domain that signals downstream caspase activation (2 , 18) . KILLER/DR5 was identified as a candidate p53 target gene, linking DNA damage signaling from p53 to downstream caspase activation and cell death (9) . The extracellular domain of TRID shares a homology with DR4 and KILLER/DR5, but it does not have a cytoplasmic death domain, and it is anchored to the membrane through a glycosyl phosphatidyl inositol linkage. TRUNDD has a substantially truncated cytoplasmic death domain. These two decoy receptors have been reported to protect cells from TRAIL-mediated apoptosis by competing with DR4 and KILLER/DR5 for binding to TRAIL (10) .
The TRAIL-mediated biochemical signaling pathway leading to apoptosis is not yet clear. Previously, it was reported that the ectopic expression of FADD-DN (dominant-negative FADD, which blocks apoptotic signaling by the Fas/APO1 death receptor) does not efficiently block apoptosis triggered by TRAIL, and that overexpression of DR4 could induce apoptosis in FADD-deficient embryonic fibroblasts (19) . These studies suggest that a FADD-independent pathway may link TRAIL to the caspase cascade (2 , 19 , 20) . Moreover, it was shown that DR4 does not efficiently recruit FADD, TNF receptor-associated death domain (TRADD) protein, receptor interacting protein (RIP), or RIP-associated ICH-1/CED-3 homologous protein (RAIDD; Ref. 10 ). Although at present there is a missing link between TRAIL death receptors and caspase activation, it is clear that once TRAIL binds to its receptors, apoptosis ensues through the activation of caspases (5 , 8 , 10) . Initiator caspases (caspases 8, 9, and 10) are composed of an N-terminal prodomain that contains the region for homotypic protein-protein interaction with adaptor molecules together with one large and one small subunit. When cells receive death-inducing signals, the prodomain is cleaved, and an active heterodimeric tetramer containing two small and two large subunits is formed. It was reported that caspases 3 and 8 became activated when HeLa cells were treated with TRAIL (21) and also that in TRAIL-sensitive breast cancer cell lines, caspase 3 cleavage was observed (22) . In addition, a recent report that T lymphocytes that have catalytically inactive caspase 10 are TRAIL-resistant implicates caspase 10 in TRAIL-mediated apoptosis (23) .
Although the efficacy and potential use of TRAIL in cancer treatment has been suggested, little is known about the factors that determine the sensitivity of cancer cells to killing by TRAIL. Recently, there were some reports on the determinants of TRAIL sensitivity in breast cancer cells (22) , melanoma (24) , and brain tumors (25 , 26) . The results have been somewhat controversial in that some reports showed no correlation between TRAIL sensitivity and the expression level of proapoptotic death receptors, whereas others demonstrated a correlation between them.
We investigated the expression level of various TRAIL receptor family members as determinants for TRAIL sensitivity and whether a DNA-damaging chemotherapeutic drug such as doxorubicin might have additive effects with TRAIL in killing cancer cells. We report here that the expression of the proapo-ptotic TRAIL receptors, in particular DR4, and the caspase activation inhibitor FLIP may be major determinants of TRAIL sensitivity. In addition to the expression level of DR4, a polymorphism found in the death domain region of DR4 prevents DR4-mediated cell killing in a dominant-negative fashion. Finally, we also report that a DNA damaging agent such as doxorubicin can sensitize cells to TRAIL-mediated cell killing. Our results provide essential preclinical information that may be useful in the design of clinical trials using recombinant TRAIL in the therapy of human cancer.
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Materials and Methods |
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Assessment of Cell Viability.
