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Immunology |
The Huntsman Cancer Institute, Division of Molecular Pharmacology, Salt Lake City, Utah 84112
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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protein as the search query. We have termed this
protein TNF-related death ligand-1 (TRDL-1). Traditional cDNA
library screening identified two additional splice variants designated
TRDL-1ß and TRDL-1
that differed from TRDL-1 by the deletion of
two small regions within the protein coding region. TRDL-1 is
identical in sequence to the recently described molecule, APRIL,
that may induce cell proliferation. We found, however, that purified,
FLAG-tagged TRDL-1 caused Jurkat cell death with kinetics that
paralleled FasL. In vitro binding experiments
demonstrated that TRDL-1 coprecipitated Fas and HVEM and suggested
TRDL-1 as an alternate ligand for these receptors. TRDL-1 localized
to chromosome 17p13.3 and its expression was widespread in normal
tissues. Examination of 48 tumor samples revealed high levels of TRDL-1
expression in several tumors, including those from the gastrointestinal
tract. Expression of TRDL-1 in COS-1 cells confirmed membrane
association of TRDL-1, typical of TNF family members. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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including
lymphotoxin (3)
, lymphotoxin ß (4)
,
CD40L (5)
, CD30L (6)
, CD27L (7)
,
OX40L (8)
, 4–1BBL (9)
, FasL
(10)
, TRAIL/APO-2 L (11
, 12)
, TL-1
(13)
, TRANCE/RANKL (14
, 15)
, LIGHT
(16)
, TWEAK (17)
, and APRIL
(18)
. These ligands are type II membrane-associated
proteins (except lymphotoxin ) that share primary structure
similarities that are confined to their COOH-terminal regions
(19)
. This ligand group interacts with a growing family of
target transmembrane receptors that are defined by cysteine-rich
extracellular domains (2)
. In addition, several members of
this receptor family, including TNF receptor 1, Fas, DR3, DR4, and DR5,
contain a region of homology termed a "death domain," which couples
receptor activation to the apoptotic cellular machinery
(20, 21, 22, 23, 24, 25, 26)
. The number of receptors contained within this
family currently exceeds the number of putative ligands and suggests
the existence of previously uncharacterized ligands. Identification of
the cognate ligand for each receptor is necessary before a complete
understanding of their roles in disease can be fully appreciated. The importance of this cytokine family to immune system function is highlighted by phenotypic alterations associated with gene knockout experiments and endogenous mutations in mice. Mutations disrupting FasL or its counterpart receptor cause lymphadenopathy and autoimmune disorders (27, 28, 29, 30, 31) . In another example, CD40 mutations lead to hyperimmunoglobulin M phenotypes, suggesting an essential role for the CD40 pathway in B-cell affinity maturation and isotype switching (32) . Inactivation of the TNF receptor type 1 pathway through knockout strategies generates mice that are highly sensitive to certain microbial infections, suggesting an important host defense role for TNF (33 , 34) . Lastly, inactivation of the murine lymphotoxin gene causes loss of peripheral lymph nodes (35) .
Diverse roles for these cytokines are likely to extend beyond strict
regulation of immune system function and into areas such as tumor
development. A number of studies have demonstrated high levels of FasL
expression in human tumors. Primary astrocytic brain tumors, colonic
adenocarcinomas, and metastases of human colonic adenocarcinomas all
show increased levels of FasL expression as compared with control
tissues (36, 37, 38)
. Overexpression of these ligands could
contribute to tumor development from two perspectives: (a)
overexpression of death-inducing ligands, such as FasL, may provide a
defense that protects tumor cells by killing intervening host immune
cells (39, 40, 41, 42)
; and (b) expression of TNF
family ligands in tumors could create a chronic inflammatory condition
that renders tumor cells resistant to immune destruction and confounds
chemotherapeutic approaches that rely on tumor cell apoptosis. Evidence
for this resistance can be seen in the observation that TNF fails to
kill many types of cancer cells. Recent evidence suggests that TNF
undermines its own killing powers by activating nuclear factor-
B, a
key molecule that can block the apoptosis pathway (43
, 44)
. Disruption of this protective mechanism may, therefore,
sensitize cells to chemotherapeutic intervention.
