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Division of Cytokine Biology, Office of Therapeutics Research and Review, and
Division of Viral Products, Office of Vaccines Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Rockville, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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hybrids, HY-1
[IFN-21a(1-75)/2c(76-165)], HY-2
[IFN-21a(1-95)/2c(96-165)], and HY-3
[IFN-2c(1-95)/21a(96-166)], were constructed, cloned, and
expressed. The hybrids had comparable specific antiviral activities on
Madin-Darby bovine kidney (MDBK)3 cells but exhibited very
different antiproliferative and binding properties on human Daudi and
WISH cells and primary human lymphocytes. Our data suggest that a
portion of the N-terminal region of the molecule is important for
interaction with components involved in binding of IFN-2b while the
C-terminal portion of IFN is critical for antiproliferative activity. A
domain affecting the antiproliferative activity was found within the
C-terminal region from amino acid residues 75–166. The signal
transduction properties of HY-2 and HY-3 were evaluated by EMSA and
RNase protection assays. Both HY-2 and HY-3 induced activation of STAT1
and 2. However, HY-2 exhibited essentially no antiproliferative effects
at concentrations that activated STAT1 and 2. Additionally, at
concentrations where no antiproliferative activity was seen, HY-2
induced a variety of IFN-responsive genes to the same degree as HY-3.
RNase protection assays also indicate that, at concentrations where no
antiproliferative activity was seen for HY-2, this construct retained
the ability to induce a variety of IFN-inducible genes. These data
suggest that the antiproliferative response may not be solely directed
by the activation of the STAT1 and STAT2 pathway in the cells
tested. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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s. Several
types of IFN-2 are currently approved for a variety of diseases
including hepatitis b and c, hairy cell leukemia, and chronic
myelogenous leukemia (2).
Recombinant techniques are useful methods for the production and
modification of IFN- proteins. The first IFN hybrid, IFN-1/2
was constructed using recombinant technology in 1981 (3).
Subsequently, a number of hybrids have been constructed and have been
informative for examining the activity of IFNs (4, 5, 6, 7, 8, 9, 10, 11).
These hybrid constructs have resulted in novel IFNs that either combine
different biological properties from the parental proteins or have
significantly different biological activity from both the parents
(3). Therefore, IFN hybrids have provided a powerful tool
for studying the structure-function
relationship of these molecules. The engineered IFN- proteins may
have important new therapeutic applications and may provide greater
insights into understanding of the clinical activities of existing
IFN-s.
To date, our laboratory has purified and characterized 22 IFN-
components produced by Sendai virus-induced human lymphoblastoid cells
(12). One of these species, component o, was found to
be noteworthy for its high antiproliferative activity and its poor
ability to compete with the IFN-2b binding site (13).
Based on our partial amino acid sequence data, component o was
indistinguishable from IFN-21a. Therefore, rIFN-21a was cloned
and expressed. It was determined to behave functionally like component
o in that it exhibits a high antiproliferative sp. act. and competes
poorly for the IFN-2b binding site (13, 14).
The goals of our studies are to determine the specific region(s) of
IFN-21a responsible for the enhanced antiproliferative activity and
to further understand the signaling mechanism it uses to elicit this
response. Three IFNA2 and IFNA21 chimeras (HY-1, HY-2, and HY-3) were
constructed using PCR technology and cloned into a pQE-30 expression
vector. The hybrid proteins were expressed in Escherichia
coli. These chimeras were selected based on the 1) amino acid
sequences of IFN-2c and IFN-21a, 2) the biological activities and
binding properties of these IFN-s, and 3) information derived from
the three-dimensional structures of human IFN- consensus and human
IFN-ß (15, 16). The three-dimensional model of a human
IFN- consensus sequence reveals that the two domains 29–35 and
123–140 are in close spatial proximity and may constitute a receptor
recognition domain. In contrast, the residues 78–95 are distant from
this region on the molecule and may influence species specificity and
differential biological activities of the human IFN-s and IFN-ß
(14).
The human IFN hybrid proteins were purified using
nickel-nitrilotriacetic acid. Agarose and mAb affinity chromatography.