Recombinant soluble human TRAIL was purchased from Kamiya Biomedical
Co. (Seattle, WA), and the anti-FLAG M2 mAb was purchased from Sigma
(Saint Louis, MI). Three thousand cells were seeded into each well of a
96-well plate. After 24 h, the cells were treated with TRAIL (200
ng/ml) and cross-linked with the anti-FLAG M2 mAb (2 µg/ml). Cell
viability was measured by using the MTT assay at 16 h after
treatment (28)
. When normal cells were treated with both
doxorubicin and TRAIL, the cells were treated with increasing
concentrations of chemotherapeutic drugs alone (doxorubicin, 0, 0.1, 1,
10, and 100 µg/ml) or in combination with TRAIL (20 ng/ml)
cross-linked with the anti-FLAG M2 Ab (2 µg/ml). To assess the
long-term effect of TRAIL, a total of 5 x
104 of each cell line were seeded in triplicate
into 24 wells, and at 24 h, cells were treated with TRAIL (50
ng/ml) and the anti-FLAG M2 Ab (2 µg/ml). The media containing TRAIL
and Ab was changed every 48 h, and the culture was maintained for
7 days, at which time the remaining cells were stained with Coomassie
Blue.
Semiquantitative RT-PCR.
Total RNA was isolated from cell lines as described (29)
.
cDNA was generated from 2 µg of total RNA in a final volume of 20
µl using SuperScript II (Life Technologies, Inc., Gaithersburg, MD)
and random primers. The sequences of specific primers used in this
experiment were as follows: DR4 F, 5'-CGATGTGGTCAGAGCTGGTACAGC-3'; DR4
R, 5'-GGACACGGCAGAGCCTGTGC-CATC-3'; KILLER/DR5 F,
5'-GGGAGCCGCTCATGAGGAAGTTG G-3', KILLER/DR5 R,
5'-GGCAAGTCTCTCTC-CCAGCGTCTC-3'; TRID F,
5'-GTTTGTTTGAAAGACTT-CACTGTG-3', TRID R,
5'-GCAGGCGTTTCTGTCTGT-GGGAAC-3'; TRUNDD F,
5'-CTTCAGGAAACCAGAGCTT-CCCTC-3', TRUNDD R,
5'-TTCTCCCGTTTGCTTATCA-CACGA-3'; GAPDH F,
5'-ACCACAGTCCATGCCATCAC-3', GAPDH R, 5'-TCCACCACCCTGTTGCTGTA-3'.
To analyze the expression level of the death receptors, 2 µl (out of
20 µl) of synthesized cDNA was amplified in a total volume of
50 µl containing 200 µM each of all four dNTPs, 2 µCi
-32P-dCTP (3000 Ci/mmol), 2 µM
each of death receptor-specific primer set along with 2
µM each of the GAPDH primers, and 1 unit of
Taq DNA polymerase (Perkin-Elmer). The cycle numbers that
showed linear growth of product were initially determined for each PCR
product by analyzing a 10-µl sample from multiple identical
amplification reactions (Fig. 2A
and data not shown). In the
case of DR4 and KILLER/DR5, 23 cycles were chosen; for TRID and TRUNDD,
24 cycles were chosen; and in the case of GAPDH, 18 cycles were chosen.
During PCR, 10 µl of the reaction were remove at the indicated cycle
numbers. PCR conditions were as follows: 1 cycle, 5 min/95°C; 23 or
24 cycles, 30 s/95°C, 30 s/55°C (for DR4, KILLER/DR5, and TRUNDD),
52°C (for TRID), or 30 s/72°C. Nondenaturing PAGE (7%) was
performed, and the gel was fixed, dried, and autoradiographed. Band
intensities were quantitated by using a Phosphorimager Storm 840
(Molecular Dynamics, Sunnyvale, CA).
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Site-directed Mutagenesis and Sequencing.
Site-directed mutagenesis was performed using a Quick change
site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to
the manufacturer’s instructions. To change a base in the death domain
region of DR4 (A to G at nucleotide 1322 of DR4), plasmids that
contained either the full-length DR4 (f/DR4 (A) in pCEP4, Invitrogen,
Carlsbad, CA) or the cytoplasmic domain of DR4 (CD/DR4 (A) in
pcDNA3.1-Myc, His; Invitrogen, Carlsbad, CA) were used as templates.