Current interventional strategies targeting TNF pathways are challenged by a lack of understanding of how these pathways are regulated and dysregulated in disease. Furthermore, an incomplete roster of ligands and receptors adds to the complexity of selecting rational points for intervention. With this in mind, we have identified and examined the biological activity of a novel death-inducing ligand that is related to TNF and that we have termed TRDL-1. This ligand was identified previously as APRIL, a putative cell proliferation-inducing ligand (18) . In contrast to this previous report, we found that APRIL/TRDL-1 stimulated Jurkat cell death and that APRIL/TRDL-1 binds to existing members of the TNF receptor family including, FAS and HVEM.
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MATERIALS AND METHODS |
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TRDL-1 Cloning and Sequence Determination
Clones corresponding to potential TNF- matches were obtained from
Genome Systems, Inc. IMAGE clone 727332 (TRDL-1) was used to screen
a human leukocyte cDNA library (Clontech Laboratories, Inc.). After an
initial titering of the library, a total of 500,000 plaques were
lifted in duplicate onto nylon filters. Phage DNA was denatured with
the following wash protocol: 2x SSC for 1 min; 1.5
M NaCl, 0.5 N NaOH for 2 min; 1.5 M
NaCl, 0.5 M Tris-HCl (pH 8.0) for 2 min; and 2x SSC for 2
min. Filters were air dried and UV cross-linked using a Stratalinker
(Stratagene). Filters were prehybridized in 6x SSC, 1x Denhardt’s
solution, 100 µg/ml denatured salmon sperm DNA, and 0.5% SDS for
2.0 h at 65°C to block nonspecific DNA binding sites.
The probe was labeled with [-32P]dCTP
(Amersham) to a specific activity of greater than 1 x 109 cpm/µg of DNA using an RTS RadPrime DNA
labeling System (Life Technologies, Inc.). Unincorporated nucleotides
were removed by passage over Chroma-Spin-30 (Clontech Laboratories,
Inc.) size exclusion columns. Hybridization was performed in the same
buffer as prehybridization for 12–14 h at 65°C with a probe
concentration of 1 x 106 cpm/ml.
After hybridization, unbound probe was removed by washing filters twice
for 20 min each in 1x SSC, 0.1% SDS at room temperature, followed by
two washes for 20 min each in 0.1x SSC, 0.1% SDS at 65°C. Filters
were exposed to X-ray film for 12–14 h, with intensifying screens at
-70°C. Plaques that showed duplicate hybridization signals were
selected into an appropriate phage buffer and titered. Each positive
plaque was then subjected to two additional rounds of hybridization
screening to completely isolate the positive plaques from other species
and to eliminate false positives. DNA was isolated, and the insert size
was determined from plaques resulting from tertiary screens. Clones
were sequenced by the Huntsman Cancer Institute Core DNA Sequencing
Facility using ABI Prism BigDye Terminators and cycle sequencing with
Taq FS DNA polymerase. DNA sequence was collected and analyzed on an
ABI Prism 377 automated DNA sequencer (PE Applied Biosystems Division,
Foster City, CA).
Northern and Dot Blot Analyses
Multiple human tissue mRNA blots I and II, a human cancer cell line
blot, and a human RNA dot blot were purchased from Clontech
Laboratories, Inc. A multiple tumor mRNA blot was obtained from
Biochain Institute, Inc. The membranes were prehybridized in ExpressHyb
(Clontech Laboratories, Inc.) for 2.5 h at 65°C and then
hybridized with a random-primed, 32P-labeled
TRDL-1 probe for 2.5 h at 65°C. Unbound probe was removed by
washing twice at room temperature in 2x SSC/0.05% SDS and twice at
50°C in 0.1x SSC/0.1% SDS. To quantify hybridization signals, blots
were exposed to a PhosphorImager (Molecular Dynamics) screen for 6–24
h.
