Three human IFN-α hybrids, HY-1 [IFN-α21a(1-75)/α2c(76-165)], HY-2 [IFN-α21a(1-95)/α2c(96-165)], and HY-3 [IFN-α2c(1-95)/α21a(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-α2b 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.

Interferons are cytokines that have been shown to affect a wide variety of cellular functions, including inhibition of cellular proliferation, antiviral activity, immune regulatory functions, and activation of multiple cellular genes (1). Antiviral and antiproliferative activities are clinically important properties of IFN-αs. Several types of IFN-α2 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-α proteins. The first IFN hybrid, IFN-α1/α2 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-α proteins may have important new therapeutic applications and may provide greater insights into understanding of the clinical activities of existing IFN-αs.

To date, our laboratory has purified and characterized 22 IFN-α 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-α2b binding site (13). Based on our partial amino acid sequence data, component o was indistinguishable from IFN-α21a. Therefore, rIFN-α21a 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-α2b binding site (13, 14).

The goals of our studies are to determine the specific region(s) of IFN-α21a 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-α2c and IFN-α21a, 2) the biological activities and binding properties of these IFN-αs, and 3) information derived from the three-dimensional structures of human IFN-α consensus and human IFN-β (15, 16). The three-dimensional model of a human IFN-α 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-αs and IFN-β (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-α 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-α is critical for antiproliferative activity. The EMSA and RNase protection assays of the hybrid IFN-αs 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.

Recombinant human IFN-α2b (obtained from Schering, Kenilworth, NJ) has an antiviral sp. act. of 2 × 108 IU/mg protein. IFN-α2c (cDNA was cloned into the vector pBluescript; Stratagene, La Jolla, CA) and IFN-α21a (cDNA from Genentech, South San Francisco, CA) proteins were expressed and purified from E. coli strains transformed with pQE30 as described below. IFN-α2b was labeled with 125I-Bolton-Hunter reagent (Amersham, Arlington Heights, IL) as previously described (13).

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).

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-α21a 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-α21a 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).

Hybrid IFN cDNAs were constructed by PCR technology (19). PCR primers for the construction of HY-1 [IFN-α 21a(1-75)/IFN-α 2c(76-165)], HY-2 [IFN-α 21a(1-95)/IFN-α 2c(96-165)], and HY-3 [IFN-α 2c(1-95)/IFN-α 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.

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 DH5αF’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-β-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 × 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-α consensus (Dr. Bruce Altrock, unpublished observations).

Purified recombinant protein concentrations were determined using the coomassie plus protein assay (Pierce, Rockford, IL). Purity of the rIFN-αs were assessed by SDS-PAGE and HPLC analysis (data not shown).

The antiproliferative activities of the IFN-α 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-αs 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).

FIGURE 2.

Inhibition of cell proliferation by IFN hybrids HY-1 (▵), HY-2 (○), HY-3(*), as well as parent IFNs IFN-α2c (□) and IFN-α21a (▿). 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-α2c (□) and IFN-α21a (▿) and the HY- 2 (○) and HY-3 (∗) chimeras. Incubation with IFNs is same as for Daudi and WISH cells. Here direct cell counts were performed by analysis on a Coulter counter. The experiment was performed in triplicate and the results of the mean are represented.

FIGURE 2.

Inhibition of cell proliferation by IFN hybrids HY-1 (▵), HY-2 (○), HY-3(*), as well as parent IFNs IFN-α2c (□) and IFN-α21a (▿). 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-α2c (□) and IFN-α21a (▿) and the HY- 2 (○) and HY-3 (∗) chimeras. Incubation with IFNs is same as for Daudi and WISH cells. Here direct cell counts were performed by analysis on a Coulter counter. The experiment was performed in triplicate and the results of the mean are represented.

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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 × 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.

Cells were treated with the various IFNs at the concentrations and times indicated (see Fig. 4) and centrifuged at 1500 × 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 × 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%, 0.25× Tris-borate-EDTA gel for 4 h. The probes used consisted of the γ response region (GRR) found within the promoter of the FcγRI gene (5′-AGCATGTTTCAAGGATTTGAGATGTATTTCCCAGAAAAG-3′) and the IFN-stimulated response element (ISRE) of the IFN-stimulated gene 15 (5′-GATCCATGCCTCGGGAAAGGGAAACCGAAACTGAAGCC-3′).

