Laboratory of Molecular Biology, Flanders Interuniversity Institute for Biotechnology and University of Ghent, B-9000 Ghent, Belgium
The various biological activities of tumor necrosis factor (TNF) are mediated by two receptors, one
of 55 kD (TNF-R55) and one of 75 kD (TNF-R75). Although the phenotypic and molecular responses elicited
by TNF in different cell types are fairly well characterized, the signaling pathways leading to them are so far only partly understood. To further unravel these processes, we focused on TNF-R55, which is responsible
for mediating most of the known TNF effects. Since
several studies have demonstrated the importance of
receptor clustering and consequently of close association of the intracellular domains for signaling, we addressed the question of whether clustering of the intracellular domains of TNF-R55 (TNF-R55i) needs to
occur in structural association with the inner side of the
cell membrane, where many signaling mediators are
known to reside. Therefore, we investigated whether induced intracellular clustering of only TNF-R55i
would be sufficient to initiate and generate a full TNF
response, without the need for a full-length receptor
molecule or a transmembrane region. Our results provide clear evidence that inducible forced trimerization of either TNF-R55i or only the death domain elicits an
efficient TNF response, comprising activation of the
nuclear factor B, induction of interleukin-6, and cell
killing.
Tumor necrosis factor (TNF)1 is a multifunctional cytokine mainly produced by activated macrophages.
It was originally identified by its antitumoral activity in vivo and its selective cytotoxic/cytostatic action on
some transformed cell lines (Carswell et al., 1975 TNF-R55 is the receptor responsible for mediating most
of the effects exerted by TNF, including activation of the
nuclear factor TNF signaling is initiated by receptor clustering, either
by the trivalent ligand TNF or by cross-linking monoclonal
antibodies (Engelmann et al., 1990 From these and other results, we may conclude that
TNF-R55 clustering is necessary and sufficient for initiating the signaling cascade. As many receptor-initiated signaling pathways are directly linked to membrane-associated components (such as G proteins, Grb, Sos, PKC, and
Ras), we inquired whether the membrane juxtaposition of
TNF-R55i is also important and necessary for signaling.
Therefore, we developed a system for the controlled expression in the cytoplasm of L929 cells of a trimerized
hTNF-R55i complex, obtained by fusion of the intracellular receptor domain to the cytosolic, trimeric chloramphenicol acetyltransferase (CAT) protein. Our results demonstrate that, while the induced intracellular expression of
hTNF-R55i as such in L929sA cells could not initiate TNF
effects, induced expression of the trimeric construct could
indeed elicit effectively the various TNF-signaling pathways leading to NF- Cytokines, Antibodies, and Reagents
mTNF and hTNF were produced in Escherichia coli and purified to at
least 99% homogeneity in our laboratory. The specific activity of the
preparations was tested in a standard cytotoxicity assay. Recombinant human interferon (hIFN)- htr-13, a murine monoclonal antibody directed against hTNF-R55i, was
a generous gift from Dr. M. Brockhaus (Hoffmann-La Roche, Basel, Switzerland; Brockhaus et al., 1990 1-deoxy[dichloroacetyl-1-14C] chloramphenicol (55 mCi/mmol) and
[ LiCl was purchased from Sigma Chemical Co. A 1 M stock solution was
made in water and filter-sterilized. Dilutions were freshly made in culture
medium. Actinomycin D (ActD) was purchased from Boehringer Mannheim (Mannheim, FRG); a 1 mg/ml stock solution was made in ethanol.
Cbz-Ile-Glu(O-t-Bu)-Ala-leucinal (PSI) was from Peptide Institute (Osaka, Japan).
Cells
TNF-sensitive murine fibrosarcoma L929sA cells (Vanhaesebroeck et al.,
1991 Plasmids
pUChTNFR55, containing the hTNF-R55 cDNA sequence, was a generous gift from Dr. W. Lesslauer (Hoffmann-La Roche; Loetscher et al.,
1990 hTNF-R55 Mutagenesis
To convert the hTNF-R55i cDNA sequence to a separate coding entity,
we engineered an ATG initiation codon (see Fig. 1 A). Part of the cDNA
sequence of hTNF-R55
Construction of a Bacterial hTNF-R55i
Expression Vector
The hTNF-R55i cDNA sequence was isolated from pMahTNFR55fm using the newly created NcoI site and a COOH-terminal BamHI site and introduced into the multicloning site of pET11d (Rosenberg et al., 1987 Bacterial Expression and Detection of the
hTNF-R55i Polypeptide
The bacterial pEThTNFR55i vector was transformed into E. coli K12
strain 2580/pT7pol26. This strain contains the T7 RNA polymerase gene on
the compatible plasmid pT7pol26, under control of the isopropyl These bacteria were also induced in the presence of [35S]methionine,
with and without rifampicin in the growth medium. E. coli 2580/pT7pol26
bacteria (Mertens et al., 1995 Construction of an Inducible Eukaryotic hTNF-R55i
Expression Vector pMxhTNFR55i
First, a universal eukaryotic expression plasmid was made by introducing
an SV-40 DNA fragment, containing the small t-splice sequences and polyadenylation signals, into the multicloning site following the Mx promoter
of pSP64Mx, thus creating pSP64MxpA. Subsequently, the cDNA coding
for hTNF-R55i was isolated from pEThTNFR55i as an XbaI fragment and
cloned into the same site of the multicloning site of pSP64MxpA. In this
way, a vector for hIFN- Construction of Eukaryotic CAT (Fusion)
Expression Plasmids
The cDNA coding for CAT was transferred from pBLCAT5 as an SauAI
fragment into the multicloning site of pSP64MxpA, creating pMxCAT.
The cDNA of hTNF-R55i was recovered from pMxhTNFR55i and introduced into pMxCAT, in-frame at the 3 Stable Transfection of Eukaryotic Expression Plasmids
into L929sA Cells
The plasmids carrying the cDNA sequences for hTNF-R55i and the different CAT fusions were transfected into L929sA cells using the calcium-phosphate precipitation method (Graham and van der Eb, 1973) and the
neomycin gene, coded for by pSV2neo, as a selection marker. G418-resistant clones were picked up after 10-12 d of selection with 400 µg/ml of
G418.