Expression of FLAG/TRDL-1 in Cos-1 Cells
Full-length TRDL-1 was cloned into pFLAG-CMV-2 (Kodak) for
expression in Cos-1 cells. Transient transfections were performed using
Lipofectamine reagent (Life Technologies, Inc.), according to the
manufacturer’s protocol. Briefly, 10 µg of DNA were combined with 50
µl of Lipofectamine in 5.0 ml of serum-free Optimem (Life
Technologies, Inc.). After DNA/Lipofectamine complex formation, the
transfection mixture was added to 80% confluent Cos-1 cells in a
100-mm culture dish. After 4 h incubation under standard
conditions, 5 ml of DMEM/10% fetal bovine serum were added, and cells
were allowed to recover overnight. On the following day, the medium was
removed and replaced with fresh, complete culture medium. TRDL-1
expression was monitored at 
48 h after transfection by analyzing
cellular lysates for FLAG epitope.
Membrane Solubilization and Purification of FLAG/TRDL-1
Cos-1 cells transiently transfected with full-length TRDL-1 were
incubated for 1 h at 4°C in hypotonic lysis buffer [50
mM Tris (pH 7.4), 50 mM NaCl, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml
aprotinin]. Cell lysates were centrifuged at 500 x g for 7 min to remove the nuclei. The supernatant was then
spun at 3000 x g for 15 min to pellet the
membranes. Membrane fractions were washed twice with cold PBS and
solubilized in a buffer consisting of 1% Triton-X-100, 50
mM Tris (pH 7.4), 150 mM
NaCl, and 10% glycerol overnight at 4°C. Particulate was removed by
centrifugation at 12,000 x g for 10 min.
Protein samples were loaded onto an anti-FLAG (M2) affinity resin
(Kodak) at a flow rate of 0.2 ml/min. Columns were washed with 5 volume
equivalents of PBS/0.1% Tween 20 and 5 volume equivalents of 50
mM Tris (pH 7.4), 150 mM
NaCl, 1 mM CaCl2, and 10%
glycerol. FLAG-TRDL-1 was eluted off the column by competition with 0.1
M FLAG peptide (Sigma). Fractions were assayed
for the presence of TRDL-1 by immunoblotting. Fractions containing
TRDL-1 were pooled and concentrated via a Centricon-10 (Amicon). Purity
of the eluate was determined by SDS-PAGE and silver staining (Bio-Rad).
Generation of TRDL-1 Antibodies
A synthetic 15-amino acid peptide corresponding to residues 121–135 of
TRDL-1 was used to generate rabbit polyclonal antibodies. The
peptide (CPINATSKDDSDVTE) was conjugated to keyhole limpet hemocyanin,
and rabbits were immunized by Quality Controlled Biochemicals, Inc.
Test bleeds were titered by ELISA and tested by Western blot against a
whole-cell lysate from Cos-1 cells expressing recombinant FLAG/TRDL-1.
Sera showing good cross-reactivity were affinity purified to produce 5
mg of purified anti-TRDL-1 antibody.
Immunoblot Analysis
Samples were assayed for protein concentration using Bio-Rad protein
assay kit. Protein samples (5–10 µg) were analyzed by SDS-PAGE on
10% tricine gels (Novex) and electrotransferred to PVDF membranes
(Gelman Sciences). Blots were incubated for 1 h at room
temperature in blocking buffer (5% powdered milk/PBS/0.1% Tween 20)
and then with a monoclonal antibody to the FLAG epitope (Kodak) diluted
1:10,000 in blocking buffer for 1 h at room temp or overnight at
4°C. Blots were washed 3x in PBS/0.1% Tween and then probed with a
secondary antibody conjugated to horseradish peroxidase. After washing
3x in PBS/0.1% Tween, FLAG protein was detected using a
chemiluminescent substrate (DuPont NEN), according to manufacturer’s
instructions.
Receptor Binding Assays
All steps were carried out at 4°C. Sepharose beads coated with M2
antibody (anti-FLAG; Kodak) were blocked in 1% BSA for 1 h. After
blocking, lysates (see above) from TRDL-1-transfected Cos-1 cells and
untransfected cells were incubated with the blocked beads for 1 h.