The antiviral, antiproliferative, binding, signal transduction, and
RNase protection properties of the IFN- hybrids were analyzed and
compared. Our data suggest that a portion of the N-terminal region of
the molecule is important for interaction with receptor components,
while the C-terminal region of IFN- is critical for
antiproliferative activity. The EMSA and RNase protection assays of the
hybrid IFN-s suggest that the antiproliferative response may not be
directed by STAT1 and 2 activation alone in either a nonhematopoietic
cell line, a hematopoietic cell line, or in a primary human cell
type.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2b
Recombinant human IFN-2b (obtained from Schering, Kenilworth,
NJ) has an antiviral sp. act. of 2 x 108
IU/mg protein. IFN-2c (cDNA was cloned into the vector pBluescript;
Stratagene, La Jolla, CA) and IFN-21a (cDNA from Genentech, South
San Francisco, CA) proteins were expressed and purified from E.
coli strains transformed with pQE30 as described below. IFN-2b
was labeled with 125I-Bolton-Hunter reagent
(Amersham, Arlington Heights, IL) as previously described
(13).
Cell and cell culture
Human Daudi cells were obtained from Dr. P. Grimley (Department of Pathology, Uniformed Services University of the Health Sciences, Bethesda, MD). Cells were grown in suspension using RPMI 1640 with 10% FCS, 2 mM glutamine, and 0.2% gentamicin. WISH cells (American Type Culture Collection (ATCC), Manassas, VA) were grown as monolayer cultures using Eagle’s minimal essential medium supplemented with 10% FCS and gentamicin (50 µg/ml). The cultures were incubated at 37°C in a humidified atmosphere containing 5% CO2. All cultures were determined to be free of mycoplasma. We obtained primary human lymphocytes from normal donors by centrifugal elutriation after Ficoll-Hypaque sedimentation (lymphocyte separation medium package insert, Organon Teknika, Durham, NC). The resultant cells were resuspended in RPMI 1640 media supplemented with 10% FCS and fungizone (250 ng/ml amphotericin B, 100 U/ml penicillin, and 100 µg/ml streptomycin).
Construction of pQE30/A21
A pair of oligonucleotides, 5'-TCCGGATCCTGTGATCTGCCTCAGAC-3'
(sense) and 5'-GAGCTCGCATGCTCATCATTCCTTCCTCCTTAATCT-3' (antisense),
with BamHI and SphI restriction sites were
synthesized based on the cDNA coding region for mature human IFN-21a
protein and were used as primers. Human IFNA21 cDNA was used as the
template. PCR was used to amplify an entire coding region for mature
human IFN-21a protein. The PCR reactions were conducted by standard
procedures (17), and the resulting products were cleaved
with restriction endonucleases BamHI and SphI and
cloned into the E. coli expression vector pQE30 (purchased
from Qiagen, Chatsworth, CA). The final construct was confirmed by DNA
sequencing (18).
Construction of IFN- hybrids DNA
Hybrid IFN cDNAs were constructed by PCR technology
(19). PCR primers for the construction of HY-1 [IFN-
21a(1-75)/IFN- 2c(76-165)], HY-2 [IFN- 21a(1-95)/IFN-
2c(96-165)], and HY-3 [IFN- 2c(1-95)/IFN- 21a(96-166)]
include: primer 1, 5'-TCC GGA TCC TGT GAT CTG CCT CAG ACC-3' (for HY-1,
2, and 3); primer 2, 5'-AGC AGA TGA GTC CTT TGT GCT GAA GAG-3' (for
HY-1); primer 3, 5'-CTC TTC AGC ACA AAG GAC TCA TCT GCT-3' (for HY-1);
primer 4, 5'-GAG CTC GCA TGC TCA TCA TTC CTT ACT TCT TAA ACT-3' (for
HY-1 and 2); primer 5, 5'-CAC GCA GGC CTC GAG GTC ATT CAG-3' (for HY-2
and 3); primer 6, 5'-CTG AAT GAC CTC GAG GCC TGC GTG-3' (for HY-2 and
3); and primer 7, 5'-GAG CTC GCA TGC TCA TCA TTC CTT CCT CCT TAA TCT-3'
(for HY-3). Primers 2, 3, 5, and 6 were used as inside primers for all
three hybrids, and primers 1 and 4 (HY-1 and HY-2) or 1 and 7 (HY-3)
were used as outside primers. The BamHI restriction site is
on the 5' outside primer (primer 1), and the SphI
restriction site is on the 3' outside primers (primers 4 and 7). The
DNA sequences of IFNA2 and IFNA21 are identical in regions recognized
by primer 1, hence, this primer was used in the construction of both
IFNA2 and IFNA21 fragments. Plasmid DNAs Bluescript/A2 and pQE30/A21
were used as the templates for the first PCR reactions for all hybrids.