FIGURE 4.

Competitive binding of IFN-α hybrids, IFN-α2c and IFN-α21a on Daudi (A) and WISH (B) cells. The concentration of 125I-IFN-α2b used in these experiments was 0.25 nM. Hybrids HY-1(▵), HY-2(○), HY-3(∗) and parent IFNs IFN-α2c(□) and IFN-α21a(▿) were used as competitors. The experiments were performed in duplicate.

FIGURE 4.

Competitive binding of IFN-α hybrids, IFN-α2c and IFN-α21a on Daudi (A) and WISH (B) cells. The concentration of 125I-IFN-α2b used in these experiments was 0.25 nM. Hybrids HY-1(▵), HY-2(○), HY-3(∗) and parent IFNs IFN-α2c(□) and IFN-α21a(▿) were used as competitors. The experiments were performed in duplicate.

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This assay was performed as described previously (23).

IFN binding assays were performed as previously described on human Daudi (13) and WISH (24) cells.

Human IFN-α2c, IFN-α21a, 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-αs 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 × 106 to 4.5 × 106 IU/mg protein on MDBK cells. The IFN-αs were further purified by 4F2 or NK2 mAb affinity chromatography (12). After the two purification steps, each IFN-α 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-αs are shown in Table I; they ranged from 2 × 108 to 3.7 × 108 IU/mg protein on MDBK cells and 0.1 × 108 to 1.9 × 108 IU/mg protein on WISH cells. The antiviral sp. act. of the three hybrids were similar to each other and to IFN-α2c and IFN-α21a on MDBK cells (2.0 × 108 IU/mg to 3.7 × 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-α2c, or IFN-α21a on WISH cells.

FIGURE 1.

The amino acid sequences of IFN-α2c, IFN-α21, and IFN-α hybrids HY-1, HY-2, and HY-3. The amino acid leader sequence for all of these sequences is MRGSHHHHHHGS.

FIGURE 1.

The amino acid sequences of IFN-α2c, IFN-α21, and IFN-α hybrids HY-1, HY-2, and HY-3. The amino acid leader sequence for all of these sequences is MRGSHHHHHHGS.

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Table I.

Summary of antiproliferative, antiviral, and binding activities of hybrid and parent IFNs

IFNAntiproliferative Activity (ng/ml)aSpecific Antiviral Activity (IU/mg)Binding Activity (competes with 125I-IFN-α2b) (ng/ml)b
Daudi cellsWish cellsRatio wish/DaudiMDBK cellsWish cellsRatio MDBK/WishDaudi cellsWish cells
IFN-α2c 0.005 80 1.6 × 104 3.3 × 108 1.9 × 108 1.7 35 25 
IFN-α21a 0.0008 95 1.2 × 105 3.7 × 108 0.7 × 108 5.3 >400 >200 
HY-1 0.5 110 2.2 × 102 2.0 × 108 0.1 × 108 20.0 >400 >200 
HY-2 1.0 >1000 >1 × 103 3.0 × 108 0.1 × 108 30.0 >400 >200 
HY-3 0.0001 8 × 104 2.0 × 108 0.7 × 108 2.9 30 30 
IFNAntiproliferative Activity (ng/ml)aSpecific Antiviral Activity (IU/mg)Binding Activity (competes with 125I-IFN-α2b) (ng/ml)b
Daudi cellsWish cellsRatio wish/DaudiMDBK cellsWish cellsRatio MDBK/WishDaudi cellsWish cells
IFN-α2c 0.005 80 1.6 × 104 3.3 × 108 1.9 × 108 1.7 35 25 
IFN-α21a 0.0008 95 1.2 × 105 3.7 × 108 0.7 × 108 5.3 >400 >200 
HY-1 0.5 110 2.2 × 102 2.0 × 108 0.1 × 108 20.0 >400 >200 
HY-2 1.0 >1000 >1 × 103 3.0 × 108 0.1 × 108 30.0 >400 >200 
HY-3 0.0001 8 × 104 2.0 × 108 0.7 × 108 2.9 30 30 
a

Concentration of IFN species that inhibits cell growth by 50%.

b

Concentration of IFN species that inhibits binding of 125I-IFN-α2b by 50%.