The beads were washed to remove detergent and unbound protein and then
incubated with 250 ng of recombinant purified receptors fused to Fc
(R&D Systems) for 2 h at 4°C in buffer containing 25
mM HEPES (pH 7.5), 50 mM NaCl, 1 mM
CaCl2, and 1% BSA. After incubation, unbound
proteins were removed by four consecutive washes with binding buffer
lacking BSA. The remaining proteins were eluted by boiling in SDS-PAGE
sample buffer and were loaded onto 10% SDS-PAGE gels. After
electrophoresis, proteins were transferred to PVDF membranes, and the
blots were probed using an antibody specific for the Fc portion of the
receptor fusions. Signals were quantified by scanning densitometry.
Cell Death Assays
Jurkat cells were resuspended in RPMI/0.5% FBS at 5 x 104 cells/well in a 96-well culture dish and
treated with vehicle alone, 50 ng/ml FasL (Upstate Biotechnologies,
Inc.), 50 ng/ml TNF- (R&D Systems), or 1.0 µg/ml purified TRDL-1.
After 15 h of incubation at 37°C, cells were visualized by light
microscopy and photographed. YO-PRO-1 dye (Molecular Probes) was then
added at a final concentration of 1 µM. After an
additional 3 h at 37°C, cells were analyzed on a fluorescence
plate reader (Cytofluor II; Perceptive Biosystems) at excitation and
emission wavelengths of 485 and 530 nm, respectively.
To determine a concentration curve, log dilutions of TRDL-1 ranging from 1000 to 0.1 ng/ml were added to cells as described above. After 48 h, cell numbers were quantified using a Coulter counter.
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RESULTS |
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, TRDL-1ß, and
TRDL-15 and the BLAST (basic local alignment
search tool) algorithm to query the dbEST database
(45)
. Two sequences, AA405973 and AA477087, displayed
interesting homology with TNF- in regions where TNF- is also
similar to FasL, TRAIL, and other members of the TNF cytokine family
(data not shown; Refs. 46
and 47
). Ten
additional expressed sequence tags with sequences that overlapped
AA405973 and AA477087 defined a cluster that continued to share
sequence similarity with the TNF family. Nucleotide sequence analysis
of each clone confirmed our initial, computer-based alignments and
strengthened the assignment of this cDNA into the TNF family. Of the
clones examined, clone 727332 contained 1260 bp and a single,
continuous open reading frame that predicted a 250-amino acid protein
with a molecular mass of 27,432 daltons (Fig. 1).
Primary alignment of the predicted open reading frame with TNF-
extended the critical homology regions, and the clone was renamed
TRDL-1 to identify it as a new member of the TNF family.
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) that appeared to differ in organization
from TRDL-1. Sequence determination of clones representing each of
these variants confirmed their identity with TRDL-1 and revealed
potential alternative splicing products (Fig. 1)
. TRDL-1ß was a
1684-bp clone that contained an open reading frame that predicted a
protein of 234 amino acids and a molecular mass of 25,677 daltons. By
comparison, TRDL-1 was composed of 1,607 bp and predicted a
257-amino acid protein with a molecular mass of 27,057 daltons.
TRDL-1ß and TRDL-1 each contained a polyadenylation signal and a
poly(A)+ tail. Fig. 1
shows the aligned, predicted amino acid sequences
of TRDL-1, TRDL-1ß, and TRDL-1. TRDL-1ß was identical to
TRDL-1, with the exception of a 48-bp deletion that removed 16 amino
acids corresponding to residues 113 through 128 of TRDL-1. TRDL-1
was also largely identical to TRDL-1 but contained a 3' deletion of
181 bp that results in substitution of the four COOH-terminal residues
of TRDL-1 with a single leucine residue.
Tissue Distribution
We next examined the tissue distribution of TRDL-1 by performing
Northern analyses on mRNA from various human tissues. Hybridization
with TRDL-1 identified mRNA species in most tissues. Highest levels
of expression were seen in peripheral blood leukocytes, with
intermediate levels of expression noted in pancreas, colon, small
intestine, prostate, and ovary. There was little expression in skeletal
muscle, thymus, or testis. Although most tissues expressed a 1.8-kb
mRNA, peripheral blood leukocytes and lung expressed a message of 1.6
kb. Dot blot analysis of 34 additional tissues showed low levels of
TRDL-1 expression in most tissues (data not shown).