Purified DNA fragments from the first PCRs were mixed as templates, and
primers 1 and 4 (for HY-1 and 2) or primers 1 and 7 (for HY-3) were
used for the secondary PCR reactions.
Confirmatory DNA sequencing on the final constructs was performed using a DNA sequencing kit (Amersham) as previously described (18). The following are the GenBank accession codes assigned to the hybrids: HY-1 AF085803, HY-2 AF085804, and HY-3 AF085805.
Expression and purification
IFNA2, IFNA21, and hybrids HY-2, HY-3 plasmid DNAs were
individually transformed into E. coli strain
SG13009[pREP4] (Qiagen), and HY-1 plasmid DNA was transformed into
E. coli strain DH5F’IQ (Life Technologies, Gaithersburg,
MD). Bacteria were grown in Luria-Bertani broth containing 100 µg/ml
ampicillin (HY-1) or 100 µg/ml ampicillin and 25 µg/ml kanamycin
(HY-2, HY-3, IFNA2, and IFNA21) in a 37°C shaker incubator overnight.
The cultures were diluted 1:50 in Luria-Bertani broth containing the
appropriate antibiotic(s) and incubated at 37°C with shaking to an
A600 of 0.8–0.9. Protein expression was induced
by 2 mM isopropyl-1-thio-ß-D-galactopyranoside.
The bacteria were then incubated at 30°C for 4–5 h, after which
cells were harvested and sonicated. The cell lysate was clarified by
centrifugation at 10,000 x g for 30 min at 40°C. IFN
purification was performed by nickel-nitrilotriacetic acid. Agarose
resin metal-affinity chromatography (20, 21) and 4F2 mAb
affinity chromatography (12). The mAb 4F2 recognizes amino
acid residues 113–149 of IFN- consensus (Dr. Bruce Altrock,
unpublished observations).
Protein determination
Purified recombinant protein concentrations were determined
using the coomassie plus protein assay (Pierce, Rockford, IL). Purity
of the rIFN-s were assessed by SDS-PAGE and HPLC analysis (data not
shown).
Antiproliferative assay
The antiproliferative activities of the IFN- constructs were
analyzed using human Daudi and WISH cells and primary human
lymphocytes. The assays on Daudi cells were performed as previously
described (13). The assays on WISH cells were performed by
incubating the cells with various IFN-s at the indicated
concentrations for 72 h at 37°C. Fifty microliters of 2 mg/ml
MTT was added into each well and incubated for 4 h at 37°C.
Then, 10% SDS, 0.01 N HCl (250 µl) was added to each well and
incubated overnight at 37°C. The OD570 of each
well was determined, and the percentage of growth inhibition was
calculated by comparing the control cultures (no IFN) with the
IFN-treated cultures. Primary human lymphocytes were treated with PHA
(Promega, Madison, WI) at 1 µg/ml overnight. The resultant PHA blasts
were then treated for 72 h with the various IFNs at the
concentrations indicated (see Fig. 2
). Percent inhibition was
calculated from direct cell counts that were done by Coulter counter
analysis (Coulter, Palo Alto, CA).
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Antiviral activity was determined as previously described using MDBK cells (ATCC) and WISH cells (ATCC) (12). All IFN units are expressed with reference to the National Institutes of Health human lymphoblastoid IFN standard Ga 23-901-532.
The Edmonston strain of measles virus (low passage, human embryonic kidney 7, VERO 5) was plaque purified and used to infect 1 x 106 primary human lymphocytes (in triplicate) following no treatment or pretreatment with 100 ng/ml of parental or hybrid IFN for 24 h before infection as previously described (22). Cells were infected with measles virus at 0.1–1.6 multiplicity of infection or mock infected with virus-free medium and harvested 72 h postinfection. Cell-associated virus and supernatants were titrated on VERO cell monolayers. Measles virus cytopathic effect was evaluated microscopically after 6 days and confirmed by staining with crystal violet. The results reported are a comparison of the mean virus in supernatants harvested from the untreated group with supernatant virus in each treated group.