The sequences of the primer pairs used for changing the base were as
follows: DR4DDMUT F,
5'-GGAAGAGAGACATGCAAGAGAGAAGATTCAGGA-CC-3'; DR4DD MUT R,
5'-GGTCCTGAATCTTCTCTCTTGCATGTCTCTCTTCC-3'. The sequences of
the mutagenized plasmids were confirmed. Sequencing of expression
plasmids was performed using a T7 DNA sequencing kit (United States
Biochemicals, Cleveland, OH) according to the manufacturer’s
instructions.
The mutagenized f/DR4 or CD/DR4 was used for transfection into SW480 colon cancer cells as previously described (30) . After 24 h of transfection, cell lysates were prepared from each transfectant followed by Western immunostaining for confirmation of expression after mutagenesis.
Evaluation of Cell Death Induced by Transfected DR4.
For cell death evaluation, cotransfection of the CMV-ß-gal marker
gene and the DR4 mutant constructs generated was performed as
previously described (31)
. Briefly, 1 x
105 of SW480 cells were plated per well in
24-well plates and transfected with 2 µg of the corresponding
parental vectors, f/DR4 (A), CD/DR4 (A), f/DR4 (G), or CD/DR4 (G), with
CMV-ß-gal at 10% of the total amount of DNA. At 24 or 48 h
later, cells were fixed and stained with
5-bromo-4-chloro-3-indolyl-ß-galactopyranoside to quantify the
number of blue cells. To determine whether polymorphic DR4 has a
dominant-negative effect on cell killing, SW480 cells were transfected
with variable ratios of CD/DR4 (A) to CD/DR4 (G), f/DR4 (A) to CD/DR4
(G), or f/DR4 (A) to f/DR4 (G) (4:1, 1:1, and 1:4) along with
CMV-ß-gal.
Abs and Western Blot Analysis.
Western blot analysis was carried out as previously described
(32)
. Blotted membranes were immunostained with anti-PARP
(1:2000; Boehringer Mannheim, Mannheim, Germany), anti-caspase 3
(E-8, 1:500; Santa Cruz Biotechnologies, Inc., Santa Cruz, CA),
anti-caspase 7 (1:500; PharMingen, San Diego, CA), anti-caspase 8
(C-20, 1:500; Santa Cruz Biotechnologies, Inc.), anti-caspase 9
(1:500; IIMGENEX, San Diego, CA), anti-caspase 10 (N-19, 1:500; Santa
Cruz Biotechnologies, Inc.), anti-caspase 2 (H-19, 1:500; Santa Cruz
Biotechnologies, Inc.), anti-DR4 (1:500, PharMingen), anti-DR5 (1:500;
IMGENEX,) anti-FLIP (1:500; IMGENEX), anti-Myc (9E10, 1:500; Santa Cruz
Biotechnologies, Inc.), or antiactin (I-19, 1:200; Santa Cruz
Biotechnologies, Inc.).
Statistical Analysis.
The statistical correlation between the expression level of TRAIL death
receptors and TRAIL-mediated apoptosis was performed using regression
analysis and the correlation between the expression of FLIP and TRAIL
sensitivity, or the expression of DR4 and TRAIL sensitivity was
performed using the 2 test.
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Results |
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and B).
In contrast, cancer cells showed a variable response to TRAIL (Fig. 1)
.
HCT116, H460, PA1, SKBr3, and SW480 were sensitive to TRAIL. TRAIL
sensitivity was defined as a <75% cell viability at 16 h after
TRAIL treatment is measured by the TRAIL MTT assay. A875, FADU, J82,
and SKOV3 cells were found to be resistant to TRAIL. Human
Papillomavirus E6-expressing HCT116, H460, and PA1 cells were
relatively more resistant to TRAIL than the neocounterparts (Fig. 1A)
. Long-term (7 days) TRAIL treatment of cell lines (Fig. 1B)
showed nearly the same result as the short-term (16 h)
MTT assay results. Based on the observations from the long-term TRAIL
treatment assay, certain fractions of cells showed resistance to TRAIL,
although the majority of the cells were killed by TRAIL treatment.