To assess a potential role for TRDL-1 in tumor development, we next
examined its expression in cancer cell lines (Fig. 2A)
. Strong expression of TRDL-1 (1.8-kb species) was observed
in mRNA from HeLa and SW480 cells. Expression was undetectable in the
other cancer cell lines examined and suggested the potential for cell
type-specific regulation of TRDL-1 in cancers. We extended the cancer
cell-based observations by surveying 48 human tumor biopsies compared
with normal tissues for the expression of TRDL-1 (Fig. 2, B and C)
. A number of tumors showed higher levels of TRDL-1
expression as compared with adjacent normal tissues. Of note were
gastrointestinal tumors, including rectum, duodenum, colon, stomach,
and esophagus.
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in COS-1 cells. To accomplish
this, FLAG/TRDL-1 was transiently expressed in COS-1 cells, and its
distribution in cytosolic or membrane fractions was examined by Western
analysis using antibodies specific for the FLAG epitope or TRDL-1. Fig. 3A
shows the majority of FLAG and TRDL-1 cross reactivities in
the membrane fractions of cells transfected with TRDL-1.
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from 1 x 108 COS-1 cells that had been transfected with
pFLAG/TRDL-1. After a 96-h incubation, cell membranes were prepared
by hypotonic lysis and differential centrifugation. Portions of
membrane preparations were then combined with a solubilization buffer
containing 10% glycerol and 1% Triton X-100. Greater than 90% of the
TRDL-1 protein was solubilized under these conditions. Solubilized
TRDL-1 was then passed over the anti-FLAG M2 affinity gel to facilitate
purification. The FLAG-tagged TRDL-1 protein was completely removed by
the affinity matrix and was successfully eluted with 0.1 M
FLAG peptide. Fig. 3B
shows a silver-stained gel of purified
FLAG/TRDL-1 and cross-reactivity of the purified protein with
antibodies specific for the FLAG epitope and TRDL-1. We estimate a
purification of FLAG/TRDL-1 at >80%.
Association with TNF Family Receptors in Vitro
Because TRDL-1 showed structural similarities to TNF family members, we
reasoned that it may bind to TNF family receptors. We, therefore,
examined the ability of TRDL-1 to interact with purified TNF family
receptors in vitro. This was performed by capturing
FLAG/TRDL-1 onto anti-FLAG affinity beads and combining these beads
with purified TNF receptor 1/Fc, FAS/Fc, HVEM/Fc, TR1/Fc, TR2/Fc, and
TR3/Fc fusion proteins. Precipitation of each receptor was assessed by
immunoblotting for the Fc portion of the receptor fusions. Fig. 4A
shows FLAG/TRDL-1-mediated precipitation of known TNF
family receptors. Although there was detectable binding to all
receptors above background, densitometric scanning revealed strongest
binding of FLAG/TRDL-1 to Fas (10.1-fold above background) and HVEM
(11.2-fold above background) as compared with control beads and with
the other receptors (Fig. 4B)
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could bind to TNF family
receptors in vitro, we next assessed whether it stimulated
apoptosis in Jurkat cells. Fig. 5A
shows the morphological consequences of exposing Jurkat
cells to vehicle alone, 50 ng/ml FasL, 50 ng/ml TNF-, or 1.0 µg/ml
TRDL-1 for 16 h. FasL and TRDL-1 each caused clumps of Jurkat
cells to disperse, as compared with vehicle-treated cells or cells
treated with TNF-. The dispersal of Jurkat cells parallels cell
death markers like caspase activation and annexin staining and suggests
activation of similar pathways by TRDL-1 and FasL. Further inspection
of the FasL and TRDL-1-treated cells showed typical apoptotic markers,
including nuclear condensation, membrane blebbing, and cell shrinkage.
In contrast, vehicle- and TNF-treated cells showed few of these
characteristics at 16 h after treatment. In these same cells,
viability was assessed by YO-PRO-1 dye uptake. Fig. 5B
shows
stimulation of YO-PRO-1 dye uptake in cells treated with FasL, TRDL-1
for 12 h but not in cells treated with vehicle and TNF-.
Finally, depletion of TRDL-1 by immunoprecipitation using the
TRDL-1-specific antisera eliminated the death-inducing capability and
confirmed TRDL-1 as the active protein in the preparation (data not
shown).