EMSA
Cells were treated with the various IFNs at the concentrations
and times indicated (see Fig. 4) and centrifuged at 1500 x
g at 40°C for 10 min. After being washed once in ice-cold
PBS, the cells were recentrifuged at the above conditions. The cell
pellet was then resuspended in lysis buffer (20 mM HEPES, pH 7.5, 1 mM
EDTA, 1 mM MgCl2, 150 mM NaCl, 1% Triton X-100,
3 mM sodium orthovanadate, 10 mM B-glycerophosphate, 20 mM DTT, and 2
mM PMSF) followed by centrifugation for 5 min at 14,000 x
g. EMSA were then performed. The clarified whole-cell
lysates containing 10 µg of total protein were diluted 1:1 in a
binding buffer containing 20% glycerol, 2 mM DTT, 150 mM KCl, 2 µg
polydeoxyinosine-deoxycytosine, 0.5 mM EDTA, and 0.5 mM
MgCl2 and incubated for 15 min on ice. One
nanogram of 32P-end labeled probe was then added
to the reaction and allowed to sit an additional 5 min on ice. For the
supershift experiments, rabbit anti-STAT1 or anti-STAT2 serum
or normal rabbit serum was then added at this point at a 1:200 final
dilution (in binding buffer) and was allowed to incubate for an
additional 30 min on ice. The samples were then subjected to
electrophoresis on a native 6%, 0.25x Tris-borate-EDTA gel for 4
h. The probes used consisted of the 
response region (GRR) found
within the promoter of the FcRI gene
(5'-AGCATGTTTCAAGGATTTGAGATGTATTTCCCAGAAAAG-3') and the IFN-stimulated
response element (ISRE) of the IFN-stimulated gene 15
(5'-GATCCATGCCTCGGGAAAGGGAAACCGAAACTGAAGCC-3').
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This assay was performed as described previously (23).
Binding assay
IFN binding assays were performed as previously described on human Daudi (13) and WISH (24) cells.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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s and hybrids
Human IFN-2c, IFN-21a, and three IFN hybrids, HY-1, HY-2,
and HY-3, were expressed in E. coli using a pQE30 expression
system. The sequences of the IFN-s deduced from their cDNA sequences
are shown in Fig. 1. Initial purification
of the IFNs on a Ni-NTA-Agarose column yielded partially purified
material, with antiviral sp. act. ranging from 3 x
106 to 4.5 x 106
IU/mg protein on MDBK cells. The IFN-s were further purified by 4F2
or NK2 mAb affinity chromatography (12). After the two
purification steps, each IFN- hybrid appeared as a single band with
an apparent m.w. of 20,000 on reducing SDS-PAGE. The antiviral sp. act.
of the purified IFN-s are shown in Table I; they ranged from 2 x
108 to 3.7 x 108
IU/mg protein on MDBK cells and 0.1 x 108
to 1.9 x 108 IU/mg protein on WISH cells.
The antiviral sp. act. of the three hybrids were similar to each other
and to IFN-2c and IFN-21a on MDBK cells (2.0 x
108 IU/mg to 3.7 x
108 IU/mg). However, the antiviral sp. act. of
HY-1 and HY-2 were, at least, 7-fold lower than that of HY-3,
IFN-2c, or IFN-21a on WISH cells.
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hybrids and the parental IFN-s to inhibit
the growth of Daudi, WISH, and primary human lymphocytes was also
examined. The inhibition curves are shown in Fig. 2
. The concentrations of IFN-s that
inhibited Daudi and WISH cell growth by 50% are shown in the Table I.
Hybrid HY-3 always exhibited a higher antiproliferative sp. act.
compared with the other hybrids and IFN-2c and IFN-21a on all
three cell populations. In comparison, hybrid HY-2 had a lower
antiproliferative sp. act. than either of the other hybrids or either
of the parental IFN-s on the same cell populations. HY-2 displayed
10,000-fold lower antiproliferative sp. act. compared with HY-3 on
Daudi cells and >1,000-fold less sp. act. on WISH and primary human
cells compared with HY-3. The hybrid HY-1 had a 2- to 8-fold greater
antiproliferative sp. act. than HY-2 on Daudi and WISH cells. [The
relative ability of HY-2 and HY-3 to induce an antiviral state in a
primary cell was determined by examining fresh human lymphocytes primed
with PHA]. The PHA blasts were treated with concentrations of HY-2 and
HY-3 known to induce STAT1 and STAT2 and subsequently infected with
measles virus. Fig. 3 shows that for two
separate normal donors at concentrations of HY-2 in which no
antiproliferative effects were seen and STAT1 and STAT2 activation was
readily apparent, there was suppression of release of measles virus.