The ability of the IFN-α hybrids and the parental IFN-αs 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-αs 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-α2c and IFN-α21a 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-αs 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.

FIGURE 3.

Antiviral studies of IFN-α2c, IFN-α21a, and the IFN-α hybrids using primary human lymphocytes. Comparison of no IFN (□, No IFN) vs treatment with parental (▨, α2C and ▩, α21a) and hybrid (▧, HY2 and ▦, HY3) IFN on measles virus replication in primary human lymphocytes. A total of 1 × 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.

FIGURE 3.

Antiviral studies of IFN-α2c, IFN-α21a, and the IFN-α hybrids using primary human lymphocytes. Comparison of no IFN (□, No IFN) vs treatment with parental (▨, α2C and ▩, α21a) and hybrid (▧, HY2 and ▦, HY3) IFN on measles virus replication in primary human lymphocytes. A total of 1 × 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.

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To examine the cell-surface binding properties of the hybrid molecules, competitive binding analysis was performed with IFN-α2c, IFN-α21a, the hybrids, and IFN-α2b. Fig. 4 shows the ability of IFN-α2c, IFN-α21a, and the three hybrids (HY-1, HY-2, HY-3) to compete for IFN-α2b binding sites on Daudi and WISH cells, respectively. IFN-α2c and the hybrid HY-3 inhibited the binding of 125I-IFN-α2b to Daudi and WISH cells very effectively, while hybrids HY-1 and HY-2 like IFN-α21a, poorly inhibited 125I-IFN-α2b binding on both cell lines. These data suggest that the amino terminal region of the IFN-α molecule is important in binding to its receptor.

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-α 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.

FIGURE 5.

HY-2 and HY-3 both activate STAT1- and STAT2 -containing DNA binding complexes. EMSA of extracts from HY-2- and HY-3-treated Daudi, Wish, and primary human lymphocytes are shown. Nuclear extracts (10 μg/lane) were prepared from 2 × 107 cells treated for 30 min with either the HY-2 or HY-3 IFN construct at concentrations chosen from the antiproliferative profiles of each of the cells. For the Daudi cells, this was 0.1 pg/ml for HY-3 and 10 pg/ml for HY-2; for the Wish cells, this was 1 ng/ml for HY-3 and 10 ng/ml for HY-2; and for the primary human lymphocytes, this was 1 ng/ml for HY-3 and 100 ng/ml for HY-2. The extracts were incubated with radiolabed GRR oligonucleotide previously shown to specifically bind IFN-α-activated STAT1 (A) or the ISRE oligonuleotide shown to specifically bind the IFN-α activated STAT2-containing ISGF3 complex (B).

FIGURE 5.

HY-2 and HY-3 both activate STAT1- and STAT2 -containing DNA binding complexes. EMSA of extracts from HY-2- and HY-3-treated Daudi, Wish, and primary human lymphocytes are shown. Nuclear extracts (10 μg/lane) were prepared from 2 × 107 cells treated for 30 min with either the HY-2 or HY-3 IFN construct at concentrations chosen from the antiproliferative profiles of each of the cells. For the Daudi cells, this was 0.1 pg/ml for HY-3 and 10 pg/ml for HY-2; for the Wish cells, this was 1 ng/ml for HY-3 and 10 ng/ml for HY-2; and for the primary human lymphocytes, this was 1 ng/ml for HY-3 and 100 ng/ml for HY-2. The extracts were incubated with radiolabed GRR oligonucleotide previously shown to specifically bind IFN-α-activated STAT1 (A) or the ISRE oligonuleotide shown to specifically bind the IFN-α activated STAT2-containing ISGF3 complex (B).

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FIGURE 6.

Supershift analysis of EMSA of HY-2 and HY-3 IFNs. EMSA of whole-cell lysates from HY-2- and HY-3-treated primary human lymphocytes are shown and experiments performed as in Fig. 4 except that rabbit anti-STAT1 (top), anti-STAT2 (bottom), and normal rabbit serum Abs were added to the reaction and allowed to incubate on ice for 30 min before the addition of either radiolabeled GRR oligonucleotide (top) or radiolabeled ISRE oligonucleotide (bottom).

FIGURE 6.