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illustrates the concentration-dependent decline in cell number in
response to TRDL-1. Replotting of this data revealed that
TRDL-1-induced death is saturable (Fig. 6B)
. Maximal
activity of TRDL-1 was seen at concentrations >100 ng/ml, with TRDL-1
showing an ED50 of 50 ng/ml.
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DISCUSSION |
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. The rapid rate of death in response to TRDL-1 (within
12 h) is similar to death induced by FasL and suggests a potential
interaction of TRDL-1 with Fas on Jurkat cells; and (b) we saw in
vitro binding of TRDL-1 to several receptors of the TNF family.
TRDL-1-coated beads precipitated purified Fas/fc, TNF receptor 1/fc,
HVEM/fc, TR1/fc, and TR2/fc fusion proteins. Of these, TRDL-1
preferentially bound Fas/fc and HVEM/fc. This suggests TRDL-1 as an
alternate ligand for activating Fas. Although we saw binding of TRDL-1
to HVEM, HVEM lacks an intracellular death domain characteristic of
apoptosis-inducing receptors (48)
. It is unlikely,
therefore, that HVEM mediates the death-inducing effects of TRDL-1 in
Jurkat cells. Future studies will be necessary to define the cognate
receptor for TRDL-1 and to determine under what conditions TRDL-1 may
activate Fas or HVEM.
While the manuscript was in preparation, Hahne et al.
(18)
reported the sequence of APRIL, a molecule that is
identical in sequence to TRDL-1. Using a strategy similar to ours,
these authors concluded that APRIL is a new member of the TNF family
based on structural analyses. In contrast to our data, however, these
authors reported that APRIL induced cell proliferation and that this
proliferative signal could promote tumor cell growth. They propose that
this activity is mediated through a novel receptor in that APRIL was
incapable of binding purified TNF family receptors in vitro.
Although reconciliation of the differences between our observation and those of Hahne et al. (18) will require additional experimentation, differences in strategies for production of bioactive APRIL/TRDL-1 protein may account for the discordant conclusions. APRIL was produced as a soluble protein that lacked 110 amino acids from its NH2 terminus (18) . After our initial identification of TRDL-1, we expressed and purified a soluble construct that lacked 53 amino acids from its NH2 terminus. This truncation removed the membrane-spanning region and allowed for more convenient purification. Addition of truncated TRDL-1, however, to a number of cell lines including Jurkat cells failed to elicit apoptotic responses, even at concentrations of TRDL-1 as high as 1.0 µg/ml (data not shown). We, therefore, turned to production of full-length TRDL-1 and witnessed the appearance of death-inducing activity as reported herein.
The observed lack of activity of truncated TRDL-1 could result from the
inability of these molecules to form homotrimers. Trimerization of TNF
family members could be central to the ability of the molecules to
efficiently bind to and activate target receptors. Zhang et
al. (49)
have shown that residues near the
membrane-spanning helix of TNF- are critical to trimerization of
these molecules and biological activity. Further, Schneider et
al. (50)
demonstrated that the apoptotic activity of
soluble FasL was reduced 1000-fold as compared with the membrane bound
form of FasL. However, soluble FasL retained its ability to interact
with Fas, and restoration of its cytotoxic activity was achieved, both
in vitro and in vivo, with the addition of
cross-linking antibodies (50)
. This suggests that the
truncated form of FasL is not able to form trimers. Because we see both
structural and functional similarities between TRDL-1 and FasL, it is
possible that deletion of a significant portion of the APRIL/TRDL-1
NH2 terminus could alter its activity. It is also
possible that truncation of APRIL/TRDL-1 could alter receptor binding
specificity and, therefore, elicit different responses. It will be
critical to the final assignment of APRIL/TRDL-1 function to determine
the trimerization state and receptor binding capabilities of the
full-length molecule compared with various
NH2-terminal truncations.