These data indicated that the antiviral pathway was operational.
However, there was no effect on measles virus replication when titers
of treated and untreated cell-associated virus were compared.
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2c, IFN-21a,
the hybrids, and IFN-2b. Fig. 4 shows
the ability of IFN-2c, IFN-21a, and the three hybrids (HY-1,
HY-2, HY-3) to compete for IFN-2b binding sites on Daudi and WISH
cells, respectively. IFN-2c and the hybrid HY-3 inhibited the
binding of 125I-IFN-2b to Daudi and WISH cells
very effectively, while hybrids HY-1 and HY-2 like IFN-21a, poorly
inhibited 125I-IFN-2b binding on both cell
lines. These data suggest that the amino terminal region of the IFN-
molecule is important in binding to its receptor. IFN signaling pathways and transcription analysis
Because HY-3 has the highest antiproliferative activity and HY-2
has the lowest on Daudi and WISH cells and primary lymphocytes (Fig. 2), these two constructs were examined to explore potential signaling
differences that could explain the dissimilarity seen in their
antiproliferative activity. One signaling pathway that is activated by
IFN- is the JAK/STAT signaling pathway. Both STAT1 and 2 are
normally activated after IFN receptor occupancy. Therefore, to
understand the mechanism governing this activity, we compared the
ability of HY-2 and HY-3 to induce STAT1 and 2 activation by EMSA. Two
32P-labeled double-stranded oligonucleotide
probes were used. One contains a GRR element that specifically binds
STAT1, while the second contains an ISRE that binds STAT2. The data are
shown in Fig. 5. A and
B show the results using Daudi and WISH cells and primary
human lymphocytes that were treated for 30 min with a concentration of
IFN that showed a clear antiviral effect for HY-3 and very little or no
antiproliferative effect for HY-2. To validate that the IFN-inducible
complex seen in Fig. 5 contained STAT1 or STAT2 specifically,
supershift experiments using anti-STAT1 and anti-STAT2 Abs were
performed. The ability of the anti-STAT1 Ab to shift the complex is
seen in Fig. 6, indicating that this
complex does, in fact, contain STAT1. When anti-STAT2 Ab is used,
the complex induced by either of the IFNs tested is knocked out, which
can occur when the Ab recognizes the DNA-binding domain as an epitope.
Normal rabbit serum was used as a negative control for all of the
supershift experiments and did not effect the mobility of the
IFN-inducible complex. The results indicated that, in all three cell
types, HY-2 induced the optimal activation of STAT1 and STAT2 at
concentrations at which essentially no antiproliferative activity was
observed. These data suggest another pathway may also be activated in
addition to the activation of STAT1 and STAT2 to elicit the
antiproliferative activity.
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). The
concentrations of HY-2 and HY-3 used were identical with those used in
the EMSA experiments and were at levels where no antiproliferative
activity was seen for HY-2 but good antiproliferative activity was
noted for the other IFNs. In addition, the ability of the IFNs to
activate IFN-induced gene expression was also identical for the ISG54,
IRF1, GBP, and IP10 genes (data not shown). These results indicate that
at concentrations where no antiproliferative activity was seen for
HY-2, this construct retained the ability to induce a variety of
IFN-inducible genes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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s is their ability to inhibit
cell growth, which has also been suggested to be of major importance in
determining antitumor action. Grander et al. (25)
indicated that IFNs can exert profound anticellular effects in vitro,
caused by either cell cycle arrest and/or induction of apoptosis. Our
studies show human IFN-2c, IFN-21a, and the hybrids HY-1, HY-2,
and HY-3 exhibit a broad range (1,000–10,000) of differences in their
antiproliferative sp. act. on Daudi and WISH cells and human
lymphocytes (see Table I and Fig. 2). HY-3 exhibited the highest
antiproliferative sp. act. The concentration of HY-3, which inhibits
cell growth by 50%, is 0.0001ng/ml on Daudi cells and 8.0 ng/ml on
WISH cells. However, these IFN-s exhibited comparable antiviral sp.
act. on MDBK cells and sp. act. within 7-fold of each other on WISH
cells. Similarly, in primary human lymphocytes, the antiviral activity
of HY-2 and HY-3 are essentially the same for the concentrations
examined (see Fig. 6). Thus, the biological effects of HY-2 and HY-3
provide evidence for a divergent mechanism by which IFN-s induce
their antiproliferative and antiviral effects. Therefore, the IFN-s
may turn on several signaling pathways to different degrees
(5). Recently, it has been reported that the
antiproliferative effects of IFN- are mediated by components of TCR
signaling pathways and that activation of the JAK-STAT pathway alone is
insufficient for the induction of the antiproliferative activity of
IFN- (22). Our observations not only support that data
but expand it beyond a cell line and a hematopoietic-based cell system.