Supershift analysis of EMSA of HY-2 and HY-3 IFNs. EMSA of whole-cell lysates from HY-2- and HY-3-treated primary human lymphocytes are shown and experiments performed as in Fig. 4 except that rabbit anti-STAT1 (top), anti-STAT2 (bottom), and normal rabbit serum Abs were added to the reaction and allowed to incubate on ice for 30 min before the addition of either radiolabeled GRR oligonucleotide (top) or radiolabeled ISRE oligonucleotide (bottom).

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RNase protection assays were used to assess the ability of the parental and hybrid IFNs to induce several IFN-responsive genes. No differences were seen among the parental and hybrid IFNs in their ability to induce the ISG15 gene in Daudi or WISH cells, and the induction was comparable for HY-2 and HY-3 in PBLs (Fig. 7). 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.

FIGURE 7.

RNase protection assays using IFN-α2c, IFN-α21a, and IFN-α hybrids. The parental and hybrid IFN constructs activate an IFN-α-inducible gene to the same extent at concentrations where antiproliferative activities of HY-2 are not observed. An RNase protection assay was performed using a radiolabeled riboprobe for the ISG15 gene (top) as a marker for the potential of each of the IFNs to induce transcriptional activation of an IFN-regulated gene. Then, 15 μg of total cellular RNA was isolated from cells treated for 16 h with the various IFNs at concentrations of 50 pg/ml, 5 ng/ml, and 100 ng/ml for the Daudi cells, Wish cells, and the primary PHA-treated human lymphocytes, respectively. A probe for GAPDH was included as a housekeeping gene control (bottom).

FIGURE 7.

RNase protection assays using IFN-α2c, IFN-α21a, and IFN-α hybrids. The parental and hybrid IFN constructs activate an IFN-α-inducible gene to the same extent at concentrations where antiproliferative activities of HY-2 are not observed. An RNase protection assay was performed using a radiolabeled riboprobe for the ISG15 gene (top) as a marker for the potential of each of the IFNs to induce transcriptional activation of an IFN-regulated gene. Then, 15 μg of total cellular RNA was isolated from cells treated for 16 h with the various IFNs at concentrations of 50 pg/ml, 5 ng/ml, and 100 ng/ml for the Daudi cells, Wish cells, and the primary PHA-treated human lymphocytes, respectively. A probe for GAPDH was included as a housekeeping gene control (bottom).

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One prominent effect of the IFN-αs 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-α2c, IFN-α21a, 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-αs 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-αs induce their antiproliferative and antiviral effects. Therefore, the IFN-αs may turn on several signaling pathways to different degrees (5). Recently, it has been reported that the antiproliferative effects of IFN-α 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-α (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-β and -α (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-α faces toward one of the subunits of the receptor and are part of one of two binding sites of IFN-α. 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-αs and IFN-β 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-αs and affecting the differential specificity of action between human IFN-αs and IFN-β. It is intriguing to speculate that these residue positions may constitute specific recognition sites associated with an accessory component of the IFN-α receptor. Our studies suggest that region 75–95 is very important for the antiproliferative activity of human IFN-α, with maximal activity observed when the construct has the α2c sequence from 75–95 and the α21a sequence from 96–166.

Our competitive binding experiments show that the hybrids or IFN-α with the α2c domain at the N terminus (residues 1–95; like IFN-α2c and HY-3) compete well with 125I-IFN-α2b binding. In contrast, hybrids or IFN-α with the IFN-α21a domain at the N terminus (residues 1–95; IFN-α21a, HY-1, HY-2) show reduced ability to compete with 125I-IFN-α2b binding. These data suggest that the N terminus is involved in IFN receptor binding interaction. Several reports have proposed two binding sites for IFN-α, 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 [α2c(75-95)/α21a(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.

We thank Joan C. Enterline for performance of antiviral assays, Valerie Calvert for elutriation of lymphocytes, and Raj Puri and Barbara Rellahan for review of the manuscript.

1

The sequences presented in this article have been submitted to GenBank under accession numbers HY-1 AF085803, HY-2 AF085804, and HY-3 AF085805.

3

Abbreviations used in this paper: MDBK, Madin-Darby bovine kidney; GRR, γ response region; ISRE, IFN-stimulated response element.

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