Cytotoxicity Tests
L929sA cells were seeded in 96-microtiter plates at 15,000 cells/well on
day Alternatively, cells were seeded in bacterial-grade Petri dishes (Sterilin, Feltham, UK), under which conditions they no longer adhered to the
plastic surface and remained in suspension. At appropriate time intervals
after induction, samples were subjected to a propidium iodide (PI) exclusion assay as described (Grooten et al., 1993 Northern Blot Analysis
Induction of mRNA expression in parental or transfected L929sA cells
was assessed by Northern blot analysis. In brief, cells were grown in 9-cm
dishes. At confluency, they were treated with 1,000 IU/ml hIFN- IL-6 Assay
IL-6, secreted in the medium of the expressing cells, was quantified by its
growth-stimulating activity on 7TD1 cells, measured by colorimetric determination of the hexosaminidase levels (Landegren, 1984 NF- Activation of NF- CAT-ELISA
To quantify the amount of CAT protein present in cell lysates, a sandwich
immunosorbent assay was optimized in our laboratory (Vanden Berghe et
al., 1993) based on the original protocol (Boehringer Mannheim).
CAT Enzyme Assay
Confluent monolayers of parental or transfected L929sA cells were
treated with 1,000 IU/ml hIFN- Bacterial Expression of hTNF-R55i cDNA
We converted the cDNA sequence corresponding to hTNF-R55i into an independently translatable cistron by creating
an ATG codon at the beginning of the hTNF-R55i-coding
sequence, following a previously described strategy (Nakamaye and Eckstein, 1986 The hTNF-R55i cDNA gene was recloned in the bacterial expression vector pET11d (Rosenberg et al., 1987
Induced Expression of hTNF-R55i in the Cytoplasm of
L929sA Cells
In pMxhTNFR55i, the coding sequence for hTNF-R55i is
under control of the murine Mx promoter, which is selectively inducible by type I IFN and which has been shown
to result in tightly controlled heterologous gene expression in VERO cells (Lleonart et al., 1990 Murine fibrosarcoma L929sA cells were transfected with
pMxhTNFR55i using the calcium phosphate procedure.
Several G418-resistant clones were isolated and tested for
TNF effects after induction with 1,000 IU/ml hIFN- Since TNF has been shown to induce IL-6 expression in
L929s cells (Defilippi et al., 1987 We concluded that expression of hTNF-R55i in L929sA
cells under control of the Mx promoter was not sufficient
to initiate TNF signalization. In all likelihood, the low induced expression level in the clones obtained did not allow
us to demonstrate the presence of the protein and furthermore did not lead to an effective aggregation or clustering
required for signaling. The possibility that higher expressing clones were counter-selected because of leakage expression in the noninduced state cannot be excluded.
Induced Expression of Trimerized hTNF-R55i Elicits
Cytotoxicity in L929sA Cells
In view of the negative results with solitary expression,
we developed an inducible system designed to force direct
clustering of the hTNF-R55i domains. This consisted of the
hTNF-R55i domain fused to CAT, a cytosolic, trimeric protein (Leslie, 1990 Both constructs were transfected in L929sA cells. From
repeated experiments, it soon became apparent that after selection, the number of clones transfected with
pMxCAThTNFR55i was considerably lower than that obtained with pMxCAT(stop)hTNFR55i. This observation indicated a strong counter-selection pressure in the transfection experiment producing CAT-R55i molecules (see
below). Several G418-resistant clones were examined for
cell killing after addition of 1,000 IU/ml hIFN-
IFN- In summary, the hIFN- Induced Expression of Trimerized hTNF-R55i Results
in NF- Since TNF activates NF-
To test for IL-6 gene expression, medium of hIFN- Taken together, expression of trimerized hTNF-R55i
molecules in L929sA cells elicited a full TNF response, including cell killing and gene induction effects.
Transfection with pMxCAThTNFR55i Is Subject to
Severe Counter-Selection, and as a Consequence,
Trimerized hTNF-R55i Is Only Expressed in Low
Quantities in Surviving Clones
To demonstrate the presence of induced proteins in transfected cells, various approaches were tried. Although the
CAT protein produced by induced pMxCAT(stop)-
hTNFR55i-transfected cl 15 could easily be demonstrated
and quantified by CAT-ELISA (30 to 50 ng/mg protein)
or visualized by immunofluorescence, CAT-R55i fusion protein of induced pMxCAThTNFR55i-transfected cl 2 could not be revealed unambiguously with any of these
techniques. Furthermore, attempts to immunoprecipitate
the fusion protein also failed, although the CAT protein in
the control cells could easily be demonstrated.
Therefore, mRNA expression levels of the previously
selected, inducible CAT-R55i-expressing cl 2 and CAT-expressing cl 15 were checked after induction with hIFN- Yet, we were able to demonstrate and to quantify the
CAT-R55i fusion protein in the transfected cells by making use of the enzymatic activity of the CAT protein in
front of the receptor domain. The results of this assay, performed on total protein extracts, clearly demonstrated inducible enzymatic CAT activity in CAT-R55i cl 2 and of
course in cl 15 (Fig. 8 A). In agreement with the very different levels of expression revealed by Northern blot analysis, the level of enzymatic activity in cl 2 cells was much
lower than the quantities in cl 15 cells. This again indicates that only low CAT-R55i-expressing cells survived the
transfection/selection procedure, i.e., presumably only those
with no leakage of the Mx promoter.
To ascertain that the enzymatic activity observed in
CAT-R55i cl 2 extract corresponded to intact CAT-R55i
protein and, for example, not to a proteolytically derived
CAT protein, we immunoprecipitated lysates from cl 2, cl 15, or parental L929sA cells with htr-13, an antibody
that specifically recognizes hTNF-R55i; these immunoprecipitates were used for an enzymatic CAT assay. CAT
enzymatic activity was clearly demonstrated with immunoprecipitates from cl 2 after induction, while immunoprecipitates from cl 15 or parental L929sA cells were negative (Fig. 8 A). These results unambiguously demonstrate
that the enzymatic activity derived from cl 2 was effectively generated by an intact CAT-R55i fusion protein.
The possibility that additional mechanisms may have
contributed to the occurrence of only limited amounts of
the lethal fusion protein, even after induction with hIFN- Induced Expression of Trimerized CAT-DD Fusion
Protein Results in IL-6 Synthesis and Cytotoxicity in
L929s Cells
Since the DD of hTNF-R55 was reported to be sufficient
for induction of cytotoxicity, we investigated whether
forced trimerization of only the DD of hTNF-R55 by fusion to the CAT gene (CAT-DD) would also induce cytotoxicity. A first expression vector pMxCAThTNFDD was constructed such that the sequence coding for the DD
was linked almost directly to the COOH terminus of the
CAT gene (Fig. 1 B). This construct was transfected into
L929sA cells, in parallel with the pMxCAThTNFR55i and
the pMxCAT(stop)hTNFR55i constructs as a reference.