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Correlation between TRAIL Receptor Expression and TRAIL
Sensitivity.
To determine whether there is any correlation between TRAIL sensitivity
and the expression level of TRAIL receptors, a semiquantitative RT-PCR
assay was performed (Fig. 2)
. The number
of PCR cycles required for linear amplification and detection was
initially determined for each death receptor (Fig. 2A)
.
KILLER/DR5 was expressed in all cell lines tested (Fig. 2, B and C)
, and its mRNA expression level did not correlate with
TRAIL sensitivity (Fig. 3B)
.
In contrast, the expression level of DR4 varied among different cell
lines (Fig. 2B)
. For example, in normal fibroblast cells,
DR4 expression was very low or not detectable (Fig. 2B
,
Lanes 1 and 2). Cancer cell lines except J82 and
SKOV3 that expressed DR4 were sensitive to TRAIL regardless of p53
status (Fig. 1
, Fig. 2B
, and Fig. 3A
; see below).
PA1, A875, and FADU cells did not express detectable DR4 protein (Fig. 2B
, Lanes 5, 6, and 9). DR4 protein
was highly expressed in HCT116, H460, and SW480 cells (DR4 in Fig. 4
, Lanes 3, 4, and
7), and they were the most sensitive cell lines to TRAIL
(Fig. 1, A and B)
. The antiapoptotic TRAIL
receptors, TRID and TRUNDD, were also expressed in cancer cells. TRID
was expressed in all of the cell lines except PA1 cells, whereas TRUNDD
was not expressed in H460, A875, SKBr3, and FADU cell lines (Fig. 2B,
Lanes 3, 6, 7, and 9). The high
expression of TRID or TRUNDD in the normal cell lines HS27 or WI38 is
consistent with previous results implicating high decoy receptor
expression as a mechanism of TRAIL resistance. However, neither TRID
nor TRUNDD levels adequately explain the observed patterns of TRAIL
sensitivity in the panel of cancer cells (Fig. 3, D-F)
. The
presence of DR4 alone (r = 0.769; P =
0.006) or DR4 and KILLER/DR5 (r = 0.786,
P = 0.004) appeared to correlate better with TRAIL
sensitivity of cancer cells than the expression of decoy receptors
(Fig. 3, A and C)
.
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,
Lanes 1, 2, 8, 10, and 11) but only in one (PA1)
of five TRAIL-sensitive cell lines (FLIP in Fig. 4
, Lane 5).
These results suggest that high expression of FLIP may be the another
important determinant of TRAIL resistance (2;
P < 0.05).
K441R Polymorphism Found in the Death Domain of DR4.
Contrary to our expectation that DR4-expressing cells should be
sensitive to TRAIL, J82 and SKOV3 were resistant to TRAIL treatment.
Previously, there was a report indicating that Fas carrying a mutation
in the death domain region could act as a dominant-negative inhibitor
of Fas-induced cell killing (25)
. To investigate whether
there is a DNA sequence change in the death domain of DR4 in J82 and
SKOV3 cells, RT-PCR and DNA sequencing was performed. Sequencing
results showed that there is an A-to-G alteration in nucleotide 1322 of
DR4 both in SKOV3 and J82 cells (Fig. 5A
and data not shown). This
A-to-G transition resulted in the conversion of the amino acid lysine
(codon 441) to arginine. To determine whether this alteration is
present in normal populations, genomic DNA was isolated from total
blood drawn from 10 normal healthy volunteers, and PCR cycle sequencing
was performed. The results revealed that 2 (donor 1 and 10) of 10
(20%) normal individuals have the base change (Fig. 5B)
,
and thus, we refer to the alteration as a polymorphism. The
polymorphism was found in donors 1 and 10, and SKOV3 was heterozygous
in all cases
.