In addition our EMSA and RNase protection results suggest that the
antiproliferative response may not be directed by activation of STAT1
and STAT2 alone. The gel shift data show that STAT1 and 2 activation
and IFN-dependent gene transcription is induced by HY-2 at
concentrations where HY-2 shows no antiproliferative activity. The
antiviral activity was comparable between the two hybrids at
concentrations in which no antiproliferative activity was seen. HY-3
was seen to induce STAT1 and STAT2 activation to a greater extent than
HY-2 when the cells were treated with equivalent concentrations (data
not shown). However, the kinetic profiles of STAT1 and STAT2 activation
by HY-2 and HY-3 at the various concentrations used in the
antiproliferative assay were identical so that the differences seen
between the two hybrids cannot be explained by STAT activation off
rates (data not shown). Conversely, HY-2 exhibits little or no
antiproliferative effects at concentrations that induce STAT1 and STAT2
activation more robust than that seen with low concentrations of HY-3
(data not shown).
Comparing the sequences of HY-1 and HY-2, there are seven amino acid
differences in the 75–95 region (Fig. 1). Comparisons of published
structural and homology models of IFN-ß and - (16, 26, 27), show that the amino acid region 75–95 forms the C helix,
which is one of the most exposed helices. The amino acids in this
region may be easily accessible to react with their appropriate
receptors (28) and thus may be important for biological
activity. Di Marco et al. (29) conducted site-directed
mutagenesis in the C helix of the IFN molecule. They have reported that
the mutated amino acids (position 84, 86, 87, 90) of helix C of IFN-
faces toward one of the subunits of the receptor and are part of one of
two binding sites of IFN-. Mutations markedly decreased the
biological activity on murine cells compared with the unmutated
protein. Korn et al. (15) and Fish (30) have
proposed that there may be sufficient complexity in the type 1 IFN
receptor to account for the differential sensitivities between IFN-s
and IFN-ß that may be associated with a residue difference in the
region 78–95. The IFN sequence 78–95 has been implicated as
influencing species specificity among the murine and human IFN-s and
affecting the differential specificity of action between human IFN-s
and IFN-ß. It is intriguing to speculate that these residue positions
may constitute specific recognition sites associated with an accessory
component of the IFN- receptor. Our studies suggest that region
75–95 is very important for the antiproliferative activity of human
IFN-, with maximal activity observed when the construct has the
2c sequence from 75–95 and the 21a sequence from 96–166.
Our competitive binding experiments show that the hybrids or IFN-
with the 2c domain at the N terminus (residues 1–95; like IFN-2c
and HY-3) compete well with 125I-IFN-2b
binding. In contrast, hybrids or IFN- with the IFN-21a domain at
the N terminus (residues 1–95; IFN-21a, HY-1, HY-2) show reduced
ability to compete with 125I-IFN-2b binding.
These data suggest that the N terminus is involved in IFN receptor
binding interaction. Several reports have proposed two binding sites
for IFN-, one which is located in the N-terminal region and would
determine the binding to high-affinity receptors. The second site would
be in the C-terminal region of the IFN molecule and would influence
low-affinity binding to cells (3, 31, 32). Extensive
evidence suggests that this receptor system is complex, possibly
consisting of either multiple receptors or a multisubunit receptor
(13, 33).