Comparison of the number of antibiotic-resistant clones
obtained indicated again an evident counter-selection for cells transfected with pMxCAThTNFR55i, but not for cells
transfected with pMxCAThTNFDD or pMxCAT(stop)-
hTNFR55i. This was a first indication that the CAT-DD
protein was not as active in cell killing as was the CAT-R55i fusion protein. Indeed, after induction with hIFN-
From these results it is clear that the membrane-proximal part of hTNF-R55i has no essential signaling function
in cytotoxicity, NF- Many hormone and cytokine receptors are clustered by
their respective ligands as an essential and initial step for
subsequent intracellular signaling. In the case of TNF signaling, receptor clustering is also crucial. This was first
demonstrated by several groups using agonistic antibodies
against the extracellular domain of the TNF receptors to
mimic TNF actions (Engelmann et al., 1990 Whether trimerization is really necessary, or whether
dimerization might be sufficient to initiate the signaling
cascade, is still a controversial issue and is not answered by
our results. Several groups reported effective signaling
with chimeric TNF receptors containing an extracellular
domain known to form bimolecular complexes after binding of the appropriate ligand, e.g., PDGF or EPO (Adam
et al., 1995 We have obtained inducible, forced trimerization by fusion of hTNF-R55i to the bacterial CAT protein. CAT is a
trimeric enzyme of identical subunits of ~25 kD, oriented
in a parallel fashion, such that their COOH-terminal
ends are ~55 Å apart. Linkage of hTNF-R55i to the
CAT COOH-terminal end automatically places the former
domains in close proximity to each other, which allows clustering and subsequent signaling. In this respect, it is remarkable that the fusion protein CAT-DD, where the DD
is linked directly to the CAT COOH-terminal end, could
not elicit TNF effects. Considering the three-dimensional
structure of the CAT protein, this inability to initiate signaling may be explained by the insufficient accessibility of
the respective DDs to each other due to the spatial configuration of the trimeric CAT. Support for this notion is
given by the finding that the signaling capacity of the fusion protein could indeed be restored by adding extra
amino acids between the CAT and the DD polypeptide
chains, as is the case in the CATlinkDD fusion. This linker
allows a greater flexibility for the DD tails to interact, resulting in full signaling capacity.
Many receptor signaling systems that have been studied
in detail involve associated factors and/or signal transducers that are constitutively or conditionally associated with
the inner side of the cell membrane. This topologically
constrained, structural interaction between receptor and
signal transducer allows optimal functioning. However, in
the system here described, this is not the case, as the fusion
proteins are expressed as cytoplasmic proteins, and no obvious signals for membrane anchorage are known in the
intracellular receptor domain. Therefore, the fact that signaling by the trimeric CAT-R55i is as effective as ligand-induced hTNF-R55 clustering means that the exact juxtaposition of the hTNF-R55i trimer relative to the membrane
is not essential for function. It is known that the CAT protein is soluble in the cytosol; however, because of the low
expression level of CAT-R55i, we still have to ascertain
the localization of the latter polypeptide in the cell. We
can of course not exclude the possibility that associating factors link it to the membrane, but even so it still would
be in a structurally quite different constellation as compared to the natural, TNF-induced TNF-R55 clusters.
Remarkably, since CATlinkDD fusion proteins expressed in L929sA are as efficient in gene induction and
cell killing as the total intracellular receptor domain fusion
proteins, the intracellular region upstream of the DD does
not seem to play a role in signal transduction leading to
IL-6 gene induction and cytotoxicity. Nevertheless, besides a purely structural role in creating the proper setting
for the DDs to interact as evidenced in this study, the upstream region has been shown to be implicated in activation of neutral sphingomyelinase (Wiegmann et al., 1994 Our results clearly show that minute amounts of CAT-R55i are fatal to a sensitive cell. This was already clear
from the strong counter-selection observed during the
transfection/selection procedure with pMxCAThTNFR55i
as compared to pMxCAT(stop)hTNFR55i. Moreover, the
expression level of the fusion product in the G418-resistant clones obtained was so low that we could not demonstrate
the generated protein by standard techniques, but it still
was sufficient to cause biological effects, including NF- In conclusion, by using the strictly controlled Mx promoter system, we were able to obtain cells producing a
structural equivalent of the activated hTNF-R55. After induction, synthesis of very low amounts of the trimeric
hTNF-R55i was sufficient to elicit typical TNF responses,
including gene induction and cytotoxicity. Both responses
were also obtained after forced clustering of only the receptor DDs (CATlinkDD). Induction of IL-6 requires at
least two signaling pathways, one involving activation of
NF-). Since
then, it has become clear that TNF exerts a wide variety of
biological activities on both transformed and untransformed cells. TNF is an important mediator in inflammation and cellular immune responses and is also believed to
play a key role in the pathology of many diseases (for reviews see Vassalli, 1992
; Fiers, 1995
; Aggarwal and Natarajan, 1996
). In its biologically active form, TNF is a trimeric protein consisting of three identical 17-kD subunits
(Wingfield et al., 1987
; Jones et al., 1989
). Two TNF receptors with molecular masses of 55 kD (TNF-R55) and 75 kD (TNF-R75) have been identified, cloned, and characterized for several species (for review see Vandenabeele et
al., 1995
). They are present on nearly all cell types and
both belong to a growing TNF receptor superfamily, which
covers more than 10 receptor proteins (for review see
Smith et al., 1994
). Nearly all family members are type I
membrane proteins that show sequence homology only in
their extracellular domains, consisting of cysteine-rich
pseudorepeats. Their respective cytoplasmic domains,
however, generally lack sequence homology among themselves (except for a small homologous region present in
the intracellular parts of TNF-R55 [TNF-R55i] and Fas
antigen), which suggests major differences in signaling mechanisms. Moreover, these receptors do not contain sequences suggestive of intrinsic catalytic activity.