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and
B). Upon transfection, we found that polymorphic DR4 was
less effective in cell killing than its wild-type counterpart (Fig. 6, C and D)
. In addition, polymorphic DR4 showed an
inhibitory effect toward cell killing by wild-type DR4. A potent
dominant-negative effect of the K441R polymorphism was observed when
the cytoplasmic DR4 (CD/DR4) was expressed. The CD/DR4 (G) rather than
f/DR4 (G) showed a nearly complete inhibition of DR4-mediated cell
killing (Fig. 6D)
.
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Cell Killing by Combination of Doxorubicin and TRAIL in
TRAIL-resistant Cell Lines.
Normal cells such as HS27 and WI38 are resistant to TRAIL in part due
to a low or undetectable expression of DR4, a high expression level of
decoy receptors, and a high expression level of FLIP (Fig. 2
and Fig. 4
). However, when these cells were treated with the combination of
doxorubicin and TRAIL, viability was dramatically reduced (Fig. 7A)
and PARP cleavage became
evident (Fig. 7B)
. Western immunostaining (Fig. 7C)
showed that there was a significant induction of
KILLER/DR5 protein expression. This induction of KILLER/DR5 by
doxorubicin may sensitize normal cells to TRAIL-mediated cell killing.
These results suggest that an increase in the ratio of expression
between proapoptotic and antiapoptotic molecules may reset the
responsiveness of the cells from resistant to sensitive. There was no
change in the level of DR4 or FLIP expression after doxorubicin
treatment (Fig. 7C)
.
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Lanes 4, 8, 12, and 16).
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and morphological data not shown).
In doxorubicin-sensitive FADU cells, caspase 8 was activated by
doxorubicin treatment alone (caspase 8 in Fig. 8C
, Lane 7).
Caspase 9 was also activated by doxorubicin treatment alone in FADU
cells (caspase 9 in
, Lane 7). Unexpectedly, however,
although there was activation of caspases 8 and 9 ("initiator"
caspases) in doxorubicin-treated FADU cells, we did not observe
complete procaspase 3 ("executioner" caspase) depletion (caspase 3
in
, Lane 7). In the doxorubicin-resistant cell lines
(A875, J832, and SKOV3), caspase activation was not observed after
exposure to either doxorubicin alone or TRAIL alone (Fig. 8C)
. Interestingly, caspases 8, 9, and 3 became activated
after exposure to the combination of doxorubicin and TRAIL (caspases 8,
9, and 3 in Fig. 8C,
Lanes 4, 12, and
16). In contrast to TRAIL-resistant cancer cells, cleavage
of caspases 8, 9, and 3 was observed after TRAIL treatment of the
TRAIL-sensitive HCT116 colon cancer cell line (Fig. 8A)
.
When HCT116 was treated with TRAIL, PARP cleavage was evident by
4 h after TRAIL addition, and caspases 8, 9, 3, and 7 became
activated at approximately the same time point (4 h after the TRAIL
addition; Fig. 8B
).
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Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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or Fas have not been successful in clinical trials when
administered systemically because of toxicity (3
, 4)
.
Our results provide novel basic information relevant to TRAIL therapy
of cancer in the following respects. First, we report that TRAIL
resistance is mainly determined by the expression of its proapoptotic
death receptors, especially DR4 (r = 0.769,
P = 0.006). In fact, cell lines that were resistant to
TRAIL were found to have a relatively low or undetectable expression
level of DR4. Normal cell lines, such as HS27 and WI38, which are
resistant to TRAIL, have extremely low expression of DR4 mRNA or
protein (Fig. 2B
, Fig. 3A
, and Fig. 4
), and a
subgroup of TRAIL-resistant cells also have low or undetectable DR4
expression (Fig. 2B
and Fig. 4
). For DR4 expression alone, a
2 analysis revealed that this parameter is a
highly significant predictor of TRAIL sensitivity when expression is
high versus low or undetectable (P < 0.01).