TRDL-1 expression was fairly widespread in normal tissues and is
similar to that reported previously for TNF, TRAIL, and TWEAK
(11
, 17) . We saw the highest levels of expression in
peripheral blood leukocytes that also displayed a message size that was
unique as compared with other normal tissues. This unique message size
could arise from alternate splicing of TRDL-1 mRNA. The possibility of
alternate splicing was confirmed by our identification of TRDL-1ß and
TRDL-1. These two cDNAs were obtained by screening a leukocyte cDNA
library with TRDL-1. TRDL-1ß and TRDL-1 each contained specific
deletions as compared with TRDL-1 that would result in translation
of TRDL-1, TRDL-1ß, and TRDL-1 messages into three distinct
proteins. The size differences in mRNAs we saw by Northern analysis
correspond closely with size differences between TRDL-1/TRDL-1ß
and TRDL-1 cDNAs. Assessment of the functional consequences of
alternative splicing warrants further investigation.
We saw differential expression of TRDL-1 in tumor tissues that suggests a potential role for TRDL-1 in tumor development and maintenance. Our survey of 48 different tumors compared with adjacent normal tissues showed increased expression of TRDL-1 in a number of tumors, particularly those derived from the gastrointestinal tract. Tumors from rectum, duodenum, colon, stomach, and esophagus showed higher levels of TRDL-1 expression than those seen in adjacent normal tissues. This increased expression in tumors is similar to that seen for expression of FasL. Elevated levels of FasL have been observed in a variety of tumors, including those from the gastrointestinal tract (37 , 38) . The role of FasL in these tumors is unclear but it may provide a tumor defense against the host immune system or stimulate a chronic inflammatory condition (39, 40, 41 , 43 , 44) . The role for TRDL-1 in tumor development is uncertain but may parallel those proposed for FasL.
In summary, we are presenting data supporting a role for APRIL/TRDL-1 as a death-inducing ligand of the TNF family. The ability of full-length APRIL/TRDL-1 to kill Jurkat cells and its overexpression in human tumor samples may offer a molecular mechanism for tumor cells to evade elimination by host immune cells. The potential for TRDL-1 to contribute to tumor development warrants further study that will focus on demonstrating TRDL-1 activity in tumor specimens.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by a grant from the Huntsman Cancer
Foundation. We thank the DNA Sequencing Facility at the University of
Utah, supported in part by National Cancer Institute Grant
5p30CA42014. 
2 These authors contributed equally to this
work.
3 To whom requests for reprints should be
addressed, at Huntsman Cancer Institute, Division of Molecular
Pharmacology, 2000 East North Campus Drive, Room 564, Salt Lake City,
UT 84112. E-mail: david.jones{at}hci.utah.edu
4 The abbreviations used are: TNF, tumor necrosis
factor; APRIL, a proliferation-inducing ligand; TRDL, tumor necrosis
factor-related death ligand; HVEM, herpes virus entry mediator; PVDF,
polyvinylidene difluoride; FasL, Fas ligand.
5 TRDL-1, TRDL-1ß, and TRDL-1 have the
GenBank accession numbers AF114011, AF114012, and AF114013,
respectively.
Received 6/16/99. Accepted 12/ 7/99.
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S. Shulga-Morskaya, M. Dobles, M. E. Walsh, L. G. Ng, F. MacKay, S. P. Rao, S. L. Kalled, and M. L. Scott B Cell-Activating Factor Belonging to the TNF Family Acts through Separate Receptors to Support B Cell Survival and T Cell-Independent Antibody Formation J. Immunol., August 15, 2004; 173(4): 2331 - 2341. [Abstract] [Full Text] [PDF]
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 D. R. Patel, H. J. A. Wallweber, J. Yin, S. K. Shriver, S. A. Marsters, N. C. Gordon, M. A. Starovasnik, and R. F. Kelley Engineering an APRIL-specific B Cell Maturation Antigen J. Biol. Chem., April 16, 2004; 279(16): 16727 - 16735. [Abstract] [Full Text] [PDF]