In conclusion, our data demonstrates that a portion of the N-terminal
region of the molecule may be important for the receptor binding
activities. Furthermore, a domain critical for antiproliferative
activity appears to exist within the C-terminal region from amino acid
residues 75–166. In our studies the IFN region
[2c(75-95)/21a(96-166)] appears to be responsible for the
higher antiproliferative sp. act. seen with HY-3. In addition, our data
define IFN domains that dissociate anitiviral and antiproliferative
activities. The hybrids (HY-1, HY-2, and HY-3) had similar antiviral
sp. act. on MDBK cells and primary human lymphocytes but different
antiproliferative and binding properties on Daudi, WISH, and primary
human cells. Analysis of gel shift gene induction and biological
responses suggest that the antiproliferative response requires critical
signals in addition to those that activate STAT1 and STAT2.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Renqui Hu, Center for Biologics Evaluation and Research, Food and Drug Administration, 1401 Rockville Pike, Rockville, MD 20892. E-mail address:
3 Abbreviations used in this paper: MDBK, Madin-Darby bovine kidney; GRR, response region; ISRE, IFN-stimulated response element.
Received for publication December 7, 1999. Accepted for publication April 26, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
therapy. Semin. Oncol. 25:(Suppl. 1):9.[Medline]
produced in E. coli and of hybrid molecules derived from them. Proc. Natl. Acad. Sci. USA 78:2848. hybrids. Pharmacol. Ther. 66:507.[Medline]
B/D hybrids on human tumor cell lines. Cancer. Res. 47:2020.. Antiviral Res. 22:121.[Medline]
/ß and their receptors: place in the hierarchy of cytokines. APMIS 105:161.[Medline]
. J. Biol. Chem. 267:15210.. J. Biol. Chem. 268:12591. consensus sequence. J. Interferon Res. 12:1.
requires components of T-cell receptor signalling. Nature 390:629.[Medline]
interferon to a high affinity cell surface binding site on bovine kidney cells. J. Biol. Chem. 257:4695. subtypes IFN-conI, IFN-8 and IFN-1 derived from the crystal structure of IFN-2b. Semin. Oncol. 24:S52.
2. Proteins Struct. Funct. Genet. 17:62.[Medline]
. Biochem. Biophys. Res. Commun. 202:1445.[Medline]
. J. Interferon Res. 12:257.[Medline]
. J. Interferon Res. 9:97.[Medline]
2c and their role in binding to cells. Mol. Immunol. 28:1289.[Medline]
receptor. Pharmacol. Ther. 52:227.[Medline]
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B. Nardelli, L. Zaritskaya, M. Semenuk, Y. H. Cho, D. W. LaFleur, D. Shah, S. Ullrich, G. Girolomoni, C. Albanesi, and P. A. Moore Regulatory Effect of IFN-{kappa}, A Novel Type I IFN, On Cytokine Production by Cells of the Innate Immune System J. Immunol., November 1, 2002; 169(9): 4822 - 4830. [Abstract] [Full Text] [PDF]
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 P. Harle, V. Cull, M.-P. Agbaga, R. Silverman, B. R. G. Williams, C. James, and D. J. J. Carr Differential Effect of Murine Alpha/Beta Interferon Transgenes on Antagonization of Herpes Simplex Virus Type 1 Replication J. Virol., June 5, 2002; 76(13): 6558 - 6567. [Abstract] [Full Text] [PDF]
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R. Hu, J. Bekisz, H. Schmeisser, P. McPhie, and K. Zoon Human IFN-{alpha} Protein Engineering: The Amino Acid Residues at Positions 86 and 90 Are Important for Antiproliferative Activity J. Immunol., August 1, 2001; 167(3): 1482 - 1489. [Abstract] [Full Text] [PDF]
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), HY-2 (
),
HY-3(*), as well as parent IFNs IFN-
) and IFN-
). A, Daudi cells.
B, WISH cells. Cells were incubated in the presence of
indicated concentrations of IFN for 3 days at 37°C. Results are
expressed as a percentage of growth relative to a control culture to
which medium without IFN was added. Experiments were performed in
triplicate (Daudi) and duplicate (WISH). C, Primary
human lymphocytes. Antiproliferative effects of the parental IFN
constructs IFN-
, 
, 
, HY2 and 
, HY3) IFN on measles virus replication in primary
human lymphocytes. A total of 1 x 106 cells were
resuspended in RPMI 1640 ± 105 pg/ml IFN 24 h
prior and during infection with measles virus at multiplicity of
infection of 0.1–1.6 TCID50/ml. Measles virus-infected and
control cells were harvested at 72 h postinfection. Supernatants
were titrated on VERO cell monolayers. All experiments were performed
in triplicate, and the results are expressed as percent of control;
100% for donors 1 and 2 were 4.95 and 5.7 TCID50/ml
log10, respectively.