For the 2 analysis, high expression was
defined as DR4/GAPDH > 50 as shown in Fig. 2C
. It is
important to note that mRNA levels do not always correlate with protein
levels and that the strength of the correlation between DR4 expression
and TRAIL sensitivity (Fig. 2
and Fig. 3
) might be stronger or weaker
if the measured DR4 protein levels (Fig. 4)
were actually quantitated.
The expression of KILLER/DR5, however, does not correlate well with
TRAIL sensitivity (Fig. 2
and Fig. 3B)
. Our observation is
supported by a recent report that TRAIL sensitivity in melanoma cells
correlates well with the expression level of DR4 (24)
.
Contrary to our observation, J82 and SKOV3 expressed DR4 (Fig. 2B
and Fig. 4
) but were resistant to TRAIL treatment. A
previous report that mutation in the death domain region of Fas can act
as in a dominant-negative fashion in cell killing (25)
prompted us to examine the death domain region of DR4 in J82 and SKOV3
cells. Indeed, J82 and SKOV3 have an A-to-G alteration at codon 441 in
the death domain region of DR4 (Fig. 5A)
. However, that
change is also found in 20% (2 of 10) of a normal population and thus,
we refer to the DR4 K441R alteration as a polymorphism. Polymorphic DR4
acted in a dominant-negative manner in DR4-mediated cell killing (Fig. 6, C and D)
. We make no claim about any disease
susceptibility associated with the K441R polymorphism in the DR4 gene.
However, the presence of the K441R DR4 polymorphism in cancers may
reduce their sensitivity to TRAIL, at least in vitro.
It is important to note the differences observed when full-length
versus cytoplasmic domain expression constructs were used to
express DR4. In particular, Fig. 6, C
and D
demonstrates that the cytoplasmic domain of DR4 does not itself induce
cell death when it contains 441R. In addition, this variant of the
cytoplasmic is capable of completely inhibiting death induced by the
441K allele. However, full-length DR4 containing the K441R
mutation does not share these properties. Instead, full-length DR4
containing the 441R allele induces apoptosis in 
50% of transfected
cells and poorly inhibits killing by the full-length 441K allele (Fig. 6D, right)
. These results suggest that the polymorphic 441R
allele may contribute but cannot alone explain the observed resistance
to TRAIL in certain cancer cell lines (J82 and SKOV3). These cell lines
express somewhat increased levels of FLIP (Fig. 4)
, which may also
contribute to their resistance to TRAIL (see below).
Second, the inhibitor of caspase activation FLIP may confer resistance
to TRAIL at a point downstream of the death receptors. We found that
83% (five of six cell lines) of TRAIL-resistant cell lines showed a
detectable expression of FLIP, whereas only one of five (20%)
TRAIL-sensitive lines expressed FLIP (Fig. 4
;
2; P < 0.05). However, the
fact that FLIP-expressing PA1 cells are sensitive to TRAIL suggests
that even in the presence of FLIP, cells can be killed if there is
enough of an input signal for inducing apoptosis.
We measured the expression level of five genes (DR4, KILLER/DR5, TRID,
TRUNDD, and FLIP) and tested for correlations with TRAIL sensitivity.
The expression of two of the parameters (DR4 and FLIP) appeared to
independently correlate with TRAIL sensitivity. From the regression
analysis shown in Fig. 3
, the P value for the DR4
correlation with TRAIL sensitivity is 0.006 (see legend of Fig. 3
).
Thus, we would have had to test 167 variables to reach the 0.006 level
of significance at random for DR4 due to the effect of multiple
testing. Moreover, the design of our study was hypothesis driven, with
a biological basis giving a reasonable pretest probability of certain
correlations. For example, we tested biologically plausible
determinants of TRAIL sensitivity. One of the concerns with multiple
correlations arises when one tests a very large number of
variables (without a hypothesis), such as in a questionnaire with
several hundred questions or perhaps a query of an expression of
several thousand genes on a DNA microarray chip, and then develops the
hypothesis based on any observed correlations at the P < 0.05 level. Of course, if one tests enough variables, there
is a random chance that a few will appear to be significant but will
actually be meaningless. Thus, because we believed that correcting for
multiple testing artifacts would not significantly alter our
Ps or conclusions, we have not corrected our calculations
for the effects of multiple comparisons. Thus, there is a small chance
that our analysis may be limited by the effects of multiple
comparisons, and it remains to be seen if others will find a similar
significance of DR4 and FLIP expression levels using larger sample
sizes and testing fewer variables.