B (NF-
B), gene induction, synthesis of
prostaglandins, and cytotoxicity. TNF-R55i lacks intrinsic
kinase activity and has no SH2 domain. TNF-R55i contains two functional domains: a COOH-terminal region of ~ 80 amino acids, called death domain (DD), which is responsible for NF-
B activation and signaling cytotoxicity,
and an NH2-terminal region involved in induction of NO
synthase (Tartaglia et al., 1993
) and activation of several kinases and phospholipases (Wiegmann et al., 1994
; Belka
et al., 1995
; Adam et al., 1996b
). A region homologous to
the DD is present in the intracellular part of the fas receptor (Itoh and Nagata, 1993
). Recently, genes coding for
several proteins that can associate with members of the
TNF receptor superfamily or are involved in the signaling
pathway have been cloned and their products characterized (Rothe et al., 1994
; Boldin et al., 1995a
, 1996; Chinnaiyan et al., 1995
; Hsu et al., 1995
; Stanger et al., 1995
;
Muzio et al., 1996
). The majority of these proteins are directly or indirectly associated with the DD of TNF-R55.
However, a number of proteins that associate with the
NH2-terminal, membrane-proximal part of TNF-R55 have
also been identified (Boldin et al., 1995b
; Song et al., 1995
;
Adam et al., 1996a
; Adam-Klages et al., 1996
).
; Espevik et al., 1990
). It
has been demonstrated that expression of human TNF-R55 (hTNF-R55) deletion mutants, lacking most of their
intracellular domain, in murine cells failed to elicit cytotoxicity after anti-hTNF-R55 antibody treatment. Moreover,
expression of these mutant human receptors suppressed endogenous murine TNF-R55 (mTNF-R55) signaling after TNF treatment, probably by formation of receptor
clusters combining murine functional with human truncated, nonfunctional TNF-R55 (Brakebusch et al., 1992
).
B activation, gene induction, and cytotoxicity.
Materials and Methods
(A/D), which is also active on murine cells, was a
generous gift from Dr. C. Weissmann (University of Zürich, Zürich, Switzerland); it had a specific antiviral activity of 7.9 × 107 U/mg as determined on murine cells in an L929 cell/VSV assay. mIFN-
was produced
in CHO cells and had a concentration of 105 U/ml.
). Purified polyclonal rabbit anti-CAT antibody, biotinylated anti-CAT antibody, streptavidin-alkaline phosphatase,
and pure CAT enzyme were purchased from 5 Prime
3 Prime, Inc.
(Boulder, CO). In CAT-ELISA, 1/1,000 dilutions of the commercial preparations were used.
32-P]dCTP were purchased from Amersham Life Science (Amersham,
UK). Acetyl coenzyme A, of which a 9 mM stock solution was prepared in
water, was from Sigma Chemical Co. (St. Louis, MO).
) were cultured in DME, supplemented with 5% heat-inactivated newborn calf serum and 5% heat-inactivated fetal calf serum, 100 U/ml
penicillin, 0.1 mg/ml streptomycin, and 0.03% l-glutamine.
). pSP64Mx contains the murine Mx promoter, including ~25 nucleotides downstream from the TATA box, and was donated by Dr. C. Weissmann (Hug et al., 1988
). pBLCAT5 contains the CAT gene derived
from Tn 9 from E. coli and was a generous gift from Dr. G. Schütz (Institute of Cell and Tumor Biology, Heidelberg, FRG; Luckow and Schütz,
1987
; Boshart et al., 1992
). pSV2neo contains the bacterial neomycin gene
used as a selection marker in transfection experiments (Southern and
Berg, 1982
). pMa58 is a phasmid vector for site-specific mutagenesis containing the replication origin of the single-stranded DNA phage f1 (Stanssens et al., 1989
). pET11d is a powerful bacterial expression vector containing the strong bacteriophage T7 promoter and strong T7 translation
signals (ribosome-binding site and first 11 codons of the T7 gene 10 protein; Rosenberg et al., 1987
).
coding for a small portion of the extracellular
domain, the transmembrane, and the total intracellular domain
was isolated from pUChTNFR55 as a PvuII fragment and inserted in the unique
SmaI site of the phasmid pMa58. Using an oligonucleotide-directed in
vitro mutagenesis system, based on a previous method (Nakamaye and
Eckstein, 1986
), an ATG codon was created at amino acid 201 (numbering
of the entire mature hTNF-R55 polypeptide based on Loetscher et al.,
1990
), i.e., three amino acids in front of the entire hTNF-R55i, as part of a
NcoI restriction site, useful for further recloning procedures (see Fig. 1 A).
The resulting plasmid was called pMahTNFR55fm.
Fig. 1.
cDNA sequence of CAT-R55i fusion constructs. (A)
cDNA sequence of wild-type and mutated hTNF-R55. Ile 201 was mutated to Met 201 to provide a potential initiation codon.
At the same time, an extra NcoI site was created. The last amino
acid of the transmembrane region of hTNF-R55 is believed to be
Leu 203 (Loetscher et al., 1990). (B) Schematic representation of
the different CAT fusion proteins. The numbering of the amino
acids is according to Loetscher et al. (1990)
. Gray boxes represent the DD of hTNF-R55.
[View Larger Version of this Image (28K GIF file)]
).
The resulting vector was called pEThTNFR55i.
-d-thiogalactopyranoside (IPTG)-inducible LacUV5 promoter (Mertens et al.,
1995
). Expression of the hTNF-R55i gene was induced by addition of
IPTG (1 mM final concentration) to a 1/100 dilution of a saturated bacterial culture.
), transformed with pEThTNFR55i, were
grown overnight at 28°C in Luria broth, supplemented with triacillin and
kanamycin. The next morning, the culture was diluted 50-fold in Luria
broth, further grown until an A600 of 0.5 was obtained, and induced with
IPTG (1 mM final concentration). After 45 min of growth at 20°C, the culture was split; rifampicin (200 µg/ml) was added to half of the culture. 45 min later, the medium from both cultures was replaced with minimal essential medium supplemented with IPTG. After 30 min, 10 µCi [35S]methionine was added; 4 h later, the bacteria were centrifuged and lysed in
1× Laemmli solution, and the proteins were separated on a 12% SDS gel.
For immunoprecipitation, metabolically labeled bacteria were centrifuged
and frozen. After resuspension in RIPA buffer, the bacteria were sonicated and cell debris was removed. htr-13 was added to the supernatant,
and the mixture was incubated overnight at 4°C. Protein G-Sepharose
beads (Pharmacia Biotech, Uppsala, Sweden) were added to bind the antibody-antigen complex. After 4 h, proteins retained on the beads were
dissolved in 1× Laemmli solution, boiled, and separated on a 12% SDS gel.