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 E. Castigli, S. Scott, F. Dedeoglu, P. Bryce, H. Jabara, A. K. Bhan, E. Mizoguchi, and R. S. Geha Impaired IgA class switching in APRIL-deficient mice PNAS, March 16, 2004; 101(11): 3903 - 3908. [Abstract] [Full Text] [PDF]
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 E. Varfolomeev, F. Kischkel, F. Martin, D. Seshasayee, H. Wang, D. Lawrence, C. Olsson, L. Tom, S. Erickson, D. French, et al. APRIL-Deficient Mice Have Normal Immune System Development Mol. Cell. Biol., February 1, 2004; 24(3): 997 - 1006. [Abstract] [Full Text] [PDF]
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 C. Kern, J.-F. Cornuel, C. Billard, R. Tang, D. Rouillard, V. Stenou, T. Defrance, F. Ajchenbaum-Cymbalista, P.-Y. Simonin, S. Feldblum, et al. Involvement of BAFF and APRIL in the resistance to apoptosis of B-CLL through an autocrine pathway Blood, January 15, 2004; 103(2): 679 - 688. [Abstract] [Full Text] [PDF]
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A. L Gavin, D. Ait-Azzouzene, C. F. Ware, and D. Nemazee {Delta}BAFF, an Alternate Splice Isoform That Regulates Receptor Binding and Biopresentation of the B Cell Survival Cytokine, BAFF J. Biol. Chem., October 3, 2003; 278(40): 38220 - 38228. [Abstract] [Full Text] [PDF]
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 T. Miyahara, T. Kikuchi, M. Akimoto, T. Kurokawa, H. Shibuki, and N. Yoshimura Gene Microarray Analysis of Experimental Glaucomatous Retina from Cynomologous Monkey Invest. Ophthalmol. Vis. Sci., October 1, 2003; 44(10): 4347 - 4356. [Abstract] [Full Text] [PDF]
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T. Koyama, H. Tsukamoto, K. Masumoto, D. Himeji, K. Hayashi, M. Harada, and T. Horiuchi A novel polymorphism of the human APRIL gene is associated with systemic lupus erythematosus Rheumatology, August 1, 2003; 42(8): 980 - 985. [Abstract] [Full Text] [PDF]
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A. Craxton, D. Magaletti, E. J. Ryan, and E. A. Clark Macrophage- and dendritic cell--dependent regulation of human B-cell proliferation requires the TNF family ligand BAFF Blood, June 1, 2003; 101(11): 4464 - 4471. [Abstract] [Full Text] [PDF]
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V. Roschke, S. Sosnovtseva, C. D. Ward, J. S. Hong, R. Smith, V. Albert, W. Stohl, K. P. Baker, S. Ullrich, B. Nardelli, et al. BLyS and APRIL Form Biologically Active Heterotrimers That Are Expressed in Patients with Systemic Immune-Based Rheumatic Diseases J. Immunol., October 15, 2002; 169(8): 4314 - 4321. [Abstract] [Full Text] [PDF]
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B. Huard, P. Schneider, D. Mauri, J. Tschopp, and L. E. French T Cell Costimulation by the TNF Ligand BAFF J. Immunol., December 1, 2001; 167(11): 6225 - 6231. [Abstract] [Full Text] [PDF]
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S. Xu and K.-P. Lam B-Cell Maturation Protein, Which Binds the Tumor Necrosis Factor Family Members BAFF and APRIL, Is Dispensable for Humoral Immune Responses Mol. Cell. Biol., June 15, 2001; 21(12): 4067 - 4074. [Abstract] [Full Text]
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 C. F. Ware APRIL and BAFF Connect Autoimmunity and Cancer J. Exp. Med., December 4, 2000; 192(11): F35 - F38. [Full Text] [PDF]
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P. Rennert, P. Schneider, T. G. Cachero, J. Thompson, L. Trabach, S. Hertig, N. Holler, F. Qian, C. Mullen, K. Strauch, et al. A Soluble Form of B Cell Maturation Antigen, a Receptor for the Tumor Necrosis Factor Family Member APRIL, Inhibits Tumor Cell Growth J. Exp. Med., December 4, 2000; 192(11): 1677 - 1684. [Abstract] [Full Text] [PDF]
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Y. Wu, D. Bressette, J. A. Carrell, T. Kaufman, P. Feng, K. Taylor, Y. Gan, Y. H. Cho, A. D. Garcia, E. Gollatz, et al. Tumor Necrosis Factor (TNF) Receptor Superfamily Member TACI Is a High Affinity Receptor for TNF Family Members APRIL and BLyS J. Biol. Chem., November 3, 2000; 275(45): 35478 - 35485. [Abstract] [Full Text] [PDF]
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