Third, the targeted destruction of p53 to generate otherwise isogenic
cancer cell lines revealed that TRAIL sensitivity could be modulated
somewhat by p53 (Fig. 1)
. This is a preliminary observation that
requires further investigation. It is clear from our data that
wild-type p53 is not required for the apoptotic response to TRAIL.
Fourth, the combination of doxorubicin and TRAIL can kill
TRAIL-resistant cancer cells, although each treatment alone cannot
effectively kill the cells. The mechanism(s) of this additive killing
is not clear yet. We have ruled out changes in the expression level of
death receptors or FLIP as a basis for enhanced cell killing by
doxorubicin plus TRAIL (data not shown). The fact that FADU cells show
caspase 8 and 9 activation upon doxorubicin treatment suggests that the
caspase activation axis from caspase 8 through Bcl2 inhibitory protein
(Bid) to caspase 9 might be intact in FADU cells but not in other
TRAIL-resistant cell lines (Fig. 8C)
. As recently reported
(22)
and observed in our experiments, doxorubicin and
TRAIL could activate caspases in augmenting the killing effect.
However, although TRAIL resistance can be overcome by combined
treatment with doxorubicin, careful consideration should be given to
the dose of doxorubicin given the observed sensitization of normal
cells to TRAIL-mediated apoptosis (Fig. 7)
.
Fifth, among TRAIL-sensitive cancer cells, a certain fraction appears
to be resistant to TRAIL-mediated killing (Fig. 1B)
. A
recent report also showed that subclones of TRAIL-sensitive cancer
cells display a variable response to TRAIL, although the expression
level of TRAIL death receptors or FLIP was not changed
(24)
. We do not know the underlying mechanism of this
TRAIL resistance yet.
Our findings suggest that although TRAIL may be useful as a therapeutic agent in cancer, particular attention to molecular determinants of sensitivity needs to be considered to optimize such therapy. TRAIL does not appear to have harmful effects toward normal cells and can kill cancer cells irrespective of p53 status if wild-type DR4 is expressed on their cell surface. Our results also indicate that doxorubicin can sensitize cells to TRAIL-mediated cell killing in vitro, thereby raising hopes that such a strategy may be useful in cancer therapy.
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FOOTNOTES |
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1 Supported in part by NIH Grants CA75138-01 and
CA75454-01. 
2 To whom requests for reprints should be
addressed, at the Laboratory of Molecular Oncology and Cell Cycle
Regulation, Howard Hughes Medical Institute, University of Pennsylvania
School of Medicine, 415 Curie Boulevard, CRB 437A, Philadelphia, PA
19104. Fax: (215) 573-9139.
3 The abbreviations used are: TRAIL, tumor
necrosis factor-related apoptosis-inducing ligand; Ab, antibody; TNF,
tumor necrosis factor; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; RT-PCR, reverse
transcription-PCR; TRUNDD, TRAIL decoy receptor containing a truncated
death domain; TRID, TRAIL decoy receptor lacking an intracellular
domain; KILLER/DR5, p53-regulated proapoptotic KILLER/death receptor 5;
FLIP, FLICE inhibitory protein; PARP, poly ADP-ribose polymerase; FADD,
FAS-associated death domain protein; CMV-ß-gal, cytomegalovirus
ß-galactosidase; mAb, monoclonal Ab.
Received 12/15/98; revised 10/29/99; accepted 10/29/99.
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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B and protects against TRAIL-mediated apoptosis, yet retains an incomplete death domain. Immunity, 7: 813-820, 1997.[CrossRef][Medline]
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