-inducible expression of hTNF-R55i in mammalian cells was created, which was named pMxhTNFR55i.
-end of the CAT-coding sequence,
but separated by an oligonucleotide coding for a flexible spacer peptide
Gly-Gly-Ser-Gly. In one construction, pMxCAThTNFR55i, the stop
codon of the CAT gene was omitted, which resulted in a translatable
fusion protein CAT-R55i. In a parallel construction, pMxCAT(stop)-
hTNFR55i, to be used as a control, the CAT termination codon was conserved. In this way, the two vectors are expected to result in almost identical mRNAs (except for three nucleotides) and totally different protein
products. pMxCAThTNFDD was constructed by exchanging the cDNA
of the total hTNF-R55i for the cDNA of the DD, the first amino acid of
the latter being Asp 326 (see Fig. 1 B). By the cloning procedure, the last
Gly from the spacer peptide was deleted and a Met was inserted in front
of the first amino acid of the DD. pMxCATlinkDD was derived from
pMxCAThTNFDD by inserting an oligonucleotide coding for an extra
15-amino acid linker (Gly4Ser)3 in front of the cDNA sequence of the DD
and after the cDNA of the linker Gly-Gly-Ser (see Fig. 1 B).
1 in 100 µl DME growth medium. At day 0, inductions were carried
out by adding the appropriate concentration of the agonists in 100 µl medium. After the indicated time period, cell survival was measured by 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining
(Tada et al., 1986
).
).
(A/D)
for the indicated time period. Total RNA was isolated with TRIzol reagent (Life Technologies, Paisley, UK), denatured in glyoxal buffer, and
separated on a 1.2% agarose gel containing 20 mM phosphate buffer, pH
7. RNA was fixed on Hybond-N+ membranes (Amersham Life Science)
using a standard capillary blotting procedure. Membranes were hybridized with an appropriate probe: hTNF-R55i, interleukin-6 (IL-6), or glyceraldehyde-3-phosphate dehydrogenase cDNA fragment, labeled with
[
-32P]dCTP using a Random Primed Labeling Kit (Boehringer Mannheim).
).
B Activity
B was measured in an electrophoretic mobility shift assay. Confluent monolayers of transfected L929sA cells were treated with
hIFN-
or mTNF for varying periods of time. Nuclear protein extracts and
binding reactions were performed as described previously (Patestos et al.,
1993
). The core site of the double-stranded oligonucleotide, used in the
binding reaction, was the murine IL-6-
B motif -GGGATTTTCC-. It was
32P-labeled using Klenow enzyme in a terminal fill-in reaction. After purification, 4,000-8,000 cpm of the freshly labeled oligonucleotide (corresponding to ~50-100 pg or 3-5 fmol) was used for the binding assay.
or left untreated. After 6 h, cells were
washed with PBS, pelleted, resuspended in 0.25 M Tris-HCl, pH 7.8, and
subjected to four freeze-thaw cycles by alternate transfer of the suspensions between a dry ice/ethanol bath and a 37°C water bath (5 min in
each). After 15 min of centrifugation at 4°C, supernatants were assayed
for enzymatic activity. For immunoprecipitation, htr-13 was added to the
cell lysates for 2 h, followed by a 2-h incubation with protein G-Sepharose
beads. The enzymatic assay was subsequently carried out with the beads. The assay mixture contained 1 µCi of [14C]1-deoxy-chloramphenicol, 20 µg
of protein extract or immunoprecipitated lysate, and 10 µl of 9 mM acetyl
coenzyme A in a total volume of 90 µl. Controls contained CAT enzyme
instead of cell extract. All reagents, except coenzyme A, were preincubated together for 5 min at 37°C. After equilibration had been reached at
this temperature, the reaction was started by adding coenzyme A. The reaction went on for 2.5 h at 37°C and was stopped with 1 ml of ice-cold
ethyl acetate, which was used to extract the chloramphenicol. The organic layer was dried and taken up in 20 µl ethyl acetate. 5 µl of this solution
was spotted on silica gel thin-layer plates and resolved with chloroform/
methanol (95:5, ascending). Visualization of the separated acetylated form
was obtained by incubation in a Storage Phosphor Screen and quantification with Image Quant software (Molecular Dynamics, Sunnyvale, CA).
Results
). In this way, an ATG codon
was obtained at position 201 (numbering of the entire mature hTNF-R55 according to Loetscher et al., 1990
), i.e.,
three amino acids before the presumed first amino acid of the intracellular domain (Fig. 1 A). At the same time, and
by an appropriate choice of the mutator oligonucleotide,
an extra restriction site (NcoI) was created that overlapped the ATG codon and facilitated further recloning
procedures.
).
Transformation in K12 strain 2580/pT7pol26 (Mertens
et al., 1995
) and subsequent induction with IPTG clearly
revealed the production of a polypeptide with the correct
size (30 kD) after Coomassie brilliant blue staining of an
SDS gel. The same result was obtained after metabolic labeling of induced bacteria in the absence or presence of
rifampicin (Fig. 2 a). The 30-kD protein expressed in Escherichia coli could be immunoprecipitated selectively
with htr-13, a monoclonal antibody specific for hTNF-R55i
(Fig. 2 b). Since these experiments demonstrated that the
recombinant hTNF-R55i cistron gave rise to a polypeptide of the correct size, it was isolated from the bacterial expression vector and recloned in a eukaryotic expression
vector, creating the plasmid pMxhTNFR55i.
Fig. 2.
Bacterial expression of hTNF-R55i. E. coli 2580/
pT7pol26 transformed with pEThTNFR55i were grown and induced as described in Materials and Methods. (a) Autoradiogram
of a serial dilution of metabolically labeled, induced bacteria in
the absence or presence of rifampicin. (b) Immunoprecipitation
of metabolically labeled bacteria. Noninduced extracts were
treated with 2 µg htr-13 (lane 1); induced extracts were treated
with 2 and 4 µg htr-13, respectively (lanes 2 and 3).
[View Larger Version of this Image (69K GIF file)]
).
.
Since L929sA cells are very sensitive to the cytotoxic effect of TNF, we first tested for the occurrence of cell killing. However, we could not observe cell death in any of
the selected clones, even after a long induction period (up to 72 h) with hIFN-
. Therefore, the integration of hTNF-R55i cDNA into the host genome and its transcription in
the corresponding mRNA were checked by Southern and
Northern analysis, respectively. The results revealed that
all of the clones tested had indeed taken up the cDNA and
expressed low levels of hTNF-R55i mRNA after induction
with hIFN-
. The presence of the corresponding protein, however, could not be demonstrated.
; Vandevoorde et al., 1991
),
we checked whether this activity could be observed after
induced expression of hTNF-R55i. The medium of hIFN-
-induced transfectants was tested in an IL-6 bioactivity
assay; no increase in IL-6 levels as compared to the background level of noninduced transfectants could be detected.
). The resulting construct pMxCAThTNFR55i
(where the hTNF-R55i cDNA is coupled in-frame via a
tetrapeptide linker to the coding sequence of CAT) and
the control vector pMxCAT (stop)-hTNFR55i (where the continuous reading frame of the fusion product is interrupted by the natural termination codon of the CAT gene;
Fig. 1 B) should yield almost identical mRNAs (except for
the three nucleotides of the stop codon) but should give
rise to totally different protein products, namely the fusion
protein CAT-R55i and CAT, respectively.
to the medium. In the pMxCAThTNFR55i-transfected cells, at least
50% of the clones tested died within 24 h after administration of hIFN-
, while the G418-resistant cells transfected with the analogous construct pMxCAT(stop)hTNFR55i
did not die, and no change in their growth or morphology
was observed. As opposed to our previous experiments
with the solitary intracellular domain, these results definitely demonstrate that after "trimerization" of hTNF-R55i, the signaling by TNF leading to cell killing could be
reproduced by the hIFN-
-inducible system. A particular
clone, CAT-R55i cl 2, was used for detailed characterization. The hIFN-
-induced cytotoxicity in this clone (Fig. 3,
B-D) can be compared with the noncytotoxic response of
hIFN-
on a CAT-expressing clone (Fig. 3, A-C). By following the cytotoxicity as a function of time, either by
staining of surviving cells with MTT (Fig. 4) or by PI uptake by dying cells (Fig. 5), we observed that 4 to 5 h after
administration of hIFN-
, cell death in CAT-R55i-expressing cl 2 became already apparent (Fig. 4 A), while the
same treatment on CAT cl 15 (Fig. 4 B) or parental
L929sA cells had no cytotoxic effect. In addition, Northern blot analysis revealed that mRNA of CAT(stop)R55i
and CAT-R55i became detectable 2 h after induction (Fig.
6 a). The much lower level of transcript in the latter cells as compared to the former will be discussed in a later section. The observed transcription/translation kinetics correlate well with the expression pattern of other Mx-controlled genes (Lleonart et al., 1990
). As a matter of fact,
the time kinetics of cell killing after treatment of CAT-R55i
cl 2 with hIFN-
are very comparable with the kinetics obtained after treatment of the same cells or parental L929sA
cells with TNF (Fig. 4, C and D). In addition, several other
treatments that augment or decrease cytotoxicity induced
by TNF in L929s cells were also examined in CAT-R55i cl 2 after hIFN-
treatment. The combined hIFN-
/LiCl treatment resulted in a synergistic enhancement of the cytotoxicity in CAT-R55i-expressing cells (Fig. 4 E), as seen with
a combined TNF/LiCl treatment of parental L929s cells
(Beyaert et al., 1989
). Treatment of CAT-R55i cells with
hIFN-
in combination with dexamethasone leads to partial reduction of the cytotoxicity (results not shown), as
does a combined TNF/dexamethasone treatment on parental L929s cells (Suffys et al., 1987
).
Fig. 3.
hIFN--induced cytotoxicity in cells expressing CAT and CAT-R55i. CAT cl 15 (A and C) and CAT-R55i cl 2 (B and D) were
treated with 1,000 IU/ml hIFN-
. Photographs were taken 8 h (A and B) or 24 h later (C and D). Bar, 100 µm.
[View Larger Version of this Image (122K GIF file)]
Fig. 4.
Kinetics of cell
death by trimerized TNF-R55i. Cytotoxicity on parental L929sA cells (D), CAT cl
15 (B), and CAT-R55i cl 2 (A, C, E, and F) was measured at different time
points, following different induction conditions, by MTT
conversion. Inductions were
carried out using the following concentrations: 1,000 IU/ ml hIFN- (
), 1,000 IU/ml
mIFN-
(
), 10 mM LiCl
(
), 10 mM LiCl + 1,000 IU/
ml hIFN-
(
), 1 µg/ml
ActD (
), 1 µg/ml ActD + 1,000 IU/ml hIFN-
(
), 500 IU/ml mTNF (×), or 500 IU/
ml hTNF (*). The percentage
of cell survival of treated
cells was compared to the untreated cell population (
).
[View Larger Version of this Image (29K GIF file)]
Fig. 5.
Kinetics of cell death by trimerized TNF-R55i as followed by PI uptake. CAT-R55i cl 2 (A) and CAT cl 15 (B) were
induced with 1,000 IU/ml hIFN- (
), and parental L929sA cells
(C) were induced with 2,000 IU/ml mTNF (
). At the indicated
time points, cell samples were taken, stained with PI, and analyzed by flow cytometry as described. PI uptake of treated cells
was compared to untreated cells (
).
[View Larger Version of this Image (11K GIF file)]
Fig. 6.
CAT-R55i mRNA and IL-6 mRNA expression levels.
Total RNA of cells from CAT cl 15 and CAT-R55i cl 2, treated
with 1,000 IU/ml hIFN- or untreated, was isolated at different
time points and used in Northern blot analysis as described. (a)
CAT(stop)R55i mRNA expression level of CAT cl 15 untreated
or treated with hIFN-
(left); expression level of CAT-R55i
mRNA of CAT-R55i cl 2 cells under the same conditions (right).
The 32P-labeled probe consisted of the hTNF-R55i cDNA fragment. The membrane was stripped and reprobed with a labeled
IL-6 cDNA fragment. (b) IL-6 mRNA levels in CAT cl 15 untreated or treated with hIFN-
(left); results with CAT-R55i cl 2 after the same treatments (right). (c) Control hybridization of
the same filter with glyceraldehyde-3-phosphate dehydrogenase
(GAPDH).
[View Larger Version of this Image (39K GIF file)]
induces many of the same effects as type I IFNs,
but cannot, for example, induce the Mx gene. Indeed,
mIFN-
could not mimic the effects induced by hIFN-
(Fig. 4 A), and this provides further proof that the expression of CAT-R55i, leading to cytotoxicity, was an
Mx promoter-driven effect (Fig. 4, A and B). Finally, the cytotoxicity after a combined treatment with hIFN-
and
ActD, as compared to an hIFN-
treatment alone, further
showed that cell killing depended on the expression of
CAT-R55i fusion protein (Fig. 4 F). In contrast, TNF/
ActD treatment of parental L929sA cells resulted in an
augmented cytotoxicity, most probably because protective
proteins cannot be synthesized.
-driven signal for cell killing is
entirely comparable, both in a qualitative and a quantitative way, to the cytotoxicity elicited by TNF itself. This
means that the intracellular expression of trimerized hTNF-R55i polypeptide is by itself sufficient for generating cytotoxic mechanisms within a cell, without the need for direct
linkage to the transmembrane domains of the TNF receptor.
B Activation and IL-6 Gene Expression
B and induces IL-6 expression in
L929s cells (Patestos et al., 1993
), we investigated whether
the induced expression of trimerized hTNF-R55i could
also elicit these TNF effects in CAT-R55i-transfected
clones. To check for NF-
B activation, gel-shift analysis
was performed as previously described (Patestos et al.,
1993
). Selected transfectants expressing either CAT-R55i
(cl 2) or CAT (cl 15) were induced with hIFN-
; cell lysates
were made after different periods of induction, ranging from 30 min to 4 h. Activated NF-
B complex started to
appear after 2.5 h and became prominent after 4 h of
hIFN-
treatment in cells expressing CAT-R55i, but not in
transfectants producing CAT protein (Fig. 7).
Fig. 7.
NF-B activation as measured by an electrophoretic
mobility shift assay. (a) NF-
B activation in CAT-R55i cl 2 after
treatment with 1,000 IU/ml mTNF (control) or hIFN-
for periods varying from 90 to 240 min. (b) Results of similar treatments
on CAT cl 15. The lower, constitutively binding band has recently
been characterized (Plaisance et al., 1997
).
[View Larger Version of this Image (54K GIF file)]
-
induced transfectants expressing CAT-R55i or CAT was
collected at several time points after induction and analyzed for IL-6 bioactivity. CAT-R55i-expressing cells
showed elevated IL-6 expression levels as compared to
noninduced cells, hIFN-
-treated, parental L929sA cells,
and hIFN-
-treated CAT-expressing cells. IL-6 expression was already detectable 5 to 6 h after administration of
hIFN-
. Northern blot analysis with total RNA samples
extracted at different induction periods confirmed these
results (Fig. 6 b).
by Northern blot analysis using a hTNF-R55i cDNA fragment as a common probe. The results clearly reveal the
low mRNA abundance in cl 2 as compared to cl 15 and
suggest that, although the former is biologically active, there must have been selection for very low CAT-R55i-
expressing clones (Fig. 6 a). Considering the much smaller
number of transfectants with pMxCAThTNFR55i, clones
with a higher expression level probably did not survive the
2-wk selection procedure in G418-containing medium because a small leakage of the Mx promoter, generating continuously minute amounts of CAT-R55i protein, may have
killed the positive cells. In contrast, inducible expression
of a nonlethal protein, such as the CAT protein itself, did
not lead to counter-selection, as cells easily tolerated a certain degree of leakiness in promoter control.
Fig. 8.
Characterization of
the CAT-R55i protein on the
basis of its enzymatic activity.
(A) Parental L929sA, CAT
cl 15, or CAT-R55i cl 2 cells
were treated with hIFN-
(+) or untreated (
). Cell extracts were made as described. Lanes 1-3 represent
serial dilutions of pure CAT
enzyme; lanes 4 and 5 are total lysates of CAT cl 15 and
CAT-R55i cl 2 treated with
hIFN-
, respectively; lanes 6-10 represent htr-13 immunoprecipitates derived from
L929sA, CAT cl 15, or CAT-R55i cl 2 cells, treated or not
with hIFN-
as indicated.
CAT enzymatic activity was
measured by the synthesis of
acetyl-[14C]1-deoxy-chloramphenicol as described in Materials and Methods. (B)
CAT-R55i cl 2 cells were
treated with hIFN-
or
hIFN-
+ PSI, and total lysates were used in the CAT
assay.
[View Larger Version of this Image (49K GIF file)]
cannot be excluded. Indeed, several groups have reported
on the probable existence of protective mechanisms within
the cell designed to restrict the number of TNF-R55 molecules per cell, as overexpression of TNF-R55 is lethal (Brakebusch et al., 1992
; Tartaglia and Goeddel, 1992
; Boldin et al.,
1995a
). Degradation by proteasomes could be such a
mechanism (Darnay et al., 1994
). In agreement with this hypothesis, addition of the proteasome inhibitor PSI
(Figueiredo-Pereira et al., 1994
; Traenckner et al., 1994
) to
hIFN-
-induced CAT-R55i-expressing cells resulted in
an 1.8-fold higher expression level of CAT-R55i as measured by the CAT assay (Fig. 8 B) and a more rapid cell killing. This suggests that (partial) prevention of CAT-R55i protein degradation synergistically augmented the
cytotoxicity. Nevertheless, even under these conditions we
were unable to directly demonstrate the presence of CAT-R55i fusion protein by CAT-ELISA or immunoprecipitation.
of CAT-DD clones, almost no cell killing could be observed. Since the DD was demonstrated to be necessary
and sufficient for cell killing, the lack of functionality in
this CAT-DD construct could be due to structural constraints. Indeed, the COOH termini of the CAT subunits
are ~55 Å from each other (Leslie, 1990
). Hence, the trimeric DDs may have been separated too far to allow functional clustering. In the CAT-R55i chimeric protein, however, this physical constraint was probably avoided by the
upstream polypeptide region in front of the DD. Therefore, we constructed the pMxCATlinkDD vector, which
has a 15 amino acid linker, (Gly-Gly-Gly-Gly-Ser)3, inserted between the two protein domains (Fig. 1 B). After
transfection of this pMxCATlinkDD construct in L929sA
cells, a strong counter-selection was again observed, as
previously seen with the pMxCAThTNFR55i construct.
G418-resistant clones were isolated and tested for inducible cell killing. After induction with hIFN-
, 60% of the clones died within 24 h with kinetics comparable to the cell
death induced by CAT-R55i. The results obtained with
two clonal lines (cl 4 and cl 12) are shown in Fig. 9 A. IFN-
induction of this trimeric fusion protein also activated NF-
B and induced IL-6 gene expression (Fig. 9 B); note that
cl 12 died so quickly that only small amounts of IL-6 were
synthesized.
Fig. 9.
Cytotoxicity and IL-6 induction in CAT-R55i and
CATlinkDD-expressing cells. (A) CAT-R55i cl 2 (), CATlinkDD
cl 4 (
), and CATlinkDD cl 12 (
) cells were treated with 1,000 IU/ml hIFN-
. At the indicated time points, cell samples were
taken, stained with PI, and analyzed by flow cytometry. PI uptake
by treated cells was compared to untreated cells (
, CAT-R55i cl 2;
, CATlinkDD cl 4; and
, CATlinkDD cl 12). (B) Cells were
treated for 12 h either with 1,000 IU/ml hIFN-
(black bars) or with
2,000 IU/ml mTNF (gray bars), or left untreated (blank). IL-6
levels in the medium were measured as described.
[View Larger Version of this Image (17K GIF file)]
B activation, and IL-6 gene induction.
Discussion
; Espevik et al.,
1990
). Moreover, TNF is only biologically active in its trimeric form, and the extracellular domain of the receptor binds in the cleft between two subunits (Van Ostade et al.,
1991; Banner et al., 1993
), which means that TNF can bind
and thereby cluster three receptor molecules (Loetscher
et al., 1991
; Pennica et al., 1992
). This brings the intracellular receptor domains in close proximity to each other to allow signaling. It has previously been reported that expression of receptor molecules or their intracellular receptor
domains was sufficient to simulate TNF effects (Boldin et al., 1995b
). In that study, it was assumed, but not
proven, that clustering of the TNF receptor domains occurred upon overexpression of the individual molecules.
In the present paper, we show that expression of the intracellular domain of the TNF-R55 as such is not sufficient to
elicit TNF effects and clearly demonstrate that only the
"forced clustering" of these receptor domains leads to various characteristic TNF effects, without the need for the extracellular or transmembrane portion of the receptor.
The apparently different results obtained by Boldin et al.
(1995a) and ourselves are most probably related to the expression levels that can be obtained in the cell system
used. As the HeLa cells used in the former study were
much less sensitive to the cytotoxic action of TNF than the
L929sA cells, counter-selection during the stable transfection procedure may not have been so strong in the HeLa cells. Hence, after subsequent induction, the higher expressing clones might have been able to produce sufficient
levels of TNF-R55i to allow self-association and signaling.
We also have tried to express the hTNF-R55i using the tetracycline-controlled system in L929sA cells, and we obtained similar results as with the Mx promoter-driven,
inducible expression. This further proves that not the expression system as such, but rather the TNF sensitivity of
the cell system used is responsible for the apparent difference in experimental outcome. Moreover, we effectively
demonstrate that intracellular domain clustering is necessary and sufficient for receptor signaling.
; Bazzoni and Beutler, 1995
; Bazzoni et al.,
1995
). On the other hand, natural TNF is a trimeric molecule, and moreover, receptor-specific, multivalent IgM antibodies are far more potent in mimicking TNF actions than
agonistic, bivalent IgG antibodies (Espevik et al., 1990
).
Finally, it was also reported that an EPO/TNF-R75i chimera, which can only be dimerized, is deficient in signaling
(Declercq et al., 1995
). It might therefore be of interest to
test forced dimerization of cytoplasmic receptor domains
in a similar system, e.g., by expressing the TNF-R55i as a
fusion with a specific dimerizing domain.
;
Adam et al., 1996b
; Adam-Klages et al., 1996
), several kinases and phospholipases, and induction of NO synthase
(iNOS) (Tartaglia et al., 1993
). By Northern analysis, we
could demonstrate indeed that CATlinkDD-expressing cells
did not induce iNOS (even after additional stimulation
with IFN-
), while CAT-R55i-expressing cells did. Therefore, the additional signaling effected by the membrane-proximal domain and responsible for the various activities
of TNF, other than IL-6 gene expression (and other, similarly regulated genes) and cell killing, is now being studied
by induced expression of mutated fusion constructs.
B
activation, gene induction, and cytotoxicity. It seems likely
therefore that there are protective mechanisms that
strictly control the number of TNF-R55 molecules per cell,
since spontaneous clustering or overexpression of TNF-R55 is lethal to the cell. Several groups already reported
the existence of such "survival" mechanisms, e.g., degradation by proteasomes may well be involved in the elimination of excess TNF-R55. Darnay et al. (1994)
reported
that deletion of the nucleotides encoding the 39 amino acids proximal to the transmembrane region minimized the
degradation of TNF-R55i, expressed as a fusion with glutathione-S-transferase. This degradation-sensitive area is
located next to the binding region of a TNF-R55i-associating protein that shows homology to a proteasomal subunit
(Boldin et al., 1995b
). Indeed, the short half-life of the TNF
receptors is consistent with a turnover involving degradation in proteasomes. Results obtained with the proteasome inhibitor PSI are in agreement with this hypothesis.
Inhibition of CAT-R55i breakdown by addition of PSI resulted in higher expression levels, leading to an increased
cytotoxicity. Hence, TNF-R55 degradation by proteasomes
might well occur as a restricting mechanism inside the cell,
insuring that expression levels of the TNF receptors are
kept constitutively low, thus preventing unwanted activation and signaling by spontaneous association.
B and possibly mediated by TNF-R55i DD
TRADD
TRAF2 interactions (Hsu et al., 1996
), and
the other activation of p38 MAP kinase (Beyaert et al.,
1996
). The pathway leading to cytotoxicity may be mediated by TNF-R55i DD
TRADD
FADD interactions
(Hsu et al., 1996
; Chinnaiyan et al., 1996
; Boldin et al.,
1996
). The CAT fusion system is exceptionally suitable for
further dissection and identification of various functions
encoded by the hTNF-R55i and subdomains thereof, and
may allow the physical and biochemical characterization
of activated hTNF-R55i complexes as such and their association properties with other signal-transducing and/or regulatory proteins.
Received for publication 7 February 1997 and in revised form 11 April 1997.
1. Abbreviations used in this paper: ActD, actinomycin D; CAT, chloramphenicol acetyltransferase; DD, death domain; IFN, interferon; IL-6, interleukin-6; IPTG, isopropyl1. |
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